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CHARACTERIZATION OF CATHEPSIN B mRNA AND PROTEIN EXPRESSION,
ENZYMATIC ACTIVITY AND CELLULAR LOCALIZATION FOLLOWING
CONTUSION SPINAL CORD INJURY IN RATS
REBECCA CATHERINE ELLIS
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
Rebecca Catherine Ellis
This document is dedicated to my family, friends and colleagues. Their support,
encouragement and guidance have made all the difference in the world.
Numerous people deserve credit for making this experience as positive and
fulfilling as it has been and I have been very fortunate to work with so many
knowledgeable and talented individuals. There are a few people, however, that deserve to
be specially recognized for their contribution.
The members of my committee, including Ronald Hayes, Richard Moyer, Harry
Nick, Lucia Notterpek and Kevin Wang, are very deserving of my gratitude, as they have
helped mold me and guide me in many different ways. Each individual has provided me
something different, which I have incorporated into my own development. I have enjoyed
our time together, and without them, my training would simply not have been as rich.
Sue Semple-Rowland has been a most wonderful influence on my scientific
progression. She is an extraordinarily passionate and motivated scientist. Her enthusiasm
and dedication to both her own work and the development of aspiring scientists are
remarkable. I have grown tremendously from our interactions and I hope that I can be as
giving to those around me as she is. I cannot express how thankful I am for all that Sue
has done for me.
Michelle Deford has been by my side through these last few years. Michelle is an
incredibly insightful and gifted scientist and I am constantly amazed by her depth and
versatility. She has always found the time to lend me a helping hand and her assistance in
this last year has been invaluable. She is truly an asset to any organization and I am
blessed to have her as my friend.
Wilbur O' Steen has also played an integral part of my progression. He has been an
endless source of information regarding so many different aspects of the research process
including my initial surgical training, the nuances of animal care and with understanding
a mountain of procedural guidelines. Wilbur is an irreplaceable resource and he has
always been willing to lend a helpful hand. I appreciate his kindness through these years.
He has made the lab a fun place to be and I am happy to have him as my friend.
Lastly, there is Dr. Douglas K. Anderson, my dissertation mentor. Dr. A has been
wonderful throughout this process. He has been thoughtful and caring (and delightfully
witty) as we worked toward the completion of these experiments. Through his incredible
work ethic and dedication to his numerous responsibilities, Doug has been a very positive
role model not only in my scientific endeavors but also in every aspect of my life. I have
enjoyed being his student and look forward to being his colleague. I am honored to have
called him "mentor."
On a personal note, I would like to thank my family. I could not have asked for a
more loving and supportive family than the one I have. Above all else, my mother
Barbara has been an inspiration for me throughout my life. She is unwavering in her
support and I love and I respect her more than I can ever express. My mother is my rock
and I am so happy to share this moment with her and with my entire family. Lastly, I
want to thank Shane McFadyen for all his support, particularly during this last year. He is
a constant source of love and encouragement and I am extremely thankful to have him in
TABLE OF CONTENTS
A C K N O W L E D G M E N T S ................................................................................................. iv
ABSTRACT .............. .................. .......... .............. xi
INTRODUCTION .......................... ........ .. ... .... ........ ...............
Spinal Cord Injury D em graphics ...................................... ......................... .......... .
Spinal Cord Injury Pathophysiology ........................................ ........................ 4
Cell D eath after Spinal Cord Injury........................................ .......................... 7
Proteases and Spinal Cord Injury ........................................ ........................... 8
M atrix M etalloproteinases....................................................... 9
C a lp a in ........................................................................................................... 1 0
C asp cases ................................................................... 12
C athepsins ............................................. 15
C a th e p sin B ........................................................................................................... 1 6
G en eral B ack g rou n d ...................................................................................... 16
Role in Peripheral Pathologies ................................................. 18
Role in Central Nervous System Pathologies......................... ............. ..19
Cath B and Apoptosis...................................... ......... 22
Cath B in SCI Pathophysiology........................... .......... 23
M A TERIA L S A N D M ETH O D S................................................................................. 24
S u rg e ry ......................... ..................................4
T issu e H arvesting ................................................................................................ 24
mRNA, Protein and Enzymatic Activity ......................................................24
Immunohistochemistry .............. .......... ............... 25
Analysis of Cath B mRNA Expression ..........................................25
RNA Isolation and cDNA Synthesis .......................................25
P rim er D e sig n ................................................................................ 2 6
R e al-tim e P C R ............................................................................................... 2 6
Analysis of Cath B Protein Expression ..........................................27
Tissue Lysis and Protein Purification........................ .... .......... 28
Immunoblotting .......................... .................28
Analysis of Cath B Enzymatic Activity................................ .... ......... 29
Sample Preparation.................................... ......... 29
Enzymatic Activity Assay ................... ......... .......... 30
Immunohistochemical Localization of Cath B ........................................ ....30
Inhibition of C ath B A activity ................... ................... ...................1.......
In h ib ito r .................................................................................................. 3 1
In Vitro Inhibitor A ssay ......................................................... .............. 31
In Vivo Inhibitor Treatm ent...................................................... .... ........... 32
Statistical A naly ses ......................... ... ............................................... .... .. .. 33
mRNA Expression and InVitro Enzymatic Activity .......................................33
Cath B Protein Expression Levels ............................. ....................33
InVivo Enzym atic A activity ............................................................................ 34
R E S U L T S ................................................................................ 3 5
Analysis of Cath B m RN A Expression ........................................ ............... .... 35
Contusion-Spinal Cord Injury Increased Cath B mRNA Levels.........................35
E effects of Sham -Injury ............................................................. .....................35
Effects of Contusion-SCI ............................................................................. 36
A analysis of Cath B Protein Expression ......................... .............. ............... .... 38
Cath B Protein Expression is Elevated following Spinal Cord Injury ................38
Increases in Cath B mRNA Expression and Cath B Proenzyme Expression are
C orrelated ................... ............................................... ................ 4 2
Analysis of Cath B Enzymatic Activity Levels.....................................................42
Contusion-Spinal Cord Injury Increases Cath B Enzymatic Activity .................42
Increases in Cath B Protein Expression and Cath B Activity Levels are
C orrelated ................................................................................... ......... 4 5
Im munohistochem ical Analysis of Cath B ......................... ...............................45
Cath B Immunoreactivity Appears Restricted to Neurons in the Normal Spinal
C o rd ........................................................................ .. 4 5
Contusion-Spinal Cord Injury Increases Cath B Immunoreactivity and Alters
Cath B Localization ....................... ..... ...... ..... ...............47
Inhibition of Cath B Enzym atic A ctivity............................................. .................. 50
CA-074 was an Effective Inhibitor of Cath B Enzymatic Activity In Vitro .......50
CA-074 is an effective inhibitor of Cath B enzymatic activity in vitro .............51
In vivo CA-074 Treatment is Ineffective at Reducing Contusion-Injury Induced
Increases in Cath B Enzymatic Activity Levels ...........................................52
D ISCU SSION ............................................................... ..... ..... ......... 54
Characterization of Cath B following Contusion-Injury ........................................55
m R N A E expression ......... .......................................................... .. .... ........55
Protein E expression ......................... ....................... ....... ...... ............. 56
Levels of Cath B Enzymatic Activity.....................................................57
The Importance of Inflammatory Cells ............................................................57
The Effects of Sham -Injury ........................................ ........................... 59
Suppression of Cath B Enzymatic Activity .............. ......................................60
C conclusion ............................................................... ..... ..... ........ 62
F future D directions .......................................................................63
L IST O F R EFE R E N C E S ............................................................................. ............. 64
B IO G R A PH IC A L SK E T C H ...................................................................... ..................85
LIST OF FIGURES
1-1: Schematic of the Cath B protein........................... .......... ..................... 17
3-1: Sham- and contusion-spinal cord injury induce expression of Cath B mRNA at the
injury epicenter ............................................................................................. .......36
3-2: Sham- and contusion-spinal cord injury induce Cath B mRNA expression in the
rostral and caudal tissue segm ents. ........................................ ....... ............... 37
3-3: Sham- and contusion-injury increases levels of all forms of the Cath B protein.......38
3-4: Contusion-injury increases Cath B protein levels at the injury site .........................40
3-5: Contusion-injury increases Cath B protein levels in adjacent segments of the spinal
c o rd ............................................................................... 4 1
3-6: Cath B enzymatic activity levels are increased following contusion-injury..............43
3-7: Contusion-injury does not increase Cath B enzymatic activity levels in adjacent
spinal cord segm ents. ..................... ...... .......... ............... .... ....... 44
3-8: Cath B staining is localized to neurons in the gray matter of the normal spinal cord.45
3-9: Cath B is staining is distinguished by its lysosomal localization............................. 46
3-10: Other cell types in the normal spinal cord do not appear to express Cath B............47
3-11: Cath B immunoreactivity is increased and altered following SCI .........................48
3-12: Cath B immunopositive cells (A) in the injury epicenter are inflammatory in origin.48
3-13: Cath B expression increases in both neuronal and non-neuronal cells in tissue
segments adjacent to the injury site............................. ......... ..... ............. 49
3-14: CA-074 potently inhibits Cath B enzymatic activity in vitro..............................50
3-15: Increases in Cath B enzymatic activity are suppressed by CA-074.......................51
3-16: In vivo CA-074 does not inhibit contusion-injury induced increases in Cath B
activ ity lev els................................................... ................ 5 3
3-17: The suppression of Cath B enzymatic activity with in vivo CA-074 treatment is not
rep ro d u c e d ........................................................................ 5 3
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
CHARACTERIZATION OF CATHEPSIN B mRNA AND PROTEIN EXPRESSION,
ENZYMATIC ACTIVITY AND CELLULAR LOCALIZATION FOLLOWING
CONTUSION SPINAL CORD INJURY IN RATS
Rebecca Catherine Ellis
Chair: Douglas K. Anderson
Major Department: Neuroscience
Mechanical spinal cord injury (SCI) initiates a cascade of pathochemical and
pathophysiological events, collectively known as the secondary injury. These processes
disrupt normal homeostasis and include a prolonged inflammatory response, a decrease in
energy metabolites, ionic gradient imbalances and the loss of vascular regulation.
Significant tissue loss via apoptotic and necrotic cell death ensues, resulting in cavitation
around the initial injury site. The subsequent loss of voluntary motor function and
sensation below the injury site is accompanied by a poor prognosis for recovery.
There has been significant interest in understanding the activation and involvement
of proteases in the secondary injury. Several proteases including calpain, the caspases and
matrix metalloproteinases are activated by injury-induced perturbations to the spinal
cord. These proteases have been linked to cell death in models of central nervous system
(CNS) injury. Cathepsin B is no exception, having been associated with a brain tumor
metastasis and progression, Alzheimer's disease, multiple sclerosis, amyotrophic lateral
sclerosis and stroke.
Collectively, our studies represent the first in-depth characterization of cathepsin B
mRNA and protein expression, enzymatic activity and cellular localization in the
contusion-injured spinal cord. Sham-injury was a sufficient perturbation to the spinal
cord to induce a transient increase in cathepsin B mRNA expression but did not affect
protein expression or enzymatic activity. Contusion-injury, however, elicited significant
increases in both cathepsin B expression and enzymatic activity levels and altered its
cellular localization. Furthermore, the immunohistochemical analyses revealed that these
increases were due to the invasion of inflammatory cells into the injured spinal cord.
In light of these findings, the powerful and indiscriminate hydrolytic properties of
Cath B make it an attractive candidate for involvement in the secondary injury cascade.
Thus, the reduction of SCI-induced increases in cathepsin B expression and enzymatic
activity may ameliorate the secondary tissue damage, thereby providing some degree of
neuroprotection to injured spinal cord tissue. However, the inhibitor used in these
experiments did not suppress cathepsin B enzymatic activity in vivo following contusion-
injury. Nevertheless, the effectiveness of cathepsin B inhibitors in other CNS insults
suggests that they should continue to be tested in models of SCI.
Spinal Cord Injury Demographics
Currently, there are approximately 243,000 people living in the United States
(1,000,000 worldwide) with spinal cord injury (SCI). Approximately 11,000 new injuries
are sustained each year, the bulk (53%) of which is sustained by younger individuals
(average age of 32.6 years). It is an injury that predominantly affects males (81.6%), a
statistic that likely reflects life style choices. Currently, motor vehicle accidents (40.9%),
falls (22.4%) and acts of violence (21.6%) are the leading causes of SCI. However, these
trends change with increasing age. For example, injuries due to violent acts decrease with
advancing age, and after age 45, falls overtake motor vehicle accidents as the leading
cause of SCI. Furthermore, the percentage of persons 60+ years old at the time of injury
has risen from 4.7% in the 1970s to 11.4% today (Spinal Cord Injury 2003). This age
related increase most likely reflects the fact that the median age of the general population
has increased from 27.9 years to 35.3 years during this same time period.
There are several terms (e.g., tetraplegia/paraplegia, complete/incomplete) used by
clinicians and researchers to designate the severity of an injury. Paraplegia results from a
primary injury to the thoracic, lumbar and/or sacral regions of the spinal cord. It generally
does not affect the sensory or motor function of the upper body. Tetraplegia occurs when
at least one of the eight cervical segments is injured, resulting in paralysis of all four
limbs. An incomplete injury indicates that some motor and sensory function below the
injury site is still present while a complete injury is one in which the patient is devoid of
discernible voluntary movement and sensation below the injury site. Although the most
severe functional deficits usually occur when the cord is transected, the vast majority of
SCIs are not complete transactions (Bunge et al., 1993; Bracken et al., 1990). The most
common type of SCI is incomplete tetraplegia (30.8%), followed by complete paraplegia
(26.6%), incomplete paraplegia (19.7%) and complete tetraplegia (18.6%). However,
clinicians and researchers have noted a decreasing trend in injury severity, "Trends over
time indicate an increasing proportion of persons with incomplete paraplegia and
decreasing proportion of persons with complete tetraplegia" (Spinal Cord Injury 2000).
