Citation
Identification and characterization of intermediate filament binding proteins in the nervous system

Material Information

Title:
Identification and characterization of intermediate filament binding proteins in the nervous system
Creator:
Errante, Laura Diane, 1965-
Publication Date:
Language:
English
Physical Description:
xv, 191 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Amino acids ( jstor )
Antibodies ( jstor )
Axons ( jstor )
Cells ( jstor )
Gels ( jstor )
Neurons ( jstor )
Phosphorylation ( jstor )
Rats ( jstor )
Spinal cord ( jstor )
Vimentin ( jstor )
Carrier Proteins -- chemistry ( mesh )
Carrier Proteins -- isolation & purification ( mesh )
Central Nervous System -- chemistry ( mesh )
Department of Neuroscience thesis Ph.D ( mesh )
Dissertations, Academic -- College of Medicine -- Department of Neuroscience -- UF ( mesh )
Glyceraldehyde-3-Phosphate Dehydrogenases ( mesh )
Intermediate Filaments ( mesh )
Rats ( mesh )
Research ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1994.
Bibliography:
Bibliography: leaves 174-190.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Laura Diane Errante.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
002335852 ( ALEPH )
50543647 ( OCLC )
ALT9594 ( NOTIS )

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Full Text









IDENTIFICATION AND CHARACTERIZATION OF INTERMEDIATE
FILAMENT BINDING PROTEINS IN THE NERVOUS SYSTEM
















By

LAURA DIANE ERRANTE


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1994




IDENTIFICATION AND CHARACTERIZATION OF INTERMEDIATE
FILAMENT BINDING PROTEINS IN THE NERVOUS SYSTEM
By
LAURA DIANE ERRANTE
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1994


To my parents, with whom I realized how much I could accomplish if only I try,
and to my dearest Gator, who would never let me lose sight of my dreams.


ACKNOWLEDGEMENTS
Although it is now time to begin anew, I will never forget the people I have
met that have made the past six years a learning experience. Foremost I would
like to thank Dr. Gerry Shaw, my advisor, who showed me, through his example,
that although it may be difficult at times to do bench work, that this is why we
become scientists in the first place. In addition, I appreciate his patience,
especially in allowing me to develop my own sense of independence. I would like
to acknowledge my committee members, Dr. Barabara Battelle, Dr. Kevin
Anderson, and Dr. Daniel Purich, for their insight and support on my doctoral work.
I would like to credit Dr. Luttge for having the wisdom 5 years ago to let me know
that although I was having difficult times, I still had the ability to become a good
scientist. I only hope that the present and future graduate students in the
department will benefit from his wisdom as I have over these years. It is easiest
to remember to thank the people who have had direct input on your research;
however, I would like to give special thanks to Dr. Roger Heintz, my undergraduate
advisor at SUNY Plattsburgh, for instilling in me the basics of learning techniques,
especially column chromatography which has been essential to my dissertation
research, and for his encouragement to enter a field of study that can be
intimidating at times but is always fascinating and rewarding.
There are a number of graduate students and staff that have made an


indulible impression on me. In particular I will always treasure my friendships with
Paul Bao, Michele Davda, Bunnie Powell, and Margaret Tremwel. I would like to
acknowledge my family, especially my mom and brother, Wayne, who may not
have an understanding of what I have been doing the past six years but have
supported me just the same, no matter how infrequently they heard from me.
It has been a long and sometimes twisted road to reach this point, and I
am fortuitous to have found a true friend in Rob Friedman. Even when I thought
all was lost, his constant and unconditional support as well as critical assessment
of my research, enable me to envision the "light at the end of tunnel" and make
my dissertation a reality.
This work was supported by a NIH Predoctoral Training Fellowship
MH15737 through the Center for Neurobiological Sciences at the University of
Florida and NIH NS22695 (to Dr. Gerry Shaw). In addition, I would like to thank
the Center for Neurobiological Sciences at the University of Florida for providing
funds to attend scientific meetings.
IV


TABLE OF CONTENTS
ACKNOWLEDGEMENTS iii
LIST OF TABLES viii
LIST OF FIGURES lx
LIST OF ABBREVIATIONS xii
ABSTRACT xiv
CHAPTERS
1 INTRODUCTION AND BACKGROUND 1
Cytoskeleton 1
Intermediate Filament Family of Proteins 2
Neurofilament Assembly 8
NF Phosphorylation 11
Interactions Between Neurofilaments and Microtubules 18
Neurofilaments Interact with Other Cellular Components .... 21
Relationship of NFs to Axonal Transport and Caliber 26
NF Expression After Axotomy 30
Role of NFs in Disease States 32
Overview of Dissertation 34
2 GENERAL METHODS 36
Biochemical Techniques 36
Protein Assay 36
SDS Polyacrylamide Gels 37
Electroblotting of Proteins 38
Immuno-Blot Analysis 39
Protein Cleavage with Cyanogen Bromide 40
Isolation of Neurofilament Proteins 41
Crude Intermediate Filament Preparation 41
Neurofilament Preparation 42
Purification of Individual Neurofilament Subunits 43
v


Anatomical Localization Experiments 44
Dorsal Root Ganglion Cell Cultures 44
Pheochromocytoma Cells (PC12 Cells) 45
Immunofluorescent Studies on Fresh Frozen Tissue 46
Immunocytochemistry on Formaldehyde Fixed Tissue .... 47
3 THE DISTRIBUTION OF PLECTIN, AN INTERMEDIATE
FILAMENT BINDING PROTEIN, IN THE ADULT RAT
CENTRAL NERVOUS SYSTEM 49
Introduction 49
Methods 50
Results 52
Immunoblot Studies 52
Comparison of Plectin to IFAP-300 56
Localization Studies 59
Discussion 70
Notes 78
4 CO-LOCALIZATION OF PLECTIN AND INTERMEDIATE
FILAMENTS IN THE RAT NERVOUS SYSTEM 79
Introduction 79
Methods 80
Results 82
Immunoblot Studies 82
Co-localization in Cultured Cells 82
Co-localization Studies Along the Rat Neural Axis 90
Effects of Peripheral Nerve Axotomy 97
Discussion 105
Notes 115
5 IDENTIFICATION OF NF BINDING PROTEINS 116
Introduction 116
Methods 117
Pig Spinal Cord Cytosolic Preparation 117
Affinity Column Preparation 117
Binding of Cytosolic Proteins to Affinity Columns 118
Production of Polyclonal and Monoclonal Antibodies .... 121
Results 122
Discussion 138
VI


6 CHARACTERIZATION OF GAPDH BINDING TO NFs
144
Introduction 144
Methods 145
Co-sedimentation Experiments 145
Immunofluorescence Studies 146
Antibodies 146
Results 147
Co-sedimentation Experiments 147
Immunofluorescence Studies in Cultured Cells 154
Discussion 161
7 OVERALL DISCUSSION 165
General Considerations 165
Criteria for Classifying IF Binding Proteins 165
Relationship between Plectin and NF Proteins 166
The Role of GAPDH in the Nervous System 168
Future Direction 170
Conclusions 173
REFERENCES 174
BIOGRAPHICAL SKETCH 191
VII


LIST OF TABLES
Table 1-1 Mammalian intermediate filament protein family based
on the classification scheme of Steinert and Roop (1988) .... 2
Table 1-2 Protein kinases which phosphorylate neurofilaments in vitro . 14
Table 1-3 Interactions of neurofilaments with other cellular components 22
Table 3-1 The distribution of plectin in different cell types of the adult
rat central nervous system 76
Table 4-1 Summary of the distribution of plectin in different cell types
of the adult rat nervous system with respect to
intermediate filament proteins 111
Table 5-1 Relative molecular weight (kD) of candidate proteins which
bound to the five different types of NF affinity columns .... 123
Table 5-2 Amino acid composition for candidate neurofilament binding
proteins with the data represented as nanomole percent .... 127
Table 5-3 Results from the FINDER program to identity NFL-38 131
Table 5-4 Comparison of NFL-38 to GAPDFI with data represented
as nanomole percent for each amino acid 132
VIII


LIST OF FIGURES
Figure 1-1 Schematic of neural intermediate filament protein sequences 5
Figure 3-1 Identification of a 300 kD protein in bovine and rat crude
IF spinal cord preparations 55
Figure 3-2 Comparison of amino acid composition of plectin and
IFAP-300 proteins 58
Figure 3-3 Plectin immunoreactivity (ID8) of astrocytes in white and
gray matter of the telencephalon and diencephalon 61
Figure 3-4 Plectin immunoreactivity (ID8) in the rostral hypothalamus
surrounding the third ventricle (3V) as visualized with DAB . 64
Figure 3-5 Plectin immunoreactivity (IA2 and ID8) in the cerebellum ... 66
Figure 3-6 Plectin immunoreactivity (ID8) in the caudal brainstem 69
Figure 3-7 Plectin immunoreactivity (ID8) in the cervical spinal cord .... 72
Figure 3-8 Plectin immunoreactivity (p21) in choroid plexus and
blood vessels 74
Figure 4-1 Characterization of peripherin polyclonal and monoclonal
antibodies 84
Figure 4-2 Co-localization of plectin (p21) and vimentin (V9) in
non-neuronal cells from DRG cultures 87
Figure 4-3 Co-localization of plectin (ID8) and NF subunits in
DRG cells and PC12 cells 89
Figure 4-4 Co-localization experiment with antibodies to plectin
(p21) (A,C,E,G) and vimentin (V9) (B,D,F,H) 93
Figure 4-5 Co-localization experiment with antibodies to plectin
(A,C,E) and GFAP (B,D,F) 95
Figure 4-6 Co-localization experiment with antibodies to plectin
(ID8) (A.C.E) and NFs (polyclonal) (B,D,F) 99
Figure 4-7 Co-localization experiment with antibodies to plectin
(IA2) (A,C) and peripherin (R19) (B,D) 101
IX


Figure 4-8 Effects of a unilateral facial nerve axotomy on plectin
immunoreactivity (1A2) 104
Figure 4-9 Co-localization experiment with plectin (IA2) (A,C)
and NF-FI (R14) (B,D) after a unilateral facial nerve
axotomy 107
Figure 4-10 Co-localization experiment with plectin(IA2) (A,C)
and peripherin (R20) (B,D) after a unilateral facial
nerve axotomy 109
Figure 5-1 Outline of the method used for identifying candidate
NF binding proteins 120
Figure 5-2 Comparison of candidate 38 kD NF binding protein
to rod domain of IF proteins 126
Figure 5-3 Comparison of amino acid composition data between
NFL-38 and IF-38, and to that of the average amino
acid composition in Genbank 130
Figure 5-4 Comparison of CNBr cleavage fragments of NFL-38
and GAPDFI 135
Figure 5-5 Immunoblot analysis using antibodies to GAPDH to
determine if the 38 kD proteins binding to various NF
affinity columns is GAPDFI 137
Figure 5-6 Graphical representation of the amino acid composition
data for NFL-16, and possible matches as determined
by the FINDER program 140
Figure 5-7 Graphical representation of the amino acid composition
data for NFL-62, and to that of the average amino acid
composition in Genbank 142
Figure 6-1 Co-sedimentation experiment with GAPDFI and NF subunits 149
Figure 6-2 Co-sedimentation of GAPDFI and NF-L with different
concentrations of GAPDFI 151
Figure 6-3 Co-sedimentation of GAPDFI and NF-L with different
concentrations of sodium chloride 153
Figure 6-4 Composite of GAPDFI antibody labelling (ID4) of a DRG
cultured neuron 156
Figure 6-5 Co-localization experiment in DRG cell cultures with
antibodies to GAPDH (ID4) (A,C) and NF-H (R14) (B,D) ... 158
x


Figure 6-6 Co-localization experiment in differentiated PC12
cell cultures with antibodies to GAPDH (ID4) (A,B)
and NF-M (R9) (C) 160
Figure 6-7 Co-localization experiment in DRG cell cultures with
antibodies to GAPDH (ID4) (A) and actin (polyclonal) (B) .. 163
XI


LIST OF ABBREVIATIONS
Amino Acids
ALA or A
-
alanine
ARG or R
-
arginine
ASN or N
-
asparagine
ASP or D
-
aspartic acid
ASX or Z
-
asparagine and/or aspartic acid
CYS or C
-
cysteine
GLN or Q
-
glutamine
GLU or E
-
glutamic acid
GLX or B
-
glutamine and/or glutamic acid
GLY or G
-
glycine
HIS or H
-
histidine
ILE or 1
-
isoleucine
LEU or L
-
leucine
LYS or K
-
lysine
MET or M
-
methionine
PHE or F
-
phenylalanine
PRO or P
-
proline
SER or S
-
serine
THR orT
-
threonine
TRP or W
-
tryptophan
TYR or Y
-
tyrosine
BBB
- blood-brain barrier
BSA
- bovine serum albumin
CBB
- Coomassie brillant blue R-250
CNBr
- cyanogen bromide
CNS
- central nervous system
CSF
- cerebrospinal fluid
DAB
- diaminobenzidine tetrahydrochloride
DRG
- dorsal root ganglion
DTT
- dithiothreitol
EDTA
- ethylenediamine tetraacetic acid
XII


FBS
- fetal bovine serum
GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
GFAP
- glial fibrillary acidic protein
GST
- glutathione S-transferase
IF
- intermediate filament
IFAP
- intermediate filament associated protein
kD
- kiloDaltons
MES
- 2-(4-morpholino)-ethanesulfonic acid
MT
- microtubule
NF
- neurofilament
NF-H
- neurofilament high molecular weight subunit
NF-M
- neurofilament middle molecular weight subunit
NF-L
- neurofilament low molecular weight subunit
TBS
- tris buffered saline
PBS
- phosphate buffered saline
PMSF
- phenylmethyl-sulfonyl fluoride
PNS
- peripheral nervous system
PVDF
- polyvinylidine difluoride
SDS
- sodium dodecyl sulfate
TAME
- Na-p-toluenesulfonyl-L-arginine methyl ester
XIII


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
IDENTIFICATION AND CHARACTERIZATION OF INTERMEDIATE
FILAMENT BINDING PROTEINS IN THE NERVOUS SYSTEM
By
LAURA DIANE ERRANTE
April 1994
Chairperson: Gerard P. J. Shaw
Major Department: Neuroscience
Neurofilaments (NFs), microfilaments (MFs) and microtubules (MTs) are the
major structural proteins that form the neuronal cytoskeleton. Much more is
understood about the function of MFs and MTs than is known for NFs. This
dissertation research characterized, plectin, a known intermediate filament
associated protein (IFAP) in the nervous system, and searched for candidate NF
binding proteins in order to examine possible roles for NFs.
Plectin distribution was examined throughout the rat CNS. Plectin was
localized to non-neuronal cells with particularly strong immunoreactivity in cells
forming ventricular and pia barriers. In addition, plectin immunoreactivity was
observed in select motoneurons. Double-label studies with plectin and IF proteins
demonstrated that plectin's distribution most closely resembled that for vimentin;
XIV


however, the staining patterns were not mutually inclusive. In select motoneurons,
plectin colocalized with the neural IF proteins, NF triplet proteins and peripherin.
Since plectin and NFs co-exist in the same cell, the previously described in vitro
plectin/NF interactions may be functionally important.
The second part of the dissertation project involved identifying a 38 kD
protein which bound to NF affinity columns. Using a computer program, FINDER,
and biochemical analysis, this protein was identified as glyceraldehyde-3-
phosphate dehydrogenase (GAPDF1). The strength of binding between GAPDFI
and NF-L subunit was shown to be relatively weak with a dramatic decrease in the
amount of protein co-pelleting at 0.05 M sodium chloride. The possible in vivo
interaction between NFs and GAPDFI was examined using fluorescent light
microscopy which showed that GAPDFI was localized throughout the cell body and
processes of dorsal root ganglion cells in culture and differentiated PC12 cells.
Although GAPDFI immunoreactivity colocalized in part with NF antibody labelling,
the immunofluorescent labelling patterns were distinct.
A number of proteins were identified which bound with varying specificity to
NFs in vitro and have a potential of interacting with NFs in vivo. Finding that NFs
may interact, in vivo, in some neurons with plectin and GAPDFI suggests that
certain NF proteins may act as important structural elements during neuronal
injury, and as docking substrates for the localization of glycolytic enzymes.
xv


CHAPTER 1
INTRODUCTION AND BACKGROUND
Cytoskeleton
The cytoskeleton is a three-dimensional network of fibrous proteins found
throughout the cytoplasm. The neuronal cytoskeleton is composed of microtubules
(MTs), microfilaments (MFs) and neurofilaments (NFs). MTs and MFs have been
well characterized in both neuronal and non-neuronal cells, and both have been
shown to have a variety of roles in cell structure and motility. In contrast, very little
is understood about the functional role of NFs (Fliegner and Liem, 1991; Shaw,
1991). NFs are primarily thought of as static structures ensuring mechanical
stability to the neuron and governing the diameter of the axon (Hoffman et al.,
1984). However, changes in phosphorylation state of NFs during development and
regeneration indicate that NFs may play a more active role in neuronal function.
This chapterwill describe pertinent information concerning NF structure, assembly,
phosphorylation, interactions with other proteins, and NFs involvement in axonal
transport and caliber. The last part of this chapter will discuss the effects on NF
proteins of axotomy and neuronal diseases which may indicate possible functions
for these neural intermediate filaments.
1


2
Intermediate Filament Family of Proteins
NFs belong to a family of intermediate filament (IF) proteins which are 10
nm diameter. The IF family is currently grouped into 6 classes of proteins based
on protein sequence similarity and intron placement (Table 1-1).
Table 1-1. Mammalian intermediate filament protein family based on the
classification scheme of Steinert and Roop (1988).
Class Name
MW (kD)
Cellular Location
I
Keratin (acidic)
40-60
epithelial cells and epidermal
II
Keratin (basic)
50-70
derivatives (eg. nails and hair)
ii ii
III
Desmin
52
muscle cells
Vimentin
57
cells of mesenchymal origin
GFAP*
51
astrocytes; some schwann cells
Peripherin
57
neurons
IV
NF-triplet proteins
NF-H11
115
neurons
NF-M§
95
ii
nf-l¥
60
ii
a-Internexin
66
ii
V
Nuclear Lamins
60-70
nuclear lamina of all cells
IV
Nestin
200
neural epithelial stem cells
GFAP: glial fibrillary acidic protein; NF-H:
NF high molecular weight protein;
§NF-M: NF middle molecular weight protein;
:Â¥NF-L: NF low molecular weight protein
A number of IF proteins are localized to neurons. The classical NFs, NF
triplet proteins, are found in varying concentration in most nerve cells. (Liem et al.,
1978; Yen and Fields, 1981; Shaw et al., 1981). In addition to the NF triplet
protein, a-internexin and peripherin are localized to a more select set of neurons


3
and have been shown to co-localize with NF-triplet protein (Kaplan et al., 1990;
Parysek et al., 1991). a-lnternexin is distributed predominantly in the central
nervous system (CNS) (Pachter and Liem, 1985; Chiu et al., 1989). Peripherin is
localized mainly in the peripheral nervous system (PNS) (Portier et al., 1984a,
1984b), but is found also in select fiber tracks and perikarya of neurons throughout
the CNS (Parysek and Goldman, 1988; Brody et al., 1989). Although vimentin is
primarily thought to associate with non-neuronal cells, it is localized in developing
neurons and is expressed in mature neurons in the olfactory epithelium (Schwob
et al., 1986) and adult retina (Draeger, 1983). Nestin, a newly described protein
assigned to class VI, is widely expressed in neuroepithelial stem cells which are
the precursors of CNS neurons and glia (Hockfield and McKay, 1985; Frederiksen
and McKay, 1988; Lendahl et al., 1990).
IFs, in general, have a fundamental structure comprised of 3 domains: (1)
amino-terminal head; (2) central a-helical rod domain; and (3) variable carboxyl-
terminal tail (Figure 1.1). The amino-terminal head domain is typically a short
globular peptide sequence of 5-7 kD containing numerous basic amino acids
(Geisler et al.,1982). Flowever, the amino-terminal domain of nestin is only 11
amino acids long and is slightly acidic (Lendahl et al., 1990). The central rod
domain (39 kD) contains a highly conserved heptad repeat of hydrophobic amino
acids that allows for the formation of an a-helical coiled-coil structure (Geisler et
al., 1982). The distinct primary amino acid sequence of the rod domain is
proposed to be the driving force of IF protein self-assembly (Steinert and Roop,


Figure 1-1. Schematic of neural intermediate filament protein sequences.
Intermediate filaments can by divided into 3 domains: (1) amino-terminus head domain; (2) a-
helical rod domain; and (3) variable carboxyl-terminus domain. Each of these domains can be
divided into regions based on repeated sequence or abundance of a amino acids in that region.
These regions are denoted with the single letter code for the amino acids. E: glutamic acid; K:
lysine; P: proline.


Vi menti n
Peri pheri n
a-lnternexin
NF-L
NF-M
NF-H
Nesti n
n
Coil 1a Coil 1b Coil 2 Tail
Coil 1a Coil 1b Coil 2 Tail
Ooil 1a Coil 1b Coil 2 Tail a E S KE segments
Coil 1a Coil 1b Coll 2 Tall a E segment
EHTIW-C
Colli Coil2 Taila E1 KSP1 E2 KSP2 KE s SP segments
Coil 1 Coil 2 Tail a KEQP segment
N-terminal Head
(5-7 kD)
a-Helical Rod
(39 kD)
Variable C-terminal Tail
(5-70 kD)
ui


6
1988). One unifying feature of all IFs is a highly conserved epitope at the end of
the rod domain (coil II) which is recognized by the anti-intermediate filament
antibody (a-IFA) (Pruss et al., 1981). Most IF subunits, such as vimentin, have a
5-10 kD globular tail sequence at the extreme C-terminus of the molecule.
Flowever, the class IV and V IF proteins differ from other IFs in that they have long
carboxyl-terminal extensions which contain several distinct types of unusual amino
acid sequences.
The NF carboxyl-terminal tails appear to have very little a-helical or 3-sheet
structure and are thus thought to consist of largely random coils (Geisler et al.,
1985b). The NF tail domain can be divided into 4 regions: (1) tail A; (2) glutamic
rich segment (E segment); (3) lysine-serine-proline repeat segment (KSP
segment); and (4) lysine and glutamic rich segment (KE or KEP segment). It has
yet to be determined if these sequence specific regions are actually functional
domains. In contrast to the other IF subunits, nestin's carboxyl-terminal tail domain
has a distinct repeat segment (KEQP) which is repeated 35 times (Lendahl et al.,
1990). The abundance of glutamic acid residues in the carboxyl-terminal of nestin
is the only similarity with the carboxyl-terminal of class IV IF proteins (Fliegner and
Liem, 1991).
The latter two segments of NF-M and NF-H carboxyl terminal tails (KSP and
KE or KEP) are thought to correspond to the rodlets protruding from the core
filament seen in ultra-structural studies (Hisanaga and Hirokawa, 1988). The
sequence motif of K-SP or K-SP is also present in microtubule associated proteins


7
tau and MAP2 (Shaw, 1989; Shaw, 1991). The K-SP and K-SP sites in pig NF-M
and K-SP site in rat NF-M are phosphorylation sites in vivo (Geisler et al., 1987;
Xu et al., 1989). At the extreme terminus of NF-M is a highly conserved region
known as the KE segment which is rich in lysine and glutamic acid. This highly
charged segment may be important in interactions with other neuronal components
since it is highly conserved and a closely related sequence is also present in the
extreme C-terminus of a-internexin (Shaw, 1991).
NF-H is somewhat similar to NF-M but has a distinct tail domain. In
contrast to NF-M, NF-H has a KSP segment that contains far more repeats of the
KSP motif. In rat, this KSP motif is repeated 60 times. In addition, the KSP repeat
sequences in NF-H are distinct from those found in NF-M (Shaw, 1991). Instead
of a KE region at the C-terminus, NF-H has a segment rich in lysine, glutamic acid
and proline (KEP) which is evolutionarily more loosely conserved (Shaw, 1991).
Recently, the different tail segments of NF-M and NF-H have been
dissected out molecularly and expressed as fusion proteins (Harris, et al., 1991).
These specific segments of the high MW NF tail regions can be used to examine
the functional significance NF through examining their interactions with NFs, other
neuronal components and by using these defined protein segments as substrates
for protein kinase assays.


8
Neurofilament Assembly
The way in which individual NF subunits interact and assemble into NFs is
not clearly understood. NF-L appears to be the core structure of assembled 10 nm
filaments since urea solubilized NF-L monomers self assemble into homopolymeric
structures when urea is removed (Geisler and Weber, 1981). NF-M and NF-H
appear to be incapable of forming long 10 nm filaments, however, when NF-L is
present, both NF-M and NF-H are readily incorporated into the 10 nm filament
(Geisler and Weber, 1981; Liem and Hutchison, 1982). Antibody studies (Willard
and Simon, 1981; Hirokawa et al., 1984) and rotary shadowing experiments
(Hisanaga and Hirokawa, 1988) support the idea that NF-L is the central core of
the filament, and NF-M and NF-H are incorporated into this core by their rod
domains where the carboxyl-terminal tail domains of NF-M and NF-H protrude from
the central core of the filament. Recently, rotary shadowing experiments with
antibodies to the tail domain of NF-M and NF-H confirm that the tail regions of NF-
M and NF-H correspond to the protrusions seen ultrastructurally with NFs (Mulligan
et al., 1991). A basic question which remains is how individual IF subunits, in
general, interact to form 10 nm filaments.
The majority of research on IF assembly has focused on class III IFs,
desmin and vimentin, which can form homopolymers in vivo. When IFs form
dimers it is believed that the hydrophobic portions of the a-helical coil regions of
two polypeptide chains interact in a parallel fashion and form a coiled-coil structure
(Parry et al., 1982). In addition to satisfying hydrophobic considerations, the coil-


9
coil type structure allows for favorable charge interactions along the rod domain.
The dimers are then thought to form four chain tetramers either in a parallel (Ip,
1988; Hisanaga et al., 1990), anti-parallel (Geisler et at., 1985a) or staggered anti
parallel manner (Stewart et al., 1989). The differences in tetrameric formation may
be related to the different IF subunits used or the way in which tetramers interact
with the intact filament network (Steinert and Roop, 1988). Higher oligomers are
then believed to form by a staggering of the tetramers which then results in the
formation of intermediate filaments (Coulombe and Fuchs, 1990; Ip et al., 1985).
Eight tetramers are thought to comprise a single 10 nm filament.
Recently the assembly process of NFs was studied using low angle rotary
shadowing. These studies showed that the basic building block of IF proteins, NF-
L and GFAP, were an eight chain structure formed from two parallel tetramers
(Hisanaga et al., 1990), where four octamers were proposed to comprise a single
10 nm filament. Differences were also found in the way in which the filaments
were reassembled. Hisanaga et al. (1990) showed that NF-L can assemble into
either four chain or eight chain complexes depending on the pH of a low ionic
strength buffer. In preceding experiments, alkaline buffer (pH 8.5) produced
tetramers (Huiatt et al., 1980; Geisler and Weber, 1982; Ip et al.,1985; Hisanaga
et al., 1990); whereas low salt buffer (pH 6.8) favored the formation of octamers
(Hisanaga et al., 1990).
Hisanaga and Hirokawa (1990a) also examined how NFs assemble end to
end to form long 10nm filaments. They found that in the polymerization of NF-L


10
monomers, sodium ions were important for end-t- end association, whereas
magnesium ions were involved in stabilizing lateral associations. When NFs were
reassembled using NF-L, NF-M and NF-H, the assembly conditions remained
unchanged; however, there was a structural change. With addition of either NF-M
or NF-FI, rod-like projections were seen emanating from the central core of the
filament similar to those previously seen using native NFs (Hisanaga and
Hirokawa, 1988).
The domains of individual NF subunits have been examined to determine
which portions of NF sequence are necessary for assembly into 10 nm filaments.
Genetic deletion of the amino terminal head domain and carboxyl terminal tail
domain of the mouse NF-L gene showed that deletions larger than 30% from the
head domain and 90% from the tail domain prevented incorporation of these
proteins into the intermediate filament network (Gill et al., 1990). In contrast, when
deletions were made in NF-M gene, up to 70% of the head domain and 90% of the
tail domain could be missing and NF-M would still be incorporated into filaments
(Wong and Cleveland, 1990). However, deletions into either amino- or carboxyl-
terminal region of the a-helical rod domain of NF-M prevented assembly of this
gene product into the filament network. Therefore, the intact rod domain was
determined to be critical for NF assembly.
Phosphorylation of NF subunits were found to effect filament assembly.
Gonda et al. (1990) demonstrated that protein kinase C phosphorylated a number
of serine residues in the N-terminal head domain of NF-L and that an increase in


11
phosphate incorporation decreased the rate of polymerization of NF-L monomer
into filaments. In addition, cAMP dependent protein kinase was shown to
phosphorylate NF-L and was found to inhibit assembly of NF-L monomers into
filaments at phosphorylation levels of 1 mol/mol of protein (Nakamura et al., 1990).
Finally, phosphorylation of NF-L filaments resulted in a slow disassembly which
took up to 6 hours. Nakamura and co-workers proposed that controlled
phosphorylation may modulate the dynamic equilibrium between assembly and
disassembly in vivo.
Neurofilament Phosphorylation
The NF triplet proteins are phosphorylated in vivo and the relative amount
of phosphorylation in rat spinal cord was determined by one group to be 3, 6 and
13 moles of phosphate/mole of NF-L, NF-M and NF-H, respectively (Julien and
Mushynski, 1981; Julien and Mushynski, 1982; Xu et al., 1990). It is important to
note that the relative amount of phosphate associated with NFs is variable and
dependent on a number of factors including species, protein preparations, and area
of the nervous system examined. What is generally known is that NF-H appears
to contain the most phosphate per mole of polypeptide followed by NF-M then NF-
L. In the case of NF-H and NF-L the phosphate is associated with serine residues,
whereas, NF-M has phosphorylation sites on both serine and threonine residues
in the rat (Julien and Mushynski, 1982).


12
Phosphorylation sites on NF triplet proteins are localized to particular
regions on the individual NF subunits. In the case of rat NF-L, phosphorylation
sites are found in both the amino head and carboxyl tail domains (Sihag and
Nixon, 1989) with the major phosphorylation site located in the middle of carboxyl-
terminal tail domain (Xu et al. (1990). In addition, in vitro phosphorylation
experiments with NF-L and protein kinase C show that there are 3 phosphorylation
sites on serine residues in NF-L amino-head domain (Gonda et al., 1990). A
detailed study of NF-M phosphorylation sites of rat neurons in tissue culture
demonstrated that there are six sites in NF-M carboxyl-terminus, four of which
have the KSP motif, one with the K--SP motif, and one motif with a serine residue
followed by glutamic acids (Xu et al., 1989). For NF-H the only known
phosphorylation sites are in the multiple KSP repeats, though it is likely that other
phosphorylation sites exist in other regions of this large molecule (Geisler et al.,
1987: Lee et al.,1988).
Three major questions still remain with respect to NF phosphorylation: (1)
what kinase activities are phosphorylating these different regions of NF subunits
in vivo; (2) are these kinases unique to NFs and/or are they previously
characterized protein kinases; and (3) what is the function of NF phosphorylation?
A number of laboratories have examined NF phosphorylation (Table 1.2). The
research with known kinases has focused primarily on NF-L subunit; whereas, NF-
M and NF-H have been utilized to search for novel protein kinase activities
responsible for phosphorylating the KSP repeat motif of NFs.


13
Three classical protein kinases were shown to phosphorylate NFs in vitro
to various degrees (Table 1-2): (1) calcium/calmodulin protein kinase II (CaM
kinase II) (Vallano et al., 1985); (2) cyclic AMP-dependent protein kinase (protein
kinase A) (Tanaka, 1984; Dosemeci and Pant, 1992); and (3) protein kinase C
(Sihag et al., 1988). In vivo phosphorylation experiments examining NF-L and NF-
M demonstrated that most of the fragments that are phosphorylated with one of the
known protein kinases are located in the amino terminal head domain (Sihag and
Nixon, 1989, 1990).
The search for the protein kinase which phosphorylates the KSP repeat
motif in NF-M and NF-H has focused on characterizing the protein kinase activities
which bind strongly to NFs during crude purification procedures (Table 1 -2). These
kinase activities are extracted from NF preparations using high salt buffer and have
been shown to contain a number of protein kinases: (1) CaM kinase II; (2)
nucleotide-dependent kinase; (3) calcium-phosphatidylserine-diglyceride-dependent
kinase; and (4) a regulator independent kinase (Dosemeci et al, 1990). The
regulator independent kinase phosphorylated all three NF subunits as well as a-
casein where the level of phosphate incorporated per mole of protein was in the
following order: a-casein > NF-M > NF-H = NF-L. This kinase activity was shown
to be related to casein kinase I based on: (1) similarities in specificity of protein
kinase inhibitors; (2) the ability of casein kinase I to phosphorylate NFs in a similar
manner to the NF-associated kinase; and (3) the molecular weight range for casein


14
Table 1-2. Protein kinases which phosphorylate neurofilaments in vitro.
Protein Kinase
Substrate
MW* (kD)
Reference
cAMP dependent
(rat brain)
NF-H, -M, -L
40
Tanaka et al., 1984
Dosemeci and Pant, 1992
Ca27Calmodulin
dependent (rat)
NF-H, -M, -L
640
Vallano et al., 1985
Protein Kinase C
(mouse)
NF-H, -M, -L
77
Sihag et al., 1988
Casein Kinase I
NF-H, -M, -L
30-42
Floyd et al., 1991
cdk1 Kinase
NF-H, -M
34
Hisanaga et al., 1991
nclk Kinase11
NF-H, -M
33
Lew et al., 1992
NF-Kinase (1983)
(rat)
NF-H, -M, -L
GFAP, vimentin
MAPs, tau
40
Toru-Delbauffe & Pierre, 1983
Toru-Delbauffe et al., 1986
NF-Kinase: 1989
(bovine)
NF-H, -M
histone H1
67
Wible et al., 1989
NF-Kinase (1990)
(bovine)
NF-H, -M, -L
casein
Dosemeci et al., 1990
NF-Kinase (1991)
(squid)
NF-H, -M, -L
casein
17-44
Floyd et al., 1991
* molecular weight expressed in kiloDaltons
11 proline directed kinase


15
kinase I (Hathaway and Traugh, 1982) was comparable to that of NF-associated
kinase (Floyd et al., 1991).
Using the salt extraction technique, other laboratories have isolated
additional NF protein kinase activities, where their molecular weight and substrate
specificity suggest that these NF kinase are distinct from each other. Toru-
Delbauffe and Pierre (1983) were able to recover two endogenous protein kinase
activities, one which phosphorylated NFs, and the other which phosphorylate
casein and was identified as casein kinase I. The NF specific protein kinase was
purified by phosphocellulose chromatography and resulted in major band at 40 kD
and a minor band at 200 kD on SDS polyacrylamide gels, and appeared to be co
factor independent (Toru-Delbauffe et al., 1986). Wible et al. (1989) partially
purified a 67 kD doublet on SDS polyacrylamide gels and, in contrast to the
previous kinases, their kinase showed more substrate specificity with a preference
for partially dephosphorylated NF-H.
Even though some of the kinases had a preference for the
dephosphorylated form of NF-H, phosphorylation by these various NF kinase
activities did not result in the expected molecular weight shift if the
dephosphorylated NF-H was being phosphorylated in the KSP repeat sequence.
Previous work has demonstrated that the two high molecular weight NF subunits,
NF-H and NF-M, mobility on SDS polyacrylamide gels is greatly retarded due to
the large number of phosphates in the carboxyl-terminal tall domain. Hisanaga
and co-workers (1991) demonstrated that the cdc kinase from starfish, p34cdc2,


16
phosphorylated both NF-H and NF-M in vitro. Furthermore, after phosphorylation
of enzymatically dephosphorylated NF-H, NF-H migration on SDS polyacrylamide
gels was similar to that observed for the native condition where NF-H is heavily
phosphorylated. Similar results were also obtained with a cdc2 kinase,
p34cdc2/p58cyc'mA) isolated from FM3A mouse mammary carcinoma cells (Guan et
al., 1992). These findings appear to be significant since cdc kinases are proline
directed kinases which specifically phosphorylate the serine residue on the
sequence S/T-P making the repeat KSP related sequences in NFs likely
substrates. However, cdc kinases are associated with the regulation of cell cycle
and have not been localized to neuronal cells which leads one to question the
relevance of in vitro phosphorylation of NF by cdc2 kinases.
Recently, a proline directed protein kinase was isolated from bovine brain
that consisted of two subunits, 33 kD and 25 kD (Lew et al., 1992a). This kinase
was shown to phosphorylate NFs, where the 33 kD subunit displayed a high
sequence homology to p34cdc2 kinase (Lew et al., 1992b). Independent, cloning
techniques identified a neuronal cdc2-like kinase (nclk) identical to that isolated by
Lew et al. (1992b) which had a 58% homology to mouse p34cdc2 kinase (Hellmich
et al., 1992). In situ hybridization experiments demonstrated that unlike p34cdc2
kinase, nclk was expressed in high levels in terminally differentiated neurons
(Hellmich et al., 1992).
Complexities in studying NF phosphorylation seems to revolve around the
phosphorylation state of the NF subunits. Most of these studies use NFs which


17
are already phosphorylated in vivo. When NFs are used in this form, the in vivo
phosphorylating sites for a specific kinase may already be phosphorylated. In
addition, the phosphorylation state of NFs may effect the activity of a NF kinase
(Wible et al., 1989). Another problem with complete enzymatic dephosphorylation
is that some phosphate groups are more susceptible to a phosphatase than others,
thus all of the phosphates may not be removed. And finally, the number of
potential phosphorylation sites that exist in NF subunits make it difficult to
determine where a specific NF-kinase is acting.
Since NFs are phosphoproteins and seemingly contain a number of
potential sites for phosphorylation, the next obvious question is what is the role of
NF phosphorylation. The distribution of phosphorylated NFs differs within the
neuron. NFs are heavily phosphorylated in the axon, whereas the dendrites and
cell body appear to contain little or no phosphorylated NFs (Sternberger and
Sternberger, 1983). Even when considering that NF phosphorylation may be
involved in calcium buffering during nerve excitation and regulation of NF
assembly/disassembly, the functional significance of the phosphorylation
differences within the neuron is still not understood.
Ultrastructural difference between NFs found in axons and dendrites was
apparent with deep-etch freeze fracture technique (Hirokawa, 1982; Hirokawa et
al., 1984). NFs appeared to be spaced farther apart in axons then they were in
dendrites. These studies also revealed what appeared to be extensive cross-links
between the NFs in axons which were not seen with other intermediate filaments.


