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©2011
Audrey Pham Le
ALL RIGHTS RESERVED
REGULATION OF PRONGF PROCESSING AND ITS EFFECTS ON
P75NTR-MEDIATED CELL DEATH FOLLOWING SEIZURES
By Audrey P. Le
A dissertation submitted to the
Graduate School-Newark
Rutgers, The State University of New Jersey
in partial fulfillment of the requirements
for the degree of
Doctor of Philosophy
Graduate Program in
Integrative Neuroscience
written under the direction of
Dr. Wilma J. Friedman
and approved by
________________________
________________________
________________________
________________________
Newark, New Jersey
May, 2011
ii
ABSTRACT OF THE DISSERTATION
Regulation of proNGF Processing and its
Effects on p75NTR-Mediated Cell Death Following Seizure
By Audrey Pham Le
Thesis Director: Dr. Wilma J. Friedman
Nerve growth factor (NGF) has been known to play critical roles in
neuronal survival and differentiation during development. Recent studies have
discovered that its immature form, proNGF, is a ligand for the p75 neurotrophin
receptor (p75NTR). proNGF binding to p75NTR activates apoptotic signaling, and
this binding occurs with a five-fold higher affinity than that of mature NGF (Lee et
al., 2001). This binding preference, along with the increased prevalence of
proNGF after injury (Harrington et al., 2004), creates a cellular environment
susceptible to cell death; thus, the balance between levels of pro- and mature
NGF may be a key factor in determining whether a neuron lives or dies (Lee et
al., 2001; Volosin et al., 2006). Using both in vitro and in vivo methods, this
thesis examined the mechanisms that regulate the extracellular processing of
proNGF and the consequences of that processing on p75NTR-mediated cell death
following injury. The results discussed here demonstrate that 1) proNGF binding
leads to cell death via the p75NTR signaling pathway; 2) after injury, proNGF is
upregulated and preferentially secreted in a functional manner capable of
activating the p75NTR-mediated apoptotic pathway; 3) the enzymes plasmin and
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MMP7 extracellularly cleave proNGF, 4) after injury, plasmin activation and
MMP7 activity are reduced, leading to increased proNGF-induced apoptosis, and
5) restoring plasmin or MMP7 activity following brain injury reduces proNGF
levels and consequently, p75NTR-mediated apoptosis. Overall, these data
suggest that increased cell death following injury may be mediated in part by a
change in the balance between extracellular proNGF and the activity of its
processing enzymes, leading to increased cell death via p75NTR.
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Table of Contents
I. Abstract………………………………………………………………………….ii
II. Table of Contents…………………………………………………………......iv
III. List of Illustrations…………………………………………………………....vi
IV. List of abbreviations………………………………………………………....ix
V. Introduction………………………………………………………………….....1 1. The neurotrophins and their receptors……………………………......2
1.1 Nerve growth factor……………………………………….…..2 1.2 Neurotrophin receptors: trks and
p75NTR……………………………………….…………………5 2. Enzymatic regulation of proneurotrophin cleavage………………..10
2.1 Matrix metalloproteases and tissue inhibitors of metalloproteases…………………………………………….11
2.2 The tissue plasminogen activator (tPA)-plasmin cascade ……………………………………………………..…………..15
3. CNS injury and kainic acid-induced seizures………………………18 4. Significance…………………………………………………………….21
VI. Research Aims………………………………………………………………..23
1. Determine the mechanisms of cell death following kainic acid treatment.……………………………………………..………………..23
2. Examine the expression and activity of MMP7 and TIMP1, and their effects on proNGF processing, in the hippocampus following kainic acid-induced injury.……………………………...…………………….23
3. Determine the expression and activity of the tPA-plasmin cascade in the hippocampus following kainic acid-induced injury ..........................................................................................………...24