Among the many factors contributing to this trend are 1) improved roadside care, 2)
aggressive blood pressure and respiratory maintenance (Vale et al., 1997; Levi et al.,
1993), 3) the administration of methylprednisolone following injury (Bracken et al.,
1997, 1992, 1990; Bracken & Holford 1993) and 4) well-defined rehabilitation programs.
In addition to the loss of function and sensation below the injury site, SCI patients
can experience a host of systemic complications. Injured persons are at greater risk of
experiencing bladder, bowel and sexual dysfunction, neuropathic pain and muscle
spasticity. Furthermore, SCI patients have special concerns regarding the development of
cardiovascular disease (Garstang 2001), deep vein thrombosis (McKinney & Garstang
2001) and osteoporosis (Weiss 2004). Prior to World War II, most spinal cord injured
persons died within weeks of injury due to urinary dysfunction, respiratory infection or
bedsores. Renal failure was also a leading cause of death. However, since the inception of
the National Spinal Cord Injury Database in 1973, the greatest contributors to the reduced
life span of SCI patients are pneumonia, pulmonary embolism and septicemia. In addition
to diminishing the quality of life of SCI patients, these systemic concerns contribute to a
significantly shortened life span. For example, if a 20 year old sustains a paraplegia
inducing SCI, his/her life expectancy is approximately 65.3 years as compared to the 77.8
years of an uninjured individual. The discrepancy is higher for more severely injured
people. The life expectancy of a person with a high C1-C4 injury decreases to 56 years.
Social and economic hardships also result from SCI. As the majority of SCIs are
sustained by people aged 16-30 (50% of which are single), the probabilities of both
getting married and sustaining that marriage are reduced. For those who are married at
the time of injury, the likelihood of the marriage remaining intact is slightly lower than in
the uninjured population. The financial burden associated with SCI is also overwhelming,
costing the taxpayers alone over 12 billion dollars per year. A person sustaining a high
C1-C4 injury, resulting in either complete or incomplete tetraplegia, requires
approximately $626,588 of medical care during the first year post-injury. The cost of care
in each subsequent year is estimated at $112,237, which excludes indirect costs such as
the loss of wages, fringe benefits and productivity. Typically, indirect costs average
-$52,915 per year although this number varies tremendously with injury severity and
pre-injury employment status. Similarly, an individual with paraplegia has a first year
financial burden of $228,955, followed by $23,297 in subsequent years. Although these
figures are directly related to injury severity, a 25-year-old individual with a high C1-C4
injury may likely incur estimated lifetime medical bills exceeding $2,393,507 (Spinal
cord injury 2003).
It is clear that SCI, although not the most prevalent CNS injury/disease in today's
society, is certainly one of the most physiologically complex and financially and socially
burdensome. There is no cure. As previously indicated, advances in roadside and clinical
care coupled with rapid pharmacological treatment and improved rehabilitative strategies
have helped to foster a trend towards less severe injuries, thereby improving quality of
life and reducing the financial and social obstacles faced by injured persons. Further
clinical improvements are dependent upon identifying and understanding the
pathophysiological processes that comprise the secondary injury cascade. Improved
communication between clinicians and researchers and a continuous influx of funding are
indispensable necessities in the battle to develop a viable and promising treatment
Spinal Cord Injury Pathophysiology
Spinal cord injury is an extremely complex injury comprised of a mechanical
primary injury and a delayed, prolonged secondary injury cascade. The primary injury
can be contusive, shearing, stretching or concussive in nature. Most humans suffer
anterior or circumferential cord compression from fracture or dislocation of a closed
vertebral system (Amar & Levy 1999). The primary injury not only damages vascular
and skeletal components, but also harms neuronal cell bodies, axons and
oligodendrocytes. The secondary injury cascade is made up of a series of uncontrolled
endogenous biochemical reactions that, together, prolong the injury process and can
extend the tissue damage to segments rostral and caudal to the original site of injury.
Ultimately, significant cell death occurs, leading to loss of motor and sensory function
below the injury site. At the present time, the prognosis for substantial functional
recovery in the vast majority of SCI individuals is extremely poor.
Research in animal models of SCI demonstrates mechanical trauma causes
vasospasm of the superficial cord vessels (Ducker & Assenmacher 1969). Within 30
minutes post-injury, petechiae form at the injury epicenter and eventually expand and
converge to replace most of the spinal gray matter (Means & Anderson 1987; Ducker et
al., 1971). Disruption of vascular elements and structures leads to the breakdown of the
blood-spinal cord barrier (BSCB) following SCI (Jaeger & Blight 1997; Popovich et al.,
1996; Goodman et al., 1976; Griffiths 1976; Griffiths & Miller 1974), release of
vasoactive molecules such as cytokines, kinins, prostaglandins, leukotrienes,
thromoboxane, catecholamines, platelet activating factor (Pan et al., 2002; Streit et al.,
1998; Anderson & Hall 1993, Xu et al., 1991) and loss of vascular system autoregulation
(Mautes et al., 2000; Amar & Levy 1999; Anderson & Hall 1993). Vascular disruption
and blood-spinal cord barrier breach (reviewed by Mautes et al., 2000) allow the rapid
invasion of neutrophils into the injury epicenter, followed by the recruitment of activated
microglia and T cells (Streit et al., 1998; Popovich et al., 1997). These events are closely
linked with edema, ischemia and decreased tissue 02 levels, all of which contribute to
spinal cord ischemia (Hall & Wolf 1986; Hickey et al., 1986). By the end of the first
week post-injury, the tissue in the injury epicenter has been transformed into a large mass
of eroded tissue filled with inflammatory cells.
There are many processes some immediate, some delayed and others cyclic-that
contribute to destruction of spinal cord tissue following injury. Within minutes of SCI,
activation of membrane phospholipases and lipases leads to the release of fatty acids
followed by the subsequent production of eicasonoids, e.g., prostaglandins and
leukotrienes (Anderson & Hall 1993; Demediuk et al., 1987, 1985; Horrocks et al.,
1985). Eicosanoids are known mediators of inflammation and vascular permeability.
Nonenzymatic (or free radical induced) lipid peroxidation also occurs very shortly after
SCI (Anderson & Hall 1993; Hall & Braughler 1986; Demopoulos et al., 1982), as
evidenced by the rapid decrease in tissue vitamin E levels and membrane cholesterol
(Saunders et al., 1987; Anderson et al., 1985; Demopoulos et al., 1982). The metabolic
composition of the spinal cord places it at greater risk for free radical mediated damage
than other tissues because 1) it is poor in antioxidant enzymes, 2) it produces a greater
number of free radicals relative to its size than other tissues and 3) it is rich in
polyunsaturated fatty acids, which are particularly vulnerable to oxidation. Thus, SCI-
induced over production of free radicals overwhelms the endogenous scavenging systems
to damage proteins, lipids and DNA and, in the process, alters the structure and functions
of cellular (neuronal, glial, vascular) and mitochondrial membranes. Membrane
disruption, in turn, precipitates the loss and/or malfunction of Ca2+ and Na+/K+-ATPase
ionic pumps, transporters and receptors. The resulting loss of osmotic regulation, coupled
with electrolyte imbalance, interrupts cellular signaling and neurotransmitter clearance
(Young & Constantini 1993; Kwo et al., 1989; Goldman et al., 1983). A number of
phosphatases, phospholipases, kinases and endonucleases (Goldman et al., 1983; Stokes
et al., 1983; Yashon et al., 1975) and other proteases (Taoka et al., 1998; Iwasaki et al.,
1987; Banik et al., 1986; lizuka et al., 1986) are activated following SCI. The increase in
both Ca2+-activated and non-Ca2+-activated protease activity, which will be discussed in
more depth later, results in the degradation of cytoskeletal, myelin and extracellular
components essential for normal cellular function and survival.
It is clear that SCI is a dynamic and complex injury. The damage sustained from
the primary injury and the spreading damage inflicted by the aberrant post-injury
biochemical processes ultimately determine the cytoarchitecture of the surviving spinal
cord tissue as well as the extent of preserved motor and sensory function. Usually, the
injury will involve spinal cord segments both rostral and caudal to the primary injury.
Although it may take more than a month (depending on the species) to establish the final
three-dimensional configuration of the lesion, eventually the injury site consists of either
a single or multilocular fluid filled cyst. This cyst can be sculpted for months post-injury
(Quencer & Bunge 1996).
Cell Death after Spinal Cord Injury
As indicated above, most cases of SCI result in cavitation of the gray matter, a
process that is reached through extensive cell death. Previously, the cell death observed
following SCI was believed to be solely necrotic (Balentine 1978a, 1978b). Necrotic cell
death, which is characterized by a loss of ionic homeostasis, organelle and cellular
swelling, membrane disruption, cell lysis, internal content spillage and inflammation, is a
consequence of the primary injury and is difficult to prevent. In many cases, 50% of the
spinal cord at the injury site is necrotic by 4 h post-injury (reviewed by Velardo et al.,
1999). Within 2 h post-injury, inflammatory cell invasion into the injury epicenter begins,
exposing the damaged tissue to additional insult via the release of reactive oxygen
species and active proteases (Bethea 2003; Babior 2000; Bartholdi & Schwab 1997;
Means & Anderson 1987). While necrosis plays a substantial role following the primary
insult, studies now show that apoptosis also significantly contributes to SCI-related cell
death (Hayashi et al., 1998; Lou et al., 1998; Mackey et al., 1997). Apoptosis is a very
orderly process that consists of well-characterized biochemical cascades that take cells
from an intact, metabolically active state to a state of cellular breakdown, e.g., the
contents of the cell being neatly packaged into membrane bound apoptotic bodies that are
digested. Over the past decade, several studies indicate that apoptotic cell death occurs
within the injured spinal cord. In models of both contusion- (Yong et al., 1998; Liu et al.,
1997) and compression-SCI (Li et al., 1999) in the rat, apoptotic cell death has been
observed in neurons, astrocytes, oligodendroglia and microglia. Although the peak period
of apoptosis differed for the various cell types (Liu et al., 1997), certain populations
continued to undergo apoptosis for 2-3 weeks post-injury (Yong et al., 1998; Crowe et
al., 1997; Liu et al., 1997). It is now clear that both necrotic and apoptotic cell death
occurs following SCI and contributes to the loss of viable tissue and the resulting
decrease in motor and sensory function.
It appears that while necrosis may be a result of the primary injury, the secondary
injury cascade, or certain components of it, is somehow responsible for the apoptosis
observed in the injured populations of cells. The observation that apoptotic cell death
occurs following SCI is important because it not only improves our understanding of SCI
pathophysiology but more important, it also provides a possible avenue for therapeutic
intervention. The prolonged time period during which cells undergo apoptosis increases
the potential therapeutic window to treat at risk cellular populations following SCI.
Proteases and Spinal Cord Injury
Among the many pathophysiological processes that occur following SCI, one
process is the activation and release of lytic enzymes such as proteases, phosphatases,
lipases and endonucleases. While some proteases like the matrix metalloproteinases
(MMPs) and calpains are calcium activated, others, like the caspases and cathepsins, are
not. Because all of these proteases are widely expressed and extremely powerful,
elaborate cellular mechanisms have evolved to regulate their expression and activity.
However, following SCI, it is likely that these regulatory controls are disturbed and/or
overwhelmed, thereby leading to the over-expression, over-activation and possibly
aberrant release of a number of proteases. Furthermore, invading inflammatory cells may
also release these enzymes exacerbating vascular, neuronal and glial damage. The
relatively ubiquitous distribution of these proteases and their documented role in the
proteolysis of vital substrates make them strong candidates for involvement in the
secondary injury cascade.
Matrix metalloproteinases (MMPs) are zinc and calcium dependent endopeptidases
capable of degrading all extracellular matrix (ECM) components. As such, they play a
critical role in matrix remodeling, development and wound healing and repair (Yong et
al., 1998). Nearly two dozen members of the MMP family have been identified and all
share a common propeptide and N terminus catalytic domain. Over the past decade, the
role of MMPs in the pathogenesis of several diseases has become clearer. MMPs are
believed to participate in tumor invasion and metastasis (Noel et al., 2004; Khasigov et
al., 2003), rheumatoid arthritis, periodontal disease (Smith et al., 2004; Ejeil et al., 2003),
atherosclerosis (Katsuda & Kaji 2003; Jones et al., 2003) and in CNS pathologies
including multiple sclerosis (Maeda & Sobel 1996, Opdenakker & Van Damme 1994),
amyotrophic lateral sclerosis (Lim et al., 1996), Alzheimer's disease (Deb & Gottschall
1996), malignant glioma invasion (Stetler-Stevenson & Yu 2001) and breakdown of the
blood-brain-barrier (Aoki et al., 2002; Rosenberg et al., 1998; Chandler et al., 1997).
MMPs have now been implicated in SCI pathophysiology. Following moderate
contusion-SCI in mice, levels of MMP-9 enzymatic activity rapidly increased, peaked at
24 h post-injury and subsided by 72 h post-injury (Noble et al., 2002). MMP-9 enzymatic
activity in the injured cords of these mice was localized to blood vessels, neutrophils and
cells expressing astrocyte and macrophage markers (Noble et al., 2002). Evidence that
MMP-9 contributed to the injury response was obtained in the parallel studies of MMP-9
knockout mice. Examination of these MMP-9 deficient following SCI revealed reduced
breakdown of the BSCB, decreased neutrophil infiltration and improved locomotor
recovery (Noble et al., 2002). Similarly, Wells et al. (2003) examined the transcript
expression of 22 MMPs following compression-injury in mice and found mRNA
expression of several MMPs was upregulated. The most noticeable induction occurred for
MMP-12 (189 fold over basal levels) at 24 h post-injury, an increase that was still evident
at 14 d post-injury. As was observed in MMP-9 knockout mice, MMP-12 knockout mice
exhibited significantly improved functional recovery, decreased BSCB permeability and
reduced microglial and macrophage density following compression-injury (Wells et al.,
2003). Although these studies implicate at least two MMPs in the destructive processes
following SCI, additional studies are required to provide a more systematic examination
of MMP mRNA and protein expression and enzymatic activity following SCI, as well as
to identify the endogenous substrates of these enzymes in the post-SCI milieu.
