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

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Identification and characterization of intermediate filament binding proteins in the nervous system
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Intermediate Filaments   ( mesh )
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Thesis:
Thesis (Ph.D.)--University of Florida, 1994.
Bibliography:
Bibliography: leaves 174-190.
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by Laura Diane Errante.
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Typescript.
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Vita.

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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.














TABLE OF CONTENTS


ACKNOWLEDGEMENTS ...................

LIST OF TABLES ........................

LIST OF FIGURES .......................

LIST OF ABBREVIATIONS .................


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
NF Expression After Axotomy ................
Role of NFs in Disease States ................
Overview of Dissertation ....................

2 GENERAL METHODS .......................


....... 26
....... 30
....... 32
....... 34

....... 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








Anatomical Localization Experiments .............
Dorsal Root Ganglion Cell Cultures ............
Pheochromocytoma Cells (PC12 Cells) .........
Immunofluorescent Studies on Fresh Frozen Tissue
Immunocytochemistry on Formaldehyde Fixed Tissue

3 THE DISTRIBUTION OF PLECTIN, AN INTERMEDIATE
FILAMENT BINDING PROTEIN, IN THE ADULT RAT
CENTRAL NERVOUS SYSTEM ..................

Introduction ...............................
M ethods .................................
Results ..................................
Immunoblot Studies ........................
Comparison of Plectin to IFAP-300 .............
Localization Studies ........................
Discussion ...............................
N otes ...................................

4 CO-LOCALIZATION OF PLECTIN AND INTERMEDIATE
FILAMENTS IN THE RAT NERVOUS SYSTEM .......

Introduction ...............................
M ethods .................................
Results ..................................
Immunoblot Studies ........................
Co-localization in Cultured Cells ...............
Co-localization Studies Along the Rat Neural Axis .
Effects of Peripheral Nerve Axotomy .............
Discussion ................................
N otes ................................... .


5 IDENTIFICATION OF NF BINDING PROTEINS .

Introduction .........................
M ethods ............................
Pig Spinal Cord Cytosolic Preparation .....


Affinity Column Preparation


Binding of Cytosolic Proteins to Affinity Columns ..
Production of Polyclonal and Monoclonal Antibodies
Results ..................................
Discussion ................................


. .. 44
... 44
... 45
. .. 46
... 47




... 49

... 49
... 50
... 52
... 52
... 56
... 59
... 70
... 78


... 79


79
80
82
82
82
90
97
105
115

116

116
117
117
117
118
121
122
138


. .








6 CHARACTERIZATION OF GAPDH BINDING TO NFs ..
Introduction ...........................
M ethods ..............................
Co-sedimentation Experiments ............
Immunofluorescence Studies ..............
Antibodies ...........................
Results ..............................
Co-sedimentation Experiments ............
Immunofluorescence Studies in Cultured Cells .
Discussion ............................

7 OVERALL DISCUSSION ....................
General Considerations ...................
Criteria for Classifying IF Binding Proteins .....
Relationship between Plectin and NF Proteins .
The Role of GAPDH in the Nervous System ....
Future Direction ........................
Conclusions ...........................


REFERENCES .................................

BIOGRAPHICAL SKETCH ..........................


144
144
145
145
146
146
147
147
154
161


...... 165
...... 165
. ... 165
. ... 166
. ... 168
...... 170
...... 173


174

191













LIST OF TABLES


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


Table 1-1


Table
Table
Table


Table 4-1


Table 5-1

Table 5-2

Table 5-3
Table 5-4













LIST OF FIGURES


Figure
Figure


Figure 3-2

Figure 3-3

Figure 3-4


Figure
Figure
Figure
Figure


Figure 4-1

Figure 4-2

Figure 4-3

Figure 4-4

Figure 4-5

Figure 4-6

Figure 4-7


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








Effects of a unilateral facial nerve axotomy on plectin


immunoreactivity (1A2)


Figure 4-9 Co-localization experiment with plectin (IA2) (A,C)
and NF-H (R14) (B,D) after a unilateral facial nerve
axotom y .....................................
Figure 4-10 Co-localization experiment with plectin(IA2) (A,C)
and peripherin (R20) (B,D) after a unilateral facial
nerve axotomy .................................
Figure 5-1 Outline of the method used for identifying candidate
NF binding proteins .............................
Figure 5-2 Comparison of candidate 38 kD NF binding protein
to rod domain of IF proteins .......................
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 ......................
Figure 5-4 Comparison of CNBr cleavage fragments of NFL-38
and GAPDH ..................................
Figure 5-5 Immunoblot analysis using antibodies to GAPDH to
determine if the 38 kD proteins binding to various NF
affinity columns is GAPDH ........................
Figure 5-6 Graphical representation of the amino acid composition
data for NFL-16, and possible matches as determined
by the FINDER program ..........................
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 ..........................
Figure 6-1 Co-sedimentation experiment with GAPDH and NF subunits
Figure 6-2 Co-sedimentation of GAPDH and NF-L with different
concentrations of GAPDH .........................
Figure 6-3 Co-sedimentation of GAPDH and NF-L with different
concentrations of sodium chloride ...................
Figure 6-4 Composite of GAPDH antibody labelling (ID4) of a DRG
cultured neuron ................................
Figure 6-5 Co-localization experiment in DRG cell cultures with
antibodies to GAPDH (ID4) (A,C) and NF-H (R14) (B,D) .