VII. Materials and Methods………………………………………………………25
1. Animals and kainic acid-induced seizures………….……………….25 2. Animal surgery and microinjection……………………..…………….25 3. Analysis of CSF………………………………………………………..26 4. Brain preparation for immunohistochemistry…….…………………26 5. In situ zymography…………………………………….………………28 6. Fluoro-Jade B labeling..……………………………..………………..29 7. Organotypic hippocampal slice cultures…………..………………...29 8. Propidium iodide assay…………………………….…………………30 9. Co-immunoprecipitation..……………………………..………………31
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10. Western blot analysis…………………………………………………31 11. Statistical analysis…………………………………………………….32
VIII. Results…………………………………………………………………………33
1. Chapter 1. Mechanisms of p75NTR mediated cell death in the hippocampus following kainic acid-induced injury..........................34
1.1 Hippocampal cell death following kainic acid treatment is p75NTR-dependent…………………………………………..34
1.2 proNGF release increases after kainic acid treatment and causes cell death via p75NTR ……………………………….35
1.3 Kainic acid induces proNGF and p75-mediated apoptosis in the hippocampus in vivo…………………………………37
2. Chapter 2. Expression and activity of MMP7 and TIMP1 from hippocampal cells after kainic acid-induced injury……..................38
2.1 MMP7 and TIMP1 expression and activity following kainic acid treatment in vitro……………………………………….38
2.2 MMP7 and TIMP1 expression and activity following kainic acid treatment in vitro……………………………………….40
2.3 MMP7 protects hippocampal neurons from death following seizure in vivo………………………………………………..41
3. Chapter 3. Effects of the tPA-plasmin cascade on proNGF processing following kainic acid-induced injury……...……………..43
3.1 tPA treatment reduces proNGF production following kainic acid treatment in vitro………………………………………..43
3.2 tPA activity decreases and proNGF and cell death increase following seizure in vivo……………………….…………….44
IX. Discussion.……………………………………………………………………47
X. Conclusions and Future Directions………………………………………56
XI. Figures and Legends………………………………………………………..60
XII. References…………………………………………………………………...111
XIII. Curriculum Vitae…………………………………………………………….134
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IV. List of Illustrations
Figure 1. Schematic of cleavage sites on proneurotrophins…….…..……….60
Figure 2. Model of complex-receptor formation for pro- and mature
NGF…............................................................................................62
Figure 3. Cell death and p75NTR expression following KA treatment in
organotypic hippocampal slice cultures………………..……………64
Figure 4. Cell death following kainic acid treatment is p75NTR
dependent………………………………………………………………66
Figure 5. ProNGF is upregulated and released after kainic acid treatment in
slice culture……..………………………………………….…………..68
Figure 6. Blocking proNGF activity results in reduced cell death and caspase
3 activation……………………………………………………………..70
Figure 7. Secretion of proBDNF and proNT-3 from hippocampal cells
following KA treatment in vitro…………………………….…….……72
Figure 8. In vivo expression of p75NTR and cell death following KA-induced
seizures…………………………………………………….….………..74
Figure 9. Expression of MMP7 and TIMP1 in the CA1 following KA treatment
in vivo……………………………………………….….……….………76
Figure 10. MMP7 and TIMP1 secretion from the hippocampus following KA
treatment………………………………………………..…….………..78
Figure 11. Differential expression of cell death and extracellular proNGF in
KA-treated slice culture…………………………….….….……..……80
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Figure 12. Expression of MMP7, TIMP1 and proNGF following KA-induced
seizure in vivo………………………………………..……...…………82
Figure 13. MMP7 activity in the hippocampus…………………………………84
Figure 14. KA-induced seizure cell death in the hippocampus in
vivo……………………………………………………………………...86
Figure 15. KA-induced p75NTR expression in the hippocampus following MMP7
infusion………………………………………………………………….88