Calpain in a cytosolic cysteine protease that is expressed in every cell and tissue
studied thus far (Banik et al., 1997). Its two forms, [t-calpain and m-calpain, are activated
by micromolar and millimolar concentrations of ionic calcium, respectively. Calpain has
numerous substrates including cytoskeletal (Croall & DeMartino 1991) and membrane
(Schlaepfer & Zimmerman 1985) proteins. The very specific endogenous inhibitor,
calpastatin, normally tightly regulates calpain activity. However, the influx of Ca2+ into
the cell following SCI overactivates calpain thereby disturbing the normal balance
between calpain activation and inhibition. In fact, increased calpain mRNA and protein
expression and activity levels following SCI have been well documented (Wingrave et
al., 2003; Shields et al., 2000; Banik et al., 1997; Springer et al., 1997; Ray et al., 1999;
Li et al., 1996, 1995). Increased calpain immunoreactivity, due to elevated levels of the
protein, was detected in multiple cell types including macrophages, reactive astrocytes,
microglia and neurons in the injury epicenter following SCI (Shields et al., 2000; Li et
al., 1996). These increases were detected within minutes of injury and persisted, in some
cases, for up to 72 hours following the initial trauma (Banik et al., 1997). Furthermore,
the increases in calpain protein levels and enzymatic activity have been shown to
correlate with the degradation of several important proteins such as a-fodrin (Ray et al.,
1999), proteolipid protein (Banik et al., 1985, 1980), microtubule associated protein-2
(MAP2, Springer et al., 1997) and neurofilament (NF, Ray et al., 2000a; Schumacher et
al., 1999 Banik et al., 1997). The calpain-specific 145 kDa all spectrin breakdown
product (BDP) has also been observed to accumulate in the injury epicenter following
SCI (Wingrave et al., 2003; Springer et al., 1997).
The most convincing evidence for calpain involvement in protein degradation
and/or cell death following SCI was obtained in studies employing calpain inhibitors.
Intraparenchymal administration of the water-soluble calpain inhibitor, CEP4143, just
prior to compression-SCI in rats inhibited calpain activation, reduced degradation of
dephosphorylated NF200 and improved performance on both the inclined plane test and
the Beattie-Bresnahan-Basso (B.B.B) test for functional hindlimb recovery (Schumacher
et al., 2000). In another series of experiments, Ray et al. (2003, 2001a,b, 2000a,b)
showed that administration of the membrane permeable cysteine protease inhibitor E64-d
over a 24 h period following contusion-SCI in rats, prevented internucelosomal DNA
fragmentation and apoptosis and inhibited NFP degradation in the injury site segment.
E64-d treatment also prevented SCI-induced increases in the levels of calpain mRNA and
protein and caspase-3 mRNA expression. Furthermore, inhibitor treatment restored
transcription of proteolipid protein and myelin basic protein. Finally, intraspinal
microinjection of the cell permeable calpain inhibitor MDL28170 prior to injury inhibited
total calpain enzymatic activity and reduced spectrin and MAP2 proteolysis (Zhang et al.,
2003). Together, these studies show that calpain contributes to the breakdown and/or
modification of important cellular proteins following SCI and suggest that this protease is
also involved in the secondary injury cascade.
Caspases are intracellular cysteine proteases that play an essential role in the
initiation and execution of apoptotic cell death. At least 14 members of the caspase
family have been identified. These cysteine proteases, which cleave their substrates after
an aspartate residue, are normally present as inactive zymogens in cells. With the
discovery of apoptotic neuronal and glial cells in spinal cord tissue following SCI (Li et
al., 1999; Emery etal., 1998; Yong etal., 1998; Crowe etal., 1997; Liu etal., 1997;
Katoh et al., 1996) and the link between caspase and apoptosis (Citron et al., 2000;
Springer et al., 1999; Yuan et al., 1993), there has been heightened interest in
understanding caspase-mediated apoptosis in SCI. Several recent reports have
documented increases in caspase protein and mRNA expression and activity in both
neuronal and non-neuronal cells following SCI. Springer et al. (1999) reported increased
caspase-9 activation and cytochrome c levels within 30 minutes of contusion-SCI in rats.
These upstream events were sustained for over 24 hours. Furthermore, Keane et al.
(2001) noted that activation of caspases-2, -8 and -9 leads to apoptosis following SCI
while Takagi et al. (2003) noted that the enzymatic activity of caspases-3 and -8 were
upregulated after contusion-SCI in mice. The effects of SCI on the expression of
"executioner" caspase-3 have also been examined. Caspase-3 enzymatic activity levels in
injured spinal cord were 600% greater than control levels within 1 hour post-injury and
persisted for 24 hours (Springer et al., 1999). Increases in caspase-3 mRNA and protein
levels are highly correlated with apoptosis in the injury site and preceded
internucleosomal DNA fragmentation and the accumulation of the 120 kDa caspase-
specific aII spectrin breakdown product (Wingrave et al., 2003; Ray et al., 2001; Li et
al., 2000) and the 160 kDa caspase-specific fodrin breakdown product (Citron et al.,
2000). Thus, it appears that increased caspase protein and mRNA expression and
enzymatic activity following SCI contribute to apoptotic cell death following SCI.
As was the case with calpain, experimental use of caspase inhibitors have helped to
delineate the role that caspases have in the secondary injury cascade. Caspase inhibitors
have been shown to prevent staurosporine-, kainate- and thapsigargin-induced apoptotic
death of mature oligodendrocytes in culture (Benjamins et al., 2003). Z-VAD-fmk, a pan
caspase/cathepsin B inhibitor, was found to increase the survival of bone marrow cells
(BMCs) that had been grafted into an ischemic area caused by middle cerebral artery
occlusion (MCAo) in adult rats (Chen et al., 2002). These animals also had significantly
reduced levels of apoptosis within the transplanted BMCs and demonstrated significant
improvement in a test of somatosensory function (adhesive removal), but did not have a
significant improvement in a test of motor function (rotarod) (Chen et al., 2002).
Following MCAo in mice, intracerebroventricular (ICV) injections of Z-VAD-fmk,
reduced ischemia-induced tissue damage, brain swelling and behavioral deficits (Hara et
al., 1997). In a rat model of traumatic brain injury, ICV injections of the cell permeable
caspase-3 inhibitor, Z-DEVD-fmk reduced TBI-induced DNA fragmentation and
improved motor function (Yakovlev et al., 1997). In aggregate, these studies suggest that
caspase inhibitors may provide varying degrees of neuroprotection through attenuation of
apoptotic cell death following an ischemic or traumatic brain insult.
The utilization of caspase inhibitors following SCI is less conclusive. ICV injection
of either a non-specific caspase inhibitor or a specific caspase-3 inhibitor immediately
following contusion-SCI in mice has been shown to reduce caspase-3 enzyme activity
(Takagi et al., 2003). Intrathecal (IT) Z-DEVD-fmk injection prior to contusion-SCI in
rats was found to block cytochrome c dependent DNA fragmentation through the
cleavage of DFF45/ICAD, a process likely to occur downstream of caspase-9 activation
but involving the activation of caspase-3 (Springer et al., 1999). Mice treated with Z-
VAD-fmk soaked gelfoam placed over the injury site had significantly smaller lesions
and demonstrated significant functional improvements as compared to control animals
(Li et al., 2000). Furthermore, in a rabbit model of ischemic SCI, Lapchak et al. (2003)
showed that the IT delivery of the non-selective cell permeable caspase inhibitor,
BDFMK, decreased both the levels of caspase-3 activity and a caspase-3 cleavage
product. However, this treatment but did not improve functional recovery. Similarly,
administration of Z-VAD-fmk using a variety of delivery routes (e.g., local,
subarachnoid, intravenous and intraparenchymal) following mild contusion-SCI in rats
did not improve B.B.B. scores or reduce levels of apoptosis as compared to control
animals (Ozawa et al., 2002).
Although it is clear that caspase-mediated apoptosis contributes to the cell death
observed following SCI and therefore would appear to be a reasonable point for
therapeutic intervention, the variety of caspase inhibitors (specific/non-specific,
permeable/impermeable), injury types (mild/moderate/severe,
compression/contusion/ischemic), animal models (rat/mouse/rabbit), administration
strategies (IP/ICV/IT/IV) and outcome measures (motor/sensory) used in these studies
complicates the assessment of the neuroprotective capacity of caspase inhibitors
following SCI. Thus, better-coordinated and controlled studies are needed to determine
the effectiveness of caspases inhibitors to protect the injured spinal cord.
Several human cathepsins have now been identified and sequenced. Like other
proteases, cathepsins are synthesized as inactive zymogens and are activated by
proteolytic removal of the N-terminus propeptide. The residue serinee, cysteine, metallo
or aspartate) in the active site distinguishes the cathepsins from one another. Cathepsins
play an important role in several physiological processes including homeostatic protein
degradation (Bond & Butler 1987; Turk et al., 2000), antigen presentation (Chapman et
al., 1997), bone remodeling (Kakegawa et al., 1993) and hormone processing (Dunn et
al., 1991). Their expression varies across species, tissues and developmental stages of an
organism (Levicar et al., 2002; Pungercar et al., 2000; San Segundo et al., 1996; Shi et
al., 1994; Petanceska & Devi 1992; Qian et al., 1991), and over the past two decades,
cathepsins have been identified as contributors to several disease and injury processes. In
many of these conditions, including multiple types of cancers, asthma, arthritis,
osteoporosis, muscle wasting and pycnodysostosis, the mRNA and protein expression,
activity and localization of multiple cathepsins are altered. Furthermore, several
cathepsins have been implicated in the pathologies of Alzheimer's disease, dementia,
multiple sclerosis, Guillian-Barre syndrome, Creutzfeld-Jakob, Down syndrome and
ischemic cell death (Gan et al., 2004; Nagai et al., 2000; Mackay et al., 1997; Haas &
Sparks 1996; Bever & Garver 1995; Bernstein & Wiederanders 1994). Their relatively
ubiquitous distribution, indiscriminate hydrolytic preferences and enzymatic robustness
(e.g., activity in acidic and neutral pH) make it worthwhile to characterize cathepsin
mRNA and protein expression, enzymatic activity and cellular localization in the context
To date, the role of cathepsins in SCI pathophyisology has received little attention.
Thus, minimal information is available concerning cathepsin expression and activity in
the injured spinal cord. Microarray analyses have shown increases in the transcript
expression of many cathepsins following two CNS insults and one peripheral nervous
system injury. Three days post-spinal root avulsion, Hu et al. (2002) reported an increase
in the expression of cathepsins B, C, D, H, I, L and S. The expression of cathepsins B
(Cath B) and D were also both elevated after either sciatic nerve crush (Fan et al., 2001)
but only Cath B was upregulated following hemisection of the spinal cord (Fan et al.,
2001). These microarray studies suggest that insults to the spinal cord cause an increase
in the mRNA expression of several of the cathepsins, particularly Cath B, which was
upregulated in all three reports.
The single human Cath B gene is comprised of 12 exons (Gong et al., 1993), spans
more than 27 kilobases (Berquin et al., 1995) and is localized to chromosome 8p22 (Fong
et al., 1992; Ferrarra et al., 1990). While its regulation is not fully characterized, it has
been suggested that more than one promoter can drive the expression of human Cath B
(Berquin & Sloane 1996; Rhaissi et al., 1993). Furthermore, multiple transcripts resulting
from alternative splicing in the 5' and 3' untranslated regions (UTRs), and possibly from
the use of alternative promoter regions, have been detected (Berquin & Sloane 1996).
While putative Cath B promoters have characteristics similar to those of housekeeping
promoters, including the absence of TATA or CAAT boxes (Gong et al., 1993), high
guanidine/cytosine (GC) content (Qian et al., 1991b) and several potential binding sites
for the transcription factor Sp-1 (Yan et al., 2000), Cath B mRNA expression still
appears to vary depending on the cell type and state of differentiation.
18 333 Inactive (37 kDa)
Light Chain Single Chain Form
so 33 Active (30 kDa)
25 kDa Double Chain Form
129 Cys, 333 Act
5 kDa Light Chain
Figure 1-1: Schematic of the Cath B protein. Preprocathepsin B (333 aa) is synthesized
on the rough ER where the signal peptide is cleaved co-translationally.
Following transport to the Golgi apparatus, the enzyme is glycosylated and the
mannose-6-phosphate signal is assembled. This receptor allows for transport
to the trans-Golgi network and then onto an acidic compartment where the
pro-peptide is removed and enzyme is activated. Within the lysosome, the
active single chain form of Cath B (30 kDa) is further modified (loss of a
dipeptide) to produce the active double chain form (25 kDa and 5 kDa) held
together via disulfide bond.
As is the case with other cathepsins, Cath B is synthesized as an inactive
preproenzyme (39 kDa) in the trans-golgi, which is processed to the still inactive
proenzyme (37 kDa) in the lysosomal compartment (Mort & Buttle 1997). Removal of
the 63-residue propeptide from its N-terminus converts the inactive proenzyme into the
single chain active form (30 kDa). This single chain Cath B undergoes hydrolysis within
the lysosome, converting it to a double chain form consisting of heavy (25 kDa) and light
(5 kDa) components held together by a disulfide bond (Mort & Buttle 1997).