.... ..... .... ... .. .. .. .. .. 104


107


109

120

126


130

135


137


140


142
149

151

153

156

158


Figure 4-8







Figure 6-6



Figure 6-7


Co-localization experiment in differentiated PC12
cell cultures with antibodies to GAPDH (ID4) (A,B)
and NF-M (R9) (C) ............................... 160


Co-localization experiment in DRG cell cultures with
antibodies to GAPDH (ID4) (A) and actin polyclonall) (B) ..


163













LIST OF ABBREVIATIONS


Amino Acids
ALA or A
ARG or R
ASN or N
ASP or D
ASX or Z
CYS or C
GLN or Q
GLU or E
GLX or B
GLY or G
HIS or H
ILE or I
LEU or L
LYS or K
MET or M
PHE or F
PRO or P
SER or S
THR or T
TRP or W
TYR or Y


- alanine
- arginine
- asparagine
- aspartic acid
- asparagine and/or
- cysteine
- glutamine
- glutamic acid
- glutamine and/or g
- glycine
- histidine
- isoleucine
- leucine
- lysine
- methionine
- phenylalanine
- proline
- serine
- threonine
- tryptophan
- tyrosine


aspartic acid



lutamic acid


BBB blood-brain barrier
BSA bovine serum albumin
CBB Coomassie brilliant blue R-250
CNBr cyanogen bromide


central nervous system
cerebrospinal fluid
diaminobenzidine tetrahydrochloride
dorsal root ganglion
dithiothreitol


EDTA ethylenediamine tetraacetic acid


CNS
CSF
DAB
DRG
DTT







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













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;







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 (GAPDH). The strength of binding between GAPDH

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 GAPDH was examined using fluorescent light

microscopy which showed that GAPDH was localized throughout the cell body and

processes of dorsal root ganglion cells in culture and differentiated PC12 cells.

Although GAPDH 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 GAPDH suggests that

certain NF proteins may act as important structural elements during neuronal

injury, and as docking substrates for the localization of glycolytic enzymes.













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 chapter will 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.










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
derivatives (eg. nails and hair)
II Keratin (basic) 50-70 "
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-H1 115 neurons
NF-M' 95 "
NF-L* 60 "
a-Internexin 66 "
V Nuclear Lamins 60-70 nuclear lamina of all cells
IV Nestin 200 neural epithelial stem cells

*GFAP: glial fibrillary acidic protein; 1NF-H: NF high molecular weight protein;
ONF-M: NF middle molecular weight protein; "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). However, 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,




















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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.

However, 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 B-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.









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 al., 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-H, 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

















Table 1-2. Protein kinases which phosphorylate neurofilaments in vitro.


Protein Kinase Substrate MW* (kD) Reference


cAMP dependent
(rat brain)

Ca2 /Calmodulin
dependent (rat)

Protein Kinase C
(mouse)

Casein Kinase I


cdkl Kinase

nclk Kinase'


NF-Kinase (1983)
(rat)


NF-Kinase: 1989
(bovine)

NF-Kinase (1990)
(bovine)

NF-Kinase (1991)
(squid)


NF-H, -M, -L


NF-H, -M, -L


NF-H, -M, -L


NF-H, -M, -L


40


640


77


30-42


NF-H, -M

NF-H, -M


NF-H, -M, -L
GFAP, vimentin
MAPs, tau

NF-H, -M
histone H1

NF-H, -M, -L
casein

NF-H, -M, -L
casein


17-44


Tanaka et al., 1984
Dosemeci and Pant, 1992

Vallano et al., 1985


Sihag et al., 1988


Floyd et al., 1991

Hisanaga et al., 1991

Lew et al., 1992

Toru-Delbauffe & Pierre, 1983
Toru-Delbauffe et al., 1986


Wible et al., 1989


Dosemeci et al., 1990


Floyd et al., 1991


* molecular weight expressed in kiloDaltons
' 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,

p34Cd2/p58CycinA, 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 p34cd2 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

(Heimann 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-H 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












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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 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-H.

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. However, 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, NF'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 B,B'-

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; Dr&ger 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 (Hirano, 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-H expression resulted in a more progressive

neuropathology that occurred over a period of three to four months (C6tM 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 curve 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








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'. 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 f3-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 destaining 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.


Electroblotting 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 destined 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 -200C 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-

800C. 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.









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 (510 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 40C 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 40C. 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) or a 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 -200C.


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 MgCl2, 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 40C 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 -200C.









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 40C 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.









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 pl 10X sterile trypsin-EDTA solution (Sigma; 0.5%

trypsin, 0.2% EDTA. 0.9% NaCl) was added. The resuspended DRG were then

incubated at 370C 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 370C, pelleted as

described above, and supernatant removed. DRG were triturated in 100 pl of L-15








45

incomplete media 15 times using an Eppendorf pipet to resuspend the ganglia into

single cells. Before plating, 500 pl of L-15 incomplete media was added to each

tube, then cells were plated on 6 acid washed coverslips (100 pl 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-CO2 incubator at 370C for 24

hours.


Pheochromocytoma 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% CO2, 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 related 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 -20*C 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 370C or 24 hours at 40C 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 (-200C) 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 370C.

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; Herrmann 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 pl 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.










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 3-1a, 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 3-1a

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 3-1a, 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.









A-
H-
M-



L- UP


-ww


1 2 3 4 5 6 7 8 9






H-

M-


B








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























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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.









,8~


V.A
/~


.j A
:-








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 prm.



























-C























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 (gcl) and appears to correspond to astocytic
processes. Note the absence of plectin antibody staining in the purkinje cell layer
(pcl). 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



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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-
















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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.


















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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.













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








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 Immunocytochemistry

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 1 D8 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 dilutedd 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.



















M-*







L-
57kD- -, -
G- 2 3 4








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 (PC12) 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




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