Figure 16. ProNGF expression and release following MMP7
infusion……………………………………….…………………………90
Figure 17. Levels of proBDNF and proNT-3 in the CSF after KA-induced
seizures……………………………………………..……………….….92
Figure 18. KA-induced cell death following tPA treatment in slice
culture…………………………………………………………………...94
Figure 19. Neuroserpin expression and release in response to KA in
hippocampal slice cultures………………………..…..………………96
Figure 20. PAI-1 expression and release in response to KA treatment in
vitro………………………………………………………….................98
Figure 21. tPA activity and neuroserpin expression following KA-induced
seizure in vivo………………………………………...…………….100
Figure 22. In vivo expression of neuroserpin following KA-induced
seizures…………………………………………………..………….102
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Figure 23. PAI-1 expression in the hippocampus following KA-induced
seizures in vivo…………………………………………….…………104
Figure 24. Effect of exogenous tPA infusion on cell death in the CA1 following
KA-induced seizure………………………………………………….106
Figure 25. Levels of proNGF in the CSF following KA-induced seizures
following tPA infusion………………………………………………..109
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V. List of Abbreviations
BDNF bran-derived neurotrophic factor
cc3 cleaved caspase 3
CNQX 6-cyano-7-nitroquinoxaline-2,3-dione
CSF cerebrospinal fluid
EDTA ethylene diamine teraacetic acid
DAB 3,3’Diaminobenzidine
Erk extracellular signal regulated kinase
FJ fluoro-jade
IP intraperitoneal
IC intracerebral
HBSS Hank’s balanced salt solution
JNK c-Jun-N-terminal kinase
KA kainic acid
MEM minimum essential medium
NMDA N-methyl-d-aspartate
MAPK mitogen-activated protein kinase
MMP matrix metalloproteinase
NGF nerve growth factor
NT-3 neurotrophin 3
NT4/5 neurotrophin 4/5
NTR neurotrophin receptor
x
NgR Nogo receptor
NADE p75NTR-associated death executor
NRAGE neurotrophin receptor-interacting MAGE homolog
NRIF neurotrophin receptor interacting factor
p75NTR p75 neurotrophin receptor
PAI plamsinogen activator inhibitor
Pcsk6 proprotein convertase subtilisin/kexin type 6
PI-3k phosphoinositide 3-kinase
PLC-γ phospholipase c gamma
SE status epilepticus
serpin serine protease inhibitor
SFM serum free media
TIMP tissue inhibitor of metalloproteinases
Trk tropomyosin receptor kinase
1
VI. Introduction
Neurotrophins are a family of growth factors that is abundantly expressed
in a variety of cell populations in the nervous system. During development, they
promote neuronal survival, proper synaptic formation, differentiation, and cell
maintenance. Members of this family include nerve growth factor (NGF) (Levi-
Montalcini, 1964a), brain-derived neurotrophic factor (BDNF) (Leibrock et al.,
1989), neurotrophin-3 (NT3) (Hohn et al., 1990; Maisonpierre et al., 1990b) and
neurotrophin 4/5 (NT4/5) (Hallbook et al., 1991; Ip et al., 1992; Wong et al.,
1993). Each neurotrophin specifically binds with a particular member of the
tropomyosin receptor kinase (trk) family of receptors to activate well-defined
signaling pathways involved in cell differentiation and survival (Chao, 1992).
Additionally, all neurotrophins may interact with the p75 neurotrophin receptor
(p75NTR), particularly when in their pro-form (Lee et al., 2001b; Roux and Barker,
2002), to induce apoptosis (Chao, 1992; Friedman, 2000). Neurotrophins can be
released from cells in their mature or pro-form, suggesting that the extracellular
balance between mature and proneutrophins plays a critical role in determining
whether they preferentially interact with trk or p75NTR to promote survival or
death, respectively. Thus, elucidating the mechanisms of neurotrophin
processing is an important step towards understanding the signaling pathway
that takes a cell towards survival or death. The work in this thesis examined
changes in the extracellular environment following injury that affect the
processing of proNGF, as well as the consequences of those changes on
p75NTR-mediated apoptotic signaling.