Cath B is involved in homeostatic protein turnover and digestion of cellular debris
(Turk et al., 2000; Mort & Buttle 1997). Because Cath B is capable of hydrolyzing
complex carbohydrates, nucleic acids, lipids and ECM components under a broad pH
optimum (Lindebaugh et al., 1999; Bajkowski & Frankfater 1983a, b), it is sequestered in
the lysosomes and away from potential substrates.
Role in Peripheral Pathologies
Cath B expression and enzymatic activity are strictly regulated at several levels
including transcription, post-transcriptional processing, translation and glycosylation,
maturation and trafficking and protein inhibition (Yan et al., 1998). Increased levels of
Cath B mRNA and protein expression and enzymatic activity, as well as the intracellular
redistribution of the protein, have been reported in multiple pathologies including
rheumatoid (Codorean & Gabrielescu 1985; Lenarcic et al., 1988) and osteoarthritis
(Bayliss & Ali 1978; Martel-Pelletier et al., 1990). Furthermore, Cath B is markedly
upregulated and aberrantly localized (and/or secreted) in several human cancers including
ovarian (Scorilas et al., 2002; Warwas et al., 1997), thyroid (Kusunoki et al., 1995),
gastric (Watanabe et al., 1989), lung (Sukoh et al., 1994), prostate (Sinha et al., 1995),
breast (Yano et al., 2001; Sameni et al., 1995), colon (Campo et al., 1994), melanomas
(Sloane et al., 1981) and osteoclastomas (Page et al., 1992). In fact, staining for Cath B is
very conspicuous at the leading edge of invading tumor cells, where it is secreted and is
associated with degradation of cellular and ECM components of healthy tissue during the
infiltration of tumor cells.
Role in Central Nervous System Pathologies
The potential role that Cath B plays in cell death and peripheral tissue pathologies
makes it a protease that needs to be investigated in CNS diseases and injuries. However,
to date, no systematic characterization of Cath B following any CNS injury has been
published. While the mechanism of action remains unclear, elevated levels of Cath B
mRNA and protein, increased enzymatic activity and altered protein subcellular
localization have been documented in multiple sclerosis (Bever et al., 1995), Alzheimer's
disease (Mackay et al., 1997), amyotrophic lateral sclerosis (Kikuchi et al., 2003) and
myoclonus epilepsy (Houseweart et al., 2003; Rinne et al., 2003). In these diseases, Cath
B's roles in necrotic and apoptotic cell death and tissue loss processes are under active
Cath B has been more thoroughly investigated for its role in tumor progression in
the brain. Rempel et al. (1994) demonstrated over-expression of Cath B mRNA and
protein in glioma cell lines and in biopsy samples as compared to control samples, a
finding confirmed by Sivaparvathi et al. (1995). Increased Cath B immunostaining has
been observed in both tumor and endothelial cells of high grade brain tumors (Strojnik et
al., 1999). Similar to non-CNS tumors, invading tumor cells adjacent to malignant
gliomas also exhibited increased Cath B mRNA and protein expression (Demchik et al.,
1999; Rempel et al., 1994). Furthermore, immunohistochemial analysis of human
gliomas revealed a positive correlation between increasing intensities of Cath B staining
and the grade of malignancy and an inverse correlation with shorter survival times
(Mikklesen et al., 1995; Strojnik et al., 2001). Based on these observations, Cath B has
become useful as a prognostic marker for human gliomas.
Because of Cath B's purported role in tumor expansion, inhibitors of this protease
have been utilized in attempts to diminish the tumorigenic quality of invading cells. The
invasion of malignant glioblastoma cells into collagen IV, fibronectin and laminin
matrices has been shown to be inhibited by the Cath B inhibitors E64, CA-074 (specific
for Cath B) and CA-074 Me (cell permeable derivative of CA-074) (Levicar et al., 2002).
The Cath B inhibitor, K11017, was also found to significantly inhibit gliomal cell
invasion into matrigel and to reduce infiltration of glioma spheroids into normal brain
aggregates (Demchik et al. 1999). The administration of other inhibitors such as CA-074
and E64-c significantly blocked glioblastoma cell migration (Lah et al., 1998). Thus,
Cath B appears to play a critical role in the processes that allow for the growth of tumors
into CNS tissue.
For transient cerebral ischemia, a CNS insult more closely related to traumatic
injury, the functional role for Cath B is becoming clearer. Indeed, ischemia is a major
pathological event following SCI and contributes heavily to the tissue loss seen following
this primary injury. Thus, studies characterizing Cath B following ischemic events in the
brain could provide insight as to the post-injury effects of this protease on spinal cord
tissue. Following a 20-minute cerebral ischemic event in both monkeys (Yamashima et
al., 1996) and gerbils (Kohda et al., 1996), the CA1 region of hippocampus undergoes
complete neuronal death by post-injury day 5 and Nitatori et al. (1995) established that
the CA1 neuronal cell death in these models was apoptotic. Both calpain and Cath B were
immediately activated in the CA1 region following cerebral ischemia and that Cath B
appeared to have redistributed to the cytosol (Yamashima et al., 2003) In their 'calpain-
cathepsin hypothesis', Yamashima et al. (1998) speculate that the disruption of the
lysosomal membrane, partially mediated by calpain activity, results in the release of
powerful hydrolytic enzymes (such as Cath B) into the cytosol where they degrade
substrates vital to cell survival. Lastly, the premise of the 'calpain-cathepsin hypothesis'
has recently been confirmed in C. elegans by Syntichaki et al. (2002), where it was
demonstrated that two specific calpains (TRA-3 and CLP-1) function upstream of the
cathepsins and are required for degradation by various necrosis initiators.
To date, the neuroprotective potential of Cath B inhibitor treatment following
traumatic or ischemic CNS injury has received little attention. However, the few studies
that have been published seem to indicate that inhibition of Cath B reduces cell death in
models of cerebral ischemia. For example, the administration of CA-074 and E-64c
immediately following transient ischemia in monkeys reduced levels of Cath B
enzymatic activity and immunoreactivity and spared approximately 67 and 84%,
respectively, of the CA1 neurons from cell death (Yoshida et al., 2002; Tsuchiya et al.,
1999; Yamashima et al., 1998). Additional studies have shown that this inhibitor-
mediated neuroprotection extended to other neuronal populations as well (e.g., cortical,
caudate-putaminal, Purkinje) (Yoshida et al., 2002). In a rat MCAo model, Cath B
immunoreactivity and enzymatic activity were both increased (Seyfried et al., 1997). The
IV injection of CP-1, a member of the peptidyl diazomethane family specific for Cath B
and L but not calpain or caspase, reduced infarct volume and improved functional scores
implicating Cath B in the development of the infarct in this model (Seyfried et al., 2001).
Cath B and Apoptosis
While cathepsin participation in apoptosis has long been neglected (as lysosomes
appear to remain intact during the programmed cell death process), it is widely held that
cathepsins do participate in cellular autolysis and the damage of nearby cells during
necrosis (Nixon & Cataldo 1993). However, the emergence of new in vitro and in vivo
data has forced reconsideration of the role of cathepsins in apoptosis (reviewed by Leist
& Jaattela 2001). While the mechanisms of action differed, a role for Cath B in both
TNF- and bile salt-induced hepatocyte apoptosis was confirmed by Guicciardi et al.
(2001, 2000) and Roberts et al. (1997), respectively. Following TNF exposure (a
cytokine that is also induced following SCI), Cath B in conjunction with an unidentified
cytosolic substrate increased the release of cytochrome c, which subsequently led to
activation of caspase-9 and -3 (Guicciardi et al., 2002). Conversely, in the model of bile-
salt induced hepatocyte apoptosis, Cath B's involvement was caspase-8 dependent
(Roberts et al., 1997). Furthermore, Cath B was essential in TNF-induced apoptosis of
WEH1 fibrosarcoma cells (Foghsgaard et al., 2001) and the apoptotic cell death of HT22
hippocampal cells and cerebellar granular cells following microglial stimulation
(Kingham & Pocock 2001).
As was the case with previously discussed proteases (i.e., MMPs, calpain and the
caspases), the prevention of increases in Cath B mRNA and protein expression and
enzymatic activity also provides protection from apoptosis. Inhibition of Cath B activity
partially attenuated TNF-induced liver damage in wild type mice (Guicciardi et al.,
2001). In a parallel set of experiments, TNF-induced release of cytochrome c, caspase
activation and apoptosis of isolated hepatocytes were markedly inhibited in Cath B
knockout mice (Guicciardi et al., 2001). In another study, the administration of CA-074
Me prevented all apoptotic related events in WEH1 fibrosarcoma cells (Foghsgaard et al.,
2001). Additionally, Cath B inhibitors blocked apoptosis induced by p53 and other
cytotoxic agents (Lotem & Sachs 1996) and the treatment with antisense Cath B cDNAs
also proved to be anti-apoptotic (Lakka et al., 2004; Monaham et al., 2001). While the
role of Cath B in apoptotic cell death varies from model to model (e.g., upstream vs.
downstream, initiator vs. executioner), it appears that Cath B contributes to apoptotic cell
death and may offer a target for potential therapeutic intervention.
Cath B in SCI Pathophysiology
The role of Cath B in the cell death and tissue loss in multiple CNS pathologies
supports the hypothesis that Cath B may contribute to the secondary injury cascade
following SCI. With a broad range of substrates at both an acidic and neutral pH, the
biochemical changes following SCI may precipitate an unregulated response by Cath B,
including increased mRNA and protein expression and enzymatic activity and a
redistribution of the active protease. As Cath B is clearly capable of hydrolyzing
important components of the cytoskeleton (e.g., neurofilament, spectrin, actin), myelin
sheath (MBP) and ECM (e.g., laminin, fibronectin, proteoglycans), it is plausible that
Cath B is, at the very least, partially responsible for the damage caused by the secondary
injury cascade. Considering the number of systems that are compromised in the post-SCI
milieu, the release of pre-existing and newly synthesized active Cath B from neuronal
and non-neuronal sources (including invading inflammatory cells) may collectively
overwhelm any endogenous inhibitors in the CNS, thus causing excessive Cath B-related
damage and tissue loss through both apoptotic and necrotic cell death.
MATERIALS AND METHODS
All experimental procedures were conducted in accordance with the guidelines set
forth by the University of Florida's Institute Animal Care and Use Committee (IACUC).
Female Long-Evans rats weighing approximately 230-300 grams (Harlan,
Indianapolis, IN) were used in this study. All surgical procedures were conducted under
sterile conditions with supplemental heat. Intraperitoneal administration of Nembutal
(sodium pentobarbital 50-60 mg/kg) was used to induce anesthesia. Following a T12
laminectomy (intact dura mater), injury to the spinal cord was produced with the NYU
impactor device (i.e., 10 g dropped 25 mm onto the exposed dura). The sham-injury
animals received a laminectomy and were placed in the injury apparatus, but were not
injured. The incision was closed in layers (i.e., muscle then skin). Animals recovered in a
heated incubator with food and water available ad libitum. Bladders were manually
expressed and fluids were administered as frequently as required.
mRNA, Protein and Enzymatic Activity
Spinal cord tissue was collected after extending the laminectomy to allow three
segments of tissue to be removed (i.e., tissue from the injury site and tissue from the
regions immediately rostral and caudal to the injury site). Each tissue segment was
approximately 6 mm in length. The fresh tissue was rinsed with cold PBS and either
immediately homogenized with 500 ptL guanidinum thiocyanate salt solution (mRNA
experiments) or flash frozen with liquid nitrogen (protein and enzymatic activity
Animals prepared for immunohistochemistry were perfused intracardially initially
with 0.9% saline and then 4.0% paraformaldehyde (pH 7.4). The perfused animal was
stored overnight at 4 C, after which the cord was removed. After the dura was carefully
dissected away, the spinal cord was cryoprotected in 30% sucrose. The segment
containing the injury site was sectioned (14 [tm) and mounted onto slides (Fisher
Scientific, Pittsburgh, PA) using the Frigocut 2800 (Reichert-Jung).
Analysis of Cath B mRNA Expression
Cath B mRNA expression was measured at 2, 6, 24, 48 and 168 h post-injury in
rats that received a either a sham-injury or contusion-injury (n = 3-6 rats/time
point/group). Spinal cord tissue from 4-5 normal (i.e., not receiving any surgical
treatments and/or other manipulations) animals was also examined for Cath B mRNA
RNA Isolation and cDNA Synthesis
Total RNA isolation from the spinal cord tissue was achieved using a modified
protocol, described by Earnhardt et al., 2002, based on the guanidinium thiocyanate-
phenol-chloroform extraction developed by Chomczynski and Sacchi (1987). Final RNA
concentrations were determined via spectrophotometry and samples were stored at -20
C in diethyl pyrocarbonate (DEPC) water for future cDNA preparation.
For cDNA synthesis, 1 |tg of total RNA from all samples was used for enzymatic
conversion of mRNA to first strand cDNA using an oligo-dT primer (Invitrogen/Life
Technologies, Carlsbad, CA; SuperScriptTM First-Strand Synthesis System for RT-PCR).
DNA contamination was monitored in the RNA samples by "no reverse transcriptase"
reactions that were performed in conjunction with cDNA synthesis reaction.
Base pair designations for rat GAPDH refer to GeneBank locus AF106860. The
primers used for all GAPDH PCR reactions were: sense 5'ggtga aggtc ggtgt gaac3'
corresponding to base pairs 852-870 and antisense 5'ggcat cctgg gctac actg3'
corresponding to base pairs 1657-1675. Cath B primers were designed using GeneBank
locus NM_022597. The sense Cath B primer recognized an upstream rat Cath B specific
mRNA sequence 5'tgagg acctg cttac ctgct3' corresponding to base pairs 466-485. The
antisense Cath B primer recognized a downstream rat Cath B specific mRNA sequence
5'gcagg gagtg aggca gatag3' corresponding to base pairs 1141-1160.