18
Therefore, it was hypothesized that the carboxyl-tail domains of NF-M and NF-H
were mediating this cross-linking, and that the length of the cross-links were
regulated by the amount of phosphate incorporation in the KSP segments.
However, no effect on the structure or length of NF tail projections were observed
upon enzymatic dephosphorylation which removed around 90% of the phosphate
groups (Hisanaga and Hirokawa, 1989).
In conclusion, there appears to be a diversity of protein kinase activities
capable of phosphorylating NFs. At least some of these phosphorylation events
are under tight spatial control in the neuron. Since NF subunits contain numerous
potential phosphorylation sites, we can expect that different phosphorylation events
will have different functional significance in the neuron.
Interactions Between Neurofilaments and Microtubules
Indirect findings suggest an interaction between NFs and MTs.
Anatomically, NFs and MTs run parallel to each other in the longitudinal direction
in axons and dendrites, and there appears to be morphological cross-bridges
between these two structures (Wuerker and Palay, 1969; Burton and Fernandez,
1973; Rice et al., 1980; Hirokawa, 1982; Hirokawa et al., 1984). In addition,
biochemical evidence shows that: (1) NFs co-purify with MTs prepared by the
polymerization-depolymerization method (Berkowitz et al., 1977); and (2) both NFs
and MTs travel in the slowest component of axonal transport (Hoffman and Lasek,
1975).


19
More direct evidence demonstrating NF-MT interactions showed that NFs
inhibit tubulin polymerization. This MT assembly inhibition was found to be the
result of NFs binding to high molecular weight microtubule associated proteins
(MAPs) which are known to catalyze MT assembly (Leterrier et al., 1982). To
further examine this interaction, Aamodt and Williams (1984) compared the ability
of NF isolated from brain (NF-Brain) with that of NFs isolated from spinal cord (NF-
SC) to form viscous complexes with MTs. They demonstrated that only NF-Brain
were able to form viscous complexes with MTs. They hypothesized that NF-SC
did not have MAPs bound to them; a suggestion supported by the finding that
when purified MAPs were added to NF-SC and MTs, a viscous solution formed.
Flynn et al. (1987) went on to show that NFs bound to the carboxyl-terminal
thrombin fragment of MAP2 (28 kD) which also is the MT binding site. Solid-phase
binding techniques show that NF-L was the only NF protein to bind to MAP2
(Fleimann et al., 1985). The 28 kD fragment of MAP2 was also found to bind to
purified NF-L (Flynn et al., 1987).
To examine a possible in vivo interaction of MAP2 binding to both NF and
MT network, B,B'-iminodipropionitrile was used to segregate the two cytoskeletal
networks in peripheral nerves. Two different monoclonal antibodies to MAP2
demonstrated that one antibody to MAP2 co-localized with the NF network while
the other co-localized with MT network, suggesting that MAP2 is acting as a
crosslinker between these two fibrous networks (Papasozomenos et al., 1985).


20
In addition to MAP2 binding to NF-L, another MT associated protein, tau,
bound to reassembled NF-L. Competition studies with MAP2 and tau for binding
to NF-L demonstrated that these proteins bound to different sites on NF-L. The
in vivo significance is in question since the affinity of either MAP2 or tau was
decreased when NF-M and NF-FI were present in the filamentous form as normally
is the case in vivo (Miyata et al., 1986).
Hisanaga and Hirokawa (1990b) examined the ability of NF subunits to bind
directly to polymerized MTs, and showed that dephosphorylated NF-H bound to
polymerized MTs in vitro. Further work by Hisanaga and co-workers (1993)
suggested that the binding of dephosphorylated NF-H to MTs was not dependent
on the degree of dephosphorylation but on the removal of a particular group of
phosphates. This was demonstrated by using two different phosphatases; alkaline
phosphatase and acid phosphatase. Partially dephosphorylated NF-H co
precipitated with MTs in a similar manner even though alkaline phosphatase
removed 46 of 51 phosphates on NF-H whereas only 19 phosphates were
removed with acid phosphatase. In addition, this binding of dephosphorylated NF-
H to MTs is thought to occur at the carboxyl terminal tail region of NF-H.
NFs were shown to promote tubulin assembly in vitro (Minami et al., 1982);
however, NF preparations used in these initial studies were contaminated with
MAPs so that the MAPs may have been a factor promoting tubulin assembly. To
clarify these results, Minami and Sakai (1983) demonstrated that highly purified NF
preparations retained their ability to promote MT assembly as measured by a


21
gelation assay. Adding purified NF subunits to the assay demonstrated that NF-H
was the subunit responsible for promoting tubulin assembly. NF-M was able to
induce some MT assembly whereas NF-L had no effect on MT assembly.
Negative stain images of MT assembled in the presence or absence of NFs
showed that there was an increase in MT assembly in the presence of NFs. When
these structures were examined under the electron microscope, there appeared
to be no morphological differences between the assembled microtubules. In the
electron micrographs, the NFs appeared to make a lateral association with the MTs
which resembled a T shaped structure.
The role of NF binding to MTs is not known but it is believed that the tail
regions of the high molecular weight NF proteins are involved in some cross-linking
function. MAP2 is localized in dendrites and may be mediating some of the
interactions between NFs and MTs; whereas, tau is localized in axons where it
may be performing a similar function.
Neurofilaments Interact with Other Cellular Components
The interactions of NFs with other components of the neuron is not as well
studied as those interactions between NFs and MTs (Table 1-3). The majority of
proteins that will be discussed below bind primarily to NF-L.
Brain spectrin (fodrin) is closely associated with the plasma membrane
throughout the neuron (Levine and Willard, 1981) and is thought to play an active
role in stabilizing the membrane as well as immobilizing certain membrane proteins


Table 1-3. Interactions of neurofilaments with other cellular components.
Component
NF Subunit
Affinity (Kd)
Method
Reference
Microtubules
NF-H
gelation of tubulin
Minami & Sakai, 1983
NF-H (dephos.)
3.8 x 10'8M
co-sedimentation
Hisanaga & Hirokawa, 1990
MAP2
NF
1.0x10'7M
co-sedimentation
Leterrier et al., 1982
NF
2.0 x 10'7M
immunocytochemistry
Papasozomenos et al., 1985
NF-L
solid-phase binding
Heimann et al., 1985
NF-L
4.8 x 10'7M
co-sedimentation
Miyata et al., 1986
MAP2
(28kD fragment)
NF, NF-L
co-sedimentation
Flynn et al., 1987
Tau
NF-L
1.6x10'6M
co-sedimentation
Miyata et al., 1986
Brain Spectrin
NF-L
4.3 x 10'7M
solid-phase binding
Frappier et al., 1987
Plectin
NF-H, -M, -L
solid-phase binding
Foisner et al., 1988
Synapsin I
NF-L
solid-phase binding
co-sedimentation
Steiner et al., 1987
NAPA-73
NF
ultrastructural analysis
Ciment, 1990
Nucleic Acids
rRNA
NF-H, -M, -L
preference 18s
co-sedimentation
Traub et al., 1985
dsDNA
NF-H, -M, -L
weakly
it
ii n
ssDNA
NF-M, -L
preference for NF-L
ii
ii ii


23
(Hirokawa, 1989). Frappier and co-workers (1987) demonstrated that brain
spectrin associates with NF-L and GFAP reversibly and in a concentration
dependent manner using in vitro binding assays. Cleavage with BNPS-skatole
reagent demonstrated that the binding site for brain spectrin was localized to the
40 kD N-terminal fragment which includes the head domain of brain spectrin and
the proximal portion of the rod domain. Further cleavage of the 40 kD N-terminal
fragment demonstrated that brain spectrin bound to the 20 kD proximal portion of
the rod domain in NF-L (Frappier et al., 1991). The binding of brain spectrin to
NF-L may indicate how NFs associate with the plasma membrane.
Plectin is an intermediate filament associated protein of an apparent
molecular weight of 300 kD (Wiche and Baker, 1982). Using in vitro binding
assays, plectin was shown to bind to the a-helical rod domain of all IF proteins
examined including the NF triplet protein subunits (Foisner et al., 1988). Plectin
has been extensively studied overthe past decade in non-neural tissue (Wiche and
Baker, 1982; Wiche et al., 1983; Wiche et al., 1984) and is considered to be a
widely distributed IF associated protein. Plectin is believed to be important in the
interaction between IF proteins, and the association between IF proteins and other
components of the cytoskeleton such as the plasma membrane (spectrin), nuclear
envelop (lamins) and MTs via MAPs (Herrmann and Wiche, 1987). In addition to
these functions, plectin may be important in cell to cell interactions at tight
junctions or myotube attachment sites since both light and electron microscopic
show it associated with these regions (Wiche, 1989).


24
Synapsin I is a phosphoprotein located in the synaptic terminal and is
involved in neurotransmitter release (Llinas et al., 1985). Synapsin I is thought to
act as a cross-linker between synaptic vesicles and the cytoskeleton in synaptic
terminals thereby localizing vesicles near release sites in the terminal. It is thought
that upon influx of extracellular calcium, synapsin I is phosphorylated by CaM
kinase II and released from the cytoskeleton, thereby allowing synaptic vesicles to
associate with release sites on the presynaptic membrane (Hirokawa et al., 1989).
In vitro binding studies by Steiner et al. (1987) have shown that synapsin I binds
to NF-L. This interaction between synapsin I and NF-L can be reduced by 60%
upon phosphorylation of synapsin I with CaM kinase II. However, since NFs do
not appear to exist in polymer form at the synaptic terminal, the binding of NF-L
to synapsin I may not be functionally significant.
NAPA-73 is an avian specific 73 kD NF-associated protein that is expressed
developmentally in the nervous system and heart (Ciment et al., 1986; Ciment,
1990). In contrast to other NF binding proteins, NAPA-73 appears to associate
only with bundles of IFs (not individual subunits) suggesting that NAPA-73 is
involved in the bundling of filaments (Ciment, 1990).
NF binding to RNA and DNA was investigated by Traub et al. (1985). They
examined the binding of individual NF subunits to rRNA (total rRNA from Ehrlich
ascites tumor cells), native DNA (double stranded DNA), and heat-denatured DNA
(partially single stranded DNA) using sucrose gradients. NFs had a higher affinity
for 18S rRNA than 28S rRNA and this affinity could be abolished in the presence


25
of 6 M urea for NF-M and NF-H. In contrast NF-L interaction with 18S rRNA was
resistant to 6 M urea. Native DNA bound very weakly to NF subunits, whereas,
heat-denatured DNA bound strongly to NF subunits except in the case of NF-FI.
The specificity of binding of the different forms of nucleic acids suggested that the
association was not solely based on electrostatic interactions (Traub et al., 1985).
The relevance of NFs binding to RNA and DNA is not known. Flowever, some
researchers speculate that NFs degraded at the axonal terminal by calpain
proteases are transported back to the cell body where they could act as a
messenger for the events occurring at the nerve terminal (Traub, 1985; Schlaepfer,
1987).
Another characteristic aspect of NFs is the ability to bind calcium.
Assembled NFs in both phosphorylated and dephosphorylated states contain high
and low affinity binding sites for calcium (Lefebvre and Mushynski, 1987, 1988).
The dephosphorylation of NFs results in a 50% decrease in the low affinity calcium
binding sites which may be related to the loss of phosphate groups. Coinciding
with the decrease in low affinity calcium binding sites is an increase in the number
of high affinity binding sites. This increase in the high affinity calcium binding sites
is probably reflected by a change in the conformation of NFs after
dephosphorylation. Glutamic acid residues are known to constitute calcium binding
sites in such proteins as calmodulin (Kilhoffer et al., 1983), thus the glutamic acid
rich region in the C-terminus of NF tail regions may be the possible binding site for
calcium. Lefebvre and Mushynski (1988) found that the high affinity calcium


26
binding sites are located in the a-helical rod domain and that the low affinity
binding sites are located at the carboxyl-terminal region of NF molecule. The
functional significance of these calcium binding sites is unknown; however, two
possible functions for calcium binding to NFs have been suggested: (1) the ability
to regulate the activation of calcium binding proteases (calpain) which degrade NFs
in the synaptic terminal (Schlaepfer et al., 1985; Schlaepfer, 1988); and (2) the
buffering of calcium during nerve excitation (Abercrombie et al., 1990).
Relationship of NFs to Axonal Transport and Caliber
The transport of material from the cell body along the axon is an essential
neural function since the axon does not contain the cellular machinery for protein
synthesis. Anterograde axonal transport can be broken down into five components
on the basis of their uniform rate: (1) 250-400 mm/day (fast component); (2) 40
mm/day; (3) 15 mm/day; (4) 2-5 mm/day (slow component b); and (5) 0.2-1
mm/day (slow component a) (Willard, 1974). Cytoskeletal elements travel in slow
component a and b. Actin with its associated proteins travel in slow component
b, whereas, NFs and MTs, travel in the slow component a (Floffman and Lasek,
1975). In addition, there appears to be a slower second wave of NFs travelling at
a non-uniform rate that is 100 fold slower than slow component a (Nixon and
Logvineko, 1986). It has been proposed that newly synthesized NFs travel down
the axon as assembled NFs, and are incorporated into the stationary cytoskeletal
network at random points during transport (Nixon and Logvineko, 1986). During


27
axonal transport, IMF's overall charge become increasingly negative which
corresponds to an increase in phosphorylation state (Nixon et al., 1986). This
phosphorylation of NF-L and NF-M is short-lived; 50-60% and 35-40% of the
phosphates are removed in five days, respectively. In contrast, NF-H shows little
to no phosphate turnover during the same period (Nixon and Lewis, 1986). Lewis
and Nixon (1988) suggest that differential turnover rate of NF phosphorylation may
regulate the incorporation of NFs into the stationary NF network, and reflect a
dynamic interaction of NFs with other proteins.
Even though NFs are normally perceived as static structures, research
examining the role NF density as an intrinsic regulator of axonal caliber suggest
a more dynamic function for NFs (Hoffman et al., 1984). During development, the
increase in NF synthesis has been correlated to a decrease in NF transport
velocity and an increase in radial growth of the axon (Willard and Simon, 1983;
Hoffman et al., 1985a). This association of NF number to axonal caliber suggests
that regulation of NFs by gene expression and axonal transport largely determines
the size of the axon. Conversely, after a sciatic nerve crush, there is a decrease
in the number of NF proteins delivered to the axon which coincides temporally with
the reduction in axonal caliber proximal to the lesion in comparison to more distal
regions (Hoffman et al., 1985b). As the slow component advances at its normal
rate, the atrophy also advances (referred to as somatofugal atrophy), and when NF
synthesis returns to normal levels the atrophy is reversed (Hoffman et al.,1985b).


28
Similar results are observed with acrylamide toxicity (Gold et al., 1985), further
suggesting that NFs play an vital role in regulating axonal caliber.
In contrast, other research showed that NF density varies considerably
along the length of the axon, as well as in different axonal types, implying that a
local regulation of axonal cytoskeleton must occur (Berthold, 1982; Nixon and
Logvineko, 1986; Price et al., 1988; Szaro et al., 1990). To examine the role of
the cytoskeleton in the local modulation of axonal caliber, a dysmyelinating mutant
mouse line, Trembler, was used. The peripheral nerves of Trembler were
characterized by a reduction in axonal caliber, an increase in density of axonal
cytoskeletal elements, and a decrease in slow component axonal transport (Low
and McLeod, 1975; Low, 1976a,b; de Waegh and Brady, 1990). De Waegh and
co-workers (1992) examined the relationship of axonal caliber and NF
phosphorylation in the Trembler mouse and found that along with an increase in
NF density, there was a concomitant decrease in NF phosphorylation and axonal
caliber. Therefore, the decrease in NF phosphorylation could in turn be related to
a decrease in charge repulsion thus allowing the NFs to pack more closely
together. In contrast, De Waegh and co-workers (1992) suggested that these
alterations were regulated by the amount of myelin surrounding the axon. Grafts
of Trembler sciatic nerve placed into normal nerve showed regenerated axons with
the Trembler morphology while adjacent axons (control) had normal characteristics.
Recently, transgenic techniques were used to examine the effects of murine
NF gene overexpression on neurons and to determine if an increase in NF number


29
in the axon resulted in a concomitant increase in axonal caliber (Monteiro et al.,
1990). The transgene mRNA exceeded the endogenous NF-L mRNA by fourfold.
This resulted in a corresponding increase of the NF-L protein in the peripheral
axons of the trangenic mice. Although there was an increase in density of axonal
NFs seen morphologically, this did not result in a change in axonal caliber.
These results with overexpressed murine NF-L gene suggest that either the
other two NF subunits may be important in regulating axonal caliber or that the
correlation between axonal diameter and NF density may only be valid when there
is a decrease in NF synthesis. The latter possibility would lend support to the
theory of mechanical stability. Since NF-M and NF-H have unusually large
carboxyl terminal domains which protrude as sidearms from the core of the
filament and are highly phosphorylated in the axon, it has been proposed that NF-
M and NF-H are important in forming or regulating the spacing of NFs whether
through actual cross-bridges or charge repulsion from the highly negative carboxyl-
terminal domains. Although these data appear to contradict the active role of NF
density in controlling axonal caliber, it seems more likely that there are both
intrinsic and extrinsic factors involved and that the role of NFs seems to be more
prevalent in large myelinated axons (Hoffman et al., 1988).
Research on neuropathologies related to abnormal NF accumulation has
focused on deficits in axonal transport. Giant axonal neuropathies are
characterized as a focal accumulation of NFs along the axon (Gold et al., 1986)
which suggests the occurrence of a local interruption of NF transport resulting in


30
the accumulation of NFs. Two model systems have shown that with normal NF
synthesis, a retardation of slow component of axonal transport by 3,3'-
iminodipropionitrile (IDPN) results in proximal axonal swelling and distal axonal
atrophy (Griffin et al., 1978; Clark et al., 1980); whereas, after treatment with 2,5-
hexanedione NF transport is accelerated, resulting in proximal axonal atrophy and
distal axonal swelling (Monaco et al., 1984 and 1989).
NF Expression After Axotomy
Differential changes in cytoskeletal proteins following axotomy of peripheral
nerves is observed for both sensory and motor neurons. Overall, the synthesis of
both tubulin and actin increases after axotomy whereas the synthesis of NF triplet
proteins are down regulated (Hoffman et al., 1987; Wong and Oblinger, 1987;
Goldstein et al., 1988; Tetzlaff et al., 1988; Oblinger et al., 1989). This results in
a decrease in the amount of newly synthesized NFs transported into the axon
(Oblinger and Lasek, 1988). However, based on immunocytochemistry, expression
of NF triplet proteins appears to remain unchanged The time course for changes
in synthesis is similar for all cytoskeletal proteins in that the largest effects occur
between 7 and 14 days, and in the case of a peripheral nerve crush, the level of
synthesis returns to normal between 3 to 8 weeks post injury. If reinnervation is
prevented, NF synthesis remains at decreased levels.
Normally, phosphorylated epitopes of the high molecularweight NF subunits
are restricted primarily to the axon whereas non-phosphorylated epitopes are


31
localized to the cell body and dendrites. However, after axotomy there is a
dramatic and transient increase in phosphorylated epitopes in the cell body that is
concurrent with a decrease in expression of NF mRNA (Moss and Lewkowicz,
1983; Drger and Hofbauer, 1984; Goldstein et al., 1987; Shaw et al., 1988). As
labelling for NF phosphorylated dependent epitopes increase in the perikarya, there
is a concomitant decrease in labelling for the dephosphorylated dependent epitope
which suggests that phosphorylation is occurring at the sites that would normally
be recognized by dephosphorylated dependent NF antibodies (Goldstein et al.,
1987). In the case where axons do not normally regenerate, phosphorylated
epitopes on NF-H remain up to 60 days in the cell body after axotomy (Drger and
Hofbauer, 1984).
In contrast to the NF triplet proteins, peripherin expression is upregulated
after sciatic nerve crush in both dorsal root ganglion (DRG) cells and lumbar
motoneurons (Oblinger et al, 1989; Wong and Oblinger, 1990). In DRG cells,
peripherin is normally expressed in small diameter cells whereas NF triplet proteins
are localized primarily to the large diameter cells (Parysek and Goldman, 1988;
Parysek et al., 1988: Ferri et al., 1990; Goldstein et al., 1991). After a peripheral
nerve crush, the mRNA level for peripherin increases in the large diameter cells
(maximizes at 7 to 14 days) but remains unchanged for the small diameter cells.
This increase in mRNA levels for peripherin translates into an increase in
immunoreactivity in large diameter DRG cells. As expected, NFs show a decrease
in mRNA synthesis which is not accompanied by a change in immunoreactivity in


32
the large diameter DRG cells. Similar results are observed in lumbar motoneurons
after sciatic nerve crush for peripherin. For peripherin, the mRNA levels increase
two fold after 4 days, remain elevated for 6 weeks, and finally recover to normal
levels at 8 weeks (Troy et al., 1990).
Role of NFs in Disease States
NFs are thought to be involved in the development of various
neuropathologies. The breakdown of the normal NF organization is prevalent in
motoneuron disease (Flirano, 1991), neurodegenerative disease (Goldman and
Yen, 1986) and toxin-induced neuropathologies (Griffin and Watson, 1988). In
amyotrophic lateral sclerosis (ALS) there is selective degeneration of anterior horn
motoneurons of the spinal cord. One of the hallmark features of ALS is an
abnormal accumulation of NFs in the perikarya, axons and dendrites as well as NF
induced swelling in the axons early in the degenerative process. It is yet to be
determined whether this increase in expression of NF protein is central to this and
other neuropathologies or is a secondary consequence of another cellular disorder.
Recent transgenic technology has resulted in two animal models to study
the overexpression of NFs, one that examines the effects of NF-L overexpression
and the other that examines NF-H overexpression. Doubly transgenic mice, which
resulted in a fourfold increase the murine NF-L gene, produced dramatic
phenotypic and morphological changes (Xu et al., 1993). Phenotypically, doubly
transgenic mice were one to two-thirds the body weight of age-matched controls,


33
kinetic activity was decreased and death usually occurred within 3 weeks due to
widespread skeletal muscle atrophy. In the CNS, murine NF-L expression was
restricted to neurons; however, outside the nervous system, the expression OF
murine NF-L was observed in different areas including skeletal muscle.
Morphometrically, select motoneurons showed chromatolytic features that included
a disrupted rough endoplasmic reticulum and a displaced nucleus. The
overexpression of murine NF-L resulted in excessive accumulation of NFs in the
soma and proximal axons of the anterior horn motoneurons which were undergoing
chromatolysis. In addition to the increase in NF density in large caliber axons, the
NFs were more closely packed together and were not always observed in the
normal parallel array to the long axis of the axon. A few mice lived past three
weeks, and after 2 months the axons showed a significant decrease in NF density
that were still higher than controls. Additionally, muscle mass returned and a slow
return of normal kinetic activity was observed. Near normal phenotype at nine
months occurred concomitant with a decrease in NF accumulation which had
plateaued at three to four weeks. These results led to the hypothesis that
excessive NF-L accumulation can result in motoneuron dysfunction before
widespread neuronal loss.
In a similar study, in which transgenic mice were produced with human NF-
H, a twofold increase of NF-FI expression resulted in a more progressive
neuropathology that occurred over a period of three to four months (Ct et al.,
1993). In these transgenic mice, the expression of the human NF-H was restricted


34
to neurons. Similar to ALS, progressive abnormal accumulations of NFs were
observed in the perikarya and proximal axons of anterior horn motoneurons of the
spinal cord.
The excessive accumulations of NFs in the neuronal cell bodies in both
types of transgenic mice suggest that it is not one particular NF subunit is critical
, to prevent normal cellular activity. However, the increase in density and
disorganization of NF structure taken together could interfere with normal axonal
transport and thus result in inappropriate accumulations of NF subunits throughout
the neuron. Thus, deficits in axonal transport appear to be a major component in
motoneuron neuropathies. This may be due to indirect effects like an abnormal
increase in NF proteins in the soma that blocks normal axonal transport which may
be the case with ALS, or may be due to a direct effect of axonal transport, that
results in local accumulations of NFs, which may be the case with giant axonal
neuropathies.
Overview of Dissertation
Although NFs are one of the most abundant proteins in the nervous system,
the dynamics of their function remain undefined. As in the case of other
cytoskeletal elements, such as MTs and MFs, one way of understanding or
defining function is realized by determining what types of proteins interact with
these cytoskeletal elements. In the set of experiments described in this


35
dissertation, I have undertaken this approach to identify candidate NF binding
proteins in an attempt to assign functional roles to NF proteins.
One candidate NF binding protein is a 300 kD protein which co-purifies with
IF proteins isolated from a crude spinal cord preparation. This protein was
identified as plectin, a protein previously characterized in non-neuronal tissue. The
relationship of plectin to its potential role in the nervous system, and specifically
to neural IF proteins was examined.
Using affinity column chromatography, additional candidate NF binding
proteins were identified based on molecular weight. A number of proteins bound
to the different NF affinity columns with varying specificity. One protein in
particular was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
The in vitro binding of GAPDH to NF-L was examined as well as possible in vivo
interactions at the light and ultrastructural level.
These findings provide an initial framework for studying NF associated
proteins. NFs appear to interact with a number of proteins and these associations
may be specific to individual subunits as well as to the intact 10 nm filaments. In
addition, future studies that identify the other candidate NF associated proteins will
lead to a better understanding of NF function. Considering the differential labelling
of NFs in neurons, NF associated proteins may also show similar specificity which
may lead to important markers for different neuronal cell types along the neural
axis.


CHAPTER 2
GENERAL METHODS
This chapter describes the routine biochemical and anatomical techniques
that were performed for this dissertation project. Techniques that were specific for
a particular chapter or modifications of these routine techniques will be described
in that chapter.
Biochemical Techniques
Protein Assay
Lowry protein assay. The method of Lowry et al. (1951) was used for some
of the protein assays. Briefly, a standard cun/e from 0 to 100 pg bovine serum
albumin (BSA) to a final volume of 0.5 ml was made. The unknown concentration
of the protein solution was determined by diluting the protein solutions so it fell in
the range of the standard curve. Five milliliters of a solution containing 0.04%
copper sulfate, 0.08% potassium sodium tartrate and 2.94% sodium carbonate was
added to the BSA standards and protein samples to be assayed. The solutions
were then incubated at room temperature for 10 minutes. Next, 0.5 ml of 50%
Folin phenol reagent solution (diluted with deionized water) was added to each
tube and then each tube was incubated for 30 minutes at room temperature. The
absorbance was measured at 650 nm for each sample and the absorbance of BSA
36


37
standards was plotted against the known concentrations. The unknown protein
concentrations were determined from the linear regression line of the BSA
standard data.
Pierce protein assay. Pierce Micro BCA protein assay kit was also used in
protein concentration determination because of its reliability and ease of use.
Protein concentrations were determined in the range of 0-20 pg/ml using BSA as
the standard and following the manufacturers instructions. Pierce Micro BCA
protein assay kit uses bicinchoninic acid which is a highly sensitive and selective
detection reagent for Cu+1. The protein concentration of the unknown samples
were determined using the same methodology as described above.
SDS Polyacrylamide Gels
The sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis
technique was based on the method of Laemmli (1970) with an acrylamide to
bisacrylamide ratio of 37:1 (22.2%:0.6%). The electrophoretic apparatus used was
the Bio-Rad mini-protean II electrophoresis cell. Sample buffer containing SDS
and B-mercaptoethanol (12.5% (v/v) 1 M Tris-HCI, pH 6.8, 4% (w/v) SDS, 10%
(v/v) glycerol, and 0.006% (v/v) bromophenol blue in ethanol) was added to each
sample and then each sample was boiled for 5 minutes before protein separation
on SDS polyacrylamide gels. The SDS polyacrylamide gels were run at a constant
current of 20 mA per gel. The gels were fixed in 40% methanol and 10% acetic
acid for 30 minutes, and then stained with 0.025% Serva Blue G for 1 hour
followed by destaining with 10% acetic acid for 1 -2 hours. This method of staining


38
and destaging of gels is fast and efficient when compared to staining with
Coomassie Brilliant Blue R-250 (CBB) in methanol and acetic acid solution which
can take up to 24 hours to properly destain the background. In cyanogen bromide
(CNBr) cleavage experiments, CBB was used since the initial staining of the bands
is comparatively faster (15 to 20 minutes) and high background did not interfere
with accurately excising the gel band of interest.
Electroblottina of Proteins
Proteins were electrophoretically transferred to nitrocellulose or
polyvinylidine difluoride (PVDF) membrane using Bio-Rad Mini Trans-Blot Cell. A
submerged protein transfer system was used where the gel and membrane were
sandwiched between Whatman 3M paper and scrub pads. The transfer buffer
used in most situations contained 10 mM 2-[N-morpholino]ethanesufonic acid
(MES) pH 6.5, 0.01% SDS. The transfer lasted for 1-2 hours at 90 Volts
depending on the protein being transferred. To prevent overheating, the mini
trans-blot unit was immersed in an ice bath.
The proteins transferred onto the nitrocellulose membranes were visualized
with 0.025% Ponceau S in 40% methanol/10% acetic acid, and were used for
Western blots. Proteins transferred onto PVDF membranes were used for amino
acid analysis and were treated in a slightly different manner than nitrocellulose
membranes. Before proteins were transferred onto PVDF membrane, the
membrane was immersed in 100% methanol and then equilibrated in transfer
buffer for 20 minutes with two changes of buffer. After the transfer of proteins to


39
the PVDF membrane, the PVDF membrane was washed in deionized water for five
minutes, and stained in 0.1% CBB in 50% methanol for 5 minutes. The PVDF
membrane was partially destained in 50% methanol and 10% acetic acid for five
minutes, followed by a final wash in deionized water for 5 minutes (four changes
of water). The PVDF membrane containing the transferred proteins was dried and
stored at -20C until analysis.
Immuno-Blot Analysis
Proteins were separated on SDS polyacrylamide gel and electrophoretically
transferred onto nitrocellulose membrane as discussed previously. The membrane
was blocked with 3% bovine serum albumin (BSA) or 5% nonfat Carnation instant
milk containing 0.1% Tween 20 in Tris buffered saline (TBS: 10 mM Tris-HCI (pH
7.5), 0.9% saline). If the nonfat milk was used to block the nitrocellulose, then the
primary and secondary antibody was diluted in this solution, and all but the last
wash was done with TBS containing 0.1% Tween 20 in place of TBS. The
remaining steps were the same as those described when BSA was used to block
the nitrocellulose membrane. The membrane was incubated with the primary
antibody containing 0.1% BSA for 1 hour at room temperature followed by three
10 minute washes in TBS. Next, the membrane was incubated with secondary
antibody, alkaline phosphatase conjugated anti-mouse or anti-rabbit, for 1 hour at
room temperature followed by two 10 minute washes in TBS. A ten minute wash
in developing buffer (10 ml 0.1 M Tris-HCI (pH 9.5) containing 5 mM MgCI2 and
0.1 M NaCI) followed. The antibody binding was visualized using 33pl 50 mg/ml


40
5-Bromo-4-chloro-3-indolyl phosphate (BCIP) and 33pl 50 mg/ml Nitro Blue
Tetrazolium (NBT) in 10 ml developing buffer.
Protein Cleavage with Cyanogen Bromide
To cleave proteins at methionine residues, the cyanogen bromide (CNBr)
cleavage method was used on SDS polyacrylamide gel protein bands (Sokolov et
al., 1989). The protein to be cleaved by CNBr was separated on a SDS
polyacrylamide gel. The gel was stained with CBB (0.25% coomassie blue R-250
in 45% methanol and 10% acetic acid) and the gel band of interest was excised
from the gel. The gel band was dried down using a vacuum evaporator at 60-
80C. The volume to rehydrate the gel band was estimated by measuring the
length, height and width of the gel band before it was dehydrated, and that volume
was added to the dried protein gel band in the form of 200 mg/ml CNBr in 70%
formic acid. The gel band was incubated in the CNBr solution at 37C for at least
6 hours. The rehydrated gel band was dried down in the vacuum evaporator to
remove the remaining CNBr and its by-products. The dried cleaved gel band was
rehydrated and neutralized in 50% 2X sample buffer and 50% 1 M Tris for 15
minutes. The cleaved protein gel band was placed on 15% SDS polyacrylamide
gel to separate the protein fragments. The protein fragments were visualized using
Serva Blue G or silver stain depending on the protein concentration.


41
Isolation of Neurofilament Proteins
Crude Intermediate Filament Preparation
A crude intermediate filament (IF) preparation was used to obtain primarily
neurofilament (NF) proteins in vitro while maintaining some of their native
characteristics. Fresh spinal cord (specific species used will be discussed in the
relevant chapter) was homogenized gently using a Wheaton Dounce Type 40 glass
homogenizer for small preparations (<10 g of tissue) or a Sears blender for larger
preparations (>10 g tissue). The tissue was brought up in 5 volumes of Buffer A
(0.1 M Tris-HCI (pH 7.5), 0.1 M NaCI, and 1 mM Na-p-tosyl-L-arginine methyl ester
(TAME)), and homogenized with 3 strokes of pestle 'B' followed by 1 to 2 strokes
with pestle 'A'. For larger preparations, the tissue was homogenized in blender at
low speed (three 5 second pulses) followed by three 5 second pulses at high
speed. The homogenate was made up to 10 volumes with Buffer A and
centrifuged at 2300xg for 10 minutes at 4C in a SS34 Sorvall rotor. The pellet
was taken up in 10 volumes 1 M sucrose in Buffer A, vortexed gently to break up
the pellet, and centrifuged at 2300xg for 20 minutes at 4C in a SM-24 Sorvall
rotor (small preparation) or a SS34 Sorvall rotor (large preparation). The floating
myelin layer, containing the NF bundles, was carefully removed and the volume
was made up to 5 volumes with Buffer A containing 1 M sucrose and 1% Triton
X-100. The myelin homogenate was gently vortexed and triturated to suspension,
and then shaken on an orbital shaker for 60 minutes at 4C. This allowed the
Triton X-100 to solubilize the myelin and release the intermediate filament bundles.


42
The suspension was centrifuged at 150,000xg for 1 hour at 4C in a Beckman TL-
100 rotor (small preparation) ora T865 Sorvall rotor (large preparation) to recover
the pelleted intermediate filament bundles. The pellet was composed mainly of NF
bundles and glial filaments. The pellet was taken up in the appropriate buffer and
stored at -20C.
Neurofilament Preparation
Partially purified NFs were prepared using the method of Delacourte et al.
(1980). Fresh pig spinal cord (250 g) was homogenized in 400 ml of 0.1 M MES
(pH 6.5), 1 mM ethylenediamine tetraacetic acid (EDTA), 0.5 nM MgCI2, 1 mM
PMSF, and I mM TAME using a Sears blender at 4C. Initially, the spinal cord
was homogenized at low speed using 3 five second pulses followed by 3 five
second pulses at high speed. The crude homogenate was centrifuged at 13,500xg
for 45 minutes at 4C in a GSA Sorvall rotor. The supernatant was filtered through
8 ply gauze sponges type VII (Professional Medical Products, Inc.) to remove any
large floating debris. Next, the filtered supernatant was centrifuged at 78,000xg
for 30 minutes at 4C in a T865 Sorvall rotor. Glycerol was added to the
supernatant to form a final concentration of 20% (v/v) glycerol. The solution was
warmed to 37C for 20 minutes. The supernatant was centrifuged at 78,000xg for
45 min at 30C in a T865 Sorvall rotor. The pelleted material was combined and
homogenized in 50 ml of MES buffer and centrifuged at 147,000xg for 30 minutes
at 4C in a T865 Sorvall rotor. The pelleted material was a pale yellowish gel that
contained NFs and glial filaments. The pellet was stored at -20C.


43
Purification of Individual Neurofilament Subunits
Individual NF subunits were purified following a modified method of
Tokutake (1984). The pellet obtained from the NF Delacourte preparation was
homogenized on ice in 10 mM sodium phosphate monobasic (pH 7.0), 6 M urea,
1 mM EDTA, and 1 mM dithiothreitol (DTT) in order to solubilize the NFs into their
individual subunits. Insoluble material was removed by high speed centrifugation
(150,000xg) for 30 minutes at 4C using a T865 Sorvall rotor and the supernatant
was filtered through a 0.45 pm filter. The soluble material containing the NF
subunits was put through a DEAE cellulose column (DE-52) at a flow rate of 25
ml/hour at 4C. The DE-52 column was washed extensively to remove any
unbound proteins and before elution of bound proteins. To make sure no more
proteins were being eluted, the optical density at 280 nm of the wash buffer
coming through the column was checked. A linear gradient of 10 mM to 400 mM
monobasic sodium phosphate (500 ml) was used to elute the bound proteins.
Tokutake showed that varying the sodium phosphate concentration instead of
using a sodium chloride gradient resulted in a better separation of the individual
NF subunits. Fractions of 5 ml were collected during the elution, and 10pl of each
even number fraction was separated on 7.5% SDS polyacrylamide gels to
determine which fractions contained the NF subunits. The fractions which were
purest (those that contained 98% of only one of the subunits) were pooled and
dialyzed against the appropriate buffer. The purified NF subunits were assayed
for protein concentration (Lowry or Pierce micro assay) and stored at -20C.