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1. The Neurotrophins and their Receptors
1.1 Nerve Growth Factor
Neurotrophins are a family of growth factors that regulate a panoply of
cellular activities, including development, migration, neurite outgrowth, synapse
formation and proliferation. Mammals use four members of the neurotrophin
family: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF),
neurotrophin 3 (NT-3) and neurotrophin 4/5 (NT-4/5) (Levi-Montalcini, 1964b;
Ernfors et al., 1990; Maisonpierre et al., 1990a; Hallbook et al., 1991). Two
additional neurotrophins, neurotrophin 6 (NT-6) and neurotrophin 7 (NT-7) have
been identified in fish (Gotz et al., 1994; Lai et al., 1998). All neurotrophins share
approximately a 52% sequence identity at the amino acid level and function in
several, often overlapping, cell populations throughout the nervous system
(McDonald et al., 1991; Robinson et al., 1995; Heinrich and Lum, 2000).
NGF was the first identified neurotrophin and continues to be one of the
more extensively characterized factors. It plays crucial roles in maturation,
differentiation and survival throughout the nervous system, particularly during
development (Levi-Montalcini, 1987). Early withdrawal of NGF in the developing
embryo results in diminished limb growth and improper sympathetic ganglia
innervation (Hamburger and Levi-Montalcini, 1949). In the adult brain, NGF
retains essential functionality in various processes including learning (Koh et al.,
1989; Henriksson et al., 1992), memory (Fischer et al., 1991; Fischer et al.,
1994), and pain perception (Adler et al., 1984; Safieh-Garabedian et al., 1995;
3
Larsson et al., 2009). In addition, altered levels of NGF have been detected in
various disease conditions such as schizophrenia, glaucoma, epilepsy and
Alzheimer’s (Gall and Isackson, 1989; Mufson et al., 1991; Woolf et al., 1994;
Chen et al., 1997; Fiore et al., 1999; Zeng et al., 2004; Sposato et al., 2008).
Mature neurotrophins are synthesized as 30-35 kDa pre-pro-proteins
(Suter et al., 1991; Heymach and Shooter, 1995). The pre- domain contains a
signaling peptide at its N-terminal that targets the pre-pro-neurotrophin to the
endoplasmic reticulum where the pre- domain is removed, producing 26-28 kDa
proneurotrophins. These pro-forms may then be sorted to the Golgi where the
prodomain is glycosylated, and then undergoes further cleavage to produce 14
kDa mature neurotrophins (Bresnahan et al., 1990; Seidah et al., 1996;
Marcinkiewicz et al., 1999) (figure 1). Earlier studies examining the sorting and
release of neurotrophins found NGF, BDNF and NT-3 to be similarly sorted to a
regulated secretory pathway (Heymach et al., 1996). However, others have
found that NGF is generally sorted to the constitutive pathway, where it
undergoes intracellular cleavage by convertases such as furin or proprotein
convertase subtilisin/kexin type 6 (Pcsk6) before release (Seidah et al., 1996).
BDNF was later confirmed to be released via the secretory pathway (Mowla et
al., 1999). Because all of these studies used different overexpression systems to
examine the localization and release of neurotrophins, and because the
intracellular efficiency of neurotrophin processing, particularly BDNF, remains
controversial (Matsumoto et al., 2008; Yang et al., 2009b), further investigation is
required.
4
For many years, the pro- form of neurotrophins was thought to be merely
a transient stage in the maturation process, and the fully processed neurotrophin
believed to be the only biologically active and secreted form of the protein
(Rattenholl et al., 2001). However, investigation into neurotrophin expression
found that the pro-forms can also be released from cells (Heymach and Shooter,
1995; Lee et al., 2001b) and account for over 50% of secreted neurotrophins,
particularly from neurons (Heymach et al., 1996; Farhadi et al., 2000; Mowla et
al., 2001). Furthermore, proNGF was found to be extremely abundant in the CNS
tissues of mouse, rat and human, often where mature NGF was undetectable
(Fahnestock et al., 2001). This led to the discovery that proNGF can also be
secreted from cells, functioning as a high-affinity ligand for the p75 neurotrophin
receptor (p75NTR) to induce apoptosis (Lee et al., 2001b; Beattie et al., 2002;
Harrington et al., 2004).