The Roche LightCycler and the double-stranded DNA binding dye, SYBR Green I
dyeTM, were used to continuously monitor all PCR reactions. The LightCycler (Roche
Biochemicals) is an advanced instrument that conducts rapid thermal cycling of the
polymerase chain reaction. SYBR Green I dyeTM preferentially binds to double-stranded
DNA and emits a fluorescent signal proportional to the DNA concentration. The reaction
kinetics of this PCR reaction are represented by an amplification curve. Each
amplification curve (fluorescence vs. cycle number) is assigned a crossing point value
(CPV), which is defined as the point of intersection between the amplification curve and
the noise band. A lower CPV indicates a more rapid increase in the level of fluorescence
and, thus a larger initial concentration of message. When comparing templates, those
with lower CPVs contain more amplified message for the gene of interest than those with
For each PCR reaction the LightCycler FastStart DNA MasterPLs SYBR Green I
(Roche) reagent was used according to manufacturer's instructions in combination with
0.5 [tM primers, 10 ng cDNA template, 6% DMSO and 2 mM MgC12. After an initial 300
s, 950 C denaturation step, 40 cycles of amplification were performed (denature @ 950 C
for 5 s, anneal @ 65 C for 10 s, extend @ 72 C for 40 s). SYBR Green ITM fluorescent
detection of double stranded PCR products occurred at the end of each 720 C extension
period. The specificity of the amplified product was confirmed through the melting curve
analysis and gel electrophoresis. To generate standard curves for the Cath B primer set,
contusion-injury templates were subjected to serial dilution. Linear regression analysis of
the logarithm of the dilution factor vs. the CPV generated a standard curve for each
specific template. All standard curves were run concomitant with segment- and time-
matched unknowns (i.e., template from normal and sham-injury animals). The relative
amount of RNA in the unknown sample was extrapolated from its CPV in relation to the
Analysis of Cath B Protein Expression
The level of Cath B protein was determined via immunoblotting in the spinal cord
of both sham- and contusion-injured rats at 2, 6, 12, 24, 48, 72 and 168 h post-injury (n =
4 rats/time point/group). Spinal cord tissue from 4 normal rats was also assessed for the
levels of Cath B protein.
Tissue Lysis and Protein Purification
Lysis buffer (1 mM EDTA, 2 mM EGTA, 1 small Protease Cocktail PillTM (Roche
Molecular Biochemicals), 0.1% SDS, 1.0% IGEPAL, 0.5% deoxycholic acid, 150 mM
NaC1, 20 mM Hepes, ddH20, pH = 7.5) was added to each sample based on the mass of
the tissue (15 tiL/1 mg). The tissue was homogenized on ice with a rotary pestle, returned
to eppendorf tubes and placed at 40 C for at least 30 minutes. Samples were sonicated and
spun at 8000 g for 5 minutes at 40 C before the supernatant was collected and stored at -
The protein concentration of each tissue homogenate was determined by
bichinchoninic acid (BCA) assay (Pierce Inc., Rockford, IL). Unless otherwise noted, all
procedures were performed at room temperature. Eighteen (18) micrograms of total
protein were mixed with 2X loading buffer (IX = 125 mM Tris-HC1, 100 mM DTT, 4.0
% SDS, 0.01% Bromophenol Blue and 10.0% glycerol) and were resolved by SDS-
PAGE on 10% Tris-HCl gels (Bio-Rad, Hercules, CA). The fractionated proteins were
subsequently transferred to a 0.20 ptM nylon membrane (Bio-Rad, Hercules, CA) in
transfer buffer (192 mM glycine, 25 mM Tris HC1, 10.0% methanol). Staining with
ponceau red (Sigma, St. Louis, MO) confirmed transfer of the proteins. Blots were
blocked in 5.0% nonfat milk/tris buffered saline-tween (TBS-T) (20 mM Tris HC1, 150
mM NaC1, 0.005% Tween 20, pH 7.5). Membranes were washed with TBS-T and
incubated overnight with a polyclonal anti-Cath B antibody (1:1000; Upstate
Biotechnology Inc.). Membranes were washed and then incubated in 3.0% non-fat
milk/TBS-T with an anti-rabbit IgG horseradish peroxidase conjugated antibody (1:3000;
(Bio-Rad, Hercules, CA). After additional washing, bound antibodies were visualized
using the chemiluminescent developing reagent ECL (Amersham Pharmacia Biotech,
UK). The Cath B antibody recognized three bands: the inactive proenzyme (37 kDa), the
active single chain form (30 kDa) and the heavy component of the double chain form (25
kDa). Representative blots were stripped and reprobed with a monoclonal anti-GAPDH
antibody (gift of Dr. Gerry Shaw, University of Florida) for loading control purposes.
Data were acquired as integrated densitometry values (IDVs) by computer-assisted
densitometric scanning (Alpha Imager 2000 Digital Imaging System, San Leandro, CA).
Analysis of Cath B Enzymatic Activity
Enzymatic activity of Cath B was assessed at 1, 2, 3, 5 and 7 d in both sham- and
contusion-injured rats (n = 3-4 rats/group/time point). Enzymatic activity of Cath B was
also measured in the spinal cord of 5 normal rats.
Frozen spinal cords were crushed with a chilled mortar and pestle. Triton extraction
buffer (20 mM Tris (pH 7.4), 150 mM NaC1, 5 mM EGTA, 1.0% Triton X-100, 0.2 mM
DTT) was added to the crushed tissue, which was then placed on ice for 60 minutes.
Samples were vortexed every 15 minutes for the next hour. At the end of the second hour,
samples were centrifuged at 15,000 rpm for 10 minutes at 40 C. The supernatant was
removed and placed into a clean eppendorf. Glycerol was added to stabilize the lysates.
Protein concentrations of the tissue lysates were determined by bichinchoninic acid
(BCA) assay (Pierce Inc., Rockford, IL). All samples were equalized to a common
protein concentration for ease of handling.
Enzymatic Activity Assay
Enzymatic activity assays were carried out in 96 round-bottom well plates (Costar,
Inc., Coring, NY). Lysates from spinal cord tissue were incubated with the substrate
solution (100 mM MES (pH 5.5), 200 [tM Z-Arg-Arg-7-amido-4-methylcoumarin
hydrochloride (Sigma-Aldrich, St. Louis, MO), 20 mM DTT and water). Fluorescence
was measured (excitation @ 355 nm, emission @ 460 nm) using the Spectra Gemini XS
(Molecular Devices, Sunnydale, CA).
Immunohistochemical Localization of Cath B
Immunohistochemistry was performed in normal (n = 2) and 7 d contusion-injured
rats (n = 2).
Sections were fluorescent immunolabeled with two primary antibodies (AB) in the
following experiments: polyclonal AB ac Cath B (1:1000; Upstate Biotechnology, Inc.,
New York) and monoclonal ABs against 1) glial acidic fibrilliary protein or GFAP
(1:1000; Sternberger Monoclonals, Lutherville, MD) for astrocytes, 2) NeuN (1:1000;
Chemicon, Temecula, CA) for mature neurons, 3) lysosomal associated membrane
protein or LAMP (1:1000, Stressgen, British Columbia, Canada) for lysosomal
membranes, 4) 0-4 (1:1000; Chemicon, Temeculah, CA), CNPase (1:1000; Sigma, St.
Louis, MO) and myelin basic protein or MBP (1:1000; Chemicon, Temeculah, CA) for
oligodendrocytes, 5) OX-42 (1:1000; Serotec Inc., Raleigh, NC) for resting microglia and
activated macrophages 6) OX-8 (1:1000; Serotec Inc., Raleigh, NC) and ED-2 (1:1000,
Serotec Inc., Raleigh, NC) for T lymphocytes, most thymocytes and natural killer cells
and macrophages. The nuclear dye DAPI (in Vectashield, Vector Laboratories,
Burlingame, CA) was used to label the nuclei. The first primary antibody was incubated
at room temperature (RT) with a 10% goat serum-10% horse serum-0.2% Triton-X 100
in 0.1 M PBS (block) solution followed by overnight incubation with the second primary
antibody also in block at RT. The tissue sections were then incubated in fluorescent-
tagged secondary antibody (1:1000) (Molecular Probes, Eugene, OR) and cover-slipped.
The sections were viewed and digitally captured with a Zeiss Axioplan 2 microscope
equipped with a SPOT Real Time Slider high-resolution color CCD digital camera
(Diagnostic Instruments, Inc., Sterling Heights, MI).
Inhibition of Cath B Activity
The specific and irreversible Cath B inhibitor CA-074 [N-(L-3-trans-
propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-proline] (Sigma-Aldrich, St. Louis,
MO) was used in all experiments.
In Vitro Inhibitor Assay
To test the efficacy of CA-074 as an inhibitor of Cath B activity, purified Cath B
from the bovine spleen (Sigma-Aldrich, St. Louis, MO) was incubated with various
concentrations (0 [tM 5000 ptM) of CA-074. Enzymatic activity assays were then
conducted as previously described. Furthermore, in a second set of in vitro experiments,
CA-074 (25 ptM) was added to the reaction vessel to confirm the increases in
fluorescence within the tissue lysates from the injury site were due to Cath B hydrolysis
rather than autofluorescence or non-specific substrate hydrolysis. In both in vitro
inhibitor experiments, the levels of Cath B enzymatic activity levels were determined as
Two experimental paradigms were employed to inhibit Cath B in vivo. In the first
in vivo inhibitor experiment, three treatment groups (i.e., one saline control and two CA-
074 treated groups; n = 5-6 rats/group) were utilized for each of three different routes of
administration (bolus intraperitoneal (IP), bolus intravenous (IV) and repeated dose IP).
In the second in vivo inhibitor experiment, a bolus injection of either CA-074 or saline (n
= 4-6 rats/dose of CA-074 or saline) was delivered by a single route of administration
(bolus IV). All animals were sacrificed at 72 h post-injury.
In Vivo Inhibitor Treatment
Animals were weighed just prior to surgery in order to determine the correct dose
of inhibitor. CA-074 was reconstituted fresh daily in 0.9 % saline and the total injection
volume was consistently maintained throughout the experiments.
As indicated above, two separate inhibitor treatment experiments were performed.
In the first experiment, saline and CA-074 were given either as an IP or IV (femoral)
injection. In order to visualize the femoral vein, the injection site was exposed prior to the
laminectomy and spinal cord injury. Bolus injections were administered immediately
after closing the incision exposing the spinal cord. Animals in the repeated dose IP group
received a total of three IP injections including an initial injection immediately after SCI
and additional injections at 24 h and 48 h post-injury. For the second experiment, CA-074
was delivered only as a single IV injection immediately after closure of the spinal cord
In the first experiment, both the route of administration and the dose of CA-074
were varied. Three experimental groups were utilized: 1) saline control, 2) 12 mg/kg CA-
074 and 3) 36 mg/kg CA-074. In the second experiment, only the dose of CA-074 was
varied. The three experimental groups were: 1) saline control, 2) 6 mg/kg CA-074 and 3)
12 mg/kg CA-074. In both experiments, net Cath B enzymatic activity levels were
measured. To generate these net enzymatic activity levels, the background fluorescence
(injury site lysates plus exogenous CA-074) was subtracted from the contusion-injury
induced increase in fluorescence (injury site lysates without exogenous CA-074).
mRNA Expression and In Vitro Enzymatic Activity
The average level of Cath B mRNA expression and enzymatic activity for the
group of normal animals was determined. The activity levels of individual sham- and
contusion-injured animals were then normalized to this averaged value. All normalized
levels within the sham- and contusion-injury groups were then averaged to generate a
Fold Increase vs. Normal value ( SEM) for that group. All induction values are reported
in terms of Fold Increase vs. Normal. A two-way ANOVA with Tukey's post-hoc test
(SigmaStat Statistical Software, SPSS Inc., Chicago, IL) was used to detect statistically
significant (p < 0.05) differences between the two groups.
Cath B Protein Expression Levels
Levels of Cath B protein in the spinal cord of normal animals (n = 4) were
essentially the same. Thus, the protein expression of individual sham-injured and
contusion-injured animals was normalized to an animal in the normal group. These
normalized values were then averaged within a group (sham- or contusion-injury) to
produce a Fold Increase vs. Normal ( SEM) value. These induction values were also
analyzed via two-way ANOVA with Tukey's post-hoc test (SigmaStat Statistical
Software, SPSS Inc., Chicago, IL). An independent analysis was completed for each of
the three forms of Cath B the 37 kDa inactive proenzyme, the active 30 kDa and 25
In Vivo Enzymatic Activity
Net Cath B enzymatic activity (i.e., activity levels without CA-074 activity levels
with CA-074) was measured in both in vivo CA-074 treatment experiments. In the first
experiment, a two-way ANOVA with Tukey's post-hoc test detected significant (p <
0.05) differences in enzymatic activity levels of the three treatment groups (saline, 12
mg/kg CA-074 and 36 mg/kg CA-074) within a particular route of administration (either
bolus IV, bolus IP, repeated dose IP). Differences between routes of administration were
not assessed. As only one route of administration (bolus IV) was used in the second in
vivo experiment, a one-way ANOVA (Tukey's post-hoc test) was utilized to detect any
significant (p < 0.05) differences among the enzymatic activity levels of the three
treatment groups (saline, 6 mg/kg CA-074 and 12 mg/kg CA-074).