44
Anatomical Localization Experiments
Dorsal Root Ganglion Cell Cultures
New born rat pups (postnatal day 1) were killed by decapitation and the
body was pinned at the extremities to a piece of styrofoam. All instruments were
sterilized with 95% ethanol. The skin over the spinal cord was removed and a
longitudinal cut, using a scalpel, was made through the dorsal portion of the
vertebrae. With scissors the dorsal vertebral column was trimmed and the spinal
cord removed. The dorsal root ganglia (DRG) were located between the vertebrae
and resembled small white spheres. The DRG were removed one by one with fine
forceps from one side of the vertebral column and placed in a sterile microfuge
screw cap containing Liebowitz L-15 media (Sigma) with 10 ml/liter penicillin
(10,000 Units/ml), streptomycin (10,000 mag/ml) and amphotericin B (25 mag/ml)
(Gibco) (L-15 incomplete media). This was repeated for the contralateral side and
for the remaining rat pups so that each tube contained approximately 10 to 15
DRG. The ganglia were pelleted in a clinical centrifuge (setting 5) for 30 seconds.
To the pelleted ganglia, 240 pi 10X sterile trypsin-EDTA solution (Sigma; 0.5%
trypsin, 0.2% EDTA. 0.9% NaCI) was added. The resuspended DRG were then
incubated at 37C for 40 minutes to dissociate the ganglia. Then, the DRG were
centrifuged for 30 seconds at setting 5 in the clinical centrifuge, the trypsin
containing supernatant was removed, and 6-8 drops of fetal bovine serum (FBS;
Gibco) was added. The DRGs were incubated for 10 minutes at 37C, pelleted as
described above, and supernatant removed. DRG were triturated in 100 pi of L-15


45
incomplete media 15 times using an Eppendorf pipet to resuspend the ganglia into
single cells. Before plating, 500 pi of L-15 incomplete media was added to each
tube, then cells were plated on 6 acid washed coverslips (100 pi per plate) placed
in Petri dishes (35x10 mm). Two milliliters of L-15 complete media (L-15
incomplete media, 10% FBS, 0.6% glucose, 2 mM L-glutamine and 0.3% A4M
methylcellulose) was added to each Petri dish. Nerve growth factor (NGF) (Sigma
7S mouse submaxillary gland NGF) was added to result in a final concentration of
200ng/ml. The DRG cells were grown in a non-C02 incubator at 37C for 24
hours.
Pheochromocvtoma Cells (PC12 Cells)
A stock solution of PC12 cells was thawed rapidly at 37C, pelleted in a
clinical centrifuge, and resuspended in RPMI media containing 85% RPMI 1640
medium (Gibco); 10% heat inactivated horse serum, 5% FBS and 10 ml/liter of
penicillin (10,000 Units/ml), streptomycin (10,000 mag/ml) and amphotericin B (25
mag/ml) (Gibco). PC12 cells were plated on a collagen coated Petri dish (60x10
mm), grown at 37C in a water-saturated atmosphere containing 8.1% C02, and
fed with new media every 2-3 days. When the cultures reached confluence, they
were subcultured at a ratio of 1:3 to 1:4. To generate neurite-bearing PC12 cells,
50 ng/ml 7S-NGF was added to each culture and the total serum concentration
was reduced to 1.5% from 15%. PC12 cells were feed every two days with RPMI
media containing 1.5% serum and 50 ng NGF. After about a week in culture,
PC12 cells produced neurites and were ready to be replated on collagen coated


46
coverslips for immunocytochemistry. The PC12 cells were removed from the Petri
dish by trituration with a Pasteur pipette and pelleted in clinical centrifuge at low
speed where the supernatant was discarded (contains cellular debris including
detached neurites). The pelleted cells were resuspended in RPMI media (1.5%
serum) with 50 ng NGF, and plated onto coverslips. The PC12 cells produced
neurites within 8 to 16 hours and by 2 to 3 days the PC12 cells have formed
extensive neuritic processes.
Immunofluorescent Studies on Fresh Frozen Tissue
Adult rats, either Sprauge Dawley or Long Evans Hooded strains, were
anesthetized with 0.5 ml pentobarbital i.p. and sacrificed by decapitation. Nervous
system tissue was dissected out, frozen on dry ice for 1 hour or quickly frozen in
isopentane cooled with liquid nitrogen, and stored at -70C until sectioning. The
tissue of interest was cut at a thickness of 6 to 10 pm using a cryostat, placed on
petroleum ether cleaned glass slides, and stored at -20C until they were labelled
with antibodies. Prior to incubation with primary antibodies, the tissue was fixed
in acetone (-20C) for ten minutes. Tissue sections were air dried 10 to 15
minutes and encircled with a PAP pen (The Binding Site) to make a well for the
antibodies. The two primary antibodies, one polyclonal and the other monoclonal,
were mixed together in buffer to obtain the appropriate final concentration for each
antibody and then were placed on the section. The tissue sections were then
incubated at 37C for 30 minutes or 4C for 24 hours, followed by three 10 minute
washes in PBS. Following primary antibody, sections were incubated for 30


47
minutes at 37C or 24 hours at 4C with secondary antibodies, FITC-conjugated
goat anti-mouse (1:40) and Texas Red conjugated donkey anti-rabbit (1:200).
After three 10 minute washes in PBS, the sections were coverslipped with anti
bleaching agent (1.0 mg/ml para-phenylenediamine (Sigma) in 20 mM Tris-HCI pH
7.9, 90% glycerol). For tissue culture immunofluorescence, the DRG and PC12
cells were fixed in cold methanol (-20C) for 5 minutes. The remaining antibody
steps were as described above. The fluorescently labelled tissue sections were
visualized and photographed using a Zeiss Axiophot microscope system.
Immunohistochemistry on Formaldehyde Fixed Tissue
Adult rats, either Sprauge Dawley or Long Evans Hooded strains, were
anesthetized with either 0.5 ml pentobarbital or 80 mg/kg ketamine and 10 mg/kg
xylazine, and perfused with saline (0.9% NaCI) containing 0.05% (v/v) heparin and
0.05% (w/v) sodium nitroprusside followed by a fixative solution of 4%
paraformaldehyde in Sorensen's Buffer, pH 7.4 (19 mM monobasic sodium
phosphate, 81 mM dibasic sodium phosphate). The rat nervous system was
dissected out and placed in the above fixative solution overnight or PBS depending
on how well the tissue was fixed. Before sectioning, the tissue was blocked and
placed in phosphate buffered saline for at least 4 hours before being cut in 30 to
50 pm sections using a vibratome.
For immunocytochemistry, the tissue sections were incubated at in 0.3%
hydrogen peroxide in PBS for 30 minutes at room temperature to remove any
endogenous peroxidase activity. The tissue sections were washed three times for


48
10 minutes and mounted on chrome-alum coated slides and allowed to air dry for
at least one hour before beginning antibody staining procedure. Vectastain elite
ABC kit (KIT) was used to visualize the labelling of the primary antibody. The
tissue sections were blocked for 20 minutes in dilute normal serum (KIT) at 37C.
Excess blocking serum was shaken off and the primary antibody in 0.25% Triton-
X-100 was placed on the tissue section and incubated for 30 minutes at 37C or
24 hours at 4C. The tissue sections were washed three times for 10 minutes
followed by incubation with biotinylated secondary antibody (KIT) for 30 minutes
at 37C or 24 hours at 4C. The tissue sections were washed 3 times for ten
minutes followed by incubation with the avidin/biotin reagent (KIT) for 30 minutes
at 37C or 4 hours at room temperature. The tissue sections were washed 3 times
for ten minutes in PBS before being developed with freshly prepared
diaminobenzidine tetrahydrochloride (DAB) solution (0.05% DAB (Sigma),
containing 0.01% hydrogen peroxide in 0.1 M Tris, pH 7.2). The reaction was
monitored using an inverted microscope in order to determine when to stop the
reaction. The reaction was stopped by placing the tissue sections in tap water for
five minutes. The tissue sections were dehydrated using ascending alcohol
concentrations: 70%, 90%, 95%, 100%, 100%; and cleared in xylene and
coverslipped with permount.


CHAPTER 3
THE DISTRIBUTION OF PLECTIN, AN INTERMEDIATE FILAMENT BINDING
PROTEIN, IN THE ADULT RAT CENTRAL NERVOUS SYSTEM
Introduction
Although intermediate filament proteins (IFs) are found with various
polypeptides known as intermediate filament associated proteins (IFAPs), IFAPs
remain much less characterized than microtubule and microfilament associated
proteins. At present only a few proteins are known to be specific IF-binding
proteins, (Foisner and Wiche, 1991) such as plectin (Wiche, 1989), IFAP-300
(Yang et al., 1985; Lieska et al., 1985), NAPA-73 (Ciment et al., 1986), filensin
(Merdes et al., 1991), and filaggrins (Haydock and Dale, 1990).
One protein in particular, plectin, has been studied extensively in non-neural
tissue. Immunofluorescence studies of various non-neuronal tissues demonstrated
that plectin was localized to a number of different cell types, and has a cytoplasmic
distribution within the cell with a distinct tendency to be concentrated at the cellular
periphery near the plasma membrane (Wiche et al., 1983). Later work
demonstrated that plectin bound to immobilized IF proteins in vitro, including the
NF triplet proteins (Foisner et al., 1988). This interaction was found to involve the
a-helical rod domain in IFs and the a-helical rod domain of plectin (Foisner et al.,
1988; 1991). Originally, plectin was identified from rat glioma C6 cells as a major
49


50
component of Triton X-100 extracts with an apparent molecular weight of 300 kD
as determined from mobility on SDS-polyacrylamide gels (Pytela and Wiche, 1980).
Recent cloning and sequencing of rat plectin has shown that the actual
molecular weight of plectin is 527 kD (Wiche et al., 1991). Portions of plectin's
primary sequence are related to desmoplakin and the bullous-pemphigoid antigen,
both of which are found in association with IFs at the plasma membrane (Wiche
et al., 1991). Overall, plectin is postulated to function as a crosslinking protein,
mediating interactions between IF proteins and other components of the cell
(Foisner and Wiche, 1991) such as the plasma membrane via spectrin, the nuclear
envelope via nuclear lamins and microtubules via microtubule-associated proteins
(Koszka et al., 1985; Plerrmann and Wiche, 1987).
As discussed earlier, previous research on plectin has focused on non-neural
tissue, and although plectin binds NF subunits in vitro, the in vivo significance of
this observation is not clear. This chapter focuses on demonstrating that plectin
is present in crude insoluble IF extracts from neural tissue and showing the
localization of plectin in the adult rat central nervous system.
Methods
Experimental Tissue
A crude intermediate filament (IF) preparation from bovine and rat spinal cord
was obtained using a modified axonal floatation technique, details of this method
were described in Chapter 2 (Shelanski et al., 1971; Shaw and Hou, 1990).
Bovine spinal cord (50 g) was obtained from the slaughter house ~1 hour


51
postmortem and prepared using the large preparation method. Rat spinal cord (~0.2
g) was obtained rapidly (>5 minutes) after decapitation and processed using the
small preparation method.
Partial purification of the 300 kD protein was accomplished using a Sephacryl
S-400 (Bio-Rad) column (1.5 cm x 100 cm). Bovine IF pellet was resuspended in
Buffer B (6 M Urea, 10 mM sodium phosphate (monobasic, pH 7.5), 1 mM EDTA,
1 mM TAME), and centrifuged at 150,000xg in a Sorvall T865 rotor for 45 minutes
at 4C to remove any insoluble material. The supernatant was filtered through a
0.8 pm filter followed by a 0.45 pm filter, then placed in a dialysis bag and
concentrated to a final volume of 2 ml by placing dry Sephadex G-50 around the
dialysis bag. The concentrated bovine IF material was separated on a Sephacryl
S-400 column which was equilibrated with Buffer B containing 5 mM DTT. The
flow rate was 15 ml per hour and 4 ml fractions were collected. Five pi from the
even number fractions were separated on 6% SDS polyacrylamide gels.
Immuno-Blot Analysis
Proteins were separated on SDS polyacrylamide gel and transferred to
nitrocellulose using 10 mM MES (pH 6.8) and 0.01% SDS for 2 hours at 90 volts
(constant). For the crude IF preparation from rat spinal cord, approximately 3% of
final preparation was used per lane. A detailed description for the methods used
here were given in Chapter 2.


52
Antibodies
Monoclonal antibodies to plectin (1D8 and 1A2) and polyclonal serum to
plectin (p21) were previously characterized (Wiche and Baker, 1982; Foisner et al.,
1991). Monoclonal antibodies were from tissue culture supernatant and were used
either undiluted or diluted 1:2. The polyclonal serum, p21, was used at 1:50.
Alkaline phosphatase conjugated anti-mouse IgG secondary antibody was
purchased from Sigma and used at 1:1000 dilution. Biotinylated secondary
antibodies were obtained from Vector Laboratories and diluted according to the
manufacturers directions. Fluorescent secondary antibodies were obtained from
Jackson Laboratories and used at 1:100 dilution.
Anatomical Procedures
Both paraformaldehyde fixed and fresh frozen acetone fixed rat neural tissue
were used in this study. Plectin was localized in the paraformaldehyde fixed tissue
using the Vectastain Elite ABC kit. For fresh frozen acetone fixed tissue, plectin
was localized using the indirect immunofluorescence procedure. Both of these
anatomical methods were described in detail in Anatomical Localization section in
Chapter 2.
Results
Immunoblot Studies
Besides the major IF proteins bands in a crude IF preparation from spinal
cord, there were a number of proteins that copurify with IFs (Figure 3-1 a, b lanes


53
1). Since these proteins were present after salt and detergent extractions, it was
hypothesized that these proteins may functionally interact with IFs.
In particular, one high MW protein band at 300 kD on SDS polyacrylamide
gel, a relatively minor component in this IF preparation, was proposed to
correspond to plectin, a known 300 kD protein that interacts with IFs in vitro
(Foisner et al., 1988). The 300 kD protein from crude IF preparation of bovine
spinal cord was partially purified using Sephacryl S-400 column. Figure 3-1 a
shows the crude bovine IF preparation (lane 1) and Sephacryl S-400 column
fractions 16-28 (lanes 2-8, respectively) in which lane 2 contains the 300 kD
protein. Immunoblot of fraction 16 (Figure 3-1 a, lane 9) demonstrated that the
plectin monoclonal antibody ID8 labelled a 300 kD protein.
Since rat neural tissue was to be utilized in the immunocytochemical
localization of plectin in the CNS, I investigated whether plectin was present in rat
neural tissue and whether the plectin antibody cross-reacted with any of the other
proteins present in the crude IF preparation. Immunoblot of crude rat IF
preparation demonstrated that plectin was present in rat spinal cord, and the
antibody did not cross-react with any of the other proteins present in the rat
preparation (Figure 3-1 b). The weak smearing just below the major plectin band
which did not correspond to any protein band in the protein stained gel (Figure 3-
1b, lane 1) was probably a degradation product of plectin (Wiche, 1989). The
relatively low amount of plectin immunoreactivity detected may reflect a difficulty
in electrophoretically transferring plectin as a result of plectin's very high molecular


Figure 3-1. Identification of a 300 kD protein in bovine and rat crude IF spinal cord
preparations.
(A) Partial purification of 300 kD protein (arrow) from crude IF preparation from
bovine spinal cord. Lane 1 is a 7.5% SDS polyacrylamide gel showing the IF
preparation before purification on a Sephacryl S-400 gel filtration column. Lanes
2-8 represent even number fractions (16-28) from the gel filtration column in which
fraction 16 contained the partially purified 300 kD protein. Lane 9 is an
immunoblot of fraction 16 labelled with monoclonal antibody to plectin (1D8). (H:
NF-H; M: NF-M; L: NF-L; G: GFAP).
(B) Immunoblot of rat spinal cord IF preparation showing plectin antibody staining.
Lane 1 is a 6% SDS polyacrylamide gel of a spinal cord cytoskeletal extract
stained with Serva Blue G. Lane 2 is an immunoblot strip labelled with plectin ID8
showing that the monoclonal antibody labels a 300 kD protein.


55
L-
G-
1
2


56
weight, at 527 kD. Even under optimal transfer conditions, when most of the other
high molecular weight proteins were transferred completely, a substantial portion
of the 300 kD protein band remained in the gel after electroblotting (data not
shown). In addition, it was possible that the fairly prominent 300 kD band seen in
SDS-PAGE contained other proteins (Lieska et al., 1985; Goldman and Yen, 1986)
besides plectin since ion exchange chromatography demonstrates that proteins
with a relative molecular weight of 300 kD are eluted off a DEAE ion exchange
column at different salt concentrations (data not shown).
Comparison of Plectin to IFAP-300
In addition to plectin, there was another IFAP identified which has similar
mobility on SDS polyacrylamide gel known as IFAP-300 (Lieska et al., 1985;
Goldman and Yen, 1986). It was of interest to compare the amino acid analysis
of these two proteins, since both plectin and IFAP-300 have similar mobilities on
SDS polyacrylamide gels (300 kD), staining distribution in non-neuronal cells, and
both bind to IF network. IFAP-300 amino acid composition data were taken from
Lieska et al. (1985) and the plectin amino acid composition was determined from
the published sequence (accession #S21876) and are presented in both table and
graphical form (Shaw, 1992) in Figure 3-2. Superficially, these two proteins appear
similar; however, differences were observed in the percent composition of a
number of amino acids Although these two proteins do not appear to be closely
related in amino acid composition, these results do not exclude the possibility that
there may be some sequence similarities, and since these two proteins have


Figure 3-2. Comparison of amino acid composition of plectin and IFAP-300 proteins.
A) The percent nanomole (% nmole) for plectin and IFAP-300 is listed. Plectin amino acid
composition was determined from the published sequence (Wiche et al., 1991). The amino acid
composition for IFAP-300 was obtained from published paper (Lieska et al., 1985) and the
values were corrected for quantitation errors using a correction factor (Shaw, 1991).
B) The amino acid composition data are represented graphically using the Star program (Shaw,
1991).


Amino Acid Composition (% nmole)
Amino Acid
Plectin
IFAP-300
ASX
5.36
9.81
THR
5.21
5.59
SER
6.68
7.19
GLX
19.53
17.08
PRO
3.29
6.57
GLY
6.44
8.99
ALA
11.08
9.87
VAL
5.33
3.82
MET
1.35
1.29
ILE
3.32
1.97
LEU
11.38
9.11
TYR
2.24
2.43
PHE
1.79
2.69
HIS
1.57
1.91
LYS
5.53
5.20
ARG
9.90
6.48
B
Plectin
asx
ARG THR
LYS
SER
HIS
IK GLX
PHE
PRO
TYR
GLY
LEU
ALA
ILE UAL
MET
LYS
IFAP-300
ASX
ARG THR
SER
HIS
/ \ GLX
PHE
PRO
TYR
GLY
LEU
ALA
I LET
MET
UAL
UT
00


59
similar molecular weights, binding and tissue localization characteristics, plectin
and IFAP-300 may belong to a family of high molecular weight IFAP similar to that
observed with the high molecular weight microtubule associated proteins.
Localization Studies
Overall, plectin immunoreactivity in the adult rat central nervous system was
predominantly associated with non-neuronal cells. In addition, plectin antibody
staining was present in a subset of neurons in the brainstem and spinal cord.
Immunoreactivity was generally strongest at the periphery of cells.
Telencephalon and diencephalon. Plectin immunoreactivity was absent in
neurons of the telencephalon and diencephalon. The only cells which labelled with
the plectin antibody in the cortical gray matter of the telencephalon were astrocytes
located in the ventromedial portion of the temporal cortex near the pial surface.
The density of astrocytic labelling with plectin antibodies decreased rapidly away
from the pial surface (Figure 3-3a). Although this labelling pattern may be
attributed to edge effect, a similar staining pattern was not observed in any other
cortical regions near the pia mater.
In the diencephalon, plectin positive astrocytes were observed at the
ventrolateral portion of the hypothalamus (Figure 3-3b). Plectin immunoreactivity
was found prominently in astrocytes throughout the white matter fiber tracts
including the optic tract (Figure 3-3c), corpus callosum, internal capsule, fornix, and
stria medullaris. Pronounced plectin immunoreactivity was observed in the
hypothalamus surrounding the third ventricle. Both ependymal cells lining the


Figure 3-3. Plectin immunoreactivity (ID8) of astrocytes in white and gray matter
of the telencephalon and diencephalon.
(A) The ventro-medial portion of the temporal cortex was the only cerebral cortical
area where plectin positive astrocytes were localized as visualized with DAB
(arrows). Scale bar: 25 pm.
(B) The ventro-lateral portion of the hypothalamus showed diffuse plectin
immunoreactivity of astrocytes(open arrow). Scale bar: 25 pm.
(C) White matter regions in the rat nervous system demonstrated plectin antibody
labelling of astrocytes as shown in the optic tract as visualized with
immunoflurescent secondary antibodies (open arrow). Scale bar: 25 pm.


61


62
dorsal portion and tanycytes, specialized ependymal cells, lining the ventral portion
of the third ventricle were plectin positive (Figure 3-4a). The plectin antibody
labelling of ependymal cells showed strong staining at the plasma membrane,
diffuse staining throughout the cytoplasm, and dense staining between the
ependymal cells (Figure 3-4b). The ependymal labelling pattern was observed in
all ventricles. A network of astrocytic fibers labelled with plectin antibodies was
seen in the subependymal layer (Figure 3-4b). In tanycytes, plectin
immunoreactivity was associated with the periphery of the cell body as well as with
the fibrous process (Figure 3-4c).
Cerebellum. Plectin immunoreactivity in the cerebellum was observed in
Bergmann glial fibers in the molecular layer, and in astrocytes in the granular cell
layer and white fiber tract layer (Figure 3-5a). The entire extent of Bergmann glial
processes were labelled with plectin antibodies; however, the strongest staining
was associated with Bergmann fibers terminal endings at the pial surface. The
relative intensity difference between the knob-like endings and processes of
Bergmann glia fibers was observed distinctly in a immunofluorescent higher power
view of molecular layer of cerebellum (Figure 3-5b). With respect to the terminal
endings of the Bergmann fibers which forms the external glial limiting membrane
in the cerebellum, it was difficult to determine whether these subpial structures
which were plectin positive formed conical and/or foot type endings (Palay and
Chan-Palay, 1974). The staining pattern once again showed strong labelling at the
periphery of the cells.


Figure 3-4. Plectin immunoreactivity (ID8) in the rostral hypothalamus surrounding
the third ventricle as visualized with DAB (3V).
(A) Cells lining the third ventricle are plectin positive and are of two types:
ependymal cells in the dorsal portion and tanycytes in the ventral portion. Scale
bar: 100 pm
(B) High power view of ependymal cells which demonstrates plectin antibody
staining throughout the cytoplasm with highest density of labelling at the cell
membrane (arrowheads). Light astrocytic staining is observed in the
subependymal cell layer just lateral to the ependymal cells. Scale bar: 25 pm.
(C) High power view of tanycytes which shows plectin immunoreactivity solely
around the plasma membrane (arrowheads) and radial process of the tanycyte
(arrows). Scale bar: 25 pm.


64


Figure 3-5. Plectin immunoreactivity (IA2 and ID8) in the cerebellum.
(A) In the cerebellum plectin antibody (IA2) labels the full extent of the Bergmann
glia process (arrow) in the molecular layer (ml) with pronounced labelling at the pia
surface (arrowhead) as visualized with DAB. Weak plectin immunoreactivity is
present in the granular cell layer (gel) and appears to correspond to astocytic
processes. Note the absence of plectin antibody staining in the purkinje cell layer
(pci). Scale bar: 100 pm.
(B) Higher power view of the cerebellum molecular layer showing the prominent
fluorescent labelling of the Bergmann glia terminal processes using ID8 antibody
(arrows) as visualized with immunoflurescent secondary antibodies. In comparison
the Bergmann glia fibers are weakly labelled with plectin antibody (open arrows).
Scale bar: 25 pm.


66


67
Brainstem and spinal cord. The general pattern of the plectin antibody
staining pattern in the brainstem and spinal cord localized plectin primarily to non
neuronal cells in the white matter tracts and to a subset of motoneurons. At low
magnification of the brainstem at the caudal medulla, plectin antibody labelling was
localized to astrocytes in the periphery and motoneurons in the nucleus ambiguus
(Figure 3-6a). A high magnification view of the ventrolateral aspect showed plectin
immunoreactivity in the inferior cerebellar peduncle and in the spinal tract of cranial
nerve V (Figure 3-6b). Plectin antibody labelling was pronounced at the outer
boundary of the inferior cerebellar peduncle as well as in glial fibers traversing the
inferior cerebellar peduncle. Throughout this region plectin positive astrocytes
were observed. Plectin staining was not observed in neurons associated with the
spinal nucleus of cranial nerve V (Figure 3-6b). In contrast, intense plectin
immunoreactivity was observed in motoneurons of nucleus ambiguus (Figure 3-6c).
However, there appeared to be a gradient in plectin antibody staining in
motoneurons such that some motoneurons labelled strongly while others stained
lightly (Figure 3-6c). This selective type of motoneuron staining was also observed
in the facial nucleus. In the pyramidal tract, plectin positive astrocytes were seen
as a network of fibers located adjacent to the pial surface (Figure 3-6d).
In the spinal cord, plectin immunoreactivity was observed at all levels with a
similar distribution. Plectin antibody labelling was observed in ependymal cells
lining the central canal, in astrocytes in the dorsal columns, in glial fibers
transversing the white matter, and in motoneurons in the ventral horn (Figure 3-


Figure 3-6. Plectin immunoreactivity (ID8) in the caudal brainstem.
(A) Ventrolateral quadrant of the medulla shows plectin antibody labelling both neurons and glia.
Arrow is pointing to the nucleus ambiguus.(spt: spinal tract trigeminal nerve; icp: inferior
cerebellar peduncle; pt: pyramidal tract). Scale bar: 100 pm.
(B) Spinal tract of V and inferior cerebellar peduncle (icp) labelled with the plectin antibody.
Prevalent plectin immunoreactivity is observed at the outer boundary of icp and in glial fibers
transversing icp (arrows). Scale bar: 50 pm.
(C) Motoneurons in nucleus ambiguus label with plectin antibody. There appears to be a
gradient of staining with some motoneurons staining strongly (arrows) while others stain lightly
(arrowhead). Around motoneuron cell bodies there is a dense particulate staining. Scale bar:
50 pm.
(D) At the periphery of the pyramidal tracts (pt), a network of plectin positive astrocytes are
concentrated near the ventral boundary (arrowheads). Scale bar: 50 pm.


69


70
7a). At a higher magnification, the ventral white matter showed plectin antibody
staining of glia cells which radially span from the pial surface to the gray matter
(Figure 3-7b). A closer examination of motoneurons in lamina IX of the spinal cord
gray matter showed that plectin immunoreactivity was associated with the cell body
and dendrites in a subpopulation of motoneurons (Figure 3-7c). Plectin staining
appeared to be excluded from the nucleus. In the vicinity of plectin positive
motoneurons, particulate staining was observed which may be cross-sections of
dendrites (Figure 3-7c.).
Choroid plexus and blood vessels. Choroidal epithelial cells showed
prominent plectin immunoreactivity (Figure 3-8a). Labelling with plectin antibodies
was observed throughout the cytoplasm with particularly intense staining at the
periphery and at points of contact between adjacent cells Differential plectin
antibody staining patterns were observed with blood vessels. Plectin
immunoreactivity was predominantly associated with endothelial cells of large blood
vessels (Figure 3-8b) but also was observed sporadically with smaller blood
vessels (Figure 3-8c).
Discussion
These experiments were initiated in an attempt to identify a salt and detergent
resistant 300 kD protein band found in spinal cord cytoskeleton preparations which
could represent a NF binding protein. Biochemically, plectin was at least a
constituent of this 300 kD protein band, and was found in crude IF preparation of


Figure 3-7. Plectin immunoreactivity (ID8) in the cervical spinal cord.
(A) Low magnification view of the cervical spinal cord that demonstrates plectin
antibody labelling of astrocytes in the dorsal columns (dc), radially oriented glia in
white matter (wm) and motoneurons in the ventral horn (vh). Scale bar: 100 pm
(B) High magnification view of lateral white matter in which the intensity of plectin
antibody staining is graded. The strongest labelling is near the periphery (arrows).
Scale bar: 50 pm.
(C) High magnification view of motoneurons in lamina IX showing that plectin
immunoreactivity is associated with the cell body and processes of motoneurons
in the ventral horn. Particulate staining is observed in the vicinity of motoneurons
(arrowheads). Scale bar: 50 pm.


4-
O


Figure 3-8. Plectin immunoreactivity (p21) in choroid plexus and blood vessels.
(A) Choroidal epithelial cells show diffuse plectin antibody staining throughout the
cytoplasm and concentrated staining at the periphery of the cell (arrow). Scale
bar: 25 pm.
(B) Pericollosal artery labels strongly with the plectin antibody. Staining is localized
predominantly to the inner membrane of endothelial cells (open arrow). Scale bar:
25 pm.
(C) A smaller blood vessel in the temporal cortex labels positive with plectin
antibody. Note the astrocytic processes surrounding the blood vessel are labelled
with the plectin antibody. Scale bar: 25 pm.


74


75
the central nervous system.
One approach to begin to understand the role of a particular protein in the
nervous system is to examine the cellular distribution. Plectin immunoreactivity
was observed in a number of different cell types in the central nervous system with
varying staining intensities (Table 5-1). To summarize these data, prominent
plectin antibody staining was localized to the periphery of ependymal cells lining
the ventricles, to tanycytes, to choroidal epithelial cells, to endothelial cells and to
pia mater cells. In addition, dense plectin immunoreactivity was observed at
junctions between choroidal epithelial cells which may be related to tight junctions
forming part of the barrier between the blood and cerebral spinal fluid (CSF)
(Peters et al., 1991). Previous research by Wiche and co-workers (1989)
demonstrated that plectin was associated to various types of junctional zones. An
interesting and rather unexpected finding was that plectin antibodies labelled the
perikarya of a few neurons in the central nervous system. Generally, the staining
pattern of the plectin antibodies in non-neuronal cells of the nervous system
coincided well with previous work on the distribution of plectin outside the nervous
system (Wiche, 1989) and was similar to the vimentin staining pattern in the central
nervous system (Shaw et al., 1981; Pixley et al., 1981; Yen and Fields, 1981).
The previous studies of plectin concluded that plectin was widely but not
ubiquitously distributed with the peripheral regions of cells, with some overlap with
the IF pattern and submembraneous component. The cellular distribution of plectin
in the CNS appeared to be similar to that observed in non-neural tissue in that


Table 3-1. The distribution of plectin in different
cell types of the adult rat central nervous system.
Cell Type
Staining
Neuronal Cells
cortical pyramidal
-
cortical interneurons
-
cerebellar granule
-
cerebellar Purkinje
-
brain stem motoneurons
+++/++/+/-
spinal cord motoneurons
+++/++/+/-
Ependyma
choroid plexus
+++
columnar epithelia
+++
tanycytes
+++
subependyma
++
Glial Cells
astrocytes (white matter)
+++
astrocytes (gray matter)
+/-
Bergmann fibers
++
Other
endothelial linings
+++/++/+/-
+: indicates that plectin was localized in these cells, and
number of + reflects intensity of staining; indicates that
plectin was not observed in these cells


77
plectin immunoreactivity was usually associated with the plasma membrane of the
cells.
A relationship of plectin with the blood-brain barrier (BBB) is suggested by a
tendency for plectin to be concentrated at all three of the elements of the BBB,
namely the pia, the walls of blood vessels and the linings of ventricles. However,
plectin immunoreactivity was not associated with all blood vessels and was only
infrequently associated with glial endfeet surrounding blood vessels. The BBB,
therefore, is not invariably associated with plectin immunoreactivity. Consequently
one can conclude that plectin is not absolutely required in all parts of this barrier.
In contrast, there is a consistent and strong layer of plectin immunoreactivity
associated with the ependymal layer including the choroid plexus, suggesting that
plectin may mechanically stabilize the submembraneous cytoskeleton and IF
components to form the three-dimension structure of the choroid plexus. In line
with this idea, previous ultrastructural studies have demonstrated a tendency for
plectin to be localized at interfaces between different tissues and especially
between tissues and fluid filled cavities. For example plectin is heavily
concentrated at the surfaces of kidney glomeruli, liver bile canaliculi, bladder
urothelium and gut villi (Wiche et al., 1983).
The selective staining pattern of plectin to subsets of motoneurons in the
brainstem and spinal cord is intriguing. One possible explanation for this unique
finding is that plectin may be associating with a particular NF subunit. Of the five
specific NF subunits, peripherin appears to be the best candidate. Peripherin has


78
a more selective distribution than the NF-triplet proteins and in contrast to cortical
localization of a-internexin, peripherin is localized to motoneurons of the spinal
cord and brainstem (Portier et al., 1984; Parysek and Goldman, 1988; Greene,
1989; Goldstein et al., 1991).
A common theme in this distinct plectin staining pattern is that plectin may be
associating with class 3 IF proteins (based on Steinert and Roop classification
scheme, 1988). Since peripherin and vimentin are similar in structure, it is likely
unlikely that plectin could be interacting at a similar site present in both proteins.
To shed some light on the selective cellular interactions of plectin in the nervous
system, Chapter 6 focuses on examining the distribution of plectin with IFs found
in the neural tissue.
Notes
Portions of this chapter are from a paper entitled "The Distribution of Plectin,
an Intermediate Filament Associated Protein, in the Adult Rat Central Nervous
System" by L.D. Errante, G. Wiche, G. Shaw. Journal of Neuroscience Research
37: 515-528. Copyrighted 1994 by Wiley-Liss. Text is used with the permission
of Wiley-Liss, a division of John Wiley & Sons, Inc.


CHAPTER 4
CO-LOCALIZATION OF PLECTIN AND INTERMEDIATE
FILAMENTS IN THE RAT NERVOUS SYSTEM
Introduction
Intermediate filaments (IFs) are a family of fibrous proteins found in most
eukaryotic cells and comprise a major component of the cytoskeletal network. In
addition to being organized into 6 classes based on sequence analysis (Steinert
and Roop, 1988), IFs can be categorized based on the types of cells they are
localized to. The strict cell-type specific expression of IFs, coupled with their high
abundance has permitted the extensive use of IF antibodies as convenient cell-
type specific markers. For example, in the nervous system, NF-triplet proteins, a-
internexin and peripherin are localized exclusively to neurons, and different neuron
types tend to have distinctly different IF composition (Shaw et al., 1981; Yen and
Fields, 1981; Portier et al., 1984; Pachter and Liem, 1985; Parysek and Goldman,
1988; Chiu et al., 1989). GFAP is localized to specific glia cell types (Yen and
Fields, 1981; Shaw et al., 1981) while vimentin can be found in subsets of glia,
ependyma and endothelia as well as some unusual neurons (Yen and Fields,
1981; Shaw et al., 1981; Draeger, 1983; Shaw et al., 1983; Schwob et al., 1986).
IF proteins are similar to the other major cytoskeletal proteins, microtubules
and actin, in that IFs have a number a polypeptides associated with them. Unlike
79


80
microtubule- and microfilament-associated protein, intermediate filament associated
proteins (IFAPs) are not well characterized especially in neural tissue. Due to the
lack of function associated with IFs in general, proteins have been classified as
specific IFAPs based on minimal criteria such that only one of the following
conditions need to be satisfied: (1) co-purification with IFs in vitro\ (2) cellular co
distribution with IFs; (3) binding to IFs or subunit proteins in vitro; and (4) have an
effect on filament organization or assembly (Foisner and Wiche, 1991).
Plectin is one of the few IFAP to have satisfied more than one of these
criteria including solid phase binding to all IF proteins examined and co-localization
with vimentin (Wiche, 1989). In the nervous system, plectin has been shown to
co-purify with crude IF preparations and to be localized in specific cell types that
contain IF proteins (Chapter 3). To address possible in vivo interactions between
plectin and IF proteins in the nervous system, we examined the co-distribution of
plectin with NF triplet proteins, peripherin, vimentin, and GFAP.
Methods
Experimental Tissue and Immunocytochemistrv
For immunofluorescent studies, adult rats were sacrificed by decapitation.
Neural tissue was dissected out immediately, quickly frozen and stored at -70C.
Immunofluorescent procedures were described in Chapter 2.
For facial nerve axotomy, adult rats were anesthetized with methoxyflurane.
An incision was made just behind the right ear, the major branch of the facial nerve
was cut, and the incision was closed with surgical staples. Six to seven days post-


81
lesion rats were anesthetized with pentobarbital, and animals were killed by
decapitation or after intracardial perfusion with 4% paraformaldehyde as described
in detail in Chapter 2. In the case of the paraformaldehyde fix tissue, the
brainstem was blocked, immersed in 30% sucrose in PBS until it sunk and then
frozen on dry ice for 1 hour. Tissue was cut at 10 pm and placed in PBS and
treated in the same manner as previously described for paraformaldehyde tissue
(Chapter 2).
Antibodies
Plectin monoclonal antibodies, clones 1D8 and IA2 (diluted 1:2), and rabbit
polyclonal serum, p21 (diluted 1:50), were previously characterized (Wiche and
Baker, 1982; Foisner et al., 1991). NF-M rabbit polyclonal sera, R9 (diluted 1:200),
to the KE (lysine-glutamic acid) region of NF-M was made using NF fusion protein
RM:677-845. NF-H rabbit polyclonal antibody, R14 (diluted 1:200), to the KSP
(lysine-serine-proline) region of NF-H was made using NF fusion protein RH:559-
794 (Harris et al., 1991). Monoclonal antibodies against glial fibrillary acidic protein
(GFAP), clone GA5 (diluted 1:1000) and vimentin, clone V9 (diluted 1:200), were
purchased from Sigma (Debus et al., 1983; Osborn et al., 1984). Rabbit polyclonal
antibody to GFAP (diliuted 1:1000) was a gift from Dr. P. J. Reier. Peripherin
rabbit polyclonal sera, R19 and R20 (diluted 1:200) and mouse monoclonal
antibody, 8G2 (undiluted), were produced in our laboratory using a trp-E fusion
protein construct in the pATH expression vector which was a gift of Dr. Edward Ziff
(see Gorham et al., 1990). The fusion protein was expressed in Escherichia coli


82
in bulk and purified from inclusion body preparations by DEAE-cellulose ion
exchange chromatography as recently described for other trp-E fusion proteins
(Harris et al., 1991). The purified fusion protein was injected into rabbits and mice
to raise polyclonal and monoclonal antibodies using standard procedures (see
Chapter 2).
Results
Immunoblot Studies
Initial characterization of peripherin antibodies were performed on crude IF
preparations from rat spinal cord to make sure that the antibody was not cross
reacting with any other cytoskeletal proteins. Both peripherin monoclonal antibody
(8G2) and polyclonal antibody (R20) labelled, cleanly and specifically, a single
protein band at -57 kD. The peripherin protein band migrated at a slightly higher
apparent molecular weight than vimentin in crude IF spinal cord preparation (Figure
4-1, lanes 2-4, respectively). Peripherin polyclonal antibody R19 also showed the
same labelling of a single band at 57 kD (data not shown).
Co-localization in Cultured Cells
Dorsal Root Ganglion (DRG) Cells. Plectin antibodies labelled most of the
cell types in DRG cultures isolated from postnatal day 1 rat pups. The non
neuronal cells or satellite cells consisted of two basic types; flat ameboid shaped
and spindle shaped which are illustrated in Figure 4-2. Plectin immunoreactivity
was localized throughout the cell body and the processes of the spindle shaped


Figure 4-1. Characterization of peripherin polyclonal and monoclonal antibodies.
Lane 1 shows a serva blue stained 6% SDS polyacrylamide gel of crude IF
preparation for rat spinal cord. Lanes 2 and 3 are immunoblots of this material
labelled with 8G2 and R20, respectively. These results show that both the
monoclonal and polyclonal antibodies to peripherin labelled a single protein band
at -57 kD. Lane 4 is an immunoblot of the same material labelled with vimentin
monoclonal antibody (V9) which demonstrates that the vimentin protein band is
located just below peripherin on a 6% SDS polyacrylamide gel.