Since then, increased proNGF levels have been detected in human cortex
during the prodromal stage of Alzheimer’s Disease (Pang et al., 2004), after
spinal cord injury in vitro (Beattie et al., 2002), in the basal forebrain after kainic
acid-induced seizure (Volosin et al., 2006) and in the hippocampus after
pilocarpine-induced seizure (Volosin et al., 2008). Furthermore, disrupting the
interaction between proNGF and p75NTR after corticospinal axotomy (Harrington
et al., 2004) or seizure (Volosin et al., 2008) results in significantly reduced cell
death, confirming that endogenous, secreted proNGF is a death-inducing ligand
for p75NTR-mediated apoptotic signaling after injury.
5
1.2 Neurotrophin Receptors: Trks and p75NTR
Neurotrophins can bind to two types of receptors: tropomyosin receptor
kinases, or trks, and p75NTR, a member of the tumor necrosis factor receptor
(TNFR) superfamily (Friedman and Greene, 1999). In their processed, mature
form, each neurotrophin preferentially binds particular trk receptors with high
affinity. NGF binds trkA (Kaplan et al., 1991b), and cleavage of proNGF to NGF
is required for trkA activation (Boutilier et al., 2008). BDNF and NT-4/5 bind trkB
(Klein et al., 1991), and NT-3 preferentially activates trkC (Lamballe et al., 1991),
but may also bind trkB (Glass et al., 1991; Squinto et al., 1991). NT-3 and NT-4/5
can also bind to trkA, but with a much lower affinity than NGF (Kaplan et al.,
1991a). Binding to trk receptors results in receptor dimerization and the
phosphorylation of tyrosine residues in the cytoplasmic domain (Jing et al.,
1992). Following phosphorylation, trk signaling may activate PI-3 kinase,
MAPK/Erk or PLC-γ, signaling pathways well known for their roles in mediating
neuronal differentiation and survival (Kaplan and Miller, 2000; Patapoutian and
Reichardt, 2001).
Neurotrophin signaling through trk receptors is a hallmark of proper
development, while trophic withdrawal functions in parallel as a necessary cell
death mechanism (Ernfors et al., 1992; Ringstedt et al., 1993; Deppmann et al.,
2008). However, p75NTR–induced signaling has also been shown to initiate
programmed cell death during development, serving as an alternate method of
neuronal pruning that is activated upon a lack of proper trk expression and
6
cognate neurotrophic support (Frade and Barde, 1999; Frade et al., 1999;
Majdan and Miller, 1999; Singh et al., 2008).
Mature neurotrophins can bind p75NTR, but with a much lower affinity than
to their respective trk receptor, a characteristic that led to p75NTR being initially
named “the low-affinity neurotrophin receptor” (Chao et al., 1986). However,
neurotrophin binding affinity can be modulated by trk interaction with p75NTR. For
example, NGF binds with increased specificity to a trkA-p75NTR complex than to
trkA alone (Hempstead et al., 1991; Benedetti et al., 1993; Esposito et al., 2001).
Furthermore, following the discovery that the pro-forms of neurotrophins carry
biologically active functions, it was revealed that proneurotrophins bind to p75NTR
with a five-fold higher affinity compared to their respective trk receptors (Lee et
al., 2001a; Chao and Bothwell, 2002) due to the formation of a complex with the
co-receptor sortilin (Nykjaer et al., 2004; Tauris et al., 2011). Sortilin binds the
pro-region of neurotrophins while p75NTR interacts with the mature domain,
creating a ternary death receptor complex and greatly increasing the affinity
between neurotrophins and p75NTR (Nykjaer et al., 2004; Teng et al., 2005).
Sortilin knockout mice display reduced neuronal apoptosis, similar to levels
observed in p75NTR knockout mice (Jansen et al., 2007), demonstrating that this
receptor complex formation is critical for proper regulation of p75NTR-mediated
programmed cell death during development (Koshimizu et al., 2009; Yang et al.,
2009a). Moreover, sortilin signaling has also been shown to be essential for cell
death following corticospinal lesion and age-dependent neurodegeneration
7
(Jansen et al., 2007), suggesting sortilin is a general p75NTR signaling partner in
proneurotrophin-induced apoptosis (figure 2).