Analysis of Cath B mRNA Expression
Contusion-Spinal Cord Injury Increased Cath B mRNA Levels
Before examining the expression of Cath B, the samples were tested for template
integrity using the housekeeping gene GAPDH. Within each set of reactions, the CPVs
for the three experimental groups (normal, sham- and contusion-injury) are not
significantly different from one another at any of the experimental time points (data not
shown). Having confirmed template integrity, standard curves were used to ascertain the
level of Cath B mRNA expression of the templates within each group. In all three
segments and at nearly every time point examined, Cath B mRNA expression is increased
following contusion spinal cord injury (Figure 3-1, 3-2).
Effects of Sham-Injury
Sham-injury increases the expression of Cath B mRNA although to a lesser degree
than the contusion-injury. At both the injury site (Figure 3-1) and the segment rostral
(Figure 3-2, top panel) to the injury site, the sham-injury induced increase in Cath B
mRNA expression peaks at 48 h post-injury (5.6 fold and 4.1 fold, respectively) and then
returns to near normal levels by the last time point examined (168 h). While the pattern of
Cath B mRNA expression is similar in the injury site and rostral segments following
sham-injury, the response in the caudal (Figure 3-2, bottom panel) segment is different.
Here, sham-injury induction of Cath B mRNA expression appears to be more robust than
in the other two segments. Specifically, in the caudal segment the increase in mRNA
expression is greater than 5 fold at the majority of post-injury time points.
0 -0-, -- r
2 6 24 48 168
Figure 3-1: Sham- and contusion-spinal cord injury induce expression of Cath B mRNA
at the injury epicenter. Increases in Cath B mRNA expression induced by
sham- (yellow bars) and contusion-injury (blue bars) are presented here as
Fold Increase vs. Normal. Cath B mRNA expression is transiently induced
following sham-injury, returning to base line levels by 168 h post-injury.
Following contusion-injury, Cath B mRNA expression increases across the
experimental time line and is significantly (**p < 0.01) greater than the
sham-injury level at 168 h post-injury, where the highest level of mRNA
expression (> 20 fold) of the study is recorded.
Effects of Contusion-SCI
As indicated above, contusion-injury also induces Cath B mRNA expression. At
the injury site, fold increases of 4.2, 10.7 and 19.2 are seen at 24, 48 and 168 h post-
injury, respectively (Figure 3-1). The increase in Cath B mRNA expression following
contusion-injury is significantly greater than sham-injury at 168 h post-injury and is the
highest level of Cath B mRNA expression seen in this study. Within the rostral segment
(Figure 3-2, top panel), contusion-injury significantly increases Cath B mRNA
expression over that seen after sham-injury at 48 and 168 h post-injury. The maximum
contusion-injury induced increase in expression occurs at 48 h post-injury (6.4 fold) and
remains elevated (6.0 fold) at 168 h post-injury. Caudal to the injury site (Figure 3-2,
bottom panel), contusion-injury induces the expression of Cath B mRNA that, like the
sham-injury animals, is generally more robust than observed in the rostral segment. Cath
B mRNA expression is elevated at 24, 48 and 168 h post-injury (> 10 fold), although the
only significant increase over the sham-injury level occurs at 48 h post-injury.
I 0 F .--1-I
2 6 24 48 168
s 1 r
Figure 3-2: Sham- and contusion-spinal cord injury induce Cath B mRNA expression in
the rostral and caudal tissue segments. Increases in Cath B mRNA expression
induced by sham- (yellow bars) and contusion-injury (blue bars) are presented
as Fold Increase vs. Normal. Following contusion-injury, the expression of
Cath B mRNA is significantly (**p < 0. 01) greater than the sham-injury level
at 48 and 168 h post-injury in the rostral segment (top panel). In the caudal
segment (bottom panel), Cath B mRNA expression peaks at 24 h post-injury
but remains nearly as elevated at 48 and 168 h post-injury. A significant
increase is present at 48 h post-injury.
Analysis of Cath B Protein Expression
Cath B Protein Expression is Elevated following Spinal Cord Injury
Figure 3-3 displays representative immunoblots (probed with anti-Cath B antibody)
for the rostral (top panel), injury (middle panel) and caudal (bottom panel) segments of
the spinal cord at 168 h post-injury. Also shown are the GAPDH loading controls.
Normal spinal cord samples produce faint bands while sham- and contusion-injury yield
broader and more intense bands for the proenzyme and the two active forms of Cath B.
37 kDa -- ProCath B
30 kDa -- Single Chain
25 kDa -- Heavy Chain
GAPDH (Loading Control)
37 kDa -- ProCath B
30 kDa --Single Chain
25 kDa -- Heavy Chain
GAPDH (Loading Control)
37 kDa -- ProCath B
30 kDa -- Single Chain
25 kDa -- Heavy Chain
GAPDH (Loading Control)
168 h Post-Injury
Figure 3-3: Sham- and contusion-injury increases levels of all forms of the Cath B
protein. Immunoblots containing normal (N), sham- (S) and contusion-(I)
injury (168 h post-injury) samples are shown. Three bands are detected using
an anti-Cath B antibody: the inactive proenzyme at 37 kDa and two active
mature forms, 30 kDa (single chain) and 25 kDa (double chain). Normal
spinal cord samples produce very faint bands. In the injury site (middle), the
sham-injury animals yield only slightly more dense bands. The contusion-
injury animals, however, have noticeably more intense and broad bands.
These increases also extend to segments rostral (top) and caudal (bottom) to
the injury site. The GAPDH loading control blots are also shown for each
In all three spinal cord segments examined, sham-injury elicits a minimal increase
in Cath B protein levels that rarely exceeds 2 fold. In fact, sham-injury elicits a greater
than 2 fold increase (maximum value was a 2.3 fold) at only 3 of 63 total time points, two
of which occur at the site of the injury. Contusion-injury, however, significantly increases
Cath B protein levels in all three segments, although to different magnitudes. The level of
Cath B proenzyme (37 kDa) at 48, 72 and 168 h post-injury at the injury site (Figure 3-4)
and at 24, 48, 72 and 168 h post-injury in the caudal segment (Figure 3-5, bottom panel)
is significantly elevated which may be indicative of ongoing protein synthesis and/or the
presence of inflammatory cells containing Cath B. No significant change in the level of
proenzyme is seen in the rostral segment (Figure 3-5, top panel). The expression of the 30
kDa active form of Cath B is significantly higher following contusion-injury than sham-
injury at 168 h post-injury in the rostral segment (Figure 3-5), and at 72 and 168 h post-
injury at both the injury site (Figure 3-4) and in the segment caudal to the injury site
(Figure 3-5). The expression of the active 25 kDa form of Cath B in contusion-injured
spinal cord is significantly elevated over sham-injury at 168 h post-injury in the rostral
segment (Figure 3-5) and at the injury site (Figure 3-4) but not in the caudal segment.
Clearly, the largest contusion-injury induced increases in all three forms of the Cath B
protein occur at 168 h post-injury, i.e., > 6 fold for the proenzyme, > 8 fold for the 30
kDa single chain and > 11 fold for 25 kDa double chain.
2 6 12 24 48 72 168
12 30 kDa :
2 6 12 24 48 72 168
1 25kDa T"
2 6 12 24 48 72 168
Figure 3-4: Contusion-injury increases Cath B protein levels at the injury site. The
expression of all three forms of Cath B the proenzyme (37 kDa) and two
active forms of Cath B (30 and 25 kDa) following sham- (yellow bars) and
contusion- (blue bars) are presented for seven post-injury time points. Sham-
injury had little effect on Cath B protein levels. Contusion-injury, however,
significantly increases Cath B expression over sham-injury levels at several
post-injury time points (**p < 0.01). The largest contusion-injury induced
increases in Cath B expression clearly occur at 168 h post-injury where
increases are > 6 fold for the 37 kDa proenzyme, > 8 fold for the 30 kDa
active and > 11 fold for the 25 kDa active forms of the protein.
2 6 12 24 48 72 168
2 6 12 24 48
2 6 12 24 48 72 168
2o ar i r i1 ri iI
2 6 12 24 48 72 168
22 24 4 72
2 6 12 24 48 72 168
Figure 3-5: Contusion-injury increases Cath B protein levels in adjacent segments of the
spinal cord. The Fold Increase vs. Normal values for the sham-injury group
(yellow bars) are compared to the contusion-injury values (blue bars) at seven
post-injury time points. The time course of these changes in the proenzyme
(37 kDa) and two active forms of Cath B (30 and 25 kDa) are presented for
the rostral (left side) and caudal segments (right side). As is the case for the
injury site, sham-injury does not noticeably increase Cath B expression over
normal levels. The maximum increases in expression following contusion-
injury is just over 2 fold in these adjacent segments and is only observed in
only three instances. The differences between the sham- and contusion-injury
groups are significant (**p < 0.01, *p < 0.05) at several time points. No
significant differences in either the proenzyme or the 25 kDa form of Cath B
are present in the rostral and caudal segments, respectively.
Increases in Cath B mRNA Expression and Cath B Proenzyme Expression are
The relationship between the increases in Cath B mRNA and protein expression
following contusion-injury was examined using a regression analysis. Cath B mRNA
expression is the independent factor and Cath B proenzyme (37 kDa) expression is the
dependent factor. Following contusion-injury, the r2 values for the rostral, injury and
caudal segments are .941, 0.971, and 0.844, respectively, suggesting that the level of Cath
B proenzyme increases linearly with the increases in Cath B mRNA. This correlation
does not hold for the sham-injury. Thus, while it appears that sham-injury can induce
Cath B mRNA expression at the injury site to some degree, this increase does not seem to
be sufficient to initiate an upregulation of Cath B protein.
Analysis of Cath B Enzymatic Activity Levels
Contusion-Spinal Cord Injury Increases Cath B Enzymatic Activity
Cath B enzymatic activity levels were determined by the production of a
fluorescent product generated specifically through Cath B-mediated hydrolysis. Levels of
Cath B enzymatic activity following both sham- (yellow bars) and contusion-injury (blue
bars) were measured from tissue lysates of the injury site at five post-injury time points
(1, 2, 3, 5 and 7 d). Cath B enzymatic activity in sham-injured animals is minimally
elevated at every time point, never exceeding 1.18 fold (Figure 3-6). Following
contusion-SCI, Cath B enzymatic activity begins to increase at 3 d post-injury reaching
values of 5.29 and 6.57 fold above normal at 5 and 7 d post-injury, respectively. The
contusion-injury induced increases at these latter two time points are significantly greater
than sham-injury levels. Furthermore, Cath B activity following contusion-injury is not
upregulated in the segments rostral and caudal to the injury site (Figure 3-7). This lack of
an increase in activity is likely due to the smaller increases in Cath B protein and/or the
presence of a smaller number of inflammatory cells in these segments.
Tissue Lysates from Injurn Site
O Sham U Injury
Figure 3-6: Cath B enzymatic activity levels are increased following contusion-injury.
Cath B enzymatic activity levels (reported as Fold Increase vs. Normal)
following both sham- (yellow bars) and contusion-injury (blue bars) were
recorded from injury site lysates at five post-injury time points. Sham-injury
levels are minimally elevated across the experimental timeline. The first
appreciable but not significant increase in Cath B enzymatic activity following
contusion-injury is detected at 3 d post-injury (p < 0.053). Following
contusion-injury, Cath B enzymatic activity increases across the experimental
timeline reaching 5.29 and 6.59 fold at 5 and 7 d post-injury, respectively.
Both of these contusion-injury induced increases are significantly (**p <
0.01) greater than sham-injury levels.
SSham U Injury
[ Sham Injury
Figure 3-7: Contusion-injury does not increase Cath B enzymatic activity levels in
adjacent spinal cord segments. Cath B enzymatic activity levels (reported as
Fold Increase vs. Normal) are presented for lysates of tissue segments
adjacent to the injury site after both sham- (yellow bars) and contusion-injury
(blue bars) at five post-injury time points. Following both sham- and
contusion-injury, Cath B activity levels are indistinguishable from levels of
the normal spinal cord. Statistically significant differences, however, are
present at 3, 5 and 7 d post-injury in the rostral segment.
Increases in Cath B Protein Expression and Cath B Activity Levels are Correlated
Regression analysis reveals the levels of the 30 kDa and 25 kDa forms of Cath B
(Ellis et al., 2004) are positively correlated with the levels of enzymatic activity (r2 =
0.9891 and r2 = 0.9828, respectively) at the injury site.
Immunohistochemical Analysis of Cath B
Cath B Immunoreactivity Appears Restricted to Neurons in the Normal Spinal
To investigate the cellular source of Cath B, frozen sections of adult spinal cord
were double immunostained with anti-Cath B and a variety of anti-cell-specific markers.
In the normal spinal cord, Cath B immunoreactivity (Figure 3-8A) is most prominent in
neuronal cell bodies, which are identified by their morphology and by the neuronal
marker NeuN (Figure 3-8B). The co-localization of staining for both Cath B and NeuN is
presented in Figure 3-8C. These Cath B immunopositive neurons (Cath B+) are found
primarily in the spinal gray matter although an occasional Cath B+ neuron is seen in the
white matter (data not shown).
Figure 3-8: Cath B staining is localized to neurons in the gray matter of the normal spinal
cord. Cath B immunoreactivity (green, A) was localized morphologically to
neurons. Staining for the neuronal marker NeuN (red, B) also co-localized
with Cath B staining and is presented in (C). (The scale bar is equivalent to 50
Cath B immunoreactivity is distinguished by its punctate granular quality (Figure
3-9A), which is characteristic of a lysosomal localization for this protease (Demchik et
al., 1999; Berquin & Sloane 1996). We co-localized a marker specific for lysosomes (i.e.,
lysosomal associated membrane protein-1 or LAMP, Figure 3-9B) with Cath B
immunoreactivity, thereby confirming that Cath B is contained in the lysosomes of
normal healthy neurons (Figure 3-9C).