00


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IDENTIFICATION AND CHARACTERIZATION OF INTERMEDIATE
FILAMENT BINDING PROTEINS IN THE NERVOUS SYSTEM
By
LAURA DIANE ERRANTE
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1994

To my parents, with whom I realized how much I could accomplish if only I try,
and to my dearest Gator, who would never let me lose sight of my dreams.

ACKNOWLEDGEMENTS
Although it is now time to begin anew, I will never forget the people I have
met that have made the past six years a learning experience. Foremost I would
like to thank Dr. Gerry Shaw, my advisor, who showed me, through his example,
that although it may be difficult at times to do bench work, that this is why we
become scientists in the first place. In addition, I appreciate his patience,
especially in allowing me to develop my own sense of independence. I would like
to acknowledge my committee members, Dr. Barabara Battelle, Dr. Kevin
Anderson, and Dr. Daniel Purich, for their insight and support on my doctoral work.
I would like to credit Dr. Luttge for having the wisdom 5 years ago to let me know
that although I was having difficult times, I still had the ability to become a good
scientist. I only hope that the present and future graduate students in the
department will benefit from his wisdom as I have over these years. It is easiest
to remember to thank the people who have had direct input on your research;
however, I would like to give special thanks to Dr. Roger Heintz, my undergraduate
advisor at SUNY Plattsburgh, for instilling in me the basics of learning techniques,
especially column chromatography which has been essential to my dissertation
research, and for his encouragement to enter a field of study that can be
intimidating at times but is always fascinating and rewarding.
There are a number of graduate students and staff that have made an

indulible impression on me. In particular I will always treasure my friendships with
Paul Bao, Michele Davda, Bunnie Powell, and Margaret Tremwel. I would like to
acknowledge my family, especially my mom and brother, Wayne, who may not
have an understanding of what I have been doing the past six years but have
supported me just the same, no matter how infrequently they heard from me.
It has been a long and sometimes twisted road to reach this point, and I
am fortuitous to have found a true friend in Rob Friedman. Even when I thought
all was lost, his constant and unconditional support as well as critical assessment
of my research, enable me to envision the "light at the end of tunnel" and make
my dissertation a reality.
This work was supported by a NIH Predoctoral Training Fellowship
MH15737 through the Center for Neurobiological Sciences at the University of
Florida and NIH NS22695 (to Dr. Gerry Shaw). In addition, I would like to thank
the Center for Neurobiological Sciences at the University of Florida for providing
funds to attend scientific meetings.
IV

TABLE OF CONTENTS
ACKNOWLEDGEMENTS iii
LIST OF TABLES viii
LIST OF FIGURES lx
LIST OF ABBREVIATIONS xii
ABSTRACT xiv
CHAPTERS
1 INTRODUCTION AND BACKGROUND 1
Cytoskeleton 1
Intermediate Filament Family of Proteins 2
Neurofilament Assembly 8
NF Phosphorylation 11
Interactions Between Neurofilaments and Microtubules 18
Neurofilaments Interact with Other Cellular Components .... 21
Relationship of NFs to Axonal Transport and Caliber 26
NF Expression After Axotomy 30
Role of NFs in Disease States 32
Overview of Dissertation 34
2 GENERAL METHODS 36
Biochemical Techniques 36
Protein Assay 36
SDS Polyacrylamide Gels 37
Electroblotting of Proteins 38
Immuno-Blot Analysis 39
Protein Cleavage with Cyanogen Bromide 40
Isolation of Neurofilament Proteins 41
Crude Intermediate Filament Preparation 41
Neurofilament Preparation 42
Purification of Individual Neurofilament Subunits 43
v

Anatomical Localization Experiments 44
Dorsal Root Ganglion Cell Cultures 44
Pheochromocytoma Cells (PC12 Cells) 45
Immunofluorescent Studies on Fresh Frozen Tissue 46
Immunocytochemistry on Formaldehyde Fixed Tissue .... 47
3 THE DISTRIBUTION OF PLECTIN, AN INTERMEDIATE
FILAMENT BINDING PROTEIN, IN THE ADULT RAT
CENTRAL NERVOUS SYSTEM 49
Introduction 49
Methods 50
Results 52
Immunoblot Studies 52
Comparison of Plectin to IFAP-300 56
Localization Studies 59
Discussion 70
Notes 78
4 CO-LOCALIZATION OF PLECTIN AND INTERMEDIATE
FILAMENTS IN THE RAT NERVOUS SYSTEM 79
Introduction 79
Methods 80
Results 82
Immunoblot Studies 82
Co-localization in Cultured Cells 82
Co-localization Studies Along the Rat Neural Axis 90
Effects of Peripheral Nerve Axotomy 97
Discussion 105
Notes 115
5 IDENTIFICATION OF NF BINDING PROTEINS 116
Introduction 116
Methods 117
Pig Spinal Cord Cytosolic Preparation 117
Affinity Column Preparation 117
Binding of Cytosolic Proteins to Affinity Columns 118
Production of Polyclonal and Monoclonal Antibodies .... 121
Results 122
Discussion 138
VI

6 CHARACTERIZATION OF GAPDH BINDING TO NFs
144
Introduction 144
Methods 145
Co-sedimentation Experiments 145
Immunofluorescence Studies 146
Antibodies 146
Results 147
Co-sedimentation Experiments 147
Immunofluorescence Studies in Cultured Cells 154
Discussion 161
7 OVERALL DISCUSSION 165
General Considerations 165
Criteria for Classifying IF Binding Proteins 165
Relationship between Plectin and NF Proteins 166
The Role of GAPDH in the Nervous System 168
Future Direction 170
Conclusions 173
REFERENCES 174
BIOGRAPHICAL SKETCH 191
VII

LIST OF TABLES
Table 1-1 Mammalian intermediate filament protein family based
on the classification scheme of Steinert and Roop (1988) .... 2
Table 1-2 Protein kinases which phosphorylate neurofilaments in vitro . . 14
Table 1-3 Interactions of neurofilaments with other cellular components . 22
Table 3-1 The distribution of plectin in different cell types of the adult
rat central nervous system 76
Table 4-1 Summary of the distribution of plectin in different cell types
of the adult rat nervous system with respect to
intermediate filament proteins 111
Table 5-1 Relative molecular weight (kD) of candidate proteins which
bound to the five different types of NF affinity columns .... 123
Table 5-2 Amino acid composition for candidate neurofilament binding
proteins with the data represented as nanomole percent .... 127
Table 5-3 Results from the FINDER program to identity NFL-38 131
Table 5-4 Comparison of NFL-38 to GAPDFI with data represented
as nanomole percent for each amino acid 132
VIII

LIST OF FIGURES
Figure 1-1 Schematic of neural intermediate filament protein sequences . 5
Figure 3-1 Identification of a 300 kD protein in bovine and rat crude
IF spinal cord preparations 55
Figure 3-2 Comparison of amino acid composition of plectin and
IFAP-300 proteins 58
Figure 3-3 Plectin immunoreactivity (ID8) of astrocytes in white and
gray matter of the telencephalon and diencephalon 61
Figure 3-4 Plectin immunoreactivity (ID8) in the rostral hypothalamus
surrounding the third ventricle (3V) as visualized with DAB . . 64
Figure 3-5 Plectin immunoreactivity (IA2 and ID8) in the cerebellum ... 66
Figure 3-6 Plectin immunoreactivity (ID8) in the caudal brainstem 69
Figure 3-7 Plectin immunoreactivity (ID8) in the cervical spinal cord .... 72
Figure 3-8 Plectin immunoreactivity (p21) in choroid plexus and
blood vessels 74
Figure 4-1 Characterization of peripherin polyclonal and monoclonal
antibodies 84
Figure 4-2 Co-localization of plectin (p21) and vimentin (V9) in
non-neuronal cells from DRG cultures 87
Figure 4-3 Co-localization of plectin (ID8) and NF subunits in
DRG cells and PC12 cells 89
Figure 4-4 Co-localization experiment with antibodies to plectin
(p21) (A,C,E,G) and vimentin (V9) (B,D,F,H) 93
Figure 4-5 Co-localization experiment with antibodies to plectin
(A,C,E) and GFAP (B,D,F) 95
Figure 4-6 Co-localization experiment with antibodies to plectin
(ID8) (A.C.E) and NFs (polyclonal) (B,D,F) 99
Figure 4-7 Co-localization experiment with antibodies to plectin
(IA2) (A,C) and peripherin (R19) (B,D) 101
IX

Figure 4-8 Effects of a unilateral facial nerve axotomy on plectin
immunoreactivity (1A2) 104
Figure 4-9 Co-localization experiment with plectin (IA2) (A,C)
and NF-FI (R14) (B,D) after a unilateral facial nerve
axotomy 107
Figure 4-10 Co-localization experiment with plectin(IA2) (A,C)
and peripherin (R20) (B,D) after a unilateral facial
nerve axotomy 109
Figure 5-1 Outline of the method used for identifying candidate
NF binding proteins 120
Figure 5-2 Comparison of candidate 38 kD NF binding protein
to rod domain of IF proteins 126
Figure 5-3 Comparison of amino acid composition data between
NFL-38 and IF-38, and to that of the average amino
acid composition in Genbank 130
Figure 5-4 Comparison of CNBr cleavage fragments of NFL-38
and GAPDFI 135
Figure 5-5 Immunoblot analysis using antibodies to GAPDH to
determine if the 38 kD proteins binding to various NF
affinity columns is GAPDFI 137
Figure 5-6 Graphical representation of the amino acid composition
data for NFL-16, and possible matches as determined
by the FINDER program 140
Figure 5-7 Graphical representation of the amino acid composition
data for NFL-62, and to that of the average amino acid
composition in Genbank 142
Figure 6-1 Co-sedimentation experiment with GAPDFI and NF subunits 149
Figure 6-2 Co-sedimentation of GAPDFI and NF-L with different
concentrations of GAPDFI 151
Figure 6-3 Co-sedimentation of GAPDFI and NF-L with different
concentrations of sodium chloride 153
Figure 6-4 Composite of GAPDFI antibody labelling (ID4) of a DRG
cultured neuron 156
Figure 6-5 Co-localization experiment in DRG cell cultures with
antibodies to GAPDH (ID4) (A,C) and NF-H (R14) (B,D) ... 158
x

Figure 6-6 Co-localization experiment in differentiated PC12
cell cultures with antibodies to GAPDH (ID4) (A,B)
and NF-M (R9) (C) 160
Figure 6-7 Co-localization experiment in DRG cell cultures with
antibodies to GAPDFI (ID4) (A) and actin (polyclonal) (B) .. 163
XI

LIST OF ABBREVIATIONS
Amino Acids
ALA or A
-
alanine
ARG or R
-
arginine
ASN or N
-
asparagine
ASP or D
-
aspartic acid
ASX or Z
-
asparagine and/or aspartic acid
CYS or C
-
cysteine
GLN or Q
-
glutamine
GLU or E
-
glutamic acid
GLX or B
-
glutamine and/or glutamic acid
GLY or G
-
glycine
HIS or H
-
histidine
ILE or 1
-
isoleucine
LEU or L
-
leucine
LYS or K
-
lysine
MET or M
-
methionine
PHE or F
-
phenylalanine
PRO or P
-
proline
SER or S
-
serine
THR orT
-
threonine
TRP or W
-
tryptophan
TYR or Y
-
tyrosine
BBB
- blood-brain barrier
BSA
- bovine serum albumin
CBB
- Coomassie brillant blue R-250
CNBr
- cyanogen bromide
CNS
- central nervous system
CSF
- cerebrospinal fluid
DAB
- diaminobenzidine tetrahydrochloride
DRG
- dorsal root ganglion
DTT
- dithiothreitol
EDTA
- ethylenediamine tetraacetic acid
XII

FBS
- fetal bovine serum
GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
GFAP
- glial fibrillary acidic protein
GST
- glutathione S-transferase
IF
- intermediate filament
IFAP
- intermediate filament associated protein
kD
- kiloDaltons
MES
- 2-(4-morpholino)-ethanesulfonic acid
MT
- microtubule
NF
- neurofilament
NF-H
- neurofilament high molecular weight subunit
NF-M
- neurofilament middle molecular weight subunit
NF-L
- neurofilament low molecular weight subunit
TBS
- tris buffered saline
PBS
- phosphate buffered saline
PMSF
- phenylmethyl-sulfonyl fluoride
PNS
- peripheral nervous system
PVDF
- polyvinylidine difluoride
SDS
- sodium dodecyl sulfate
TAME
- Na-p-toluenesulfonyl-L-arginine methyl ester
XIII

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
IDENTIFICATION AND CHARACTERIZATION OF INTERMEDIATE
FILAMENT BINDING PROTEINS IN THE NERVOUS SYSTEM
By
LAURA DIANE ERRANTE
April 1994
Chairperson: Gerard P. J. Shaw
Major Department: Neuroscience
Neurofilaments (NFs), microfilaments (MFs) and microtubules (MTs) are the
major structural proteins that form the neuronal cytoskeleton. Much more is
understood about the function of MFs and MTs than is known for NFs. This
dissertation research characterized, plectin, a known intermediate filament
associated protein (IFAP) in the nervous system, and searched for candidate NF
binding proteins in order to examine possible roles for NFs.
Plectin distribution was examined throughout the rat CNS. Plectin was
localized to non-neuronal cells with particularly strong immunoreactivity in cells
forming ventricular and pia barriers. In addition, plectin immunoreactivity was
observed in select motoneurons. Double-label studies with plectin and IF proteins
demonstrated that plectin's distribution most closely resembled that for vimentin;
XIV

however, the staining patterns were not mutually inclusive. In select motoneurons,
plectin colocalized with the neural IF proteins, NF triplet proteins and peripherin.
Since plectin and NFs co-exist in the same cell, the previously described in vitro
plectin/NF interactions may be functionally important.
The second part of the dissertation project involved identifying a 38 kD
protein which bound to NF affinity columns. Using a computer program, FINDER,
and biochemical analysis, this protein was identified as glyceraldehyde-3-
phosphate dehydrogenase (GAPDF1). The strength of binding between GAPDFI
and NF-L subunit was shown to be relatively weak with a dramatic decrease in the
amount of protein co-pelleting at 0.05 M sodium chloride. The possible in vivo
interaction between NFs and GAPDFI was examined using fluorescent light
microscopy which showed that GAPDFI was localized throughout the cell body and
processes of dorsal root ganglion cells in culture and differentiated PC12 cells.
Although GAPDFI immunoreactivity colocalized in part with NF antibody labelling,
the immunofluorescent labelling patterns were distinct.
A number of proteins were identified which bound with varying specificity to
NFs in vitro and have a potential of interacting with NFs in vivo. Finding that NFs
may interact, in vivo, in some neurons with plectin and GAPDFI suggests that
certain NF proteins may act as important structural elements during neuronal
injury, and as docking substrates for the localization of glycolytic enzymes.
xv

CHAPTER 1
INTRODUCTION AND BACKGROUND
Cytoskeleton
The cytoskeleton is a three-dimensional network of fibrous proteins found
throughout the cytoplasm. The neuronal cytoskeleton is composed of microtubules
(MTs), microfilaments (MFs) and neurofilaments (NFs). MTs and MFs have been
well characterized in both neuronal and non-neuronal cells, and both have been
shown to have a variety of roles in cell structure and motility. In contrast, very little
is understood about the functional role of NFs (Fliegner and Liem, 1991; Shaw,
1991). NFs are primarily thought of as static structures ensuring mechanical
stability to the neuron and governing the diameter of the axon (Hoffman et al.,
1984). However, changes in phosphorylation state of NFs during development and
regeneration indicate that NFs may play a more active role in neuronal function.
This chapterwill describe pertinent information concerning NF structure, assembly,
phosphorylation, interactions with other proteins, and NFs involvement in axonal
transport and caliber. The last part of this chapter will discuss the effects on NF
proteins of axotomy and neuronal diseases which may indicate possible functions
for these neural intermediate filaments.
1

2
Intermediate Filament Family of Proteins
NFs belong to a family of intermediate filament (IF) proteins which are 10
nm diameter. The IF family is currently grouped into 6 classes of proteins based
on protein sequence similarity and intron placement (Table 1-1).
Table 1-1. Mammalian intermediate filament protein family based on the
classification scheme of Steinert and Roop (1988).
Class Name
MW (kD)
Cellular Location
I
Keratin (acidic)
40-60
epithelial cells and epidermal
II
Keratin (basic)
50-70
derivatives (eg. nails and hair)
ii ii
III
Desmin
52
muscle cells
Vimentin
57
cells of mesenchymal origin
GFAP*
51
astrocytes; some schwann cells
Peripherin
57
neurons
IV
NF-triplet proteins
NF-H11
115
neurons
NF-M§
95
ii
nf-l¥
60
ii
a-Internexin
66
ii
V
Nuclear Lamins
60-70
nuclear lamina of all cells
IV
Nestin
200
neural epithelial stem cells
•GFAP: glial fibrillary acidic protein; ’’’NF-H:
NF high molecular weight protein;
§NF-M: NF middle molecular weight protein;
:Â¥NF-L: NF low molecular weight protein
A number of IF proteins are localized to neurons. The classical NFs, NF
triplet proteins, are found in varying concentration in most nerve cells. (Liem et al.,
1978; Yen and Fields, 1981; Shaw et al., 1981). In addition to the NF triplet
protein, a-internexin and peripherin are localized to a more select set of neurons

3
and have been shown to co-localize with NF-triplet protein (Kaplan et al., 1990;
Parysek et al., 1991). a-lnternexin is distributed predominantly in the central
nervous system (CNS) (Pachter and Liem, 1985; Chiu et al., 1989). Peripherin is
localized mainly in the peripheral nervous system (PNS) (Portier et al., 1984a,
1984b), but is found also in select fiber tracks and perikarya of neurons throughout
the CNS (Parysek and Goldman, 1988; Brody et al., 1989). Although vimentin is
primarily thought to associate with non-neuronal cells, it is localized in developing
neurons and is expressed in mature neurons in the olfactory epithelium (Schwob
et al., 1986) and adult retina (Draeger, 1983). Nestin, a newly described protein
assigned to class VI, is widely expressed in neuroepithelial stem cells which are
the precursors of CNS neurons and glia (Hockfield and McKay, 1985; Frederiksen
and McKay, 1988; Lendahl et al., 1990).
IFs, in general, have a fundamental structure comprised of 3 domains: (1)
amino-terminal head; (2) central a-helical rod domain; and (3) variable carboxyl-
terminal tail (Figure 1.1). The amino-terminal head domain is typically a short
globular peptide sequence of 5-7 kD containing numerous basic amino acids
(Geisler et al.,1982). Flowever, the amino-terminal domain of nestin is only 11
amino acids long and is slightly acidic (Lendahl et al., 1990). The central rod
domain (39 kD) contains a highly conserved heptad repeat of hydrophobic amino
acids that allows for the formation of an a-helical coiled-coil structure (Geisler et
al., 1982). The distinct primary amino acid sequence of the rod domain is
proposed to be the driving force of IF protein self-assembly (Steinert and Roop,

Figure 1-1. Schematic of neural intermediate filament protein sequences.
Intermediate filaments can by divided into 3 domains: (1) amino-terminus head domain; (2) a-
helical rod domain; and (3) variable carboxyl-terminus domain. Each of these domains can be
divided into regions based on repeated sequence or abundance of a amino acids in that region.
These regions are denoted with the single letter code for the amino acids. E: glutamic acid; K:
lysine; P: proline.

Vi menti n
Peri pheri n
a-lnternexin
NF-L
NF-M
NF-H
Nesti n
Ni—U4¥iS»^c
Coil 1a Coil 1b Coil 2 Tail
Coil 1a Coil 1b Coil 2 Tail
Ooil 1a Coil 1b Coil 2 Tail a E S KE segments
Coil 1a Coil 1b Coil 2 Tall a E segment
EHTIW-C
Colli Coil 2 Tail a E1 KSP1 E2 KSP2 KE s SP segments
Coil 1 Coil 2 Tail a KEQP segment
N-terminal Head
(5-7 kD)
a-Helical Rod
(39 kD)
Variable C-terminal Tail
(5-70 kD)
U1

6
1988). One unifying feature of all IFs is a highly conserved epitope at the end of
the rod domain (coil II) which is recognized by the anti-intermediate filament
antibody (a-IFA) (Pruss et al., 1981). Most IF subunits, such as vimentin, have a
5-10 kD globular tail sequence at the extreme C-terminus of the molecule.
Flowever, the class IV and V IF proteins differ from other IFs in that they have long
carboxyl-terminal extensions which contain several distinct types of unusual amino
acid sequences.
The NF carboxyl-terminal tails appear to have very little a-helical or 3-sheet
structure and are thus thought to consist of largely random coils (Geisler et al.,
1985b). The NF tail domain can be divided into 4 regions: (1) tail A; (2) glutamic
rich segment (E segment); (3) lysine-serine-proline repeat segment (KSP
segment); and (4) lysine and glutamic rich segment (KE or KEP segment). It has
yet to be determined if these sequence specific regions are actually functional
domains. In contrast to the other IF subunits, nestin's carboxyl-terminal tail domain
has a distinct repeat segment (KEQP) which is repeated 35 times (Lendahl et al.,
1990). The abundance of glutamic acid residues in the carboxyl-terminal of nestin
is the only similarity with the carboxyl-terminal of class IV IF proteins (Fliegner and
Liem, 1991).
The latter two segments of NF-M and NF-H carboxyl terminal tails (KSP and
KE or KEP) are thought to correspond to the rodlets protruding from the core
filament seen in ultra-structural studies (Hisanaga and Hirokawa, 1988). The
sequence motif of K-SP or K-SP is also present in microtubule associated proteins

7
tau and MAP2 (Shaw, 1989; Shaw, 1991). The K-SP and K-SP sites in pig NF-M
and K-SP site in rat NF-M are phosphorylation sites in vivo (Geisler et al., 1987;
Xu et al., 1989). At the extreme terminus of NF-M is a highly conserved region
known as the KE segment which is rich in lysine and glutamic acid. This highly
charged segment may be important in interactions with other neuronal components
since it is highly conserved and a closely related sequence is also present in the
extreme C-terminus of a-internexin (Shaw, 1991).
NF-H is somewhat similar to NF-M but has a distinct tail domain. In
contrast to NF-M, NF-H has a KSP segment that contains far more repeats of the
KSP motif. In rat, this KSP motif is repeated 60 times. In addition, the KSP repeat
sequences in NF-H are distinct from those found in NF-M (Shaw, 1991). Instead
of a KE region at the C-terminus, NF-H has a segment rich in lysine, glutamic acid
and proline (KEP) which is evolutionary more loosely conserved (Shaw, 1991).
Recently, the different tail segments of NF-M and NF-H have been
dissected out molecularly and expressed as fusion proteins (Harris, et al., 1991).
These specific segments of the high MW NF tail regions can be used to examine
the functional significance NF through examining their interactions with NFs, other
neuronal components and by using these defined protein segments as substrates
for protein kinase assays.

8
Neurofilament Assembly
The way in which individual NF subunits interact and assemble into NFs is
not clearly understood. NF-L appears to be the core structure of assembled 10 nm
filaments since urea solubilized NF-L monomers self assemble into homopolymeric
structures when urea is removed (Geisler and Weber, 1981). NF-M and NF-H
appear to be incapable of forming long 10 nm filaments, however, when NF-L is
present, both NF-M and NF-H are readily incorporated into the 10 nm filament
(Geisler and Weber, 1981; Liem and Hutchison, 1982). Antibody studies (Willard
and Simon, 1981; Hirokawa et al., 1984) and rotary shadowing experiments
(Hisanaga and Hirokawa, 1988) support the idea that NF-L is the central core of
the filament, and NF-M and NF-H are incorporated into this core by their rod
domains where the carboxyl-terminal tail domains of NF-M and NF-H protrude from
the central core of the filament. Recently, rotary shadowing experiments with
antibodies to the tail domain of NF-M and NF-H confirm that the tail regions of NF-
M and NF-H correspond to the protrusions seen ultrastructurally with NFs (Mulligan
et al., 1991). A basic question which remains is how individual IF subunits, in
general, interact to form 10 nm filaments.
The majority of research on IF assembly has focused on class III IFs,
desmin and vimentin, which can form homopolymers in vivo. When IFs form
dimers it is believed that the hydrophobic portions of the a-helical coil regions of
two polypeptide chains interact in a parallel fashion and form a coiled-coil structure
(Parry et al., 1982). In addition to satisfying hydrophobic considerations, the coil-

9
coil type structure allows for favorable charge interactions along the rod domain.
The dimers are then thought to form four chain tetramers either in a parallel (Ip,
1988; Hisanaga et al., 1990), anti-parallel (Geisler et at., 1985a) or staggered anti¬
parallel manner (Stewart et al., 1989). The differences in tetrameric formation may
be related to the different IF subunits used or the way in which tetramers interact
with the intact filament network (Steinert and Roop, 1988). Higher oligomers are
then believed to form by a staggering of the tetramers which then results in the
formation of intermediate filaments (Coulombe and Fuchs, 1990; Ip et al., 1985).
Eight tetramers are thought to comprise a single 10 nm filament.
Recently the assembly process of NFs was studied using low angle rotary
shadowing. These studies showed that the basic building block of IF proteins, NF-
L and GFAP, were an eight chain structure formed from two parallel tetramers
(Hisanaga et al., 1990), where four octamers were proposed to comprise a single
10 nm filament. Differences were also found in the way in which the filaments
were reassembled. Hisanaga et al. (1990) showed that NF-L can assemble into
either four chain or eight chain complexes depending on the pH of a low ionic
strength buffer. In preceding experiments, alkaline buffer (pH 8.5) produced
tetramers (Huiatt et al., 1980; Geisler and Weber, 1982; Ip et al.,1985; Hisanaga
et al., 1990); whereas low salt buffer (pH 6.8) favored the formation of octamers
(Hisanaga et al., 1990).
Hisanaga and Hirokawa (1990a) also examined how NFs assemble end to
end to form long 10nm filaments. They found that in the polymerization of NF-L

10
monomers, sodium ions were important for end-t- end association, whereas
magnesium ions were involved in stabilizing lateral associations. When NFs were
reassembled using NF-L, NF-M and NF-H, the assembly conditions remained
unchanged; however, there was a structural change. With addition of either NF-M
or NF-FI, rod-like projections were seen emanating from the central core of the
filament similar to those previously seen using native NFs (Hisanaga and
Hirokawa, 1988).
The domains of individual NF subunits have been examined to determine
which portions of NF sequence are necessary for assembly into 10 nm filaments.
Genetic deletion of the amino terminal head domain and carboxyl terminal tail
domain of the mouse NF-L gene showed that deletions larger than 30% from the
head domain and 90% from the tail domain prevented incorporation of these
proteins into the intermediate filament network (Gill et al., 1990). In contrast, when
deletions were made in NF-M gene, up to 70% of the head domain and 90% of the
tail domain could be missing and NF-M would still be incorporated into filaments
(Wong and Cleveland, 1990). However, deletions into either amino- or carboxyl-
terminal region of the a-helical rod domain of NF-M prevented assembly of this
gene product into the filament network. Therefore, the intact rod domain was
determined to be critical for NF assembly.
Phosphorylation of NF subunits were found to effect filament assembly.
Gonda et al. (1990) demonstrated that protein kinase C phosphorylated a number
of serine residues in the N-terminal head domain of NF-L and that an increase in

11
phosphate incorporation decreased the rate of polymerization of NF-L monomer
into filaments. In addition, cAMP dependent protein kinase was shown to
phosphorylate NF-L and was found to inhibit assembly of NF-L monomers into
filaments at phosphorylation levels of 1 mol/mol of protein (Nakamura et al., 1990).
Finally, phosphorylation of NF-L filaments resulted in a slow disassembly which
took up to 6 hours. Nakamura and co-workers proposed that controlled
phosphorylation may modulate the dynamic equilibrium between assembly and
disassembly in vivo.
Neurofilament Phosphorylation
The NF triplet proteins are phosphorylated in vivo and the relative amount
of phosphorylation in rat spinal cord was determined by one group to be 3, 6 and
13 moles of phosphate/mole of NF-L, NF-M and NF-H, respectively (Julien and
Mushynski, 1981; Julien and Mushynski, 1982; Xu et al., 1990). It is important to
note that the relative amount of phosphate associated with NFs is variable and
dependent on a number of factors including species, protein preparations, and area
of the nervous system examined. What is generally known is that NF-H appears
to contain the most phosphate per mole of polypeptide followed by NF-M then NF-
L. In the case of NF-H and NF-L the phosphate is associated with serine residues,
whereas, NF-M has phosphorylation sites on both serine and threonine residues
in the rat (Julien and Mushynski, 1982).

12
Phosphorylation sites on NF triplet proteins are localized to particular
regions on the individual NF subunits. In the case of rat NF-L, phosphorylation
sites are found in both the amino head and carboxyl tail domains (Sihag and
Nixon, 1989) with the major phosphorylation site located in the middle of carboxyl-
terminal tail domain (Xu et al. (1990). In addition, in vitro phosphorylation
experiments with NF-L and protein kinase C show that there are 3 phosphorylation
sites on serine residues in NF-L amino-head domain (Gonda et al., 1990). A
detailed study of NF-M phosphorylation sites of rat neurons in tissue culture
demonstrated that there are six sites in NF-M carboxyl-terminus, four of which
have the KSP motif, one with the K--SP motif, and one motif with a serine residue
followed by glutamic acids (Xu et al., 1989). For NF-H the only known
phosphorylation sites are in the multiple KSP repeats, though it is likely that other
phosphorylation sites exist in other regions of this large molecule (Geisler et al.,
1987: Lee et al.,1988).
Three major questions still remain with respect to NF phosphorylation: (1)
what kinase activities are phosphorylating these different regions of NF subunits
in vivo; (2) are these kinases unique to NFs and/or are they previously
characterized protein kinases; and (3) what is the function of NF phosphorylation?
A number of laboratories have examined NF phosphorylation (Table 1.2). The
research with known kinases has focused primarily on NF-L subunit; whereas, NF-
M and NF-H have been utilized to search for novel protein kinase activities
responsible for phosphorylating the KSP repeat motif of NFs.

13
Three classical protein kinases were shown to phosphorylate NFs in vitro
to various degrees (Table 1-2): (1) calcium/calmodulin protein kinase II (CaM
kinase II) (Vallano et al., 1985); (2) cyclic AMP-dependent protein kinase (protein
kinase A) (Tanaka, 1984; Dosemeci and Pant, 1992); and (3) protein kinase C
(Sihag et al., 1988). In vivo phosphorylation experiments examining NF-L and NF-
M demonstrated that most of the fragments that are phosphorylated with one of the
known protein kinases are located in the amino terminal head domain (Sihag and
Nixon, 1989, 1990).
The search for the protein kinase which phosphorylates the KSP repeat
motif in NF-M and NF-H has focused on characterizing the protein kinase activities
which bind strongly to NFs during crude purification procedures (Table 1 -2). These
kinase activities are extracted from NF preparations using high salt buffer and have
been shown to contain a number of protein kinases: (1) CaM kinase II; (2)
nucleotide-dependent kinase; (3) calcium-phosphatidylserine-diglyceride-dependent
kinase; and (4) a regulator independent kinase (Dosemeci et al, 1990). The
regulator independent kinase phosphorylated all three NF subunits as well as a-
casein where the level of phosphate incorporated per mole of protein was in the
following order: a-casein > NF-M > NF-H = NF-L. This kinase activity was shown
to be related to casein kinase I based on: (1) similarities in specificity of protein
kinase inhibitors; (2) the ability of casein kinase I to phosphorylate NFs in a similar
manner to the NF-associated kinase; and (3) the molecular weight range for casein

14
Table 1-2. Protein kinases which phosphoryiate neurofilaments in vitro.
Protein Kinase
Substrate
MW* (kD)
Reference
cAMP dependent
(rat brain)
NF-H, -M, -L
40
Tanaka et al., 1984
Dosemeci and Pant, 1992
Ca27Calmodulin
dependent (rat)
NF-H, -M, -L
640
Vallano et al., 1985
Protein Kinase C
(mouse)
NF-H, -M, -L
77
Sihag et al., 1988
Casein Kinase I
NF-H, -M, -L
30-42
Floyd et al., 1991
cdk1 Kinase
NF-H, -M
34
Hisanaga et al., 1991
nclk Kinase11
NF-H, -M
33
Lew et al., 1992
NF-Kinase (1983)
(rat)
NF-H, -M, -L
GFAP, vimentin
MAPs, tau
40
Toru-Delbauffe & Pierre, 1983
Toru-Delbauffe et al., 1986
NF-Kinase: 1989
(bovine)
NF-H, -M
histone H1
67
Wible et al., 1989
NF-Kinase (1990)
(bovine)
NF-H, -M, -L
casein
Dosemeci et al., 1990
NF-Kinase (1991)
(squid)
NF-H, -M, -L
casein
17-44
Floyd et al., 1991
* molecular weight expressed in kiloDaltons
11 proline directed kinase

15
kinase I (Hathaway and Traugh, 1982) was comparable to that of NF-associated
kinase (Floyd et al., 1991).
Using the salt extraction technique, other laboratories have isolated
additional NF protein kinase activities, where their molecular weight and substrate
specificity suggest that these NF kinase are distinct from each other. Toru-
Delbauffe and Pierre (1983) were able to recover two endogenous protein kinase
activities, one which phosphorylated NFs, and the other which phosphorylate
casein and was identified as casein kinase I. The NF specific protein kinase was
purified by phosphocellulose chromatography and resulted in major band at 40 kD
and a minor band at 200 kD on SDS polyacrylamide gels, and appeared to be co¬
factor independent (Toru-Delbauffe et al., 1986). Wible et al. (1989) partially
purified a 67 kD doublet on SDS polyacrylamide gels and, in contrast to the
previous kinases, their kinase showed more substrate specificity with a preference
for partially dephosphorylated NF-H.
Even though some of the kinases had a preference for the
dephosphorylated form of NF-H, phosphorylation by these various NF kinase
activities did not result in the expected molecular weight shift if the
dephosphorylated NF-H was being phosphorylated in the KSP repeat sequence.
Previous work has demonstrated that the two high molecular weight NF subunits,
NF-H and NF-M, mobility on SDS polyacrylamide gels is greatly retarded due to
the large number of phosphates in the carboxyl-terminal tail domain. Hisanaga
and co-workers (1991) demonstrated that the cdc kinase from starfish, p34cdc2,

16
phosphorylated both NF-H and NF-M in vitro. Furthermore, after phosphorylation
of enzymatically dephosphorylated NF-H, NF-H migration on SDS polyacrylamide
gels was similar to that observed for the native condition where NF-H is heavily
phosphorylated. Similar results were also obtained with a cdc2 kinase,
p34cdc2/p58cyc'mA) isolated from FM3A mouse mammary carcinoma cells (Guan et
al., 1992). These findings appear to be significant since cdc kinases are proline
directed kinases which specifically phosphorylate the serine residue on the
sequence S/T-P making the repeat KSP related sequences in NFs likely
substrates. However, cdc kinases are associated with the regulation of cell cycle
and have not been localized to neuronal cells which leads one to question the
relevance of in vitro phosphorylation of NF by cdc2 kinases.
Recently, a proline directed protein kinase was isolated from bovine brain
that consisted of two subunits, 33 kD and 25 kD (Lew et al., 1992a). This kinase
was shown to phosphorylate NFs, where the 33 kD subunit displayed a high
sequence homology to p34cdc2 kinase (Lew et al., 1992b). Independent, cloning
techniques identified a neuronal cdc2-like kinase (nclk) identical to that isolated by
Lew et al. (1992b) which had a 58% homology to mouse p34cdc2 kinase (Hellmich
et al., 1992). In situ hybridization experiments demonstrated that unlike p34cdc2
kinase, nclk was expressed in high levels in terminally differentiated neurons
(Hellmich et al., 1992).
Complexities in studying NF phosphorylation seems to revolve around the
phosphorylation state of the NF subunits. Most of these studies use NFs which

17
are already phosphorylated in vivo. When NFs are used in this form, the in vivo
phosphorylating sites for a specific kinase may already be phosphorylated. In
addition, the phosphorylation state of NFs may effect the activity of a NF kinase
(Wible et al., 1989). Another problem with complete enzymatic dephosphorylation
is that some phosphate groups are more susceptible to a phosphatase than others,
thus all of the phosphates may not be removed. And finally, the number of
potential phosphorylation sites that exist in NF subunits make it difficult to
determine where a specific NF-kinase is acting.
Since NFs are phosphoproteins and seemingly contain a number of
potential sites for phosphorylation, the next obvious question is what is the role of
NF phosphorylation. The distribution of phosphorylated NFs differs within the
neuron. NFs are heavily phosphorylated in the axon, whereas the dendrites and
cell body appear to contain little or no phosphorylated NFs (Sternberger and
Sternberger, 1983). Even when considering that NF phosphorylation may be
involved in calcium buffering during nerve excitation and regulation of NF
assembly/disassembly, the functional significance of the phosphorylation
differences within the neuron is still not understood.
Ultrastructural difference between NFs found in axons and dendrites was
apparent with deep-etch freeze fracture technique (Hirokawa, 1982; Hirokawa et
al., 1984). NFs appeared to be spaced farther apart in axons then they were in
dendrites. These studies also revealed what appeared to be extensive cross-links
between the NFs in axons which were not seen with other intermediate filaments.

18
Therefore, it was hypothesized that the carboxyl-tail domains of NF-M and NF-H
were mediating this cross-linking, and that the length of the cross-links were
regulated by the amount of phosphate incorporation in the KSP segments.
However, no effect on the structure or length of NF tail projections were observed
upon enzymatic dephosphorylation which removed around 90% of the phosphate
groups (Hisanaga and Hirokawa, 1989).
In conclusion, there appears to be a diversity of protein kinase activities
capable of phosphorylating NFs. At least some of these phosphorylation events
are under tight spatial control in the neuron. Since NF subunits contain numerous
potential phosphorylation sites, we can expect that different phosphorylation events
will have different functional significance in the neuron.
Interactions Between Neurofilaments and Microtubules
Indirect findings suggest an interaction between NFs and MTs.
Anatomically, NFs and MTs run parallel to each other in the longitudinal direction
in axons and dendrites, and there appears to be morphological cross-bridges
between these two structures (Wuerker and Palay, 1969; Burton and Fernandez,
1973; Rice et al., 1980; Hirokawa, 1982; Hirokawa et al., 1984). In addition,
biochemical evidence shows that: (1) NFs co-purify with MTs prepared by the
polymerization-depolymerization method (Berkowitz et al., 1977); and (2) both NFs
and MTs travel in the slowest component of axonal transport (Hoffman and Lasek,
1975).