Like the other members of the TNFR superfamily, p75NTR contains an
intracellular “death domain” near the C-terminus, and cleavage of this domain
has been shown to be necessary for p75NTR -mediated apoptotic signaling
(Majdan et al., 1997; Kenchappa et al., 2006). However, p75NTR itself lacks
enzymatic activity and signals by recruiting various intracellular adapter proteins,,
many of which have been linked to apoptosis. For example, the zinc finger
protein, neurotrophin receptor interacting factor (NRIF), has been shown to
interact with p75NTR to signal cell death (Casademunt et al., 1999). In vitro,
p75NTR-mediated cell death was not detected in neurons from NRIF-/- mice
following trophic withdrawal (Linggi et al., 2005) or treatment with proNGF
(Volosin et al., 2008). Furthermore, following pilocarpine-induced seizures in
vivo, NRIF-/- mice demonstrated decreased cell death compared to wild-type
mice, at a level comparable to those of p75NTR-/- mice (Volosin et al., 2008).
Other adapter proteins that are recruited to p75NTR to initiate apoptotic signaling
include the p75NTR-associated death executor (NADE) (Yi et al., 2003) and the
neurotrophin receptor-interacting MAGE homolog (NRAGE) (Salehi et al., 2000).
Neurotrophin-stimulated p75NTR apoptotic signaling leads to the
phosphorylation of c-Jun-N-terminal kinase (JNK) as first demonstrated in
oligodendrocytes, which lack trkA (Casaccia-Bonnefil et al., 1996). However,
investigation into the downstream events following neurotrophin signaling in cells
containing both trks and p75NTR have revealed a crosstalk between the survival
8
and apoptotic pathways elicited by each receptor. For example, in
oligodendrocytes with virally expressed trkA, NGF treatment halted JNK
phosphorylation, increased the extracellular signal-regulated kinase (ERK1)
activity, and abolished p75NTR-mediated apoptosis (Yoon et al., 1998). Similarly,
in sympathetic neurons, which express trkA and p75NTR, but not trkB, NGF
treatment also prevented JNK phosphorylation and p75NTR-mediated cell death
(Bamji et al., 1998). Conversely, BDNF treatment did lead to JNK activation and
p75NTR-mediated apoptosis in the same cells, likely due to the lack of trkB and
the presence of p75NTR activation in the absence of competitive trophic signaling
(Bamji et al., 1998; Kohn et al., 1999). Thus, the outcome of neurotrophin
signaling is selectively influenced by the presence of particular trk receptors and
their ability to inhibit JNK activation.
The convergence of trk and p75NTR signaling pathways has also been
examined in basal forebrain neurons, which, unlike sympathetic neurons,
express all three trk receptors and p75NTR throughout life. Also unlike
sympathetic neurons, the activation of trk signaling alone in basal forebrain
neurons was not enough to inhibit neurotrophin-induced cell death; activation of
the PI3 kinase-Akt pathway by mature BDNF was also required to overcome
apoptotic signaling (Volosin et al., 2006). Moreover, in the presence of both
proNGF and BDNF, proNGF signaling through p75NTR suppressed BDNF-trk
activation of Akt, favoring cell death in basal forebrain neurons (Song et al.,
2010). Taken together, these results indicate that the final outcome of
neurotrophin signaling is highly dependent upon cellular context.
9
The importance of p75NTR signaling during development has also been
illustrated using knockout mice. Two groups have generated p75NTR mutant mice
that lack either exon III (Lee et al., 1992) or exon IV (von Schack et al., 2001) of
the gene, resulting in a missing NGF-binding domain. Both knockout forms
manifest posterior limb ataxia and a severe loss of peripheral sensory neurons
and nerve volume. Mice carrying the exon III deletion have also been observed
to have more cholinergic neurons in the basal forebrain (Van der Zee et al.,
1996), as well as increased basal forebrain volume (Yeo et al., 1997) compared
to wild-type mice. Furthermore, transgenic mice over-expressing p75NTR
demonstrate extensive cell loss throughout the CNS (Majdan et al., 1997). Taken
together, these studies illustrate the importance of p75NTR signaling in proper
development.