Figure 3-9: Cath B is staining is distinguished by its lysosomal localization. Cath B
immunoreactivity is characterized by its punctate granular appearance (A),
strongly resembling the staining pattern of lysosomal associated membrane
protein-1 or LAMP (red, B). A merged image of Cath B (A) and LAMP (B)
staining is shown in (C), thereby establishing a lysosomal localization for
Cath B in normal healthy neurons. Negative primary antibody controls
(absence of 1 antibodies) are shown in the insets of (A) and (B). (The scale
bar is equivalent to 50 pu)
Cath B immunoreactivity does not coincide with staining of markers for astrocytes
(GFAP, Figure 3-10A) or resting microglia (OX-42, Figure 3-10B) in normal spinal cord
tissue. The level of Cath B immunoreactivity is also below detection in oligodendroglia
in the normal spinal cord as indicated by the absence of Cath B staining with staining for
MBP (Figure 3-11C). The absence of Cath B immunoreactivity in oligodendroglia is
further confirmed by the lack of Cath B co-localization with other oligodendroglial
markers including CNPase and 04 (data not shown). Finally, there is a low basal level of
Cath B immunoreactivity in the white matter, although it does not co-localize with any of
the cell specific markers used in the study (data not shown).
Figure 3-10: Other cell types in the normal spinal cord do not appear to express Cath B.
Cath B (green) immunoreactivity is absent in non-neuronal cell types in the
normal spinal cord. Cath B staining evident in neurons but does not co-
localize with staining for the astrocytic marker GFAP (red, A), the resting
microglial marker OX-42 (red, B) or the oligodendroglial marker MBP (red,
C) in tissue sections of the normal spinal cord. (The scale bars are equivalent
to 20 pM)
Contusion-Spinal Cord Injury Increases Cath B Immunoreactivity and Alters Cath
Although we previously detected increases in Cath B protein levels following SCI
(Ellis et al., 2004), the source of these increases was not identified. For the present study,
we used immunohistochemical techniques to identify the potential sources) of the
elevation in Cath B at 7 d post-injury. Following contusion-injury, Cath B
immunoreactivity is now evident in non-neuronal cells that essentially replace the entire
spinal cord gray matter and much of the white matter (Figure 3-11B). The normal spinal
cord is presented for reference in Figure 3-11A. By 7 d post-injury, these Cath B+ cells
are so numerous that few, if any, Cath B+ neurons can be found in the injury epicenter.
Often only the central canal (stained blue with DAPI) is discernible (Figure 3-11C).
Figure 3-11: Cath B immunoreactivity is increased and altered following SCI. Cath B
(green) staining is primarily restricted to gray matter neurons in normal spinal
cord tissue (A). The injury site segment is characterized by the presence of
Cath B immunopositive cells (B) by 7 d post-injury. The deterioration of the
spinal cord is so extensive that Cath B+ neurons are absent and only the
central canal stained in blue is discernible (C). (Scale bars represent 50 pMin
panels A-B, 5 pM in panel C)
The Cath B immunopositive cells in the injury epicenter (Figure 3-12A) stain
positively for inflammatory cell markers including OX-42 (Figure 3-12B) and OX-8 and
ED-2 (data not shown). The merged image of these panels demonstrating their co-
localization is shown in Figure 3-12C and magnified for clarity in Figure 3-12D.
Figure 3-12: Cath B immunopositive cells (A) in the injury epicenter are inflammatory in
origin. Cath B+ cells in the injury epicenter stain positively with markers for
inflammatory cells including OX-42 (B). The overlay of these images with the
nuclear stain DAPI is presented in (C) and magnified in (D), thereby
confirming the inflammatory source of Cath B in the post-injured spinal cord
(Scale bars represent 50 pM in panels A-C, 20 pM in panel D)
In regions just adjacent to the injury epicenter, the number of cells
immunopositive for both Cath B and inflammatory cell markers are reduced and
generally are most prominently found in the dorsal columns (Figure 3-13A). In addition,
unlike that seen at the injury epicenter at 7 d post-injury, neurons can still be identified in
regions that are adjacent to the injury epicenter. However, some of these neurons are
more intensely Cath B immunoreactive (Figure 3-13C). In other neurons, there is a shift
in the Cath B staining from its normal punctate character (Figure 3-13B) to a more
uniform distribution throughout the neuronal cell body (Figure 3-13D). This shift in
location of Cath B immunoreactivity with concomitant alterations in LAMP staining
(Figure 3-13E) suggest that the lysosomal membrane has been compromised allowing
Cath B to escape into the cytosol (Figure 3-13F). Following contusion-injury, Cath B
immunoreactivity in astrocytes and oligodendroglia continues to be unremarkable.
Figure 3-13: Cath B expression increases in both neuronal and non-neuronal cells in
tissue segments adjacent to the injury site. Following SCI, Cath B
immunoreactivity is particularly robust in a population of cells in the dorsal
columns (A). Contusion-injury also alters the quality of Cath B staining from
its punctate granular staining in neurons of the normal spinal cord (B). While
Cath B immunoreactivity is more robust in these neurons (C), other neurons
exhibit a less granular and more diffuse pattern of Cath B immunoreactivity
(D). A similar change in LAMP staining (red) is also seen (E), indicating the
release of Cath B from its lysosomal sequestration. An overlay of (D) and (E)
is presented in (F). Morphologically, the neurons in the injured spinal cord (C,
D) also differ from neurons in the normal spinal cord (B). (Scale bars
represent 50 pM in panel A and 20 pM in panels B-F)
Inhibition of Cath B Enzymatic Activity
CA-074 was an Effective Inhibitor of Cath B Enzymatic Activity In Vitro
Cath B enzymatic activity levels were assessed by the production of a fluorescent
product generated specifically through Cath B-mediated hydrolysis. Addition of the
specific, irreversible and cell impermeable Cath B inhibitor CA-074 in a range 0-5000
[LM inhibits the activity of purified Cath B from the bovine spleen in a dose-dependent
manner, demonstrating the potency of CA-074 as a Cath B inhibitor in vitro (Figure 3-
Purified Cath B
0 0.5 5 50 500 5000
Concentration of CA-074 (LPM)
Figure 3-14: CA-074 potently inhibits Cath B enzymatic activity in vitro. Enzymatic
activity levels of purified Cath B (bovine spleen) were assessed by the
generation of a Cath B specific cleavage fluorescent product. The addition of
CA-074 to the reaction vessel in increasing concentrations nearly eliminates
the fluorescent signal generated through Cath B mediated hydrolysis. The
level of fluorescence is -65,000 units in the absence of CA-074 (0 [LM) vs.
-6,000 units in the presence of 5000 [LM CA-074.
CA-074 is an effective inhibitor of Cath B enzymatic activity in vitro
The addition of CA-074 to lysates of the tissue segment containing the injury
epicenter (Figure 3-15) essentially eliminates the contusion-injury induced increases in
Cath B enzymatic activity (previously seen in Figure 3-6). CA-074 reduces Cath B
activity in the injured spinal cord to essentially the level seen in both the normal and
sham-injured spinal cords at all post-injury time points (Figure 3-15), thereby confirming
that the fluorescent signal is from a product of Cath B mediated hydrolysis. Despite the
lack of Cath B enzymatic activity in either the sham- or contusion-injury groups
following CA-074 treatment, the difference between them is statistically significantly at 5
Tissue Lysates from Injury Site
E. OSham MInjury +CA-074
1 2 3 5 7
Figure 3-15: Increases in Cath B enzymatic activity are suppressed by CA-074. The
addition of the specific, irreversible and cell impermeable Cath B inhibitor
CA-074 (25 [tM) completely inhibits the contusion-injury induced increases in
Cath B enzymatic activity previously seen in Figure 3-6. In the presence of
CA-074, the Cath B enzymatic activity levels in the three groups are nearly
In vivo CA-074 Treatment is Ineffective at Reducing Contusion-Injury Induced
Increases in Cath B Enzymatic Activity Levels
Cath B enzymatic activity levels in saline treated animals (black bars) are
independent of the variations in timing and route of administration, indicating that these
variables have little or no effect on Cath B enzymatic activity (Figure 3-16). Conversely,
with two exceptions, treatment with CA-074 increases Cath B enzymatic activity to
levels greater than the saline controls. In both IP groups, treatment with 12 mg/kg CA-
074 (yellow bars) raises Cath B enzymatic activity, although not significantly over
controls. Similarly, animals receiving 36 mg/kg CA-074 IV (red bars) and 36 mg/kg CA-
074 IP have the highest levels of Cath B enzymatic activity in the study and are
significantly greater than their respective saline controls. As for the two exceptions, the
activity levels of the repeated dose 36 mg/kg CA-074 and saline groups are nearly
identical and the 12 mg/kg CA-074 IV dose is the only one to show a tendency to
suppress Cath B enzymatic activity in vivo (23% suppression, Figure 3-16).
Because 12 mg/kg of CA-074 given as a single IV dose was the only regimen that
appears to suppress Cath B enzymatic activity, an additional experiment was designed to
confirm this finding. A lower IV dose of CA-074 (6 mg/kg) was included to determine if
CA-074 was operating on an inverse dose response curve. However, as illustrated in
Figure 3-17, treatment with either 6 mg/kg CA-074 (blue bar) or 12 mg/kg CA-074
(yellow bar) does not have any significant effect on Cath B enzymatic activity. Thus, this
study is unable to replicate the minimal suppression of Cath B enzymatic activity seen
Tissue Lysates from Injury Site
1600 Saline 0 12 mg 36 mg
IV (Bol) IP (Bol) IP (RD)
Route of CA-074 Administration
Figure 3-16: In vivo CA-074 does not inhibit contusion-injury induced increases in Cath
B activity levels. Saline (black bars), 12 mg/kg CA-074 (yellow bars) and 36
mg/kg CA-074 (red bars) were given as a bolus (IV and IP) or a repeated dose
(IP only) injection. All animals received an injection immediately after SCI
while the repeated dose group received additional injections at 24 and 48 h
post-injury. Saline treated animals have similar levels of Cath B enzymatic
activity. Only the 12 mg/kg IV group demonstrates a tendency to suppress
Cath B activity (-23%). Asterisks denote significant differences in activity
levels (*p < 0.05, **p < 0.01) between various treatment groups.
Tissue Lysates from Injury site
Saline 6 mglkg 12 mglkg
Figure 3-17: The suppression of Cath B enzymatic activity with in vivo CA-074 treatment
is not reproduced. A bolus IV injection of saline (black bar), 6 mg/kg CA-074
(blue bar) and 12 mg/kg CA-074 (yellow bar) was given immediately after
SCI. There are no significant differences in levels of enzymatic activity
following either CA-074 treatment.
Mechanical trauma to the spinal cord initiates a complex cascade of biochemical
processes that collectively contribute to neuronal and glial cell death, tissue cavitation
and sensory and motor deficits. Currently, there is limited opportunity to improve and/or
restore function to the individual suffering a SCI. While a number of the
pathophysiological events contributing to the secondary injury have been identified,
Nixon and Cataldo (1993) suggested that lysosomal leakage or rupture represents the
greatest threat to neuronal cell survival. Since that report was published, research on the
role of lysosomal proteases in the etiology of the secondary injury cascade following SCI
has lagged and most investigations have focused on the expression and activity of other
proteases such as the calpains, caspases and most recently, the matrix metalloproteinases.
Consequently, very few studies have been conducted to directly examine the potent
lysosomal protease, Cath B, in CNS trauma. Analyses of changes in mRNA expression
following dorsal root avulsion (Hu et al., 2002), hemisection (Fan et al., 2001) and
peripheral sciatic nerve crush (Fan et al., 2001) suggest that Cath B may play a role in the
injury cascade associated with these insults. However, to date, studies examining Cath B
expression and enzymatic activity following traumatic SCI have not been carried out. The
work described in this dissertation represents the first systematic examination of changes
in Cath B expression, enzymatic activity and cellular localization in the spinal cord
following contusion-injury. In these studies, changes in Cath B were examined at several
post-injury time points, both within and adjacent to the injury site. Preliminary
experiments utilizing the Cath B inhibitor CA-074 were also conducted in an attempt to
determine if Cath B contributes to the pathology of secondary SCI.
Characterization of Cath B following Contusion-Injury
Following contusion-injury, levels of Cath B mRNA were significantly higher in
the injured spinal cord than in the sham-injured cord. Increased transcript levels were
found in all spinal cord segments examined with the largest increase (>20 fold) occurring
at the injury site itself. In general, the levels of Cath B mRNA in segments adjacent to the
injury epicenter were higher in caudal segment than in the rostral segment. While the
underlying conditions within the spinal cord that lead to the differences in Cath B mRNA
expression between these two regions are unknown, other investigators have also noted
regional differences in the response of the spinal cord to injury. For example, Yong et al.
(1998) and Citron et al. (1998) have both reported increased apoptotic cell death in
regions caudal to the injury site than in those rostral to it.
Currently, information regarding the regulation of transcription of the Cath B gene
in the injured CNS is limited. The putative promoter of the Cath B gene contains several
elements common to the promoters of housekeeping genes (e.g., absence of CAAT and
TATA boxes and Sp-1 binding sites) (Gong et al., 1993). However, unlike the ubiquitous
expression patterns observed for housekeeping genes, Cath B mRNA expression varies
considerably across tissues as a function of cell type and differentiation state (reviewed
by Berquin & Sloane 1996; San Segundo et al., 1996). The presence of variable patterns
of expression suggests that additional regulatory elements control expression of Cath B.