19
More direct evidence demonstrating NF-MT interactions showed that NFs
inhibit tubulin polymerization. This MT assembly inhibition was found to be the
result of NFs binding to high molecular weight microtubule associated proteins
(MAPs) which are known to catalyze MT assembly (Leterrier et al., 1982). To
further examine this interaction, Aamodt and Williams (1984) compared the ability
of NF isolated from brain (NF-Brain) with that of NFs isolated from spinal cord (NF-
SC) to form viscous complexes with MTs. They demonstrated that only NF-Brain
were able to form viscous complexes with MTs. They hypothesized that NF-SC
did not have MAPs bound to them; a suggestion supported by the finding that
when purified MAPs were added to NF-SC and MTs, a viscous solution formed.
Flynn et al. (1987) went on to show that NFs bound to the carboxyl-terminal
thrombin fragment of MAP2 (28 kD) which also is the MT binding site. Solid-phase
binding techniques show that NF-L was the only NF protein to bind to MAP2
(Fleimann et al., 1985). The 28 kD fragment of MAP2 was also found to bind to
purified NF-L (Flynn et al., 1987).
To examine a possible in vivo interaction of MAP2 binding to both NF and
MT network, B,B'-iminodipropionitrile was used to segregate the two cytoskeletal
networks in peripheral nerves. Two different monoclonal antibodies to MAP2
demonstrated that one antibody to MAP2 co-localized with the NF network while
the other co-localized with MT network, suggesting that MAP2 is acting as a
crosslinker between these two fibrous networks (Papasozomenos et al., 1985).

20
In addition to MAP2 binding to NF-L, another MT associated protein, tau,
bound to reassembled NF-L. Competition studies with MAP2 and tau for binding
to NF-L demonstrated that these proteins bound to different sites on NF-L. The
in vivo significance is in question since the affinity of either MAP2 or tau was
decreased when NF-M and NF-FI were present in the filamentous form as normally
is the case in vivo (Miyata et al., 1986).
Hisanaga and Hirokawa (1990b) examined the ability of NF subunits to bind
directly to polymerized MTs, and showed that dephosphorylated NF-H bound to
polymerized MTs in vitro. Further work by Hisanaga and co-workers (1993)
suggested that the binding of dephosphorylated NF-H to MTs was not dependent
on the degree of dephosphorylation but on the removal of a particular group of
phosphates. This was demonstrated by using two different phosphatases; alkaline
phosphatase and acid phosphatase. Partially dephosphorylated NF-H co¬
precipitated with MTs in a similar manner even though alkaline phosphatase
removed 46 of 51 phosphates on NF-H whereas only 19 phosphates were
removed with acid phosphatase. In addition, this binding of dephosphorylated NF-
H to MTs is thought to occur at the carboxyl terminal tail region of NF-H.
NFs were shown to promote tubulin assembly in vitro (Minami et al., 1982);
however, NF preparations used in these initial studies were contaminated with
MAPs so that the MAPs may have been a factor promoting tubulin assembly. To
clarify these results, Minami and Sakai (1983) demonstrated that highly purified NF
preparations retained their ability to promote MT assembly as measured by a

21
gelation assay. Adding purified NF subunits to the assay demonstrated that NF-H
was the subunit responsible for promoting tubulin assembly. NF-M was able to
induce some MT assembly whereas NF-L had no effect on MT assembly.
Negative stain images of MT assembled in the presence or absence of NFs
showed that there was an increase in MT assembly in the presence of NFs. When
these structures were examined under the electron microscope, there appeared
to be no morphological differences between the assembled microtubules. In the
electron micrographs, the NFs appeared to make a lateral association with the MTs
which resembled a T shaped structure.
The role of NF binding to MTs is not known but it is believed that the tail
regions of the high molecular weight NF proteins are involved in some cross-linking
function. MAP2 is localized in dendrites and may be mediating some of the
interactions between NFs and MTs; whereas, tau is localized in axons where it
may be performing a similar function.
Neurofilaments Interact with Other Cellular Components
The interactions of NFs with other components of the neuron is not as well
studied as those interactions between NFs and MTs (Table 1-3). The majority of
proteins that will be discussed below bind primarily to NF-L.
Brain spectrin (fodrin) is closely associated with the plasma membrane
throughout the neuron (Levine and Willard, 1981) and is thought to play an active
role in stabilizing the membrane as well as immobilizing certain membrane proteins

Table 1-3. Interactions of neurofilaments with other cellular components.
Component
NF Subunit
Affinity (Kd)
Method
Reference
Microtubules
NF-H
gelation of tubulin
Minami & Sakai, 1983
NF-H (dephos.)
3.8 x 10'8M
co-sedimentatlon
Hisanaga & Hirokawa, 1990
MAP2
NF
1.0x10'7M
co-sedimentation
Leterrier et al., 1982
NF
2.0 x 10'7M
immunocytochemistry
Papasozomenos et al., 1985
NF-L
solid-phase binding
Heimann et al., 1985
NF-L
4.8 x 10'7M
co-sedimentation
Miyata et al., 1986
MAP2
(28kD fragment)
NF, NF-L
co-sedimentation
Flynn et al., 1987
Tau
NF-L
1.6x10'6M
co-sedimentation
Miyata et al., 1986
Brain Spectrin
NF-L
4.3 x 10'7M
solid-phase binding
Frappier et al., 1987
Plectin
NF-H, -M, -L
solid-phase binding
Foisner et al., 1988
Synapsin I
NF-L
solid-phase binding
co-sedimentation
Steiner et al., 1987
NAPA-73
NF
ultrastructural analysis
Ciment, 1990
Nucleic Acids
rRNA
NF-H, -M, -L
preference 18s
co-sedimentation
Traub et al., 1985
dsDNA
NF-H, -M, -L
weakly
it
ii ii
ssDNA
NF-M, -L
preference for NF-L
ii
ii ii

23
(Hirokawa, 1989). Frappier and co-workers (1987) demonstrated that brain
spectrin associates with NF-L and GFAP reversibly and in a concentration
dependent manner using in vitro binding assays. Cleavage with BNPS-skatole
reagent demonstrated that the binding site for brain spectrin was localized to the
40 kD N-terminal fragment which includes the head domain of brain spectrin and
the proximal portion of the rod domain. Further cleavage of the 40 kD N-terminal
fragment demonstrated that brain spectrin bound to the 20 kD proximal portion of
the rod domain in NF-L (Frappier et al.p 1991). The binding of brain spectrin to
NF-L may indicate how NFs associate with the plasma membrane.
Plectin is an intermediate filament associated protein of an apparent
molecular weight of 300 kD (Wiche and Baker, 1982). Using in vitro binding
assays, plectin was shown to bind to the a-helical rod domain of all IF proteins
examined including the NF triplet protein subunits (Foisner et al., 1988). Plectin
has been extensively studied over the past decade in non-neural tissue (Wiche and
Baker, 1982; Wiche et al., 1983; Wiche et al., 1984) and is considered to be a
widely distributed IF associated protein. Plectin is believed to be important in the
interaction between IF proteins, and the association between IF proteins and other
components of the cytoskeleton such as the plasma membrane (spectrin), nuclear
envelop (lamins) and MTs via MAPs (Herrmann and Wiche, 1987). In addition to
these functions, plectin may be important in cell to cell interactions at tight
junctions or myotube attachment sites since both light and electron microscopic
show it associated with these regions (Wiche, 1989).

24
Synapsin I is a phosphoprotein located in the synaptic terminal and is
involved in neurotransmitter release (Llinas et al., 1985). Synapsin I is thought to
act as a cross-linker between synaptic vesicles and the cytoskeleton in synaptic
terminals thereby localizing vesicles near release sites in the terminal. It is thought
that upon influx of extracellular calcium, synapsin I is phosphorylated by CaM
kinase II and released from the cytoskeleton, thereby allowing synaptic vesicles to
associate with release sites on the presynaptic membrane (Hirokawa et al., 1989).
In vitro binding studies by Steiner et al. (1987) have shown that synapsin I binds
to NF-L. This interaction between synapsin I and NF-L can be reduced by 60%
upon phosphorylation of synapsin I with CaM kinase II. However, since NFs do
not appear to exist in polymer form at the synaptic terminal, the binding of NF-L
to synapsin I may not be functionally significant.
NAPA-73 is an avian specific 73 kD NF-associated protein that is expressed
developmentally in the nervous system and heart (Ciment et al., 1986; Ciment,
1990). In contrast to other NF binding proteins, NAPA-73 appears to associate
only with bundles of IFs (not individual subunits) suggesting that NAPA-73 is
involved in the bundling of filaments (Ciment, 1990).
NF binding to RNA and DNA was investigated by Traub et al. (1985). They
examined the binding of individual NF subunits to rRNA (total rRNA from Ehrlich
ascites tumor cells), native DNA (double stranded DNA), and heat-denatured DNA
(partially single stranded DNA) using sucrose gradients. NFs had a higher affinity
for 18S rRNA than 28S rRNA and this affinity could be abolished in the presence

25
of 6 M urea for NF-M and NF-H. In contrast NF-L interaction with 18S rRNA was
resistant to 6 M urea. Native DNA bound very weakly to NF subunits, whereas,
heat-denatured DNA bound strongly to NF subunits except in the case of NF-FI.
The specificity of binding of the different forms of nucleic acids suggested that the
association was not solely based on electrostatic interactions (Traub et al., 1985).
The relevance of NFs binding to RNA and DNA is not known. Flowever, some
researchers speculate that NFs degraded at the axonal terminal by calpain
proteases are transported back to the cell body where they could act as a
messenger for the events occurring at the nerve terminal (Traub, 1985; Schlaepfer,
1987).
Another characteristic aspect of NFs is the ability to bind calcium.
Assembled NFs in both phosphorylated and dephosphorylated states contain high
and low affinity binding sites for calcium (Lefebvre and Mushynski, 1987, 1988).
The dephosphorylation of NFs results in a 50% decrease in the low affinity calcium
binding sites which may be related to the loss of phosphate groups. Coinciding
with the decrease in low affinity calcium binding sites is an increase in the number
of high affinity binding sites. This increase in the high affinity calcium binding sites
is probably reflected by a change in the conformation of NFs after
dephosphorylation. Glutamic acid residues are known to constitute calcium binding
sites in such proteins as calmodulin (Kilhoffer et al., 1983), thus the glutamic acid
rich region in the C-terminus of NF tail regions may be the possible binding site for
calcium. Lefebvre and Mushynski (1988) found that the high affinity calcium

26
binding sites are located in the a-helical rod domain and that the low affinity
binding sites are located at the carboxyl-terminal region of NF molecule. The
functional significance of these calcium binding sites is unknown; however, two
possible functions for calcium binding to NFs have been suggested: (1) the ability
to regulate the activation of calcium binding proteases (calpain) which degrade NFs
in the synaptic terminal (Schlaepfer et al., 1985; Schlaepfer, 1988); and (2) the
buffering of calcium during nerve excitation (Abercrombie et al., 1990).
Relationship of NFs to Axonal Transport and Caliber
The transport of material from the cell body along the axon is an essential
neural function since the axon does not contain the cellular machinery for protein
synthesis. Anterograde axonal transport can be broken down into five components
on the basis of their uniform rate: (1) 250-400 mm/day (fast component); (2) 40
mm/day; (3) 15 mm/day; (4) 2-5 mm/day (slow component b); and (5) 0.2-1
mm/day (slow component a) (Willard, 1974). Cytoskeletal elements travel in slow
component a and b. Actin with its associated proteins travel in slow component
b, whereas, NFs and MTs, travel in the slow component a (Hoffman and Lasek,
1975). In addition, there appears to be a slower second wave of NFs travelling at
a non-uniform rate that is 100 fold slower than slow component a (Nixon and
Logvineko, 1986). It has been proposed that newly synthesized NFs travel down
the axon as assembled NFs, and are incorporated into the stationary cytoskeletal
network at random points during transport (Nixon and Logvineko, 1986). During

27
axonal transport, IMF's overall charge become increasingly negative which
corresponds to an increase in phosphorylation state (Nixon et al., 1986). This
phosphorylation of NF-L and NF-M is short-lived; 50-60% and 35-40% of the
phosphates are removed in five days, respectively. In contrast, NF-H shows little
to no phosphate turnover during the same period (Nixon and Lewis, 1986). Lewis
and Nixon (1988) suggest that differential turnover rate of NF phosphorylation may
regulate the incorporation of NFs into the stationary NF network, and reflect a
dynamic interaction of NFs with other proteins.
Even though NFs are normally perceived as static structures, research
examining the role NF density as an intrinsic regulator of axonal caliber suggest
a more dynamic function for NFs (Hoffman et al., 1984). During development, the
increase in NF synthesis has been correlated to a decrease in NF transport
velocity and an increase in radial growth of the axon (Willard and Simon, 1983;
Hoffman et al., 1985a). This association of NF number to axonal caliber suggests
that regulation of NFs by gene expression and axonal transport largely determines
the size of the axon. Conversely, after a sciatic nerve crush, there is a decrease
in the number of NF proteins delivered to the axon which coincides temporally with
the reduction in axonal caliber proximal to the lesion in comparison to more distal
regions (Hoffman et al., 1985b). As the slow component advances at its normal
rate, the atrophy also advances (referred to as somatofugal atrophy), and when NF
synthesis returns to normal levels the atrophy is reversed (Hoffman et al.,1985b).

28
Similar results are observed with acrylamide toxicity (Gold et al., 1985), further
suggesting that NFs play an vital role in regulating axonal caliber.
In contrast, other research showed that NF density varies considerably
along the length of the axon, as well as in different axonal types, implying that a
local regulation of axonal cytoskeleton must occur (Berthold, 1982; Nixon and
Logvineko, 1986; Price et al., 1988; Szaro et al., 1990). To examine the role of
the cytoskeleton in the local modulation of axonal caliber, a dysmyelinating mutant
mouse line, Trembler, was used. The peripheral nerves of Trembler were
characterized by a reduction in axonal caliber, an increase in density of axonal
cytoskeletal elements, and a decrease in slow component axonal transport (Low
and McLeod, 1975; Low, 1976a,b; de Waegh and Brady, 1990). De Waegh and
co-workers (1992) examined the relationship of axonal caliber and NF
phosphorylation in the Trembler mouse and found that along with an increase in
NF density, there was a concomitant decrease in NF phosphorylation and axonal
caliber. Therefore, the decrease in NF phosphorylation could in turn be related to
a decrease in charge repulsion thus allowing the NFs to pack more closely
together. In contrast, De Waegh and co-workers (1992) suggested that these
alterations were regulated by the amount of myelin surrounding the axon. Grafts
of Trembler sciatic nerve placed into normal nerve showed regenerated axons with
the Trembler morphology while adjacent axons (control) had normal characteristics.
Recently, transgenic techniques were used to examine the effects of murine
NF gene overexpression on neurons and to determine if an increase in NF number

29
in the axon resulted in a concomitant increase in axonal caliber (Monteiro et al.,
1990). The transgene mRNA exceeded the endogenous NF-L mRNA by fourfold.
This resulted in a corresponding increase of the NF-L protein in the peripheral
axons of the trangenic mice. Although there was an increase in density of axonal
NFs seen morphologically, this did not result in a change in axonal caliber.
These results with overexpressed murine NF-L gene suggest that either the
other two NF subunits may be important in regulating axonal caliber or that the
correlation between axonal diameter and NF density may only be valid when there
is a decrease in NF synthesis. The latter possibility would lend support to the
theory of mechanical stability. Since NF-M and NF-H have unusually large
carboxyl terminal domains which protrude as sidearms from the core of the
filament and are highly phosphorylated in the axon, it has been proposed that NF-
M and NF-H are important in forming or regulating the spacing of NFs whether
through actual cross-bridges or charge repulsion from the highly negative carboxyl-
terminal domains. Although these data appear to contradict the active role of NF
density in controlling axonal caliber, it seems more likely that there are both
intrinsic and extrinsic factors involved and that the role of NFs seems to be more
prevalent in large myelinated axons (Hoffman et al., 1988).
Research on neuropathologies related to abnormal NF accumulation has
focused on deficits in axonal transport. Giant axonal neuropathies are
characterized as a focal accumulation of NFs along the axon (Gold et al., 1986)
which suggests the occurrence of a local interruption of NF transport resulting in

30
the accumulation of NFs. Two model systems have shown that with normal NF
synthesis, a retardation of slow component of axonal transport by 3,3'-
iminodipropionitrile (IDPN) results in proximal axonal swelling and distal axonal
atrophy (Griffin et al., 1978; Clark et al., 1980); whereas, after treatment with 2,5-
hexanedione NF transport is accelerated, resulting in proximal axonal atrophy and
distal axonal swelling (Monaco et al., 1984 and 1989).
NF Expression After Axotomy
Differential changes in cytoskeletal proteins following axotomy of peripheral
nerves is observed for both sensory and motor neurons. Overall, the synthesis of
both tubulin and actin increases after axotomy whereas the synthesis of NF triplet
proteins are down regulated (Hoffman et al., 1987; Wong and Oblinger, 1987;
Goldstein et al., 1988; Tetzlaff et al., 1988; Oblinger et al., 1989). This results in
a decrease in the amount of newly synthesized NFs transported into the axon
(Oblinger and Lasek, 1988). However, based on immunocytochemistry, expression
of NF triplet proteins appears to remain unchanged . The time course for changes
in synthesis is similar for all cytoskeletal proteins in that the largest effects occur
between 7 and 14 days, and in the case of a peripheral nerve crush, the level of
synthesis returns to normal between 3 to 8 weeks post injury. If reinnervation is
prevented, NF synthesis remains at decreased levels.
Normally, phosphorylated epitopes of the high molecular weight NF subunits
are restricted primarily to the axon whereas non-phosphorylated epitopes are

31
localized to the cell body and dendrites. However, after axotomy there is a
dramatic and transient increase in phosphorylated epitopes in the cell body that is
concurrent with a decrease in expression of NF mRNA (Moss and Lewkowicz,
1983; Dráger and Hofbauer, 1984; Goldstein et al., 1987; Shaw et al., 1988). As
labelling for NF phosphorylated dependent epitopes increase in the perikarya, there
is a concomitant decrease in labelling for the dephosphorylated dependent epitope
which suggests that phosphorylation is occurring at the sites that would normally
be recognized by dephosphorylated dependent NF antibodies (Goldstein et al.,
1987). In the case where axons do not normally regenerate, phosphorylated
epitopes on NF-H remain up to 60 days in the cell body after axotomy (Dráger and
Hofbauer, 1984).
In contrast to the NF triplet proteins, peripherin expression is upregulated
after sciatic nerve crush in both dorsal root ganglion (DRG) cells and lumbar
motoneurons (Oblinger et al, 1989; Wong and Oblinger, 1990). In DRG cells,
peripherin is normally expressed in small diameter cells whereas NF triplet proteins
are localized primarily to the large diameter cells (Parysek and Goldman, 1988;
Parysek et al., 1988: Ferri et al., 1990; Goldstein et al., 1991). After a peripheral
nerve crush, the mRNA level for peripherin increases in the large diameter cells
(maximizes at 7 to 14 days) but remains unchanged for the small diameter cells.
This increase in mRNA levels for peripherin translates into an increase in
immunoreactivity in large diameter DRG cells. As expected, NFs show a decrease
in mRNA synthesis which is not accompanied by a change in immunoreactivity in

32
the large diameter DRG cells. Similar results are observed in lumbar motoneurons
after sciatic nerve crush for peripherin. For peripherin, the mRNA levels increase
two fold after 4 days, remain elevated for 6 weeks, and finally recover to normal
levels at 8 weeks (Troy et al., 1990).
Role of NFs in Disease States
NFs are thought to be involved in the development of various
neuropathologies. The breakdown of the normal NF organization is prevalent in
motoneuron disease (Flirano, 1991), neurodegenerative disease (Goldman and
Yen, 1986) and toxin-induced neuropathologies (Griffin and Watson, 1988). In
amyotrophic lateral sclerosis (ALS) there is selective degeneration of anterior horn
motoneurons of the spinal cord. One of the hallmark features of ALS is an
abnormal accumulation of NFs in the perikarya, axons and dendrites as well as NF
induced swelling in the axons early in the degenerative process. It is yet to be
determined whether this increase in expression of NF protein is central to this and
other neuropathologies or is a secondary consequence of another cellular disorder.
Recent transgenic technology has resulted in two animal models to study
the overexpression of NFs, one that examines the effects of NF-L overexpression
and the other that examines NF-H overexpression. Doubly transgenic mice, which
resulted in a fourfold increase the murine NF-L gene, produced dramatic
phenotypic and morphological changes (Xu et al., 1993). Phenotypically, doubly
transgenic mice were one to two-thirds the body weight of age-matched controls,

33
kinetic activity was decreased and death usually occurred within 3 weeks due to
widespread skeletal muscle atrophy. In the CNS, murine NF-L expression was
restricted to neurons; however, outside the nervous system, the expression OF
murine NF-L was observed in different areas including skeletal muscle.
Morphometrically, select motoneurons showed chromatolytic features that included
a disrupted rough endoplasmic reticulum and a displaced nucleus. The
overexpression of murine NF-L resulted in excessive accumulation of NFs in the
soma and proximal axons of the anterior horn motoneurons which were undergoing
chromatolysis. In addition to the increase in NF density in large caliber axons, the
NFs were more closely packed together and were not always observed in the
normal parallel array to the long axis of the axon. A few mice lived past three
weeks, and after 2 months the axons showed a significant decrease in NF density
that were still higher than controls. Additionally, muscle mass returned and a slow
return of normal kinetic activity was observed. Near normal phenotype at nine
months occurred concomitant with a decrease in NF accumulation which had
plateaued at three to four weeks. These results led to the hypothesis that
excessive NF-L accumulation can result in motoneuron dysfunction before
widespread neuronal loss.
In a similar study, in which transgenic mice were produced with human NF-
H, a twofold increase of NF-FI expression resulted in a more progressive
neuropathology that occurred over a period of three to four months (Cóté et al.,
1993). In these transgenic mice, the expression of the human NF-FI was restricted

34
to neurons. Similar to ALS, progressive abnormal accumulations of NFs were
observed in the perikarya and proximal axons of anterior horn motoneurons of the
spinal cord.
The excessive accumulations of NFs in the neuronal cell bodies in both
types of transgenic mice suggest that it is not one particular NF subunit is critical
, to prevent normal cellular activity. However, the increase in density and
disorganization of NF structure taken together could interfere with normal axonal
transport and thus result in inappropriate accumulations of NF subunits throughout
the neuron. Thus, deficits in axonal transport appear to be a major component in
motoneuron neuropathies. This may be due to indirect effects like an abnormal
increase in NF proteins in the soma that blocks normal axonal transport which may
be the case with ALS, or may be due to a direct effect of axonal transport, that
results in local accumulations of NFs, which may be the case with giant axonal
neuropathies.
Overview of Dissertation
Although NFs are one of the most abundant proteins in the nervous system,
the dynamics of their function remain undefined. As in the case of other
cytoskeletal elements, such as MTs and MFs, one way of understanding or
defining function is realized by determining what types of proteins interact with
these cytoskeletal elements. In the set of experiments described in this

35
dissertation, I have undertaken this approach to identify candidate NF binding
proteins in an attempt to assign functional roles to NF proteins.
One candidate NF binding protein is a 300 kD protein which co-purifies with
IF proteins isolated from a crude spinal cord preparation. This protein was
identified as plectin, a protein previously characterized in non-neuronal tissue. The
relationship of plectin to its potential role in the nervous system, and specifically
to neural IF proteins was examined.
Using affinity column chromatography, additional candidate NF binding
proteins were identified based on molecular weight. A number of proteins bound
to the different NF affinity columns with varying specificity. One protein in
particular was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
The in vitro binding of GAPDH to NF-L was examined as well as possible in vivo
interactions at the light and ultrastructural level.
These findings provide an initial framework for studying NF associated
proteins. NFs appear to interact with a number of proteins and these associations
may be specific to individual subunits as well as to the intact 10 nm filaments. In
addition, future studies that identify the other candidate NF associated proteins will
lead to a better understanding of NF function. Considering the differential labelling
of NFs in neurons, NF associated proteins may also show similar specificity which
may lead to important markers for different neuronal cell types along the neural
axis.

CHAPTER 2
GENERAL METHODS
This chapter describes the routine biochemical and anatomical techniques
that were performed for this dissertation project. Techniques that were specific for
a particular chapter or modifications of these routine techniques will be described
in that chapter.
Biochemical Techniques
Protein Assay
Lowry protein assay. The method of Lowry et al. (1951) was used for some
of the protein assays. Briefly, a standard cun/e from 0 to 100 pg bovine serum
albumin (BSA) to a final volume of 0.5 ml was made. The unknown concentration
of the protein solution was determined by diluting the protein solutions so it fell in
the range of the standard curve. Five milliliters of a solution containing 0.04%
copper sulfate, 0.08% potassium sodium tartrate and 2.94% sodium carbonate was
added to the BSA standards and protein samples to be assayed. The solutions
were then incubated at room temperature for 10 minutes. Next, 0.5 ml of 50%
Folin phenol reagent solution (diluted with deionized water) was added to each
tube and then each tube was incubated for 30 minutes at room temperature. The
absorbance was measured at 650 nm for each sample and the absorbance of BSA
36

37
standards was plotted against the known concentrations. The unknown protein
concentrations were determined from the linear regression line of the BSA
standard data.
Pierce protein assay. Pierce Micro BCA protein assay kit was also used in
protein concentration determination because of its reliability and ease of use.
Protein concentrations were determined in the range of 0-20 pg/ml using BSA as
the standard and following the manufacturers instructions. Pierce Micro BCA
protein assay kit uses bicinchoninic acid which is a highly sensitive and selective
detection reagent for Cu+1. The protein concentration of the unknown samples
were determined using the same methodology as described above.
SDS Polyacrylamide Gels
The sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis
technique was based on the method of Laemmli (1970) with an acrylamide to
bisacrylamide ratio of 37:1 (22.2%:0.6%). The electrophoretic apparatus used was
the Bio-Rad mini-protean II electrophoresis cell. Sample buffer containing SDS
and B-mercaptoethanol (12.5% (v/v) 1 M Tris-HCI, pH 6.8, 4% (w/v) SDS, 10%
(v/v) glycerol, and 0.006% (v/v) bromophenol blue in ethanol) was added to each
sample and then each sample was boiled for 5 minutes before protein separation
on SDS polyacrylamide gels. The SDS polyacrylamide gels were run at a constant
current of 20 mA per gel. The gels were fixed in 40% methanol and 10% acetic
acid for 30 minutes, and then stained with 0.025% Serva Blue G for 1 hour
followed by destaining with 10% acetic acid for 1 -2 hours. This method of staining

38
and destaging of gels is fast and efficient when compared to staining with
Coomassie Brilliant Blue R-250 (CBB) in methanol and acetic acid solution which
can take up to 24 hours to properly destain the background. In cyanogen bromide
(CNBr) cleavage experiments, CBB was used since the initial staining of the bands
is comparatively faster (15 to 20 minutes) and high background did not interfere
with accurately excising the gel band of interest.
Electroblottina of Proteins
Proteins were electrophoretically transferred to nitrocellulose or
polyvinylidine difluoride (PVDF) membrane using Bio-Rad Mini Trans-Blot Cell. A
submerged protein transfer system was used where the gel and membrane were
sandwiched between Whatman 3M paper and scrub pads. The transfer buffer
used in most situations contained 10 mM 2-[N-morpholino]ethanesufonic acid
(MES) pH 6.5, 0.01% SDS. The transfer lasted for 1-2 hours at 90 Volts
depending on the protein being transferred. To prevent overheating, the mini
trans-blot unit was immersed in an ice bath.
The proteins transferred onto the nitrocellulose membranes were visualized
with 0.025% Ponceau S in 40% methanol/10% acetic acid, and were used for
Western blots. Proteins transferred onto PVDF membranes were used for amino
acid analysis and were treated in a slightly different manner than nitrocellulose
membranes. Before proteins were transferred onto PVDF membrane, the
membrane was immersed in 100% methanol and then equilibrated in transfer
buffer for 20 minutes with two changes of buffer. After the transfer of proteins to

39
the PVDF membrane, the PVDF membrane was washed in deionized water for five
minutes, and stained in 0.1% CBB in 50% methanol for 5 minutes. The PVDF
membrane was partially destained in 50% methanol and 10% acetic acid for five
minutes, followed by a final wash in deionized water for 5 minutes (four changes
of water). The PVDF membrane containing the transferred proteins was dried and
stored at -20°C until analysis.
Immuno-Blot Analysis
Proteins were separated on SDS polyacrylamide gel and electrophoretically
transferred onto nitrocellulose membrane as discussed previously. The membrane
was blocked with 3% bovine serum albumin (BSA) or 5% nonfat Carnation instant
milk containing 0.1% Tween 20 in Tris buffered saline (TBS: 10 mM Tris-HCI (pH
7.5), 0.9% saline). If the nonfat milk was used to block the nitrocellulose, then the
primary and secondary antibody was diluted in this solution, and all but the last
wash was done with TBS containing 0.1% Tween 20 in place of TBS. The
remaining steps were the same as those described when BSA was used to block
the nitrocellulose membrane. The membrane was incubated with the primary
antibody containing 0.1% BSA for 1 hour at room temperature followed by three
10 minute washes in TBS. Next, the membrane was incubated with secondary
antibody, alkaline phosphatase conjugated anti-mouse or anti-rabbit, for 1 hour at
room temperature followed by two 10 minute washes in TBS. A ten minute wash
in developing buffer (10 ml 0.1 M Tris-HCI (pH 9.5) containing 5 mM MgCI2 and
0.1 M NaCI) followed. The antibody binding was visualized using 33pl 50 mg/ml

40
5-Bromo-4-chloro-3-indolyl phosphate (BCIP) and 33pl 50 mg/ml Nitro Blue
Tetrazolium (NBT) in 10 ml developing buffer.
Protein Cleavage with Cyanogen Bromide
To cleave proteins at methionine residues, the cyanogen bromide (CNBr)
cleavage method was used on SDS polyacrylamide gel protein bands (Sokolov et
al., 1989). The protein to be cleaved by CNBr was separated on a SDS
polyacrylamide gel. The gel was stained with CBB (0.25% coomassie blue R-250
in 45% methanol and 10% acetic acid) and the gel band of interest was excised
from the gel. The gel band was dried down using a vacuum evaporator at 60-
80°C. The volume to rehydrate the gel band was estimated by measuring the
length, height and width of the gel band before it was dehydrated, and that volume
was added to the dried protein gel band in the form of 200 mg/ml CNBr in 70%
formic acid. The gel band was incubated in the CNBr solution at 37°C for at least
6 hours. The rehydrated gel band was dried down in the vacuum evaporator to
remove the remaining CNBr and its by-products. The dried cleaved gel band was
rehydrated and neutralized in 50% 2X sample buffer and 50% 1 M Tris for 15
minutes. The cleaved protein gel band was placed on 15% SDS polyacrylamide
gel to separate the protein fragments. The protein fragments were visualized using
Serva Blue G or silver stain depending on the protein concentration.

41
Isolation of Neurofilament Proteins
Crude Intermediate Filament Preparation
A crude intermediate filament (IF) preparation was used to obtain primarily
neurofilament (NF) proteins in vitro while maintaining some of their native
characteristics. Fresh spinal cord (specific species used will be discussed in the
relevant chapter) was homogenized gently using a Wheaton Dounce Type 40 glass
homogenizer for small preparations (<10 g of tissue) or a Sears blender for larger
preparations (>10 g tissue). The tissue was brought up in 5 volumes of Buffer A
(0.1 M Tris-HCI (pH 7.5), 0.1 M NaCI, and 1 mM Na-p-tosyl-L-arginine methyl ester
(TAME)), and homogenized with 3 strokes of pestle 'B' followed by 1 to 2 strokes
with pestle 'A'. For larger preparations, the tissue was homogenized in blender at
low speed (three 5 second pulses) followed by three 5 second pulses at high
speed. The homogenate was made up to 10 volumes with Buffer A and
centrifuged at 2300xg for 10 minutes at 4°C in a SS34 Sorvall rotor. The pellet
was taken up in 10 volumes 1 M sucrose in Buffer A, vortexed gently to break up
the pellet, and centrifuged at 2300xg for 20 minutes at 4°C in a SM-24 Sorvall
rotor (small preparation) or a SS34 Sorvall rotor (large preparation). The floating
myelin layer, containing the NF bundles, was carefully removed and the volume
was made up to 5 volumes with Buffer A containing 1 M sucrose and 1% Triton
X-100. The myelin homogenate was gently vortexed and triturated to suspension,
and then shaken on an orbital shaker for 60 minutes at 4°C. This allowed the
Triton X-100 to solubilize the myelin and release the intermediate filament bundles.

42
The suspension was centrifuged at 150,000xg for 1 hour at 4°C in a Beckman TL-
100 rotor (small preparation) ora T865 Sorvall rotor (large preparation) to recover
the pelleted intermediate filament bundles. The pellet was composed mainly of NF
bundles and glial filaments. The pellet was taken up in the appropriate buffer and
stored at -20°C.
Neurofilament Preparation
Partially purified NFs were prepared using the method of Delacourte et al.
(1980). Fresh pig spinal cord (250 g) was homogenized in 400 ml of 0.1 M MES
(pH 6.5), 1 mM ethylenediamine tetraacetic acid (EDTA), 0.5 nM MgCI2, 1 mM
PMSF, and I mM TAME using a Sears blender at 4°C. Initially, the spinal cord
was homogenized at low speed using 3 five second pulses followed by 3 five
second pulses at high speed. The crude homogenate was centrifuged at 13,500xg
for 45 minutes at 4°C in a GSA Sorvall rotor. The supernatant was filtered through
8 ply gauze sponges type VII (Professional Medical Products, Inc.) to remove any
large floating debris. Next, the filtered supernatant was centrifuged at 78,000xg
for 30 minutes at 4°C in a T865 Sorvall rotor. Glycerol was added to the
supernatant to form a final concentration of 20% (v/v) glycerol. The solution was
warmed to 37°C for 20 minutes. The supernatant was centrifuged at 78,000xg for
45 min at 30°C in a T865 Sorvall rotor. The pelleted material was combined and
homogenized in 50 ml of MES buffer and centrifuged at 147,000xg for 30 minutes
at 4°C in a T865 Sorvall rotor. The pelleted material was a pale yellowish gel that
contained NFs and glial filaments. The pellet was stored at -20°C.

43
Purification of Individual Neurofilament Subunits
Individual NF subunits were purified following a modified method of
Tokutake (1984). The pellet obtained from the NF Delacourte preparation was
homogenized on ice in 10 mM sodium phosphate monobasic (pH 7.0), 6 M urea,
1 mM EDTA, and 1 mM dithiothreitol (DTT) in order to solubilize the NFs into their
individual subunits. Insoluble material was removed by high speed centrifugation
(150,000xg) for 30 minutes at 4°C using a T865 Sorvall rotor and the supernatant
was filtered through a 0.45 pm filter. The soluble material containing the NF
subunits was put through a DEAE cellulose column (DE-52) at a flow rate of 25
ml/hour at 4°C. The DE-52 column was washed extensively to remove any
unbound proteins and before elution of bound proteins. To make sure no more
proteins were being eluted, the optical density at 280 nm of the wash buffer
coming through the column was checked. A linear gradient of 10 mM to 400 mM
monobasic sodium phosphate (500 ml) was used to elute the bound proteins.
Tokutake showed that varying the sodium phosphate concentration instead of
using a sodium chloride gradient resulted in a better separation of the individual
NF subunits. Fractions of 5 ml were collected during the elution, and 10pl of each
even number fraction was separated on 7.5% SDS polyacrylamide gels to
determine which fractions contained the NF subunits. The fractions which were
purest (those that contained 98% of only one of the subunits) were pooled and
dialyzed against the appropriate buffer. The purified NF subunits were assayed
for protein concentration (Lowry or Pierce micro assay) and stored at -20°C.