In addition to trks and sortilin, p75NTR can form signaling complexes with
other receptors to mediate a wide range of biological functions, such as Nogo
receptor (NgR) to modulate neurite outgrowth or Ephrin-A to signal axon
repulsion (Wang et al., 2002; Lim et al., 2008). In the hippocampus, p75NTR
binding to proBDNF plays an important role in facilitating LTD (Woo et al., 2005).
In the PNS, p75NTR signaling has also been shown to recruit Par-3 and to form a
complex necessary for the establishment of polarity and proper Schwann cell
myelination (Chan et al., 2006).
Nonetheless, the most extensively characterized role of p75NTR is in
mediating apoptosis (Chao, 1992; Barrett and Bartlett, 1994). In addition to its
function in developmental programmed cell death, p75NTR–mediated apoptosis
10
has also been described in other systems, including oligodendrocyte (Casaccia-
Bonnefil et al., 1996) and photoreceptor cultures (Srinivasan et al., 2004), two
different seizure models (Roux et al., 1999; Troy et al., 2002), ischemia (Park et
al., 2000), multiple sclerosis (Nataf et al., 1998; Chang et al., 2000), Alzheimer’s
disease (Edwards et al., 1988; Kerwin et al., 1992), and corticospinal axotomy
(Giehl et al., 2001; Harrington et al., 2004). Not coincidentally, these conditions
correspond with those in which increased proneurotrophin levels are also
detected. These studies and others have created a large body of research
suggesting that the apoptotic consequence of p75NTR signaling in pathological
conditions is dependent upon the availability of proneurotrophins. If changes in
the proportion of pro- and mature NGF, with consequent differences in p75NTR
binding affinity, create an environment more susceptible to cell death, particularly
in disease states or after injury, it will be important to understand the
mechanisms of pro- and mature NGF production under such conditions. We
suggest that the regulation of that balance may be key in determining the
difference been cell death or survival after injury.
2. Enzymatic regulation of proneurotrophin cleavage
The pro-forms of neurotrophins are potent, high affinity ligands for the
p75NTR-mediated apoptotic pathway. In pathological conditions, proneurotrophins
are induced and released from cells, both in vitro and in vivo, suggesting that the
extracellular proteolysis of proneurotrophins to their mature forms may be a
11
critical checkpoint in determining the extent of cell death after injury. To date, two
enzymes have been identified that can extracellularly cleave proNGF to mature
NGF: matrix metalloproteinase 7 (MMP7) and plasmin (Smith et al., 1995; Lee et
al., 2001b; Watanabe et al., 2008). The existence of other enzymes capable of
cleaving proneurotrophins to their mature forms remains a possibility. This
section discusses the potential contributions of MMP7 and plasmin to
extracellular proneurotrophin processing and cell death.
2.1 Matrix metalloproteinases and tissue inhibitors of
metalloproteinases
The matrix metalloproteinases (MMPs) are a quickly expanding family of
over twenty secreted Zn2+ endopeptidases, classically described as the enzymes
responsible for degrading all of the components of the extracellular matrix
(Birkedal-Hansen, 1993; Wojtowicz-Praga et al., 1997; Malemud, 2006). They
are found in all organisms and contain conserved pro- and catalytic domains.
Each MMP is categorized into a sub-class defined by its substrate specificity (Ra
and Parks, 2007).
Most MMPs are secreted as inactive pro-proteins. Classical activation of
MMPs is referred to as the “cysteine switch,” in which the bond between a
cysteine residue on the pro-region and a zinc ion on the catalytic site is disrupted
(Springman et al., 1990). In vitro, full cleavage of the pro-domain is not always
required to activate the enzyme, only disruption of the cysteine-zinc interaction.