Factors that can influence Cath B mRNA expression include cytokines (Gerber et al.,
2000; Li & Bever 1997, 1996; Keppler et al., 1994), phorbol esters (Berquin et al., 1999;
Phillips et al., 1989) and dexamethasone (Hong & Forsberg 1995; Burnett et al., 1986).
The increase in Cath B mRNA seen following SCI may be derived from the well-
characterized inflammatory response that occurs in injured spinal cord tissue. SCI
induces rapid increases in the levels of transcripts encoding several proinflammatory
cytokines including TNF-c, IL-13, IL-6 and IL-la (Bareyre & Schwab 2003; Pan et al.,
2002; Streit et al., 1998; Bartholdi & Schwab 1997; Wang et al., 1996), which can lead to
elevated cytokines levels in the injured tissue (Segal et al., 1997). Links between
increased cytokine expression and the induction of Cath B have been established
experimentally, but the evidence has been generated in studies of non-CNS systems.
Examples include the observations that INF-y induces Cath B transcription in THP-1
cells (human macrophage-like cells; Li & Bever 1997), U-937 monocytes (Lah et al.,
1995), human muscle cells (Gallardo et al., 2001) and in macrophages (Schmid et al.,
2002). In addition, TNF-c, INF-y and IL-1 have all been found to increase secretion of
Cath B from synovial fibroblast-like cells in people with rheumatoid arthritis (Lemaire et
al., 1997; Huet et al., 1993) while IL-13 can stimulate increases in Cath B protein in
rabbit articular chondrocytes (Baici & Lang 1990). Although other regulatory
mechanisms exist, it appears that the induction of Cath B mRNA expression following
SCI may be due, in part, to contusion-injury mediated increases in cytokines.
Levels of Cath B protein (i.e., the proenzyme and both active forms) were also
increased by contusion-SCI paralleling the increases seen in Cath B mRNA expression.
Specifically, Cath B proenzyme increased linearly relative to the increases in Cath B
mRNA as confirmed by high r2 values in the rostral (r2 = .941), injury (r2 = .971) and
caudal (r2 = .844) segments. The highest levels of Cath B protein were observed at the
injury site. Significant increases in Cath B protein were also seen in the segments rostral
and caudal to the injury site, but the magnitude of these increases was minimal (-2 fold).
Importantly, the finding that the levels of both active forms of Cath B increased in the
injured tissues indicates ongoing processing of the Cath B proenzyme in the post-injury
spinal cord and/or a contribution from resident and invading inflammatory cells.
Levels of Cath B Enzymatic Activity
The observed increases in Cath B enzymatic activity at the injury site were not
surprising in view of the increases in Cath B protein expression (particularly the active
forms) described above. There were significant increases in Cath B enzymatic activity at
the injury site on post-injury days 5 and 7. Although not significant, the increases in Cath
B enzymatic activity on post-injury day 3 (>2 fold) coincided with similar increases in
the expression of both active forms of Cath B protein. The increases in Cath B enzymatic
activity were highly correlated with the increases in the 30 kDa (r2 = 0.9891) and 25 kDa
(r2 = 0.9829) forms of Cath B. Interestingly, elevated Cath B enzymatic activity was not
observed in the rostral or caudal segments. The absence of contusion-injury induced
changes in Cath B enzymatic activity in these flanking segments likely reflects the small
increases in Cath B protein (generally < 2 fold) primarily from the smaller number of
inflammatory cells observed in these segments (see below).
The Importance of Inflammatory Cells
What are the cellular sources of the increases in Cath B mRNA and protein
expression and activity in the injured spinal cord? Potential candidates include neurons
and glia within the spinal cord parenchyma and inflammatory cells that damage tissue
following injury. Immunohistochemical analysis showed that in normal, non-injured
spinal cord, Cath B was localized primarily to lysosomes of neuronal cell bodies with
little, if any, Cath B staining in astrocytes, microglia or oligodendroglia. Following
contusion-injury, however, intense Cath B immunoreactivity was seen in the large
numbers of inflammatory cells (e.g., neutrophils, macrophages, activated microglia) that
essentially filled the epicenter of the injury site. Indeed, by 7 d post-injury Cath B
inflammatory cells were the dominant cell type and were uniformly distributed through
the gray and white matter at the injury epicenter to the extent that very few, if any, Cath
B+ neurons could be found. In the segments adjacent to the injury site, smaller numbers
of Cath B inflammatory cells were seen and these appeared to be located primarily in the
dorsal columns. While neurons could be identified in these surrounding segments, many
appeared irregularly shaped with a more intense and uniform (i.e., loss of punctuate
character) Cath B staining. These findings suggest that the contusion-injury induced
increases in Cath B expression and enzymatic activity are primarily due to both
endogenous and invading inflammatory cells rather than to increased synthesis of Cath B
by resident neurons and glia. Indeed, the changes seen in Cath B immunoreactivity in
surviving neurons may signal the death of these neurons due to the release of Cath B
from lysosomes into the cytosol. However, this does not exclude the possibility that
neuronal and glial cells contribute to the increases in Cath B mRNA and protein
expression that were observed immediately following injury.
The presence of large numbers of Cath B+ inflammatory cells in the injured spinal
cord and a delocalization of Cath B in injured neurons is consisted with the proposition
that Cath B may be a contributor to the secondary injury cascade. This hypothesis is
supported by studies that show Cath B expression is significantly elevated in activated
(but not resting) macrophages (Lah et al., 1995; Kominami et al., 1988), and that Cath B
is produced by and released from activated microglia (Reddy et al., 1995; Ryan et al.,
1995). Furthermore, activated microglia produce and secrete various cytokines and
proteases and other molecules such as oxygen species that are also likely to stimulate the
production and secretion of potentially damaging proteases like Cath B (Kim & Ko 1998;
Rothwell et al., 1996; Giulian & Corpuz 1993; Giulian & Vaca 1993). Enzymes produced
by activated microglia have been shown to play a direct role in neuronophagia (Thanos
1991; Banati et al., 1993), in degradation of extracellular matrix (ECM) components
(Nakanishi 2003; Maeda & Sobel 1996; Gottschall et al., 1995) and in neuronal cell death
(Gan et al., 2004; Nitatori et al., 1996). It is of interest that Cath B remains active despite
shifts in pH (Mort et al., 1994; Buck et al., 1992; Lah et al., 1989), a property that would
allow it to continue to hydrolyze carbohydrates, proteins, nucleic acids and ECM
molecules in the post-SCI environment. Reducing or blocking the influx of neutrophils
and macrophages into the injury site following SCI decreases the extent of tissue damage
(Mabon et al., 2000; Popovich et al., 1999; Giulian & Robertson 1997; Hamada et al.,
1996; Blight 1994). Thus, our data confirm the presence of a large population of
inflammatory cells in, and to a lesser degree, around injury site. In addition, we show for
the first time that these cells are intensely Cath B immunoreactive placing this potent
protease in a position where its release could significantly amplify the secondary injury
The Effects of Sham-Injury
Previous studies from our laboratory have shown that exposing the spinal cord by
laminectomy is not a benign procedure, in that it causes a decrease in spinal cord blood
flow, alters energy metabolism and modifies membrane lipid composition (Demediuk et
al., 1987, 1985; Anderson and Means 1985; Anderson etal., 1980, 1978). In addition,
more recent findings from our laboratory demonstrated that this surgical procedure
induced expression of the MnSOD gene in spinal cord tissue (Earnhardt et al., 2002).
While every precaution was taken to minimize damage to the dura mater and underlying
spinal cord while performing the laminectomy, this procedure still cuased an elevation of
Cath B mRNA expression in and around the injury site. However, by post-injury day 7,
Cath B mRNA had returned to normal both at the injury site and rostral to the injury site.
Cath B protein expression was also affected by sham-injury; however, the increases in
protein levels were minor never becoming greater than double that seen in the normal
spinal cord. Consequently, sham-injury did not increase Cath B enzymatic activity. These
findings demonstrate that laminectomy alone can alter Cath B mRNA expression, but this
increase is not translated into increased protein levels or enzymatic activity. Thurs, the
large increases in Cath B mRNA, protein and enzymatic activity seen following SCI are
due to the contusion-injury itself rather than the laminectomy procedure.
Suppression of Cath B Enzymatic Activity
The studies described thus far represent the first in-depth characterization of Cath B
expression in spinal cord following SCI. However, we have not yet identified the specific
post-injury substrates for Cath B nor has the role of this potentially damaging protease
been defined in the secondary injury cascade. Nonetheless, because Cath B has been
reported to contribute the pathobiology of ischemic brain injury (Seyfried et al., 1997;
Yamashima et al., 1996) and other CNS (Kikuchi et al., 2003; Bever & Garver 1995;
Mikkelsen et al., 1995) and systemic (Lah et al., 2000; Campo et al., 1994; Bayliss & Ali
1978) diseases and because our studies place high concentrations of active Cath B in the
injured tissue, it remains a strong candidate for involvement in the tissue destruction that
occurs following SCI. As a first step toward attempting to establish a role for Cath B in
the secondary injury response, experiments were designed to determine if inhibiting Cath
B activity in vivo could modulate the magnitude of the injury response. Our studies were
based upon the previous use of Cath B inhibitors that reduced cell death and tissue loss in
non-SCI models of CNS insult (Seyfried et al., 2001, 1997; Yoshida et al., 2002,
Tsuchiya etal., 1999; Yamashima etal., 1998).
Based upon these previous investigations, we chose the specific Cath B inhibitor
CA-074. The rate of inactivation by CA-074 for Cath B is three orders of magnitude
greater than for other related proteases (Buttle et al., 1992). In addition, its efficacy and
specificity have been demonstrated in many in vivo cell culture applications (Premzl et
al., 2003; Levicar et al., 2003; Montaser et al., 2002; Lah et al., 2000) and in vivo whole
animal models (Ohshita et al., 1992; Towatari et al., 1991).
Our experiments re-confirmed the ability of CA-074 to inhibit Cath B enzymatic
activity. First, CA-074 inhibited the activity of purified bovine Cath B in a dose-
dependent manner. Second, the addition of CA-074 to tissue lysates of the injury site
segment eliminated the contusion-injury induced increases in Cath B enzymatic activity
levels previously described. Thus, in vitro CA-074 reduced the levels of Cath B
enzymatic activity in the injured tissue to those seen in normal and sham-injured controls.
Prompted by the success of these in vitro studies, we examined the in vivo
inhibitory potential of CA-074 in our contusion model of SCI. Initially, CA-074 and
saline were administered using a variety of dosing, timing and delivery strategies with a
working hypothesis that levels of Cath B enzymatic activity in the spinal cord of CA-074
treated animals would be lower than those measured in the saline-treated controls.
However, the degree of CA-074 inhibition of Cath B seen in vitro was not seen in vivo.
Indeed, only one experimental variation produced any discernible suppression of Cath B
enzymatic activity. In fact, with almost every experimental variation, the levels of Cath B
enzymatic activity in the CA-074 treated animals were actually higher than those in
saline-treated controls. Furthermore, the one incidence of CA-074 mediated suppression
was not reproduced in a follow-up experiment. While we have no definitive explanation
for these findings, it is possible that the lack of suppression is, in part, due to the negative
charge of CA-074, which prevents it from passively diffusing across membranes (Bogyo
et al., 2000; Wilcox & Mason 1992). Additional studies are required to test this
Collectively, these studies represent the first in depth characterization of Cath B
expression, enzymatic activity and cellular localization in the contusion-injured spinal
cord. Our data show that contusion-injury increases Cath B expression and enzymatic
activity and further suggest that these increases are due, in large measure, to the presence
of inflammatory cells at the injury site. As a powerful and indiscriminate protease, Cath
B may contribute to the tissue pathology of the secondary injury cascade. The attempt to
confirm its role in secondary spinal cord injury through in vivo inhibition of its activity
was not successful. With so little available information pertaining to the
pharmacodynamics of CA-074, the use of alternative inhibitors coupled with a different
administration protocol may prove more effective in inhibiting Cath B activity in vivo.
Our data suggest that further inhibitory experiments are necessary to implicate Cath B in
the secondary injury response following SCI.
Several other studies must be completed before Cath B can be considered a
potential target for therapeutic intervention. First, Cath B expression should be examined
over the course of a longer post-injury timeline. Second, the pharmacological properties
of CA-074 and other available Cath B inhibitors should be fully characterized using
appropriate in vitro studies. These studies would include examination of the ability of
these agents to cross cell membranes. Third, the tissue distribution and clearance rates of
these inhibitors must be determined in normal and contusion-injured rats for multiple
routes of administration including intrathecal, direct, and intravenous infusion. Finally,
the ability of CA-074 to transverse the blood-brain-barrier in both non-compromised and
compromised states must be assessed. Clearly, these compounds must be able to reach
the source of cellular increases in Cath B expression and activity.
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Rebecca Catherine Ellis was born in Derby, CT, in 1976. During her years at
Shelton High School, Rebecca was an active member of the study body serving as
National Honor Society President, Class Secretary and Treasurer of the Spanish Honor
Society. She also captained the softball and volleyball teams. After graduating with
honors in June 1994, Rebecca enrolled at the College of the Holy Cross in Worcester,
MA. At Holy Cross, she was a member of the Lady Crusader softball team and conducted
research examining the effects of steroid treatment in multiple animal models of anxiety
under the supervision of Dr. Daniel Bitran. After graduating from Holy Cross in May
1998 with a B.A. in biology, Rebecca spent the summer in the Netherlands where she
coached and played for the Islanders, the hometown team of a small village south of
Rotterdam. She returned in August of 1998 and entered the Interdisciplinary Program
(IDP) in Biomedical Sciences at the University of Florida. In May of 1999, she joined the
laboratory of Dr. Douglas K. Anderson. During her dissertation work, Rebecca has
focused on characterizing the expression and activity profiles of the powerful protease
cathepsin B following contusion-spinal cord injury.