44
Anatomical Localization Experiments
Dorsal Root Ganglion Cell Cultures
New born rat pups (postnatal day 1) were killed by decapitation and the
body was pinned at the extremities to a piece of styrofoam. All instruments were
sterilized with 95% ethanol. The skin over the spinal cord was removed and a
longitudinal cut, using a scalpel, was made through the dorsal portion of the
vertebrae. With scissors the dorsal vertebral column was trimmed and the spinal
cord removed. The dorsal root ganglia (DRG) were located between the vertebrae
and resembled small white spheres. The DRG were removed one by one with fine
forceps from one side of the vertebral column and placed in a sterile microfuge
screw cap containing Liebowitz L-15 media (Sigma) with 10 ml/liter penicillin
(10,000 Units/ml), streptomycin (10,000 mag/ml) and amphotericin B (25 mag/ml)
(Gibco) (L-15 incomplete media). This was repeated for the contralateral side and
for the remaining rat pups so that each tube contained approximately 10 to 15
DRG. The ganglia were pelleted in a clinical centrifuge (setting 5) for 30 seconds.
To the pelleted ganglia, 240 pi 10X sterile trypsin-EDTA solution (Sigma; 0.5%
trypsin, 0.2% EDTA. 0.9% NaCI) was added. The resuspended DRG were then
incubated at 37°C for 40 minutes to dissociate the ganglia. Then, the DRG were
centrifuged for 30 seconds at setting 5 in the clinical centrifuge, the trypsin
containing supernatant was removed, and 6-8 drops of fetal bovine serum (FBS;
Gibco) was added. The DRGs were incubated for 10 minutes at 37°C, pelleted as
described above, and supernatant removed. DRG were triturated in 100 pi of L-15

45
incomplete media 15 times using an Eppendorf pipet to resuspend the ganglia into
single cells. Before plating, 500 pi of L-15 incomplete media was added to each
tube, then cells were plated on 6 acid washed coverslips (100 pi per plate) placed
in Petri dishes (35x10 mm). Two milliliters of L-15 complete media (L-15
incomplete media, 10% FBS, 0.6% glucose, 2 mM L-glutamine and 0.3% A4M
methylcellulose) was added to each Petri dish. Nerve growth factor (NGF) (Sigma
7S mouse submaxillary gland NGF) was added to result in a final concentration of
200ng/ml. The DRG cells were grown in a non-C02 incubator at 37°C for 24
hours.
Pheochromocvtoma Cells (PC12 Cells)
A stock solution of PC12 cells was thawed rapidly at 37°C, pelleted in a
clinical centrifuge, and resuspended in RPMI media containing 85% RPMI 1640
medium (Gibco); 10% heat inactivated horse serum, 5% FBS and 10 ml/liter of
penicillin (10,000 Units/ml), streptomycin (10,000 mag/ml) and amphotericin B (25
mag/ml) (Gibco). PC12 cells were plated on a collagen coated Petri dish (60x10
mm), grown at 37°C in a water-saturated atmosphere containing 8.1% C02, and
fed with new media every 2-3 days. When the cultures reached confluence, they
were subcultured at a ratio of 1:3 to 1:4. To generate neurite-bearing PC12 cells,
50 ng/ml 7S-NGF was added to each culture and the total serum concentration
was reduced to 1.5% from 15%. PC12 cells were feed every two days with RPMI
media containing 1.5% serum and 50 ng NGF. After about a week in culture,
PC12 cells produced neurites and were ready to be replated on collagen coated

46
coverslips for immunocytochemistry. The PC12 cells were removed from the Petri
dish by trituration with a Pasteur pipette and pelleted in clinical centrifuge at low
speed where the supernatant was discarded (contains cellular debris including
detached neurites). The pelleted cells were resuspended in RPMI media (1.5%
serum) with 50 ng NGF, and plated onto coverslips. The PC12 cells produced
neurites within 8 to 16 hours and by 2 to 3 days the PC12 cells have formed
extensive neuritic processes.
Immunofluorescent Studies on Fresh Frozen Tissue
Adult rats, either Sprauge Dawley or Long Evans Hooded strains, were
anesthetized with 0.5 ml pentobarbital i.p. and sacrificed by decapitation. Nervous
system tissue was dissected out, frozen on dry ice for 1 hour or quickly frozen in
isopentane cooled with liquid nitrogen, and stored at -70°C until sectioning. The
tissue of interest was cut at a thickness of 6 to 10 pm using a cryostat, placed on
petroleum ether cleaned glass slides, and stored at -20°C until they were labelled
with antibodies. Prior to incubation with primary antibodies, the tissue was fixed
in acetone (-20°C) for ten minutes. Tissue sections were air dried 10 to 15
minutes and encircled with a PAP pen (The Binding Site) to make a well for the
antibodies. The two primary antibodies, one polyclonal and the other monoclonal,
were mixed together in buffer to obtain the appropriate final concentration for each
antibody and then were placed on the section. The tissue sections were then
incubated at 37°C for 30 minutes or 4°C for 24 hours, followed by three 10 minute
washes in PBS. Following primary antibody, sections were incubated for 30

47
minutes at 37°C or 24 hours at 4°C with secondary antibodies, FITC-conjugated
goat anti-mouse (1:40) and Texas Red conjugated donkey anti-rabbit (1:200).
After three 10 minute washes in PBS, the sections were coverslipped with anti¬
bleaching agent (1.0 mg/ml para-phenylenediamine (Sigma) in 20 mM Tris-HCI pH
7.9, 90% glycerol). For tissue culture immunofluorescence, the DRG and PC12
cells were fixed in cold methanol (-20°C) for 5 minutes. The remaining antibody
steps were as described above. The fluorescently labelled tissue sections were
visualized and photographed using a Zeiss Axiophot microscope system.
Immunohistochemistry on Formaldehyde Fixed Tissue
Adult rats, either Sprauge Dawley or Long Evans Hooded strains, were
anesthetized with either 0.5 ml pentobarbital or 80 mg/kg ketamine and 10 mg/kg
xylazine, and perfused with saline (0.9% NaCI) containing 0.05% (v/v) heparin and
0.05% (w/v) sodium nitroprusside followed by a fixative solution of 4%
paraformaldehyde in Sorensen's Buffer, pH 7.4 (19 mM monobasic sodium
phosphate, 81 mM dibasic sodium phosphate). The rat nervous system was
dissected out and placed in the above fixative solution overnight or PBS depending
on how well the tissue was fixed. Before sectioning, the tissue was blocked and
placed in phosphate buffered saline for at least 4 hours before being cut in 30 to
50 pm sections using a vibratome.
For immunocytochemistry, the tissue sections were incubated at in 0.3%
hydrogen peroxide in PBS for 30 minutes at room temperature to remove any
endogenous peroxidase activity. The tissue sections were washed three times for

48
10 minutes and mounted on chrome-alum coated slides and allowed to air dry for
at least one hour before beginning antibody staining procedure. Vectastain elite
ABC kit (KIT) was used to visualize the labelling of the primary antibody. The
tissue sections were blocked for 20 minutes in dilute normal serum (KIT) at 37°C.
Excess blocking serum was shaken off and the primary antibody in 0.25% Triton-
X-100 was placed on the tissue section and incubated for 30 minutes at 37°C or
24 hours at 4°C. The tissue sections were washed three times for 10 minutes
followed by incubation with biotinylated secondary antibody (KIT) for 30 minutes
at 37°C or 24 hours at 4°C. The tissue sections were washed 3 times for ten
minutes followed by incubation with the avidin/biotin reagent (KIT) for 30 minutes
at 37°C or 4 hours at room temperature. The tissue sections were washed 3 times
for ten minutes in PBS before being developed with freshly prepared
diaminobenzidine tetrahydrochloride (DAB) solution (0.05% DAB (Sigma),
containing 0.01% hydrogen peroxide in 0.1 M Tris, pH 7.2). The reaction was
monitored using an inverted microscope in order to determine when to stop the
reaction. The reaction was stopped by placing the tissue sections in tap water for
five minutes. The tissue sections were dehydrated using ascending alcohol
concentrations: 70%, 90%, 95%, 100%, 100%; and cleared in xylene and
coverslipped with permount.

CHAPTER 3
THE DISTRIBUTION OF PLECTIN, AN INTERMEDIATE FILAMENT BINDING
PROTEIN, IN THE ADULT RAT CENTRAL NERVOUS SYSTEM
Introduction
Although intermediate filament proteins (IFs) are found with various
polypeptides known as intermediate filament associated proteins (IFAPs), IFAPs
remain much less characterized than microtubule and microfilament associated
proteins. At present only a few proteins are known to be specific IF-binding
proteins, (Foisner and Wiche, 1991) such as plectin (Wiche, 1989), IFAP-300
(Yang et al., 1985; Lieska et al., 1985), NAPA-73 (Ciment et al., 1986), filensin
(Merdes et al., 1991), and filaggrins (Haydock and Dale, 1990).
One protein in particular, plectin, has been studied extensively in non-neural
tissue. Immunofluorescence studies of various non-neuronal tissues demonstrated
that plectin was localized to a number of different cell types, and has a cytoplasmic
distribution within the cell with a distinct tendency to be concentrated at the cellular
periphery near the plasma membrane (Wiche et al., 1983). Later work
demonstrated that plectin bound to immobilized IF proteins in vitro, including the
NF triplet proteins (Foisner et al., 1988). This interaction was found to involve the
a-helical rod domain in IFs and the a-helical rod domain of plectin (Foisner et al.,
1988; 1991). Originally, plectin was identified from rat glioma C6 cells as a major
49

50
component of Triton X-100 extracts with an apparent molecular weight of 300 kD
as determined from mobility on SDS-polyacrylamide gels (Pytela and Wiche, 1980).
Recent cloning and sequencing of rat plectin has shown that the actual
molecular weight of plectin is 527 kD (Wiche et al., 1991). Portions of plectin's
primary sequence are related to desmoplakin and the bullous-pemphigoid antigen,
both of which are found in association with IFs at the plasma membrane (Wiche
et al., 1991). Overall, plectin is postulated to function as a crosslinking protein,
mediating interactions between IF proteins and other components of the cell
(Foisner and Wiche, 1991) such as the plasma membrane via spectrin, the nuclear
envelope via nuclear lamins and microtubules via microtubule-associated proteins
(Koszka et al., 1985; Plerrmann and Wiche, 1987).
As discussed earlier, previous research on plectin has focused on non-neural
tissue, and although plectin binds NF subunits in vitro, the in vivo significance of
this observation is not clear. This chapter focuses on demonstrating that plectin
is present in crude insoluble IF extracts from neural tissue and showing the
localization of plectin in the adult rat central nervous system.
Methods
Experimental Tissue
A crude intermediate filament (IF) preparation from bovine and rat spinal cord
was obtained using a modified axonal floatation technique, details of this method
were described in Chapter 2 (Shelanski et al., 1971; Shaw and Hou, 1990).
Bovine spinal cord (50 g) was obtained from the slaughter house ~1 hour

51
postmortem and prepared using the large preparation method. Rat spinal cord (~0.2
g) was obtained rapidly (>5 minutes) after decapitation and processed using the
small preparation method.
Partial purification of the 300 kD protein was accomplished using a Sephacryl
S-400 (Bio-Rad) column (1.5 cm x 100 cm). Bovine IF pellet was resuspended in
Buffer B (6 M Urea, 10 mM sodium phosphate (monobasic, pH 7.5), 1 mM EDTA,
1 mM TAME), and centrifuged at 150,000xg in a Sorvall T865 rotor for 45 minutes
at 4°C to remove any insoluble material. The supernatant was filtered through a
0.8 pm filter followed by a 0.45 pm filter, then placed in a dialysis bag and
concentrated to a final volume of 2 ml by placing dry Sephadex G-50 around the
dialysis bag. The concentrated bovine IF material was separated on a Sephacryl
S-400 column which was equilibrated with Buffer B containing 5 mM DTT. The
flow rate was 15 ml per hour and 4 ml fractions were collected. Five pi from the
even number fractions were separated on 6% SDS polyacrylamide gels.
Immuno-Blot Analysis
Proteins were separated on SDS polyacrylamide gel and transferred to
nitrocellulose using 10 mM MES (pH 6.8) and 0.01% SDS for 2 hours at 90 volts
(constant). For the crude IF preparation from rat spinal cord, approximately 3% of
final preparation was used per lane. A detailed description for the methods used
here were given in Chapter 2.

52
Antibodies
Monoclonal antibodies to plectin (1D8 and 1A2) and polyclonal serum to
plectin (p21) were previously characterized (Wiche and Baker, 1982; Foisner et al.,
1991). Monoclonal antibodies were from tissue culture supernatant and were used
either undiluted or diluted 1:2. The polyclonal serum, p21, was used at 1:50.
Alkaline phosphatase conjugated anti-mouse IgG secondary antibody was
purchased from Sigma and used at 1:1000 dilution. Biotinylated secondary
antibodies were obtained from Vector Laboratories and diluted according to the
manufacturers directions. Fluorescent secondary antibodies were obtained from
Jackson Laboratories and used at 1:100 dilution.
Anatomical Procedures
Both paraformaldehyde fixed and fresh frozen acetone fixed rat neural tissue
were used in this study. Plectin was localized in the paraformaldehyde fixed tissue
using the Vectastain Elite ABC kit. For fresh frozen acetone fixed tissue, plectin
was localized using the indirect immunofluorescence procedure. Both of these
anatomical methods were described in detail in Anatomical Localization section in
Chapter 2.
Results
Immunoblot Studies
Besides the major IF proteins bands in a crude IF preparation from spinal
cord, there were a number of proteins that copurify with IFs (Figure 3-1 a, b lanes

53
1). Since these proteins were present after salt and detergent extractions, it was
hypothesized that these proteins may functionally interact with IFs.
In particular, one high MW protein band at 300 kD on SDS polyacrylamide
gel, a relatively minor component in this IF preparation, was proposed to
correspond to plectin, a known 300 kD protein that interacts with IFs in vitro
(Foisner et al., 1988). The 300 kD protein from crude IF preparation of bovine
spinal cord was partially purified using Sephacryl S-400 column. Figure 3-1 a
shows the crude bovine IF preparation (lane 1) and Sephacryl S-400 column
fractions 16-28 (lanes 2-8, respectively) in which lane 2 contains the 300 kD
protein. Immunoblot of fraction 16 (Figure 3-1 a, lane 9) demonstrated that the
plectin monoclonal antibody ID8 labelled a 300 kD protein.
Since rat neural tissue was to be utilized in the immunocytochemical
localization of plectin in the CNS, I investigated whether plectin was present in rat
neural tissue and whether the plectin antibody cross-reacted with any of the other
proteins present in the crude IF preparation. Immunoblot of crude rat IF
preparation demonstrated that plectin was present in rat spinal cord, and the
antibody did not cross-react with any of the other proteins present in the rat
preparation (Figure 3-1 b). The weak smearing just below the major plectin band
which did not correspond to any protein band in the protein stained gel (Figure 3-
1b, lane 1) was probably a degradation product of plectin (Wiche, 1989). The
relatively low amount of plectin immunoreactivity detected may reflect a difficulty
in electrophoretically transferring plectin as a result of plectin's very high molecular

Figure 3-1. Identification of a 300 kD protein in bovine and rat crude IF spinal cord
preparations.
(A) Partial purification of 300 kD protein (arrow) from crude IF preparation from
bovine spinal cord. Lane 1 is a 7.5% SDS polyacrylamide gel showing the IF
preparation before purification on a Sephacryl S-400 gel filtration column. Lanes
2-8 represent even number fractions (16-28) from the gel filtration column in which
fraction 16 contained the partially purified 300 kD protein. Lane 9 is an
immunoblot of fraction 16 labelled with monoclonal antibody to plectin (1D8). (H:
NF-H; M: NF-M; L: NF-L; G: GFAP).
(B) Immunoblot of rat spinal cord IF preparation showing plectin antibody staining.
Lane 1 is a 6% SDS polyacrylamide gel of a spinal cord cytoskeletal extract
stained with Serva Blue G. Lane 2 is an immunoblot strip labelled with plectin ID8
showing that the monoclonal antibody labels a 300 kD protein.

55
L-
G-
1
2

56
weight, at 527 kD. Even under optimal transfer conditions, when most of the other
high molecular weight proteins were transferred completely, a substantial portion
of the 300 kD protein band remained in the gel after electroblotting (data not
shown). In addition, it was possible that the fairly prominent 300 kD band seen in
SDS-PAGE contained other proteins (Lieska et al., 1985; Goldman and Yen, 1986)
besides plectin since ion exchange chromatography demonstrates that proteins
with a relative molecular weight of 300 kD are eluted off a DEAE ion exchange
column at different salt concentrations (data not shown).
Comparison of Plectin to IFAP-300
In addition to plectin, there was another IFAP identified which has similar
mobility on SDS polyacrylamide gel known as IFAP-300 (Lieska et al., 1985;
Goldman and Yen, 1986). It was of interest to compare the amino acid analysis
of these two proteins, since both plectin and IFAP-300 have similar mobilities on
SDS polyacrylamide gels (300 kD), staining distribution in non-neuronal cells, and
both bind to IF network. IFAP-300 amino acid composition data were taken from
Lieska et al. (1985) and the plectin amino acid composition was determined from
the published sequence (accession #S21876) and are presented in both table and
graphical form (Shaw, 1992) in Figure 3-2. Superficially, these two proteins appear
similar; however, differences were observed in the percent composition of a
number of amino acids Although these two proteins do not appear to be closely
related in amino acid composition, these results do not exclude the possibility that
there may be some sequence similarities, and since these two proteins have

Figure 3-2. Comparison of amino acid composition of plectin and IFAP-300 proteins.
A) The percent nanomole (% nmole) for plectin and IFAP-300 is listed. Plectin amino acid
composition was determined from the published sequence (Wiche et al., 1991). The amino acid
composition for IFAP-300 was obtained from published paper (Lieska et al., 1985) and the
values were corrected for quantitation errors using a correction factor (Shaw, 1991).
B) The amino acid composition data are represented graphically using the Star program (Shaw,
1991).

Amino Acid Composition (% nmole)
Amino Acid
Plectin
IFAP-300
ASX
5.36
9.81
THR
5.21
5.59
SER
6.68
7.19
GLX
19.53
17.08
PRO
3.29
6.57
GLY
6.44
8.99
ALA
11.08
9.87
VAL
5.33
3.82
MET
1.35
1.29
ILE
3.32
1.97
LEU
11.38
9.11
TYR
2.24
2.43
PHE
1.79
2.69
HIS
1.57
1.91
LYS
5.53
5.20
ARG
9.90
6.48
B
Plectin
asx
ARG THR
LYS
SER
HIS
k GLX
PHE
PRO
TYR
GLY
LEU
ALA
ILE UAL
MET
LYS
IFAP-300
ASX
ARG THR
SER
HIS
/ \ GLX
PHE
pro
TYR
GLY
LEU
ALA
ILE UAL
MET
UAL
UT
00

59
similar molecular weights, binding and tissue localization characteristics, plectin
and IFAP-300 may belong to a family of high molecular weight IFAP similar to that
observed with the high molecular weight microtubule associated proteins.
Localization Studies
Overall, plectin immunoreactivity in the adult rat central nervous system was
predominantly associated with non-neuronal cells. In addition, plectin antibody
staining was present in a subset of neurons in the brainstem and spinal cord.
Immunoreactivity was generally strongest at the periphery of cells.
Telencephalon and diencephalon. Plectin immunoreactivity was absent in
neurons of the telencephalon and diencephalon. The only cells which labelled with
the plectin antibody in the cortical gray matter of the telencephalon were astrocytes
located in the ventromedial portion of the temporal cortex near the pial surface.
The density of astrocytic labelling with plectin antibodies decreased rapidly away
from the pial surface (Figure 3-3a). Although this labelling pattern may be
attributed to edge effect, a similar staining pattern was not observed in any other
cortical regions near the pia mater.
In the diencephalon, plectin positive astrocytes were observed at the
ventrolateral portion of the hypothalamus (Figure 3-3b). Plectin immunoreactivity
was found prominently in astrocytes throughout the white matter fiber tracts
including the optic tract (Figure 3-3c), corpus callosum, internal capsule, fornix, and
stria medullaris. Pronounced plectin immunoreactivity was observed in the
hypothalamus surrounding the third ventricle. Both ependymal cells lining the

Figure 3-3. Plectin immunoreactivity (ID8) of astrocytes in white and gray matter
of the telencephalon and diencephalon.
(A) The ventro-medial portion of the temporal cortex was the only cerebral cortical
area where plectin positive astrocytes were localized as visualized with DAB
(arrows). Scale bar: 25 pm.
(B) The ventro-lateral portion of the hypothalamus showed diffuse plectin
immunoreactivity of astrocytes(open arrow). Scale bar: 25 pm.
(C) White matter regions in the rat nervous system demonstrated plectin antibody
labelling of astrocytes as shown in the optic tract as visualized with
immunoflurescent secondary antibodies (open arrow). Scale bar: 25 pm.

61

62
dorsal portion and tanycytes, specialized ependymal cells, lining the ventral portion
of the third ventricle were plectin positive (Figure 3-4a). The plectin antibody
labelling of ependymal cells showed strong staining at the plasma membrane,
diffuse staining throughout the cytoplasm, and dense staining between the
ependymal cells (Figure 3-4b). The ependymal labelling pattern was observed in
all ventricles. A network of astrocytic fibers labelled with plectin antibodies was
seen in the subependymal layer (Figure 3-4b). In tanycytes, plectin
immunoreactivity was associated with the periphery of the cell body as well as with
the fibrous process (Figure 3-4c).
Cerebellum. Plectin immunoreactivity in the cerebellum was observed in
Bergmann glial fibers in the molecular layer, and in astrocytes in the granular cell
layer and white fiber tract layer (Figure 3-5a). The entire extent of Bergmann glial
processes were labelled with plectin antibodies; however, the strongest staining
was associated with Bergmann fibers terminal endings at the pial surface. The
relative intensity difference between the knob-like endings and processes of
Bergmann glia fibers was observed distinctly in a immunofluorescent higher power
view of molecular layer of cerebellum (Figure 3-5b). With respect to the terminal
endings of the Bergmann fibers which forms the external glial limiting membrane
in the cerebellum, it was difficult to determine whether these subpial structures
which were plectin positive formed conical and/or foot type endings (Palay and
Chan-Palay, 1974). The staining pattern once again showed strong labelling at the
periphery of the cells.

Figure 3-4. Plectin immunoreactivity (ID8) in the rostral hypothalamus surrounding
the third ventricle as visualized with DAB (3V).
(A) Cells lining the third ventricle are plectin positive and are of two types:
ependymal cells in the dorsal portion and tanycytes in the ventral portion. Scale
bar: 100 pm
(B) High power view of ependymal cells which demonstrates plectin antibody
staining throughout the cytoplasm with highest density of labelling at the cell
membrane (arrowheads). Light astrocytic staining is observed in the
subependymal cell layer just lateral to the ependymal cells. Scale bar: 25 pm.
(C) High power view of tanycytes which shows plectin immunoreactivity solely
around the plasma membrane (arrowheads) and radial process of the tanycyte
(arrows). Scale bar: 25 pm.

64

Figure 3-5. Plectin immunoreactivity (IA2 and ID8) in the cerebellum.
(A) In the cerebellum plectin antibody (IA2) labels the full extent of the Bergmann
glia process (arrow) in the molecular layer (ml) with pronounced labelling at the pia
surface (arrowhead) as visualized with DAB. Weak plectin immunoreactivity is
present in the granular cell layer (gel) and appears to correspond to astocytic
processes. Note the absence of plectin antibody staining in the purkinje cell layer
(pci). Scale bar: 100 pm.
(B) Higher power view of the cerebellum molecular layer showing the prominent
fluorescent labelling of the Bergmann glia terminal processes using ID8 antibody
(arrows) as visualized with immunoflurescent secondary antibodies. In comparison
the Bergmann glia fibers are weakly labelled with plectin antibody (open arrows).
Scale bar: 25 pm.

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67
Brainstem and spinal cord. The general pattern of the plectin antibody
staining pattern in the brainstem and spinal cord localized plectin primarily to non¬
neuronal cells in the white matter tracts and to a subset of motoneurons. At low
magnification of the brainstem at the caudal medulla, plectin antibody labelling was
localized to astrocytes in the periphery and motoneurons in the nucleus ambiguus
(Figure 3-6a). A high magnification view of the ventrolateral aspect showed plectin
immunoreactivity in the inferior cerebellar peduncle and in the spinal tract of cranial
nerve V (Figure 3-6b). Plectin antibody labelling was pronounced at the outer
boundary of the inferior cerebellar peduncle as well as in glial fibers traversing the
inferior cerebellar peduncle. Throughout this region plectin positive astrocytes
were observed. Plectin staining was not observed in neurons associated with the
spinal nucleus of cranial nerve V (Figure 3-6b). In contrast, intense plectin
immunoreactivity was observed in motoneurons of nucleus ambiguus (Figure 3-6c).
However, there appeared to be a gradient in plectin antibody staining in
motoneurons such that some motoneurons labelled strongly while others stained
lightly (Figure 3-6c). This selective type of motoneuron staining was also observed
in the facial nucleus. In the pyramidal tract, plectin positive astrocytes were seen
as a network of fibers located adjacent to the pial surface (Figure 3-6d).
In the spinal cord, plectin immunoreactivity was observed at all levels with a
similar distribution. Plectin antibody labelling was observed in ependymal cells
lining the central canal, in astrocytes in the dorsal columns, in glial fibers
transversing the white matter, and in motoneurons in the ventral horn (Figure 3-

Figure 3-6. Plectin immunoreactivity (ID8) in the caudal brainstem.
(A) Ventrolateral quadrant of the medulla shows plectin antibody labelling both neurons and glia.
Arrow is pointing to the nucleus ambiguus.(spt: spinal tract trigeminal nerve; icp: inferior
cerebellar peduncle; pt: pyramidal tract). Scale bar: 100 pm.
(B) Spinal tract of V and inferior cerebellar peduncle (icp) labelled with the plectin antibody.
Prevalent plectin immunoreactivity is observed at the outer boundary of icp and in glial fibers
transversing icp (arrows). Scale bar: 50 pm.
(C) Motoneurons in nucleus ambiguus label with plectin antibody. There appears to be a
gradient of staining with some motoneurons staining strongly (arrows) while others stain lightly
(arrowhead). Around motoneuron cell bodies there is a dense particulate staining. Scale bar:
50 pm.
(D) At the periphery of the pyramidal tracts (pt), a network of plectin positive astrocytes are
concentrated near the ventral boundary (arrowheads). Scale bar: 50 pm.

69

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7a). At a higher magnification, the ventral white matter showed plectin antibody
staining of glia cells which radially span from the pial surface to the gray matter
(Figure 3-7b). A closer examination of motoneurons in lamina IX of the spinal cord
gray matter showed that plectin immunoreactivity was associated with the cell body
and dendrites in a subpopulation of motoneurons (Figure 3-7c). Plectin staining
appeared to be excluded from the nucleus. In the vicinity of plectin positive
motoneurons, particulate staining was observed which may be cross-sections of
dendrites (Figure 3-7c.).
Choroid plexus and blood vessels. Choroidal epithelial cells showed
prominent plectin immunoreactivity (Figure 3-8a). Labelling with plectin antibodies
was observed throughout the cytoplasm with particularly intense staining at the
periphery and at points of contact between adjacent cells Differential plectin
antibody staining patterns were observed with blood vessels. Plectin
immunoreactivity was predominantly associated with endothelial cells of large blood
vessels (Figure 3-8b) but also was observed sporadically with smaller blood
vessels (Figure 3-8c).
Discussion
These experiments were initiated in an attempt to identify a salt and detergent
resistant 300 kD protein band found in spinal cord cytoskeleton preparations which
could represent a NF binding protein. Biochemically, plectin was at least a
constituent of this 300 kD protein band, and was found in crude IF preparation of

Figure 3-7. Plectin immunoreactivity (ID8) in the cervical spinal cord.
(A) Low magnification view of the cervical spinal cord that demonstrates plectin
antibody labelling of astrocytes in the dorsal columns (dc), radially oriented glia in
white matter (wm) and motoneurons in the ventral horn (vh). Scale bar: 100 pm
(B) High magnification view of lateral white matter in which the intensity of plectin
antibody staining is graded. The strongest labelling is near the periphery (arrows).
Scale bar: 50 pm.
(C) High magnification view of motoneurons in lamina IX showing that plectin
immunoreactivity is associated with the cell body and processes of motoneurons
in the ventral horn. Particulate staining is observed in the vicinity of motoneurons
(arrowheads). Scale bar: 50 pm.

o

Figure 3-8. Plectin immunoreactivity (p21) in choroid plexus and blood vessels.
(A) Choroidal epithelial cells show diffuse plectin antibody staining throughout the
cytoplasm and concentrated staining at the periphery of the cell (arrow). Scale
bar: 25 pm.
(B) Pericollosal artery labels strongly with the plectin antibody. Staining is localized
predominantly to the inner membrane of endothelial cells (open arrow). Scale bar:
25 pm.
(C) A smaller blood vessel in the temporal cortex labels positive with plectin
antibody. Note the astrocytic processes surrounding the blood vessel are labelled
with the plectin antibody. Scale bar: 25 pm.

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the central nervous system.
One approach to begin to understand the role of a particular protein in the
nervous system is to examine the cellular distribution. Plectin immunoreactivity
was observed in a number of different cell types in the central nervous system with
varying staining intensities (Table 5-1). To summarize these data, prominent
plectin antibody staining was localized to the periphery of ependymal cells lining
the ventricles, to tanycytes, to choroidal epithelial cells, to endothelial cells and to
pia mater cells. In addition, dense plectin immunoreactivity was observed at
junctions between choroidal epithelial cells which may be related to tight junctions
forming part of the barrier between the blood and cerebral spinal fluid (CSF)
(Peters et al., 1991). Previous research by Wiche and co-workers (1989)
demonstrated that plectin was associated to various types of junctional zones. An
interesting and rather unexpected finding was that plectin antibodies labelled the
perikarya of a few neurons in the central nervous system. Generally, the staining
pattern of the plectin antibodies in non-neuronal cells of the nervous system
coincided well with previous work on the distribution of plectin outside the nervous
system (Wiche, 1989) and was similarto the vimentin staining pattern in the central
nervous system (Shaw et al., 1981; Pixley et al., 1981; Yen and Fields, 1981).
The previous studies of plectin concluded that plectin was widely but not
ubiquitously distributed with the peripheral regions of cells, with some overlap with
the IF pattern and submembraneous component. The cellular distribution of plectin
in the CNS appeared to be similar to that observed in non-neural tissue in that

Table 3-1. The distribution of plectin in different
cell types of the adult rat central nervous system.
Cell Type
Staining
Neuronal Cells
cortical pyramidal
-
cortical interneurons
-
cerebellar granule
-
cerebellar Purkinje
-
brain stem motoneurons
+++/++/+/-
spinal cord motoneurons
+++/++/+/-
Ependyma
choroid plexus
+++
columnar epithelia
+++
tanycytes
+++
subependyma
++
Glial Cells
astrocytes (white matter)
+++
astrocytes (gray matter)
+/-
Bergmann fibers
++
Other
endothelial linings
+++/++/+/-
+: indicates that plectin was localized in these cells, and
number of + reflects intensity of staining; indicates that
plectin was not observed in these cells

77
plectin immunoreactivity was usually associated with the plasma membrane of the
cells.
A relationship of plectin with the blood-brain barrier (BBB) is suggested by a
tendency for plectin to be concentrated at all three of the elements of the BBB,
namely the pia, the walls of blood vessels and the linings of ventricles. However,
plectin immunoreactivity was not associated with all blood vessels and was only
infrequently associated with glial endfeet surrounding blood vessels. The BBB,
therefore, is not invariably associated with plectin immunoreactivity. Consequently
one can conclude that plectin is not absolutely required in all parts of this barrier.
In contrast, there is a consistent and strong layer of plectin immunoreactivity
associated with the ependymal layer including the choroid plexus, suggesting that
plectin may mechanically stabilize the submembraneous cytoskeleton and IF
components to form the three-dimension structure of the choroid plexus. In line
with this idea, previous ultrastructural studies have demonstrated a tendency for
plectin to be localized at interfaces between different tissues and especially
between tissues and fluid filled cavities. For example plectin is heavily
concentrated at the surfaces of kidney glomeruli, liver bile canaliculi, bladder
urothelium and gut villi (Wiche et al., 1983).
The selective staining pattern of plectin to subsets of motoneurons in the
brainstem and spinal cord is intriguing. One possible explanation for this unique
finding is that plectin may be associating with a particular NF subunit. Of the five
specific NF subunits, peripherin appears to be the best candidate. Peripherin has

78
a more selective distribution than the NF-triplet proteins and in contrast to cortical
localization of a-internexin, peripherin is localized to motoneurons of the spinal
cord and brainstem (Portier et al., 1984; Parysek and Goldman, 1988; Greene,
1989; Goldstein et al., 1991).
A common theme in this distinct plectin staining pattern is that plectin may be
associating with class 3 IF proteins (based on Steinert and Roop classification
scheme, 1988). Since peripherin and vimentin are similar in structure, it is likely
unlikely that plectin could be interacting at a similar site present in both proteins.
To shed some light on the selective cellular interactions of plectin in the nervous
system, Chapter 6 focuses on examining the distribution of plectin with IFs found
in the neural tissue.
Notes
Portions of this chapter are from a paper entitled "The Distribution of Plectin,
an Intermediate Filament Associated Protein, in the Adult Rat Central Nervous
System" by L.D. Errante, G. Wiche, G. Shaw. Journal of Neuroscience Research
37: 515-528. Copyrighted © 1994 by Wiley-Liss. Text is used with the permission
of Wiley-Liss, a division of John Wiley & Sons, Inc.

CHAPTER 4
CO-LOCALIZATION OF PLECTIN AND INTERMEDIATE
FILAMENTS IN THE RAT NERVOUS SYSTEM
Introduction
Intermediate filaments (IFs) are a family of fibrous proteins found in most
eukaryotic cells and comprise a major component of the cytoskeletal network. In
addition to being organized into 6 classes based on sequence analysis (Steinert
and Roop, 1988), IFs can be categorized based on the types of cells they are
localized to. The strict cell-type specific expression of IFs, coupled with their high
abundance has permitted the extensive use of IF antibodies as convenient cell-
type specific markers. For example, in the nervous system, NF-triplet proteins, a-
internexin and peripherin are localized exclusively to neurons, and different neuron
types tend to have distinctly different IF composition (Shaw et al., 1981; Yen and
Fields, 1981; Portier et al., 1984; Pachter and Liem, 1985; Parysek and Goldman,
1988; Chiu et al., 1989). GFAP is localized to specific glia cell types (Yen and
Fields, 1981; Shaw et al., 1981) while vimentin can be found in subsets of glia,
ependyma and endothelia as well as some unusual neurons (Yen and Fields,
1981; Shaw et al., 1981; Draeger, 1983; Shaw et al., 1983; Schwob et al., 1986).
IF proteins are similar to the other major cytoskeletal proteins, microtubules
and actin, in that IFs have a number a polypeptides associated with them. Unlike
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80
microtubule- and microfilament-associated protein, intermediate filament associated
proteins (IFAPs) are not well characterized especially in neural tissue. Due to the
lack of function associated with IFs in general, proteins have been classified as
specific IFAPs based on minimal criteria such that only one of the following
conditions need to be satisfied: (1) co-purification with IFs in vitro\ (2) cellular co¬
distribution with IFs; (3) binding to IFs or subunit proteins in vitro; and (4) have an
effect on filament organization or assembly (Foisner and Wiche, 1991).
Plectin is one of the few IFAP to have satisfied more than one of these
criteria including solid phase binding to all IF proteins examined and co-localization
with vimentin (Wiche, 1989). In the nervous system, plectin has been shown to
co-purify with crude IF preparations and to be localized in specific cell types that
contain IF proteins (Chapter 3). To address possible in vivo interactions between
plectin and IF proteins in the nervous system, we examined the co-distribution of
plectin with NF triplet proteins, peripherin, vimentin, and GFAP.
Methods
Experimental Tissue and Immunocvtochemistrv
For immunofluorescent studies, adult rats were sacrificed by decapitation.
Neural tissue was dissected out immediately, quickly frozen and stored at -70°C.
Immunofluorescent procedures were described in Chapter 2.
For facial nerve axotomy, adult rats were anesthetized with methoxyflurane.
An incision was made just behind the right ear, the major branch of the facial nerve
was cut, and the incision was closed with surgical staples. Six to seven days post-

81
lesion rats were anesthetized with pentobarbital, and animals were killed by
decapitation or after intracardial perfusion with 4% paraformaldehyde as described
in detail in Chapter 2. In the case of the paraformaldehyde fix tissue, the
brainstem was blocked, immersed in 30% sucrose in PBS until it sunk and then
frozen on dry ice for 1 hour. Tissue was cut at 10 pm and placed in PBS and
treated in the same manner as previously described for paraformaldehyde tissue
(Chapter 2).
Antibodies
Plectin monoclonal antibodies, clones 1D8 and IA2 (diluted 1:2), and rabbit
polyclonal serum, p21 (diluted 1:50), were previously characterized (Wiche and
Baker, 1982; Foisner et al., 1991). NF-M rabbit polyclonal sera, R9 (diluted 1:200),
to the KE (lysine-glutamic acid) region of NF-M was made using NF fusion protein
RM:677-845. NF-H rabbit polyclonal antibody, R14 (diluted 1:200), to the KSP
(lysine-serine-proline) region of NF-H was made using NF fusion protein RH:559-
794 (Harris et al., 1991). Monoclonal antibodies against glial fibrillary acidic protein
(GFAP), clone GA5 (diluted 1:1000) and vimentin, clone V9 (diluted 1:200), were
purchased from Sigma (Debus et al., 1983; Osborn et al., 1984). Rabbit polyclonal
antibody to GFAP (diliuted 1:1000) was a gift from Dr. P. J. Reier. Peripherin
rabbit polyclonal sera, R19 and R20 (diluted 1:200) and mouse monoclonal
antibody, 8G2 (undiluted), were produced in our laboratory using a trp-E fusion
protein construct in the pATH expression vector which was a gift of Dr. Edward Ziff
(see Gorham et al., 1990). The fusion protein was expressed in Escherichia coli

82
in bulk and purified from inclusion body preparations by DEAE-cellulose ion
exchange chromatography as recently described for other trp-E fusion proteins
(Harris et al., 1991). The purified fusion protein was injected into rabbits and mice
to raise polyclonal and monoclonal antibodies using standard procedures (see
Chapter 2).
Results
Immunoblot Studies
Initial characterization of peripherin antibodies were performed on crude IF
preparations from rat spinal cord to make sure that the antibody was not cross¬
reacting with any other cytoskeletal proteins. Both peripherin monoclonal antibody
(8G2) and polyclonal antibody (R20) labelled, cleanly and specifically, a single
protein band at -57 kD. The peripherin protein band migrated at a slightly higher
apparent molecular weight than vimentin in crude IF spinal cord preparation (Figure
4-1, lanes 2-4, respectively). Peripherin polyclonal antibody R19 also showed the
same labelling of a single band at 57 kD (data not shown).
Co-localization in Cultured Cells
Dorsal Root Ganglion (DRG) Cells. Plectin antibodies labelled most of the
cell types in DRG cultures isolated from postnatal day 1 rat pups. The non¬
neuronal cells or satellite cells consisted of two basic types; flat ameboid shaped
and spindle shaped which are illustrated in Figure 4-2. Plectin immunoreactivity
was localized throughout the cell body and the processes of the spindle shaped

Figure 4-1. Characterization of peripherin polyclonal and monoclonal antibodies.
Lane 1 shows a serva blue stained 6% SDS polyacrylamide gel of crude IF
preparation for rat spinal cord. Lanes 2 and 3 are immunoblots of this material
labelled with 8G2 and R20, respectively. These results show that both the
monoclonal and polyclonal antibodies to peripherin labelled a single protein band
at -57 kD. Lane 4 is an immunoblot of the same material labelled with vimentin
monoclonal antibody (V9) which demonstrates that the vimentin protein band is
located just below peripherin on a 6% SDS polyacrylamide gel.

84

85
cells (Figure 4-2a, right side). In contrast the larger, flatter cells showed
particularly strong plectin immunoreactivity surrounding the nuclei and this labelling
intensity decreased dramatically at the peripheral regions of the cell (Figure 4-2a,
left side). Double labelling of these cells with vimentin revealed that plectin
immunoreactivity (Figure 4-2a) was localized discontinuously along vimentin
filamentous network (Figure 4-2b).
Plectin immunoreactivity in DRG neurons was similar to that observed for
the non-neuronal cells in tissue culture. Plectin antibodies strongly labelled the cell
body of DRG cells and weakly labelled the neuritic processes (Figure 4-3a);
whereas, peripherin labelled the cell body and neurites with similar intensity. The
peripherin antibody labelling pattern was consistent to that observed with NF triplet
protein antibodies in DRG (Shaw and Weber, 1981).
Phenocytoma ÍPC121 Cells. The distribution of plectin was compared to
NFs in both undifferentiated and differentiated PC12 cells. In undifferentiated
PC12 cells NF antibody labelling was found adjacent to the nucleus and in a tight
ball (Figure 4-3d), whereas plectin immunoreactivity was found diffusely throughout
the cell and with some overlap with the NF staining pattern (Figure 4-3c). After
exposure to nerve growth factor (NGF), PC12 cells stop dividing and differentiate
which results in the formation of neuritic-like processes as well as acquiring similar
properties as sympathetic neurons. In differentiated PC12 cells, plectin antibodies
labelled the cell body strongly with weak labelling in the processes that was similar
to that observed in the DRG cultures (Figure 4-3e). Additionally, plectin

Figure 4-2. Co-localization of plectin (p21) and vimentin (V9) in non-neuronal cells
from DRG cultures.
(A-B) Plectin immunoreactivity (A) is localized primarily in the cell body of both
types of non-neuronal cells; however, there is some labelling in the periphery of the
flat cell and in the processes of the spindle-shaped cells. Vimentin antibody
labelling (B) demonstrates a filamentous network throughout the cells with selective
overlap with the plectin staining pattern. Scale bar: 25 pm.


Figure 4-3. Co-localization of plectin (ID8) and NF subunits in DRG cells and
PC12 cells.
(A-B) In DRG cells plectin immunoreactivity (A) was almost exclusively localized
to the cell body (arrow) with only weak labelling in the neuritic processes, whereas,
peripherin antibody (R20) labelled the cell body as well as the neuritic processes
with similar intensity (B). Scale bar: 25 pm.
(C-D) In undifferentiated PC12 cells, plectin antibody labelling (C) was observed
diffusely throughout the cytoplasm with some areas of the cell body staining more
intensely than others (arrows). Double-labelling with NF antibody (D)
demonstrated that there was some overlap with the plectin labelling pattern.
Scale bar: 25 pm.
(E-F) In NGF differentiated PC12 cells the pattern of plectin (E) and NF (F)
antibody labelling was somewhat similar in the cell body (large arrow); however,
NF labelling was more intense in the proximal neurite (small arrow) and plectin
immunoreactivity was more intense along certain points of the neurite (arrowhead).
Scale bar: 25 pm.

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90
immunoreactivity was strong at particular points along the neuritic process (Figure
4-3e, arrowhead). In contrast, NF immunoreactivity was localized to the cell body
and in the initial segment of the neurite-like process (Figure 4-3f).
Co-Localization Studies Along the Rat Neural Axis
Plectin and Vimentin. Prior research localized vimentin primarily to non¬
neuronal cell: astrocytes, radial glial cells, ependymal cells, meninges and blood
vessels in the nervous system (Pixley et al., 1981; Shaw et al., 1981; Yen and
Fields, 1981). Plectin distribution in the nervous system most closely
corresponded to that of vimentin, when compared to all of the known IF subunits,
although differences in the staining pattern appear in the extent and type of
labelling within cells. A diffuse plectin antibody staining pattern was evident in
white matter astrocytes and choroidal epithelial cells in contrast to the well defined
filamentous pattern of vimentin within the same cells (Figure 4-2). A striking
difference between antibody labelling patterns was visualized in the Bergmann glial
fibers of the cerebellum (Figure 4-4 a-d). Plectin immunoreactivity was associated
predominantly with the distal tips of Bergmann glia cell process at the pial surface
whereas vimentin immunoreactivity was equally distributed throughout the process
and appears to be diminished or absent from the terminal endings. Although
plectin was localized to some blood vessels shown previously in Chapter 3 as well
as in Figure 4-4 a, it was absent from the blood vessels in certain CNS areas such
as in the diencephalon. A section through the thalamus-fornix junction double-
stained with vimentin and plectin antibodies showed that both antibodies labelled

91
the astrocytes in the fornix; however, only the vimentin antibody labelled the blood
vessels located in the thalamus (Figure 4-4 e,f). Plectin immunoreactivity
appeared to be more strongly associated with larger vessels or vessels located at
the pial surface such as the pericallosal artery (Chapter 3). In addition, plectin did
not completely co-localize with vimentin in the sciatic nerve. Both plectin and
vimentin immunoreactivities overlapped in labelling the outer portion of the myelin
sheath surrounding the axon; however, plectin polyclonal serum additionally
intensely labelled the inner portion of the myelin sheath (Figure 4-4 g,h).
Plectin and GFAP. GFAP was previously shown to be localized to
astrocytes and certain radial glial cells in the nervous system (Shaw et al., 1981;
Yen and Fields, 1981). As described above, plectin immunoreactivity was localized
to the ependymal cells and choroid plexus cells with pronounced staining at the
plasma membrane (Figure 4-5 a). In contrast, GFAP antibody staining was found
predominantly in astrocytic fibers surrounding the ventricle (Figure 4-5 b).
Frequently GFAP positive cells did not label with plectin antibodies (Figure 4-5 c,d),
although the white matter astrocytes which are vimentin positive in rat are also
plectin and GFAP positive (Figure 4-5 a,b,g,h). The plectin antibody showed
punctate staining around the axonal bundles in the caudate nucleus which was
similar to that seen in the thalamus; however, there was no distinct cellular staining
except for a few lightly stained astrocytes (Figure 4-5 c). In contrast, GFAP
immunoreactivity was observed throughout the caudate nucleus especially
surrounding the axonal bundles (Figure 4-5 d). Small diameter blood vessels in

Figure 4-4. Co-localization experiment with antibodies to plectin (p21) (A,C,E,G)
and vimentin (V9) (B,D,F,H).
(A-B) In cerebellar molecular layer, plectin immunoreactivity (A) is localized to the
Bergman glia processes with the strongest staining at the terminal endings just
beneath the pia surface. In contrast, vimentin antibody (B) uniformly labels the
entire extent of the Bergmann glia process. A blood vessel is labelled with
antibodies to both plectin and vimentin (arrowhead), ml: molecular layer; pci:
purkinje cell layer. Scale bar: 50 pm.
(C-D) A high magnification view of A and B showing the terminal endings of the
Bergman glia fibers. Plectin immunoreactivity (C) was observed most intensely at
the terminal endings in which some of these endings were double-labelled with
vimentin (open arrow) whereas others were not (arrowhead). Scale bar: 25 pm.
(E-F) At the thalamus and fornix junction, plectin antibody (E) labels astrocytes
in white matter which corresponds to vimentin immunoreactivity (F) in the fornix.
In contrast, plectin immunoreactivity (E) is completely absent from blood vessels
in the thalamus which label with vimentin antibody (arrows) (F). Scale bar: 25 pm.
(G-H) In a cross-section of sciatic nerve, similar labelling pattern is observed for
plectin (G) and vimentin (H) in the sciatic nerve outer myelin sheath layer (arrows);
however, plectin antibody also labelled the myelin membrane surrounding the axon
(arrowheads). Scale bar: 25 pm.


Figure 4-5. Co-localization experiment with antibodies to plectin (A,C,E) and GFAP
(B,D,F).
(A-B) In lateral ventricle, plectin immunoreactivity (p21) is confined primarily to
ependymal (e) and choroid plexus epithelial cells (cp) with weak labelling of
astrocytes (arrow) near the ventricle (A). In contrast, GFAP antibody (GA5) labels
predominantly the astrocytes around the ventricle (arrow, B). Scale bar: 50 pm.
(C-D) In caudate nucleus, plectin antibody (p21) labels the caudate with
indistinguishable punctate particles (C). The area where little to no plectin
immunoreactivity represents the fascicles of fiber tracts coursing through the
caudate. In contrast GFAP immunoreactivity (GA5) is localized to astrocytes
(arrows) throughout the caudate (D). Both plectin and GFAP antibodies label a
blood vessel in gray matter (arrowheads); however, the labelling patterns are
distinct and plectin antibody does not label all blood vessels (arrowhead). Scale
bar: 50 pm.
(E-F) In pyramidal tracts, both plectin (IA2) and GFAP (polyclonal) antibodies label
the astrocytes near the pia surface (arrows). Scale bar: 50 pm.

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96
the gray matter of the caudate were labelled with both plectin and GFAP antibodies
(Figure 4-5 c,d); however, the staining pattern was distinct in that plectin antibody
appeared to be directly labelling the blood vessel whereas GFAP antibody seemed
to be associated with the astrocytic end feet around the blood vessel. In the
pyramidal tracts, plectin colocalized with GFAP in the astrocyte network near the
pial surface (Figure 4-5 e,f).
Plectin and NF triplet proteins. NF triplet proteins are localized exclusively
to neurons, although the level and pattern of expression is quite variable between
different neuronal cell types (e.g. Yen and Fields 1981, Shaw et al., 1981, Portier
et al. 1984, Brody et al. 1989, Pachter and Liem 1985, Chiu et al. 1989, Kaplan
et al. 1990). Given that plectin has an ability to cross-link NFs in vitro, it is
possible that the limited plectin expression seen in certain neurons may reflect a
co-distribution with one of the NF subunits.
As expected, NF immunoreactivity was much more extensive and therefore,
for the most part, did not overlap with plectin immunoreactivity. Flowever, plectin
colocalized with NFs in motoneurons in the brainstem and spinal cord although not
all NF positive motoneurons showed plectin antibody staining (Figure 4-6 a,b). In
rostral brainstem nuclei, mesencephalic nucleus, plectin immunoreactivity was
weak and diffuse throughout the cytoplasm, with strikingly intense labelling at or
around the plasma membrane (Figure 4-6 c,d). At higher magnification, the dense
labelling of plectin antibody of the plasma membrane between two adjacent cell
bodies (arrow) did not exactly correspond to NF-H antibody labelling (Figure 4-6

97
e,f), but showed some overlap. In a fiber bundle, a distinct staining pattern of
plectin immunoreactivity was evident as shown in a cross-section of adult rat
sciatic nerve (Figure 4-6 g). Plectin antibodies labelled the Schwann cells
surrounding the myelin sheath and the inner membrane surrounding the axon. In
addition the axon itself weakly labelled with the plectin antibody, whereas NF
antibodies labelled only the axon and not the Schwann cells (Figure 4-6 h).
Plectin and Peripherin. Peripherin is a NF subunit originally identified in the
peripheral nervous system (Portier et al., 1984) but is also localized to certain
neurons and nerve fibers in the central nervous system (Brody et al., 1989). Like
NF-H, plectin co-localized with peripherin in specific motoneurons in the brainstem
(Figure 4-7 a-d) and spinal cord. However, some peripherin positive neurons were
not labelled with plectin antibodies, indicating that the expression of peripherin in
neurons was clearly independent of that of plectin (Figure 4-7 e,f). Plectin
immunoreactivity in CNS fiber tracts was similar to that observed in the sciatic
nerve (Figure 4-4 g; 4-6 g). Plectin was most closely associated with the outer
axon sheath whereas peripherin labelled the axons themselves (Figure 4-7 g,h).
Effects of Peripheral Nerve Axotomv
After peripheral nerve axotomy, expression of the major cytoskeletal proteins
are effected differentially. In the case of microtubules and actin, the mRNA for
these proteins are dramatically increased over a period of 7 to 14 days, whereas,
the NF triplet proteins show a significant decrease in mRNA expressed over the
same period of time (Hoffman et al., 1987; Wong and Oblinger, 1987; Goldstein

Figure 4-6. Co-localization experiment with antibodies to plectin (ID8) (A,C,E) and
NFs (polyclonal) (B,D,F).
(A-B) In cervical spinal cord motoneurons, plectin antibody (A) only labelled two
of the three motoneurons (arrows) which labelled with NFH-KSP polyclonal serum
(B). Scale bar: 50 pm.
(C-D) Low magnification view of mesencephalic nucleus of V double-labelled with
plectin (C) and NFH-KSP (D) showing that plectin immunoreactivity is concentrated
at the periphery of these neurons; whereas, NF antibody labelling is found
throughout the cytoplasm. Scale bar: 50 pm.
(E-F) In a higher magnification view of the mesencephalic nucleus of V, plectin
antibody (E) labelled most intensely at the periphery of the neurons (arrowhead)
while NFH-KSP antibody (F) labelling was the strongest in the cytoplasm and
decrease dramatically at the periphery of the neurons (arrowhead). Scale bar: 50
pm.
(G-H) In sciatic nerve, plectin antibody (E) staining is associated with the
outermost (open arrow) and innermost (arrowhead) plasma membrane of the
Schwann cell process. Diffuse plectin immunoreactivity is also observed within the
axon. In contrast polyclonal antibody, NFM-KE, labels only the axons (arrowhead).
Scale bar: 25 pm.


Figure 4-7. Co-localization experiment with antibodies to plectin (IA2) (A,C) and
peripherin (R19) (B,D).
(A-B) In the nucleus ambiguus, plectin (A) and peripherin (B) had similar labelling
distribution in the neurons with some slight differences in intensity of labelling.
Scale bar: 50 pm.
(C-D) In brainstem (medulla), plectin (C) and peripherin (D) had similar labelling
pattern in motoneurons. Scale bar: 50 pm.
(E-F) Although the lateral tegmental nucleus showed strong peripherin
immunoreactivity (F), this rostral brainstem nuclei lacked plectin immunoreactivity
(E) in the neurons. Scale bar: 50 pm.
(G-H) In a CNS white fiber tract in the spinal cord, plectin immunoreactivity (G)
is confined predominantly to the outer portion of the axon (open arrow); whereas,
peripherin immunoreactivity labels the axon bundles in cross-section (open arrow).
Scale bar: 25 pm.

101

102
et al., 1988; Tetzlaff et al., 1988; Oblinger et al., 1989). In contrast to NF-triplet
proteins, peripherin mRNA and protein expression is dramatically increased after
sciatic nerve axotomy in both the dorsal root ganglion cells and ventral
motoneurons (Oblinger et al, 1989; Wong and Oblinger, 1990). Since plectin's
neuronal distribution is limited to motoneurons in the brainstem and spinal cord,
and one of the primary roles of plectin appears to be related to the structure of the
cell (Wiche, 1989), it was of interest to determine if plectin's protein expression
would be altered after peripheral nerve lesion.
The facial nerve axotomy model is a popular model based upon the ease
of making the lesion and the ability to determine the location of the facial nucleus
both externally as well as in cross-section (Kreutzberg et al., 1990). Axotomy of
the facial nerve affects musculature ipsilateral to the side of the lesion, most
profoundly affecting the rat's whiskers. Figure 4-8 (a,b) shows a low power view
of the left and right ventral quadrant of the brainstem labelled with plectin antibody
after the facial nerve was transected on the right side. The facial nucleus is
outlined with arrowheads. In the control side (Figure 4-8a), the facial motoneurons
are weakly labelled with plectin antibodies which is visualized better under high
power (Figure 4-8c). On the lesion side, the facial motoneurons show a dramatic
increase in plectin immunoreactivity throughout the cell body (Figure 4-8b).
Another difference was observed in the extent of labelling. On the control side,
plectin immunoreactivity is diffuse with a small area of dense labelling (Figure 4-8c,
arrows) similar to that seen previous in nucleus ambiguus and ventral horn

Figure 4-8. Effects of a unilateral facial nerve axotomy on plectin immunoreactivity (1A2).
(A-B) A low magnification view of the of the left (A) and right (B) ventral quadrant of the
brainstem labelled with plectin antibody 7 days after the facial nerve was transected on the right
side. The facial nucleus is outlined in arrowheads. On the control side (A), the facial
motoneurons are weakly labelled with peripherin antibody. On the lesion side (B), there is a
dramatic increase in plectin immunoreactivity. Scale bar: 100 pm.
(C-D) A higher magnification view of the facial nucleus shows differences in the extent of plectin
antibody labelling within the cell body. On the control side (C), plectin immunoreactivity is weak
throughout the cell body with some areas of intense labelling (arrows). In contrast, on the lesion
side (D), plectin immunoreactivity is strong throughout the cell body. Scale bar: 50 pm.

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105
motoneurons (Chapter 3). After facial nerve axotomy, the entire facial motoneuron
cell bodies labelled densely with plectin antibodies whereas the processes
remained weakly labelled similar to the control side (Figure 4-8d).
Immunofluorescent double-label experiments with plectin and NF triplet
proteins and with plectin and peripherin were conducted to obtain a better
understanding of the relationship between plectin and the different NF subunits
after axotomy. As discussed earlier, the mRNA expression for NF triplet proteins
decreases after facial nerve axotomy while the protein expression remained
unchanged (Tetzlaff et al., 1988). On the control side only a few of the facial
motoneurons were double-labelled with both plectin and NF triplet antibodies
(Figure 4-9a,c, respectively); however, plectin immunoreactivity was extremely
weak. On the lesion side, the majority of the facial motoneurons labelled with both
plectin and NF triplet antibodies in a similar manner (Figure 4-9b,d). In the case
of peripherin, it was expected that facial motoneurons would express more
peripherin protein on the side of the lesion since that was what was observed after
axotomy of sciatic nerve. Figure 4-10 showed that there was a prominent increase
of both plectin and peripherin protein expression on the lesion side with only weak
labelling of motoneurons on the control side.
Discussion
The distribution of plectin in different cell types of the adult nervous system
with respect to neural IF proteins is summarized in Table 4-1. Previously, plectin
has been shown to bind to several different types of IF in vitro (Foisner et al.,

Figure 4-9. Co-localization experiment with plectin (1A2) (A,C) and NF-H (R14) (B,D) after
unilateral facial nerve axotomy.
(A-C) Light micrographs showing double-labelling of the facial nucleus on the control side.
Plectin antibody (A) only labels a few cells in the facial nucleus weakly (thick arrows) whereas
NF-H immunoreactivity (B) is localized in numerous cells in the facial nucleus. Scale bar: 50 pm.
(B-D) Light micrographs showing double-labelling of the facial nucleus on the lesion side.
Plectin antibody (B) labelling is stronger on the lesion side (arrows) in comparison to the control
side and labels the motoneurons with similar intensity as NF-H antibody (D; arrows). Scale bar:
50 pm.

107

Figure 4-10. Co-localization experiment with plectin (1A2) (A,C) and peripherin (R20) (B,D) after
unilateral facial nerve axotomy.
(A-C) Light micrographs showing double-labelling of the facial nucleus on the control side.
Plectin antibody (A) only labels a few cells in the facial nucleus weakly (thick arrow) which
coincides with the weak labelling of the same motoneurons with peripherin antibody (C).Scale
bar: 50 pm.
(B-D) Light micrographs showing double-labelling of the facial nucleus on the lesion side.
Plectin antibody (B) labelling is stronger on the lesion side (arrows) in comparison to the control
side (A, thick arrow) and labels the motoneurons with similar intensity as peripherin antibody (D).
Scale bar: 50 pm.

601-

110
1988) and to co-localize with vimentin in non-neuronal tissue (Wiche, 1989). In
line with these findings, plectin has similar distribution to that previously reported
for vimentin in the central nervous system (Pixley et al., 1981; Shaw et al., 1981;
Yen and Fields, 1981). Little plectin immunoreactivity is observed in gray matter
astrocytes, which, in rat at least, contain very little vimentin immunoreactivity (Shaw
et al., 1981; Yen and Fields, 1981). Similarly, more marked plectin
immunoreactivity is found in white matter astrocytes which, in rat, stain strongly for
vimentin (Shaw et al., 1981; Yen and Fields, 1981). Double-label experiments with
plectin and vimentin antibodies in non-neuronal cells in DRG cultures demonstrate
that plectin can be localized along the vimentin's filamentous network; however, the
labelling is discontinuous along the vimentin filament bundles. Overall, three major
difference exist between plectin and vimentin labelling patterns: (1) the lack of
small blood vessel labelling by plectin antibodies in gray matter; (2) the distribution
of labelling in the Bergmann glia processes; and (3) the presence of plectin
immunoreactivity in motoneurons in the brainstem and spinal cord. The few
regions in which plectin antibody labelling overlap with GFAP immunoreactivity
coincide with those found previous by for vimentin and GFAP antigen distribution
(Shaw et al., 1981; Pixley et al., 1984).
An interesting finding was plectin immunoreactivity in neurons. In DRG cells
and PC12 cells, plectin antibody staining was localized predominantly in the cell
body; however, there was some weak immunoreactivity in the neuritic processes.
Although plectin was found in postnatal day 1 DRG cultures, plectin

Table 4-1. Summary of the distribution of plectin in different cell types of the adult rat
nervous system with respect to intermediate filament proteins.
Cell Type
Plectin
NF-triplet
Peripherin
Vimentin
GFAP
Ependyma
choroid plexus
++
—
—
++
—
ependymal
++
—
—
++
+\-
subependyma
++
—
—
++
++
tanycytes
++
—
—
++
—
Glial Cells
astrocytes (white matter)
++
—
...
++
++
astrocytes (gray matter)
+/-
—
...
—
++
Bergmann fibers
++
—
—
++
++
Schwann cells
++
—
...
++
+\-
Neuronal Cells
cortical neurons
—
+/-
+\-
—
—
cerebellar granule
—
++
—
---
—
cerebellar Purkinje
—
++
—
...
---
motoneurons
+/-
++
+\-
...
---
axons
—
++
+\-
—
—
Other
endothelial cells
+/-
—
—
++
—
pia mater
++
—
—
++
—
++: indicates that the protein was localized in the majority of this cell type; +/-: indicates that the protein
labelled a subpopulation of this cell typle; —: indicates that the protein was not observed to these cells.
111

112
immunoreactivity was not found in adult rat DRG cells (data not shown). Whether
plectin antibody labelling of the DRG culture cells was related to a developmental
progression or was an artifact of tissue culture remains to be determined. In the
adult rat nervous system, plectin immunoreactivity was associated with
motoneurons in the brainstem and spinal cord. Colocalization experiments with
plectin and different NF subunits revealed that plectin positive motoneurons also
contained NF triplet proteins and peripherin. The presence of plectin in cells
containing the NF triplet proteins also showed that an in vitro interaction between
plectin and the NF triplet proteins may reflect a meaningful in vivo interaction
(Foisner et al. 1988).
The relationship between plectin and peripherin was examined since plectin
was localized only to a small number of neuron in comparison to the large number
of types of neurons that express NF triplet proteins. Previous research as well as
this present study showed that subsets of brainstem and spinal cord neurons
labelled with peripherin antibodies (Portier et al., 1984; Parysek and Goldman,
1988; Brody et al., 1989). As shown here, plectin positive neurons all expressed
peripherin so that peripherin, which is quite similar to vimentin in primary sequence
and biochemical properties, may in some functional sense replace vimentin in
these cells. In addition, we noted that although plectin positive motoneurons were
always peripherin positive, the converse was not true, indicating that while plectin
was expressed in consort with peripherin or vimentin in the nervous system, not
all cells expressing these two proteins expressed detectable amounts of plectin.

113
To further study the relationship between plectin and neural IF proteins in
motoneurons, the expression of plectin, peripherin, and NF-H, was examined
immunocytochemically after a unilateral lesion to the facial nerve. Previous
research has investigated the effects on cytoskeletal elements after a nerve
axotomy and found that tubulin, actin and peripherin mRNA were upregulated,
whereas NF triplet proteins mRNA were down regulated with maximum effects
occurring at 7-14 days depending on the model system used (Floffman et al., 1987;
Wong and Oblinger, 1987; Goldstein et al., 1988; Tetzlaff et al., 1988; Oblinger et
al., 1989). In addition, peripherin immunoreactivity was shown to increase in
ventral horn motoneurons on the lesion side and NF triplet protein antibody
labelling appeared to remain unchanged when using a phosphate insensitive
antibody. After facial nerve lesion, plectin immunoreactivity in the facial nucleus
on the lesion side dramatically increased in comparison to the control side. The
number of motoneurons that labelled with plectin did not appear significantly
different; however, the extent of labelling within the cytoplasm was dramatic. On
the control side there was diffuse plectin immunoreactivity with only some small
areas of intense immunoreactivity. The increase in plectin immunoreactivity that
was localized throughout the cytoplasm, suggested that plectin may be performing
a stabilizing function after injury to the cell. Another possibility is that the transport
of plectin into the axons may decrease resulting in an increase in plectin in the
accummultation in the cell body. However, since plectin does not appear to
localize to axons in renders this hypothesis rather unlikely. In comparing plectin's

114
expression to NF-H, plectin was weakly localized to some of the NF-H positive
motoneurons on the nonlesion side, whereas, on the lesion side the same neurons
expressed both plectin and NF-H with similar intensities. Double-label experiments
with plectin and peripherin antibodies showed that plectin and peripherin were
localized to the same motoneurons on both the non-lesion and lesion sides, and
that the intensity of staining was similar for both of the antigens.
The submembraneous labelling of neurons in the brain stem and spinal cord
suggests that plectin may stabilize the submembraneous cytoskeleton in these
cells, and also may act as a cross-linker between the plasma membrane and
cytoskeleton network, as appears to be the case in non-neuronal cells (Wiche et
al., 1983; Hermann and Wiche, 1987; Wiche, 1989). The reorganization within the
motoneuons after a peripheral nerve axotomy is extensive. In particular there is
both an up-regulation and down-regulation of mRNA for cytoskeletal proteins. The
increase in plectin immunoreactivity after peripheral nerve axotomy suggests that
plectin may function to stabilize the cytoskeleton during injury state since plectin
is known to interact with cytoskeletal elements. Future studies examining the time
course of the increase in plectin immunoreactivity, including immunoelectron
microscopy studies to determine the ultrastructural distribution of plectin in
motoneurons after peripheral nerve axotomy, should result in a better
understanding of the function of plectin.

115
Notes
Portions of this chapter are from a paper entitled "The Distribution of Plectin,
an Intermediate Filament Associated Protein, in the Adult Rat Central Nervous
System" by L.D. Errante, G. Wiche, G. Shaw. Journal of Neuroscience Research
37: 515-528. Copyrighted © 1994 by Wiley-Liss. Text is used with the permission
of Wiley-Liss, a division of John Wiley & Sons, Inc.

CHAPTER 5
IDENTIFICATION OF CANDIDATE NEUROFILAMENT BINDING PROTEINS
Introduction
NFs are abundant proteins within the nervous system, however, the role of
these proteins is not well defined. Protein sequence information has given insight
to the protein structure of the NF subunits and how NF may interact with each
other. NFs have been shown to bind to cytoskeletal elements directly as in the
case of tubulin (Minami et al., 1983) and microtubules (MTs) (Hisanaga and
Hirokawa, 1990) or indirectly by the cross-linking ability of MAP2 to both MTs and
NFs (ie. Leterrier et al., 1982; Flynn et al., 1987). Various other proteins such as
brain spectrin (fodrin) (Frappier et al., 1987), plectin (Foisner et al., 1988) and
synapsin I (Steiner et al., 1987) have been shown to bind to various forms of NFs
in vitro.
Previous studies utilizing affinity columns to identify actin (Miller et al., 1989)
and microtubule binding proteins (Kellog et al., 1989) were successful in identifying
cytoskeletal associated proteins. Here, NF affinity columns were employed to test
the hypothesis that soluble proteins bind to NF subunits, and were used as a way
to identify candidate cytosolic NF associated proteins in a partially purified form.
116

117
Methods
Pig Spinal Cord Cytosolic Preparation
The pig spinal cord was obtained from a slaughterhouse within an hour after
the pigs were killed. The connective tissue and blood vessels were removed and
the spinal cord was placed on ice. The spinal cord was homogenized at 4°C in a
Sears blender in 50 mM MES pH 6.5, 5 mM MgCI2, 1 mM DTT, 1 mM PMSF and
1 mM TAME. The tissue was homogenized with three 5 second pulses on low
speed and three 5 second pulses on high speed. The crude homogenate was
centrifuged at 9240xg for 30 minutes at 4°C in a SS34 Sorvall rotor. The
supernatant was filtered through gauze sponge type VII (8 ply). The supernatant
was re-centrifuged at 147,000xg for 1 hour at 4°C in a T865 Sorvall rotor. The
final supernatant was filtered through a Whatmann #1 filter and was referred to as
pig cytosol. The cytosol was used immediately for affinity column binding.
Affinity Column Preparation
Affinity columns were made using Affi-Gel 10 resin (Bio-Rad). The Affi-Gel
beads offer high stability bonds, high coupling efficiency and rapid coupling of
proteins to the gel support. The Affi-Gel 10 support is composed of a 10 atom
spacer arm that is linked through ether bonds to chemically cross-linked agarose
gel beads (Bio-Gel A-5m gel). The protein ligand forms a stable amide bond with
the Affi-Gel's terminal carboxyl of the spacer arm by displacing N-
hydroxysuccinimide. Seven different types of affinity columns were made: crude
IF preparation, NF triplet proteins purified using ion exchange chromatography,

118
purified subunits (NF-H, NF-M and NF-L), and control columns with either BSA or
no protein bound. The protein ligands were coupled to the gel support using 0.1
M MES pH 6.5, 5 mM MgCI2, and 1 mM DTT. The coupling reaction occurred at
4°C for at least 12 hours. The remaining active ester groups were blocked with 0.1
ml 1 M ethanolamine-FICI (pH 8) per ml of gel at 4°C for 2 hours. The columns
were then washed with coupling buffer followed by a wash with coupling buffer
containing 1 M KCI. The column was ready to use, after a final wash with coupling
buffer. Columns were stored at 4°C and used 4 to 5 times before a new column
was made.
Binding of Cytosolic Proteins to Affinity Columns
An overview of the procedure to bind cytosolic proteins to the affinity
columns is shown in schematic form in Figure 5-1. The affinity column to be used
was equilibrated with 50 mM MES pH 6.5, 5 mM MgCI, and 1 mM DTT (Binding
Buffer). Approximately 20 ml of pig spinal cord cytosol (2.5 mg/ml) was cycled
through a particular NF affinity column at a rate of 0.5 ml/minute for 2 hours at
4°C. The column flow through was collected and saved. The column was washed
with 50 ml of binding buffer to remove any unbound protein. To make sure no
more unbound protein was being eluted, the optical density at 280nm was
measured using the last milliliter of eluted wash buffer. Proteins that bound to the
affinity column were eluted using a 30 ml salt gradient; 0 to 0.5 M KCI in binding
buffer. Column fractions were precipitated with trichloro-acetic acid and
precipitated proteins were brought up in 25 pi of 1 M Tris and 25 pi of 2X sample

Figure 5-1. Outline of the method used for identifying candidate NF binding
proteins.
The top gel shows the purity of the different NF proteins used in making the affinity
columns. The purified NF triplet affinity column is a mixture of the three purified
NF triplet subunits. As an example, the bottom gel shows the cytosol preparation
before it is cycled through a column (C) and with a representative gel of the
proteins which bound to the NF-L affinity column. The horizontal markers between
the cytosol lane and the candidate NF binding protein gel represents the relative
positions of the molecular weight standards.

120
NF Affinity Columns Preparation of Pig S.C. Cytosol
5 g Pig Spinal Cord
homogenize in 50mM
MES pH=6.5 containing
5mM MgCI, 1mM DTT,
u proteases inhibitors
Centrifuge
10,000xg
150,000xg
Cycle 20ml Cytosol (2-3mg/ml)
through Column for 2 hours
(Flow Rate
0.5ml/min.
Wash Column
to remove
j unbound protein
Elute Bound Protein with
30ml Salt Gradient (0-0.5M NaCI)
collect 1 ml fractions
^ concentrate samples
Separate with SDS PAGE
I
Measure Relative MW
of Unknown Proteins
2 Purified NF-H
3 Purified NF-M
4 Purified NF-L
c

121
buffer. A sample (10 pi) of each precipitated fraction was separated on either a
10% or 12% SDS polyacrylamide gel. Proteins were visualized using the method
described in Chapter 2.
Production of Polyclonal and Monoclonal Antibodies
Polyclonal antibodies to GAPDH were produced in male Balb-C mice (n=2).
The mice initially received an intraperitoneal injection with 0.4 ml solution
containing 50% GAPDH (Sigma; 2 mg/ml) and 50% Freund's complete adjuvant.
Two weeks following the first injection the mice received a second injection
consisting of 50% GAPDH (2mg/ml) and 50% Freund's incomplete adjuvant. All
subsequent immunogen injections used this solution. The third injection followed
a week later where a test bleed was done to determine the antibody titre. Weekly
injections of the immunogen solution followed with test bleeds until an appropriate
titre level was reached (total of 4 injections). In addition, both mice were producing
fluid in the peritoneal cavity. This fluid was drained using a 16 gauge needle
inserted into the cavity and tested for presence of antibodies to GAPDH. The titre
was determined using a immuno-blot of the candidate NFL-38 protein and purified
GAPDH (Sigma). The mouse with the higher antibody titre was then used to make
monoclonal antibodies. This mouse was injected with 0.1 ml of a 1 mg/ml solution
of GAPDH five days before the mouse was brought to the hybridoma core facility
for the monoclonal antibody fusion procedure. ELISA positive clones were tested
by immunoblot analysis to determine if GAPDH was recognized.

122
Results
A number of proteins bound to the various NF affinity columns with different
specificity. Table 5-1 shows the relative molecular weight of proteins from the pig
cytosol preparation which bound consistently to the different NF affinity columns.
None of these proteins bound to control columns containing either BSA or no
protein. The individual NF columns bound the following proteins, based on relative
molecular weight, consistently and exclusively: NF-H bound 70 kD and 49 kD
proteins; NF-M bound 87 kD, 66 kD and 46 kD proteins; and NF-L bound 84 kD
and 55 kD proteins. Some of these proteins bound to the crude IF and purified NF
columns which was expected since these columns contain all 3 NF subunits. In
contrast not all the proteins which bound to the purified NF columns (84, 66 and
15 kD) bound to the crude IF affinity column. A possible reason for this selectivity
is that the binding site on the individual subunit columns may be blocked with
another protein in the crude IF affinity column. All the NF columns bound the
following proteins: 20, 29, and 38 kD. The most prominent protein band present
was a 38 kD protein which bound to all NF affinity columns, but seemed to bind
to the NF-L affinity column particularly well. The 38 kD band eluted from the
various NF affinity columns at KCI salt concentrations in the range of 0.1 M - 0.2
M, with the salt concentrations being slightly different for each column.
The characterization of the 38 kD protein began with testing the hypothesis
that the 38 kD protein could be a degradation product of intermediate filaments
(IFs) since the a-helical rod domain of IFs is approximately 39 kD. To examine

123
Table 5-1. Relative molecular weight (kD) of candidate proteins which
bound to the five different types of NF affinity columns.
Crude IF
Purified NF
Purified NF-H
Purified NF-M
Purified NF-L
88
87
87
84
68
—
70
—
65
66
60
59
60
61
54
55
50
49
—
45
46
38
37
38
40
39
29
28
30
29
29
19
21
19
20
20
16
15

124
this possibility, an immunoblot of 38 kD protein from each of the affinity columns
was done using Pruss monoclonal antibody which labels a common epitope in the
rod domain of all IFs (Pruss et al., 1981). Only the 38 kD protein which bound to
the crude IF affinity column (IF-38) labeled with the Pruss antibody (Figure 5-2a;
lane 1, lower band). The other proteins which weakly labeled with Pruss antibody
at ~50 kD were most likely keratin contamination from skin. To further examine if
the IF-38 was the rod domain of IF protein, the amino acid composition of IF-38
was compared to that of the rod domain of NF-L which demonstrated that these
proteins were not related (Figure 5-2b). This conclusion was based primarily on
the difference in leucine content which is characteristically high in the a-helical rod
domain of all IFs (10.84% for NF-L) but was low in the IF-38 candidate protein
(5.78%). This suggests that IF-38 would not form the typical IF type coiled-coil
structure.
In addition, the differential antibody staining with the Pruss antibody between
IF-38 and the other 38 kD NF binding proteins from the other NF affinity columns
was not supported by the amino acid profiles (Table 5-2; Figure 5-3) and chemical
cleavage studies (data not shown) done on IF-38 and the 38 kD protein that bound
to the NF-L affinity column (NFL-38). These results showed that these two
proteins were very similar if not identical. One plausible explanation for this was
that there were two proteins at the same relative molecular weight (38 kD) binding
to the crude IF affinity column, and the one which labeled with the Pruss antibody
was at such a low concentration that it did not make a significant contribution to

Figure 5-2. Comparison of candidate 38 kD NF binding protein to rod domain of IF proteins.
(A) Immunoblot using Pruss antibody (anti-IFA) to examine if the 38 kD protein is a degradation
product of IF rod domain. Lanes 1-5 contain fractions with the 38 kD protein from crude IF,
purified NF, purified NF-H, NF-M and NF-L affinity columns. Only the 38 kD protein which bound
to crude IF column (IF-38) labels with anti-IFA. The minor bands labelling at -50 kD are probably
due to keratin contamination.
(B) Comparison of amino acid composition for the IF-38 to that of the rod domain of NF-M. The
spokes in the wheel represent the percent nanomole for each particular amino acid. The values
for NF-L rod domain were determined from rat sequence. The prominent difference exists in
leucine content which is low in IF-38 (5.78%) but high for rod domains in general (10.84% for
NF-L).

A
1 2 3 4 5
Bl
NF-L Rod
ASX
ARG THR
ILE UAL
rtT
126

Table 5-2. Amino acid composition for candidate neurofilament binding proteins with the data
represented as nanomole percent.
Amino
Acid
NFL-38
IF-38
NFL-16
NFL-62
Result #1
Result *2
Average
Result
Result
Result
ASX
12.72
11.08
11.90
11.85
10.16
8.46
THR
7.06
5.70
6.38
6.64
7.49
3.94
SER
6.48
4.35
5.42
7.36
5.68
5.16
GLX
6.87
6.83
6.85
7.71
11.24
11.86
PRO
3.88
3.41
3.65
3.56
0.00
0.00
GLY
11.72
9.97
10.85
13.15
10.98
19.51
ALA
10.47
8.95
9.71
9.99
4.69
8.66
VAL
6.38
13.97
10.18
7.02
9.86
9.01
MET
0.86
1.97
1.41
1.03
1.52
1.63
ILE
5.38
5.84
5.61
4.75
5.24
4.92
LEU
6.04
5.97
6.00
5.78
7.67
7.17
TYR
2.65
3.44
3.04
2.50
1.56
0.00
PHE
3.93
4.99
4.46
3.27
3.81
3.33
HIS
3.51
3.49
3.50
2.74
1.81
3.24
LYS
7.75
6.99
7.37
6.70
13.04
7.83
ARG
4.29
3.06
3.67
5.92
5.26
5.27
127

128
the amino acid analysis. The amino acid composition for IF-38 and NFL-38 was
compared to the average amino acid composition of all the entries in Genbank in
order to determine if these proteins were high or low in any particular amino acid.
The amino acid data is graphically represented in Figure 5-3 in which the circle
represents the average nanomole percent for each amino acid and the spokes
represent the IF-38 and NFL-38 data normalized to the average amino acid profile
of all entries in Genbank. Spokes outside the circle show that the 38 kD protein
has more of that particular amino acid than the average amino acid, whereas,
spokes inside the circle indicate that a particular amino acid is present in a lower
concentration than that observed for the average amino acid in Genbank. The
amino acid composition of IF-38 and NFL-38 is similar to the average protein but
overall these proteins appear to be low in hydrophobic amino acids, and a little
above average for glycine and alanine. In addition, NFL-38 protein is shown to be
slightly higher in histidine content.
Preliminary identification of the 38 kD protein was accomplished using a
computer program, FINDER, which searches the protein database for proteins of
similar amino acid composition to that of an unknown protein (Shaw, 1993). The
output data from the FINDER program gave the calculated molecular weight,
isoelectric point and accession number for the known protein, as well as an amino
acid match score. The amino acid match score is a representation of how similar
the amino acid composition is between the candidate protein and a protein in the
database. The highest possible match score is 32 and a close match is 22 or

Figure 5-3. Comparison of amino acid composition data between the NFL-38 and IF-38, and to
that of the average amino acid composition in Genbank.
A) The diagrams represent the amino acid composition of NFL-38 in which the top figure
represents nanomole percent for each amino acid. The bottom figure depicts the same values
normalized to the average protein in Genbank.
B) The diagrams represent the amino acid composition of IF-38 in which the top figure
represents nanomole percent of each amino acid. The bottom figure depicts the same values
normalized to the average protein in Genbank.
Overall, there are no significant differences reflected in the amino acid composition between IF-
38 and NFL-38 proteins. There does not appear to be any significant high or low concentration
of any particular amino acid when compared to the average amino acid in Genbank. The actual
nanomole percent for each amino acid for the 38 kD protein is given in Table 5-2.

130

131
Table 5-3. Results from the FINDER program to identify NFL-38.
Best Candidates
Score
MW* (kD)
IEP11
Accession
Number
GAPDH§; human
24
36.0
7.8
DEHUGL
GAPDH; chicken
24
35.7
8.0
A22035
GAPDH; human
24
36.0
7.9
A21939
GAPDH; human
24
36.0
7.9
A31988
GAPDH; human
24
35.9
7.9
B22939
GAPDH; human
23
35.8
6.7
DEHUG3
GAPDH; chicken
23
35.7
7.9
DECHG3
GAPDH; chicken
23
35.7
7.9
A32737
GAPDH; pig
22
35.6
6.9
DEPGG3
GAPDH; hamster
22
35.7
7.9
DEHYG
GAPDH; petunia
22
36.5
6.8
DEPJG
GAPDH;
mouse-ear cress
22
36.9
6.4
JQ1287
MW: molecular weight (calculated)
IEP: isoelectric point (calculated)
GAPDH: glyceraldehyde-3-phosphate dehydrogenase

132
Table 5-4. Comparison of NFL-38 to GAPDH with data represented as
nanomole percent for each amino acid.
Amino
Acid
NFL-38
Average1
NFL-38
Corrected*
GAPDH
(human11)
NFL-38 ± 1.5 SD
Adjusted5
GAPDH
Adjusted
Score¥
ASX
11.90
11.78
11.85
16.10
±
1.36
16.12
2
THR
6.38
6.32
6.38
8.63
±
0.35
8.68
2
SER
5.42
5.77
6.38
7.88
±
0.90
8.68
2
GLX
6.85
6.59
6.08
9.00
±
0.71
8.26
1
PRO
3.65
3.17
3.65
4.34
±
1.99
4.96
2
GLY
10.85
9.86
10.03
13.48
±
1.98
13.64
2
ALA
9.71
9.34
9.42
12.76
±
0.33
12.81
2
VAL
10.18
11.70
9.73
15.99
±
1.51
13.22
1
MET
1.41
2.07
3.04
2.83
±
2.19
4.13
2
ILE
5.61
6.16
6.69
8.42
±
0.58
9.09
1
LEU
6.00
5.77
5.78
7.88
±
0.42
7.85
2
TYR
3.04
2.76
2.74
3.78
±
0.22
3.72
2
PHE
4.46
4.46
4.26
6.09
±
0.44
5.79
2
HIS
3.50
4.37
3.04
5.98
±
0.70
4.13
0
LYS
7.37
6.09
7.90
8.32
±
0.82
10.74
0
ARG
3.67
3.78
3.04
5.17
±
0.61
4.13
1
f: data represents the average of two amino acid analysis
4: data was multiplied by a correction factor which corrects for amino acid quantitation
errors
H: data for GAPDH was derived from human sequence (DEHUGL)
§: data is based on calculation initially excluding PRO, GLY, VAL and MET then calculating
these four relative to 100% for the other 12 amino acids
Â¥: a score of 2 is given if the nanomole percent is within 1.5 SD and a score of 1 if it is
within 3 SD (SD: standard deviation)

133
above. The 12 closest matches were all forms of the same protein isolated from
different species, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Table 5-
3). Comparison of the amino acid profile from 38 kD protein and GAPDH shows
that the two proteins are similar with only a slight variation in the nanomole percent
for any particular amino acid (Table 5-4).
Although the evidence presented above suggest that NFL-38 is GAPDH by
computer analysis, further biochemical characterization was necessary. Cyanogen
bromide cleavage experiments using NFL-38 and GAPDH from pig muscle
(purchased from Sigma) demonstrated that the two proteins had the same
cleavage fragment pattern (fingerprint) although the intensity of the silver stained
bands varied somewhat (Figure 5-4). Subsequently, a polyclonal antibody was
made in mouse to GAPDH using purified GAPDH from pig muscle. Figure 5-5a
demonstrates that the polyclonal antibody to GAPDH labeled both GAPDH and
NFL-38, and that the two proteins co-migrated at the same relative molecular
weight on SDS polyacrylamide gel. In addition, we made a monoclonal antibody
(mouse) to GAPDH and showed that it labelled the 38 kD protein which bound to
all NF affinity columns (Figure 5-5b).
Other candidate cytosolic NF proteins have been initially characterized;
however, identification of these proteins have met with little success. The amino
acid composition for NFL-16 protein is given in Table 5-2 and graphically
represented in Figure 5-6a. This data was then put through the FINDER program.
Three close matches were found with a score of 22/32. These proteins, myelin

Figure 5-4. Comparison of CNBr cleavage fragments of NFL-38 and GAPDFI.
The silver stained gel shows that GAPDH (lane 1) and NFL-38 (Lane 2) have
similar cleavage fragments although the intensity of staining of individual bands
was slightly different.

135
1
2

Figure 5-5. Immunoblot analysis using antibodies to GAPDH to determine if the
38 kD proteins binding to various NF affinity columns is GAPDH
(A) Immunoblot demonstrates that the polyclonal antibody to GAPDH labels the 38
kD protein which bound to NF-L affinity column (lane 2). In addition, NFL-38 co¬
migrates with GAPDH (lane 1) which provides further evidence that these two
proteins are similar.
(B) Immunoblot shows that the monoclonal antibody to GAPDH (1D4) labels the
38 kD protein which bound to all NF affinity columns. Lane 1-5 are fractions
containing the 38 kD protein which bound to the different affinity columns; crude
IF, NF-purified, NF-H, NF-M, and NF-L, respectively.

137

138
P2, fatty acid binding protein and phosphotransferase system II enzyme, are
graphically represented in Figure 5-6a, and all have a similar molecular weight to
that of NFL-16. However, on closer inspection these proteins may not be good
matches since significant amounts of a particular amino acid is present in NFL-16
but not in the possible protein matches. This NFL-16 amino acid profile was
compared to that of the average protein in Genbank and showed a very high
concentration of lysine which is a basic amino acid (Figure 5-6b).
A similar analysis was performed on NFL-62 but no close matches were
found using the FINDER program. The amino acid profile is given in Table 5-2
and graphically represented in Figure 5-6a. In addition, the amino acid
composition was compared to the average protein in Genbank and showed a high
concentration of glycine (Figure 5-6b). A potential and major problem to identify
a protein is that one needs enough protein (at least 10 nmole) to get an accurate
amino acid profile. In the case of NFL-16 and NFL-62, the amount of protein used
for the analysis was 6.65 and 3.71 nmole, respectively. This may have resulted
in no detectable amounts of proline in both NFL-16 and NFL-62, and no dectable
amounts of tyrosine in NFL-62. In these cases, the insufficient amount of protein
may have resulted in inaccurate estimates of the percent for each amino acid.
Discussion
This research represents a first attempt at identifying soluble candidate NF
binding proteins using affinity chromatography. A number of proteins, based on
relative molecular weights, were shown to bind to the various NF columns. In the

Figure 5-6. Graphical representions of the amino acid composition data for NFL-
16, and possible matches as determined by the FINDER program.
(A) NFL-16 amino acid composition values (see Table 5-2) were put throught the
FINDER program. Three protein closely matched with a score of 22/32: myelin P2,
fatty acid binding protein, and phosphotransferase enzyme. However, these three
proteins had significant differences with NFL-16 amino acid composition data which
suggest that none of these proteins may be good matches.
(B) The NFL-16 protein was compared to the average protein in Genbank and the
prominent feature that was shown is a high lysine content.

B
Comparison of NFL-16 to Average Amino
Acid Composition in Genbank
ASX
ARG THR
LEU
ALA
ILE
MET
UAL

Figure 5-7. Graphical represention of the amino acid composition data for NFL-62,
and to that of the average amino acid composition in Genbank.
The top figure is a graphical representation of NFL-62 protein amino acid data (see
Table 5-2). There was no percent nanomole given for either proline or tyrosine.
Since proline and tyrosine are normally low in most proteins, the absence of these
two amino acids may reflect the low amount of protein used in the quantitative
amino acid analysis. In comparing this protein with the average amino acid
composition of all proteins in Genbank shows that NFL-62 is particularly high in
glycine. The amino acid composition for NFL-62 was put through the FINDER
program; however, there were no close matches to help identify this protein.

142
Comparison of NFL-62 to the Average
Amino Acid Compostion in Genbank
ASX
ARG THR
ILE
MET
UAL

143
attempt to identify three of these proteins based on amino acid composition and
the FINDER program (Shaw, 1993), only the 38 kD protein was identified.
The present work firmly identifies GAPDH as an in vitro NF binding protein,
with the ability to stick to all three NF triplet proteins, although NF-L appears to
have the largest binding capacity. GAPDFI is a glycolytic enzyme which catalyzes
the oxidative phosphorylation of D-glyceraldehyde 3-phosphate as well as other
aldehydes (Lehninger, 1982). Most of the research relating to GAPDH has
focused on bacterial systems and skeletal muscle, where glycolysis is an important
mechanism (Opperdoes, 1988). In contrast, only a few papers describe a possible
role for GAPDH in the nervous system. GAPDH is found in high abundance in the
brain (Reid and Masters, 1986; Slagboom et al., 1990), presumably reflecting the
almost absolute requirement for glucose as a substrate for oxidative metabolism
in this organ. In relation to cytoskeletal elements in vitro, GAPDH has been
shown to bind to and bundle MTs (Durrieu et al., 1987; Somers et al., 1990) and
bind to actin filaments forming a gel-like, three dimensional filament network
(Clarke and Morton, 1982; Clarke et al., 1986). These interactions with MTs and
actin suggest an additional structural function for GAPDH.
GAPDH may bind to NFs to play a similar structural role as observed for the
other cytoskeletal proteins. The crucial question that remains is whether NFs and
GAPDH have the potential for interacting in vivo. The next chapter will focus on the
strength of interaction between GAPDH and NF triplet proteins, and examine if this
in vitro interaction may be relevant in vivo.

CHAPTER 6
CHARACTERIZATION OF GLYCERALDEHYDE-3-PHOSPHATE
DEHYDROGENASE BINDING TO NEUROFILAMENTS IN VITRO
Introduction
GAPDH is a soluble glycolytic enzyme that is localized to subcellular
structures in muscle (Masters, 1981; Bronstein and Knull, 1981) and particulate
fractions of whole brain homogenates (Clarke and Morton, 1982). In the adult
mouse brain the distribution between soluble and particulate GAPDH activity is
roughly equal; however, the developmental ontogeny shows that bound GAPDH
activity constantly increases from the prenatal period to adulthood, whereas, the
soluble form is high prenatally and rapidly decreases during the first postnatal
week. The soluble form then increases to a level similar to bound GAPDH activity
in the adult mouse (Reid and Masters, 1986). GAPDH is present in brain tissue
in higher amounts than the other glycolytic enzymes, and most other proteins with
the exception of the cytoskeletal proteins. This suggests a possible structural role
for GAPDH. Immunocytochemical analysis has demonstrated the presence of
GAPDH in the cytoplasm of neurons (Morgenegg et al, 1986). In additon, GAPDH
activity was shown to associate with the membrane fractions of a synaptosomal
preparation (Knull, 1978; 1980).
In the previous chapter, an in vitro interaction between NF triplet proteins
144

145
and GAPDH was demonstrated. However, before one could ask questions about
the functional role of this interaction, the potential for in vivo interaction between
NFs and GAPDH was examined.
Methods
Co-sedimentation Experiments
GAPDH from pig muscle was purchased from Sigma as a crystalline
suspension in 3.2 M ammonium sulfate solution (500 units). GAPDH was
centrifuged at 150,000xg for 15 minutes at 4°C in a Beckman TL-45 rotor and
buffer exchanged with Centricon 30 microdialysis centrifuge tubes (Fisher
Scientific) following manufacturers instructions. Purified NF subunits were
prepared using the method described in Chapter 2 and dialyzed against 50 mM
MES (pH 6.8) containing 3.4 M glycerol. Protein concentrations were determined
for the dialyzed GAPDH and purified NF subunits using the Pierce microassay as
described in Chapter 2.
GAPDH at a final concentration of 0.7 pM was incubated with NF-L (1.4
pM), NFM (1.0 pM), and NF-H (0.95 pM) for 1 hour at room temperature. The
molar ratio of GAPDH to NF-L was varied to determine the total of amount of
GAPDH that would bind to NF-L. Additionally, the NF-L concentration was held
constant while the concentration of GAPDH was varied (0.35, 0.7,1.4 and 2.8 pM).
The strength of the interaction between GAPDH and NF-L subunit was examined
by adding sodium chloride (0 M - 0.3 M) to the incubation mixture. The incubation
mixture was centrifuge at 150,000xg for 1 hour at 25°C in a Beckman TL-45 rotor.

146
The supernatant was removed and added to SDS sample buffer. The pellet was
then resuspended in 50 mM MES buffer (pH 6.8) and SDS sample buffer. Both
the pellet and supernatant were separated on a 10% SDS polyacrylamide gel and
proteins were visualized with Serva Blue G method (see Chapter 2).
Immunofluorescence Studies
DRG cultures were performed as described in Chapter 2. In addition to
using post-natal day 1 rat pups, embryonic day 21 rat pups were used; however,
no differences in the antibody staining patterns of DRG cells were observed
between these two ages. Cells were fixed in methanol (-20°C) for 5 minutes or
extracted with 0.1% Triton-X 100 for 1 minute. Immunocytochemical procedures
were as described in Chapter 2 with the following exception, a 50 mM MES buffer
(pH 6.8) was used to make all dilutions and for rinsing in between incubation steps.
PC12 cell cultures were prepared as described in Chapter 2 and fixation
was the same as described above for the DRG cell cultures.
Antibodies
GAPDH monoclonal antibody, ID4 (undiluted), was characterized in Chapter
5. NF-H polyclonal rabbit serum (R14; dilution 1:200) to the KSP region of NF-H
was made using NF fusion protein RH:559-794; NF-M polyclonal rabbit serum (R9;
dilution 1:200) to the KE region of NF-M was made using NF fusion protein
RM:677-845 (Harris et al., 1991). The polyclonal antibody to actin (dilution 1:25)
was purchased from Sigma.

147
Results
Co-sedimentation Experiments
Without NFs in the co-sedimentation experiments, the majority of GAPDH
remained in the supernatant fraction after a high speed centrifugation (Figure 6-1).
In contrast , in the presence of NF-L, most of GAPDH was found in the pellet
fraction with a ratio of 0.7 nM GAPDH to 1.4 nM NF-L subunit (Figure 6-1). The
data were not as clear for NF-H and NF-M (Figure 6-1). The reason may be that
NF-M and NF-H do not form a normal homopolymer like NF-L in vitro (Geisler and
Weber, 1981; Liem and Hutchison, 1982) and thus do not pellet well even under
high speed centrifugation conditions. In the case of NF-M, one observes more co¬
pelleting of GAPDH than with NF-H (Figure 6-1).
The maximum amount of GAPDH that would bind to NF-L was examined.
NF-L concentration was kept constant at 1.4 pM while GAPDH concentration
varied from 0.35 pM to 2.8 pM (Figure 6-2). These results demonstate qualitatively
that GAPDH binds to NF-L in a 1 to 2 molar ratio.
In the presence of sodium chloride, the interaction of NF-L with GAPDH is
dramatically affected. At low sodium chloride concentration (0.05 M), the amount
of GAPDH that pellets with NF-L decreases approximately 50% (Figure 6-3). The
interaction between NF-L and GAPDH is almost eliminated in the presence of 0.1
M sodium chloride (Figure 6-3). This interaction appears much weaker than
observed during the elution of GAPDH from the NF-L affinity column where the
eluting salt concentration ranged from 0.1 to 0.2 M potassium chloride (see

Figure 6-1. Co-sedimentation experiment with GAPDH and NF subunits.
GAPDFI remains in the supernatant (S) fraction after high speed centrifugation.
In the presence of the purified NF-L subunit, the majority of GAPDH is found in the
pellet (P) fraction and not in the supernatant fraction. In the presence of NF-M,
only some of GAPDH is localized to the pellet fraction, while in the presence of
NF-H, most of the GAPDH is localized in the supernatant.

149
NF-H -
NF-M -
NF-L -
GAPDH -
S
P
S
P
GAPDH
NF-L
NF-M
NF-H

Figure 6-2. Co-sedimentation of GAPDH and NF-L with different concentrations
of GAPDH.
The NF-L concentration was kept constant at 1.4 pM while GAPDH concentration
varied from 0.35 to 2.8 pM. These results demonstrate that maximum binding of
GAPDH occurs at approximately a 1 to 2 molar ratio of GAPDH to NF-L.

GAPDH -
S P
0.35 pM
S P S P
0.7 pM 1.4 pM
GAPDH Concentration

Figure 6-3. Co-sedimentation of GAPDH and NF-L with different concentrations of
sodium chloride.
The addition of sodium chloride results in a dramatic decrease in binding of
GAPDH to NF-L at 0.05M sodium chloride concentration, where much of the
GAPDH is found in the supernatant (S). P: pellet fraction.

153
NF-L
GAPDH -
SPSPS P SP
0 M 0.05 M 0.1 M 0.2 M
Sodium Chloride Concentration

154
Chapter 5). These results suggest that the NF-L subunit and GAPDH bind by
means of a weak electrostatic interaction.
Immunofluorescence Studies in Cultured Cells
Single labelling of DRG cells with GAPDH monoclonal antibody (ID4)
demonstrated that GAPDH was localized in the soma, neurites, and growth cones
(Figure 6-4, arrows). The labelling along the neuritic processes had a patchy
distribution. ID4 antibody appeared to label the neuritic branch points more
intensely than the neurite itself (Figure 6-4, arrowheads).
Double-labelling of DRG cell cultures with ID4 and NF-H antibody (R14)
resulted in an overlap in labelling between the two antibodies in the soma and
along the neurites of these cells (Figure 6-5 a,b; arrowhead). However, the
GAPDH antibody distribution was more extensive than the NF labelling pattern in
the growth cone where ID4 labelling extended into the growth cone and labeled the
filopodia (Figure 6-5 c,d). In addition, ID4 did not label the entire extent of the
neurite as seen with NF antibodies but labeled along the neurite in a punctate
distribution with increased labelling at neuritic branch points. In NGF differentiated
PC12 cells, the GAPDH antibody labeling pattern was similar to that observed in
DRG cells. Double-label experiments with NGF differentiated PC12 cell cultures
showed that GAPDH appeared to be associated more with the membranous
compartment of the neuron especially in the growth cone (arrow). This suggests
a complementary distribution between NF subunits and GAPDH antibodies (Figure
6-6).

Figure 6-4. Composite of GAPDH antibody labelling (ID4) of a DRG cultured
neuron.
GAPDH antibody staining pattern is localized throughout the cell body and into the
neurites and growth cones (arrows). The labelling along the neurite appears
patchy with intense labelling at neuritic branch points (arrowheads), s: satellite
cells or non-neuronal cells. Scale bar: 25 pm.

156

Figure 6-5. Co-localization experiment in DRG cell cultures with antibodies to GAPDH (ID4)
(A.C) and NF-H (R14) (B,D).
(A,B) Double-labelling of DRG cells demonstrates that the GAPDH labelling pattern (A) overlaps
with the NF labelling pattern (B) in the cell body and along the neurites, particularly at branch
points (arrowheads). In contrast, in growth cones GAPDH labels throughout the growth cone
and into the filopodia (A, arrows), whereas, NF proteins are localized only at the base of the
growth cone (B). Scale bar: 25 pm.
(C,D) A high power view of growth cones shown in (A and B) demonstrates that GAPDH (C)
and NF (D) antibody labelling patterns overlap along the neurite and into the base of the growth
cone; however, GAPDH labelling extends well into the growth cone and filopodia (arrow). Scale
bar: 12.5 pm.


Figure 6-6. Co-localization experiment in differentiated PC12 cell cultures with antibodies to
GAPDH (ID4) (A,B) and NF-M (R9) (C).
(A) GAPDH immunoreactivity is observed throughout the NGF-differentiated PC12 cell. The
strongest labelling was associated with the cell body and growth cone (arrow). In addition,
GAPDH antibody labels the neurite in discrete patches (asterisks). Scale bar: 25 pm.
(B,C) Double-labelling of NGF-differentiated PC12 cells demonstrate that the GAPDH labelling
•pattern (B) overlaps with the NF labelling pattern (C). GAPDH immunoreactivity was observed
in the filopodia and the labelling appeared to be associated with the plasma membrane. In
contrast, NF-M immunoreactivity was observed labelling a distinct fiber bundle at the base of the
growth cone (wide arrow) but no labelling was observed in the filopodia. Scale bar: 25 pm.

160
<
oo

161
Since GAPDH antibody labelling pattern was found to label the growth cone,
including the filopodia, double labelling of GAPDH with actin antibodies was
examined in DRG cell cultures. Figure 6-7 demonstrates that GAPDH co-localized
with actin throughout the soma and neurites, as well as in the growth cone.
Discussion
GAPDH was shown to co-sediment with purified NF-L subunit in vitro.
These data confirmed the results of GAPDH binding to the NF-L affinity column
(Chapter 5). The binding of GAPDH to NF-L appeared to be weak with a 50%
decrease in binding of GAPDH to the NF-L subunit when only 0.05 M sodium
chloride in 50 mM MES buffer was added to the incubation mixture. Although this
interaction between GAPDH and NF-L subunit is weak, previous research showed
that the bound form of GAPDH is released from particulate fraction of lysed nerve
endings at a 0.1 M sodium chloride concentration (Knull, 1978). This suggests that
overall there is a weak interaction between GAPDH and insoluble brain material.
Immunofluorescent studies of DRG cultured cells demonstrated the
presence of GAPDH throughout the cell body and neuritic processes. However,
GAPDH was not exclusively co-localized with NFs in DRG cells. This result was
not unexpected given that previously GAPDH was shown to bind to both MFs and
MTs in vitro (Clarke and Morton, 1982; Clarke et al., 1986; Durrieu et al., 1987;
Somers et al., 1990).
The data presented here suggest that some neuronal GAPDH could be

Figure 6-7. Co-localization experiment in DRG cell cultures with antibodies to
GAPDH (ID4) (A) and actin (polyclonal) (B).
(A,B) Double-labelling of DRG cells demonstrated that the GAPDH labelling
pattern (A) overlaps completely with the actin labelling pattern (B) in the cell body,
along the neurites and in the growth cones (arrow). Scale bar: 25 pm.

163

164
associated with NF in vivo. However, additional results suggest that GAPDH may
be more closely associate with the actin network than the NF network. In muscle
cells, a number of glycolytic enzymes bind to the actin network which allows for a
close physical association between the energy pathway and the contractile
machinery (Clarke et al., 1985). A similar mechanism in neurons would in turn
suggest that one of the functions of NF proteins may be to act as docking
substrates for the localization of GAPDH and possibly other enzymes in vivo.
Future electron microscopical studies that examine the question of the exact
localization of GAPDH in vivo may give a better idea of how GAPDH interacts with
cytoskeletal structures.
If GAPDH and NFs are shown to interact in vivo, the next step would be to
determine which part of the NF subunit is interacting with GAPDH. The NF binding
site for GAPDH may be represented by one of the homologous sequences found
in NF triplet proteins, such as the a-helical rod regions or the glutamic acid rich
segments in the carboxyl terminal. It is interesting to note that GAPDH is mildly
basic and that tubulins, which also bind GAPDH, also contain a glutamic acid rich
region. This suggests that the glutamic rich region in the carboxyl-terminal domain
of NFs may be a candidate binding site.

CHAPTER 7
OVERALL DISCUSSION
General Considerations
This dissertation research took two different approaches to identify
candidate NF binding proteins. The first approach focused on insoluble proteins
that co-purify with NFs in a salt and detergent preparation from spinal cord. This
resulted in the identification of plectin, a known IFAP characterized in non-neural
tissues which was previously shown to bind to NF triplet proteins in vitro (Foisner
et al., 1988). The second approach used different NF affinity columns to identify
candidate soluble NF binding proteins based on relative mobility on SDS
polyacrylamide gels. Although a number of proteins were found to bind with
varying specificity to the different NF affinity columns, this work focused on a 38
kD protein which was identified as glyceraldehyde-3-phosphate dehydrogenase
(GAPDH). The potential in vivo interaction was examined for both of these in vitro
NF binding proteins.
Criteria for Classifying IF Binding Proteins
A number of lines of evidence are required to classify a protein as a protein
binding to a specific target protein. Depending on the knowledge of the target
protein, this evidence will vary from weak to strong criteria. It is important to
165

166
establish an overall criteria, as well as a minimal criteria to judge your candidate
binding protein. The following represents criteria by which one can use in
identifying and classifying candidate binding proteins: (1) co-purification or co¬
sedimentation of the candidate binding protein to the target protein; (2)
colocalization of both the candidate binding protein and target protein in the same
cell types; (3) ultrastructural localization of the candidate binding protein to the
target protein; (4) an affinity or dissociation constant of the candidate protein to the
target protein, and how sensitive this interaction is to the addition of salt; (5)
stoichiometry of this interaction; and (6) functionality of the interaction between the
candidate binding protein and target protein.
In the case of IFAP a very loose criteria has been used in identifying a
protein as an IF binding protein in which only one of the above criteria needs to
shown (Foisner and Wiche, 1991). One reason for this is that not much is
understood about the function of IF proteins in general, and it is therefore difficult
to design a functional assay to determine a possible relationship between the
candidate binding protein and the target protein. The hope of research is that by
defining what proteins bind to and interact with IF proteins one will be able to
assign more functions to IF proteins.
Relationship between Plectin and NF Proteins
Based on the above criteria, plectin has been firmly established as an IFAP.
Plectin co-purifies with a number of IF proteins, and co-localizes with IF proteins
at both the light and ultrastructural level. The stoichiometry of this interaction is 1

167
plectin per 20 vimentin polypeptide chains, where plectin is thought to cross-link
IF proteins, at least in cultured cells (for review: Wiche, 1989).
One of the original reasons for examining the relationship between plectin
and NFs was to determine whether the proposed cross-linking function of plectin
could account for the marked stability of NF bundles in vitro (Shaw and Hou,
1990). Although plectin was localized to the cytoplasm of select neurons, the
distribution of plectin more closely reflected that of vimentin in non-neuronal cells
in the nervous system. In addition, the submembranous localization of plectin
observed in neurons, especially in the mesencephalic nucleus of V and in the
axonal bundles in both the CNS and PNS suggests that plectin does not
contributes to the marked stability of NF bundles. Pilot experiments that examined
crude IF bundles isolated from spinal cord found that the NF bundles were not
labeled by plectin antibodies (data not shown). Rather, plectin immunoreactivity
was associated with small punctate structures which may have been derived from
the submembranous cytoskeleton. Therefore it seems that plectin is not
responsible for a significant amount of NF cross-linking; instead, plectin may be
responsible for the interaction between NF network and plasma membrane in
select neurons.
One interesting feature of plectin's anatomical distribution was the
expression of plectin in moderate amounts in select motoneurons in the brainstem
and spinal cord. These cells were also labeled with antibodies to NF triplet
proteins and peripherin. To determine whether plectin expression corresponded

168
more closely with NF triplet proteins or peripherin, facial nerve lesions were
performed. An interesting feature after a peripheral nerve lesion is that the mRNA
of NF triplet proteins is down-regulated (Floffman et al., 1987; Wong and Oblinger,
1987; Goldstein et al., 1988; Tetzlaff et al., 1988); whereas, peripherin mRNA is
upregulated in axotomized motoneurons (Oblinger et al., 1989; Wong and Oblinger,
1990). This upregulation is reflected in an increase in peripherin immunoreactivity
in motoneurons. After peripheral nerve transection, plectin and peripherin
immunoreactivity increased in axotomized motoneurons. This suggests that plectin
may more closely be associated with the class III neural IF proteins, peripherin and
vimentin, than with the class IV neural IF proteins in vivo.
Finally, the types of motoneurons that express plectin are similar to the
motoneurons which contain abnormal NF accumulations that degenerate in
motoneuron diseases. This occurrence taken together with the dramatic increase
in plectin immunoreactivity in motoneurons after axonal injury suggest that the high
level of this known IF cross-linker may play some role in the NF accumulations
seen in some motoneuron diseases.
The Role of GAPDFI in the Nervous System
A known role of GAPDFI in the nervous system is its function in glucose
metabolism, an essential component of cellular energy production. GAPDFI is
found in high abundance in brain, more.than any other enzyme, and is present at
comparable levels, although in lower amounts, to the cytoskeletal proteins. This
suggests that GAPDFI may perform a dual function in the CNS.

169
This research demonstrated that GAPDH is a protein that binds in vitro to
NFs. Additionally, previous research has shown that GAPDH binds in vitro with
actin and tubulin. These results taken together suggest that GAPDH may also
function in a structural role in the CNS.
In muscle cells, GAPDH is one of a number of glycolytic enzymes that binds
to the actin network. This allows for a close association between the energy
pathway and contractile machinery. In the case of developing neurons, GAPDH
was shown to co-localize with actin in the growth cone. Since growth cones are
pathfinding structures and actin assemblies generate the movements of growth
cones, localization of GAPDH to actin network may be an important theme in
linking energy pathways to contractile machinery in a number of cell types. In
addition, GAPDH has been shown to assembly actin monomers in vitro to form a
gel-like matrix. This suggests that in vivo GAPDH may be structurally important in
stabilizing the newly formed actin network at the leading edge of the growth cone,
as well as acting in the same fashion in synaptic plasticity in the adult nervous
system. Finally, the intense labelling of GAPDH antibodies at neuritic branch
points may reflect GAPDH's role in structural stability, since this enzyme was
shown to bundle microtubules in vitro, in addition to assembling actin monomers.
Immunofluorescent studies suggest a potential in vivo interaction of GAPDH
and NFs; however, in some areas GAPDH appeared more closely associated to
the plasma membrane. If the interaction between GAPDH and NFs is
physiological, then a potential role for GAPDH may be to allow energy production

170
near proteins that are heavily phosphorylated in the axon. The punctate labelling
of GAPDH along the developing neurite may correspond to docking sites for this
enzyme, as well as other glycolytic enzymes, where NF phosphorylation may be
taking place along the neurite.
Based on the criteria for characterizing a protein as a binding protein,
GAPDH can be defined as an in vitro NF binding protein. GAPDH bound to the
different NF affinity columns and co-sedimented with NF-L subunit with an
approximate ratio of 1 mole of GAPDH (tetramer form) to 2 mole of NF-L subunit.
Although this interaction was shown to be relatively weak with the addition of salt,
the in vivo interaction between GAPDH and NFs may only be a transient event
where a tight interaction is not necessary. In addition, the potential for an in vivo
interaction is shown in the co-localization of GAPDH with NFs at the light level.
Future Directions
Other NF Binding Assays
In addition to the approaches taken in this dissertation to identify and
characterize IFAPs, other strategies are also available.
NF Fusion Proteins. Molecular biology techniques have allowed for high
level of protein expression which results in a higher yield of protein and easier
purification methods. One such protein expression system, glutathione S-
transferase (GST) gene fusion system, allows for rapid purification of the GST
fusion protein by taking advantage of the binding of GST to Glutathione Sepharose
4B (Kaelin et al., 1991). The GST fusion protein can be eluted from Glutathione

171
Sepharose 4B by using reduced glutathione or by cleaving the clone portion of the
GST fusion protein using site-specific proteases. This rapid and simple purification
procedure has the advantage over other fusion protein systems (ie. trpE bacterial
system) because the purification methodology is performed under non-denaturing
conditions.
In addition, one could take advantage of the tight interaction between GST
fusion protein and Glutathione Sepharose 4B and directly make an affinity column
to examine the interactions between a specific region of the protein of interest with
other proteins. This minimizes the need to utilize harsh methods used in protein
purification procedures as well as the problem of knowing whether the region of
interest is available for binding since the GST portion of the fusion protein is
binding to the immobilized matrix.
Competition Experiments. These experiments use known proteins, fusion
proteins or peptide sequences which are known to bind to NFs in vitro to determine
if your candidate NF binding protein has a similar binding domain to that of the
known NF binding protein. In addition, these types of experiments can be used
to examine the effects of co-factors such as ATP or calcium to determine if they
have any effect on the ability of proteins to bind. For example, the addition of co¬
factors could induce binding of proteins to NFs or decrease the affinity of a
particular protein to NFs.
Bio-Sensor. For studying molecular interactions in real time, the biosensor-
based technology employs a surface plasmon resonance system (Jónsson et al.,

172
1991). To covalently immobilize a protein, the amine groups of the protein are
reacted with the active esters of a polymer consisting of a carboxymethylated
dextran matrix plated on a sensor chip (a glass surface covered with gold film).
To examine the possible interaction of a particular protein or a mixture of proteins
with that of an immobilized protein, proteins are allowed to flow across the
immobilized protein complex. Interactions are measured by using polarized light
from a light-emitting diode, which is reflected by the gold film where the reflected
light is detected by a photo detector array. Changes in the refractive index, as
measured by this optical technique are directly proportional to the change in
adsorbed mass of the protein complex. The advantages of using the Biosensor
system is that allows for the detection of an interaction of a protein, normally found
in such low concentration that it would be missed in a SDS polyacrylamide gel
when using affinity chromatogaphy techniques. In addition, the binding constants
of most interactions can be determined.
The two-hvbrid system. The two-hybrid system takes advantage of
molecular biology techniques to detect protein-protein interactions in vivo (Fields
and Song, 1989; Guarente, 1993) and is available commercially from Clontech.
This system uses two reporter genes HIS3 and lacZ which are under the control
of the GAL4 responsive sequences in yeast host cells. The clone gene for the
protein of interest is put into a DNA-binding domain vector, pGBT9, which also
contains the TRP1 gene necessary for tryptophan biosynthesis, whereas, a cDNA
library or select clone genes for candidate binding proteins are placed into the

173
activation domain vector, pGAD424, which contains a gene encoding for LEU2
necessary for leucine biosynthesis. These two vectors are co-transformed into the
HF7c yeast host strain and plated on synthetic medium without tryptophan, leucine
or histidine, to select for co-transformants which contain two interacting hybrid
proteins. If the target protein interacts with a library-encoded protein then the
functional G AL4 activator is reconstituted by the interaction of the two proteins, and
HIS3 reporter gene is activated which allows for the selection of histidine producing
yeast cells. A secondary screen of the histidine positive transformants is tested
by the expression of the second reporter gene using B-galactosidase activity.
The advantages of this system are that weak or transient interactions are
readily observed, that the experiment is performed in vivo and thus proteins are
more likely to be in their native conformation, and once the interacting protein has
been identified, the gene is already cloned.
Conclusions
A great deal of work remains to fully understand the potential interaction of
plectin and GAPDH to neural IF proteins, as well as the roles of all of these
proteins in the nervous system. One step would be to examine more closely the
distribution of GAPDH and plectin within the neuron using electron microscopy.
If these in vitro interactions between NFs with plectin and GAPDH prove to be
physiologically relevant, then NF proteins will be shown to be as functionally
diverse as the other cytoskeletal elements.

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BIOGRAPHICAL SKETCH
Laura Diane Errante was born on April 9, 1965, in Binghamton, New York.
Being the youngest of 6 children, she was eager to go to school like her older
brothers and sister and was always fascinated with nature and science. She
entered SUNY Plattsburgh as a biochemistry major in 1982 and by the end of her
freshman year, she knew that she wanted to attend graduate school, and thus
began a research project with Dr. Roger Heintz which lasted until she graduated
in 1986. Here she learned that although research can be difficult at times, it was
always fascinating. Her interest in neuroscience was cultivated in an independent
reading project in neuroscience with Dr. Henry Morlock, and after an attempt at
sheep brain dissection (or mutilation?) under his guidance, Laura became intrigued
with how the brain worked. After a stint as a lab technician determining amino acid
composition of soil samples from Back Bay in Virginia, she entered the
Neuroscience Department at the University of Florida College of Medicine in 1987.
Determined to choose the best possible lab based on sound advice, she made that
critical decision based on a short film on growth cone motility. Thus she entered
the laboratory of Dr. Gerry Shaw. Although her research she conducted in his lab
went in a different direction, she learned valuable techniques and knowledge which
she will use during her postdoc on cellular mechanisms of neuronal growth cones.
191

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.
Gerard P. J. Shaw, Chairperson
Associate Professor of Neuroscience
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.
Barbara-A. Battelle
Associate Professor of Neuroscience
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.
Kevin J. ^thderson
Associate Professor of Neuroscience
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.
Daniel L. Purich
Professor of Biochemistry and
Molecular Biology

This dissertation was submitted to the Graduate Faculty of the College of
Medicine and to the Graduate School and was accepted as partial fulfillment of
the requirements for the degree of Doctor of Philosophy.
April 1994
Dean, College of Medicine
Dean, Graduate School

UNIVERSITY OF FLORIDA
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