12
However, it is believed that in vivo, MMPs must undergo additional cleavages,
including proteolysis of the pro-domain, to generate a fully processed enzyme
(Nagase et al., 1990; Suzuki et al., 1990). These extra processing steps may be
autolytic (Suzuki et al., 1990) or performed by other MMPs or serine proteases
(Nagase, 1997).
Once active, MMPs act upon a wide variety of substrates beyond
extracellular matrix components. They can process cytokines, receptors and
other peptides to modulate cell signaling in normal physiological processes such
as reproduction, embryonic development and tissue remodeling (So et al., 1992;
Harvey et al., 1995; Parks et al., 2004; Kessenbrock et al., 2010). In addition,
MMP action has also been closely associated with cell migration (Ray and
Stetler-Stevenson, 1995), tumor growth and metastatic activity (Liotta et al.,
1991; Wojtowicz-Praga et al., 1997), and neurotoxic related death (Jourquin et
al., 2003).
MMPs can also act upon secreted proneurotrophins. Specifically, proNGF
and proBDNF both contain consensus sites for cleavage by MMP7, which
effectively processes both proneurotrophins to their mature forms (Smith et al.,
1995; Lee et al., 2001b). MMP7 (also known as matrilysin) is the smallest of the
MMPs at 19 kDa due to the lack of a COOH-terminal domain. Also unlike most
other MMP family members, MMP7 is constitutively expressed in most adult
tissues (Wielockx et al., 2004). It can break down a wide range of substrates
including casein, proteoglycans, fibronectin, and elastin (Woessner and Taplin,
1988; Miyazaki et al., 1990; Fosang et al., 1992; Hardingham and Fosang, 1992;
13
Imai et al., 1995), as well as activate the pro-forms of gelatinases MMP2 and
MMP9 (von Bredow et al., 1998). It is secreted from cells in its pro-form, and
although proMMP7 can be activated by enzymes such as trypsin and MMP3 in
vitro, its mechanism of activation in vivo remains unclear (Imai et al., 1995; Ra
and Parks, 2007).
MMP activity is endogenously inhibited by tissue inhibitors of
metalloproteinases, or TIMPs. To date, four TIMPs have been identified and are
named TIMP1-TIMP4 in order of discovery. To inhibit MMP activity, TIMPs bind
MMPs in a 1:1 or 2:2 stoichiometric fashion (Nagase and Brew, 2003) forming a
complex that covers the active site cleft (Gomis-Ruth et al., 1997). TIMPs may
bind to either the mature or the pro-form of MMPs, and each TIMP has the
capability to inhibit several different MMPs. In particular, TIMP1, which is the
largest of the TIMPs at 28 kDa (Carmichael et al., 1986), can inhibit MMP7, as
well as other MMPs, including MMP1, -2, -3, -9, -13, and -14. To inhibit MMP7
activity, TIMP1 binds to its pro-form, both blocking possible substrate interaction
and disallowing the final cleavage steps required for its activation (Gogly et al.,
2009).
TIMP1 function has been linked to myriad biological processes throughout
the body, including tissue remodeling and growth factor induction, as well as
pathological conditions, including inflammation, angiogenesis and tumor cell
metastasis (Gomez et al., 1997; Crocker et al., 2006). In the CNS, TIMP1 is
associated with neuroprotection, such as maintaining the blood-brain barrier and
mediating excitotoxic stress (Tan et al., 2003). TIMP1 also manifests inhibitory
14
effects on tumor growth and angiogenesis (London et al., 2003), and transfection
of TIMP1 into tumor cells has been shown to inhibit invasive metastatic activity in
cell lines (Kawamata et al., 1995; Jee et al., 2006). Due to their modulating
effects on malignant cell progression, the use of MMP inhibitors has been widely
hypothesized as a possible treatment for cancer (Yoon et al., 2003).
However, TIMP1 expression is not always associated with beneficial
consequences. TIMP1 mRNA and protein levels are strongly upregulated in the
forebrain and hippocampus following kainic acid-induced seizure, and this
expression coincides with maximal levels of cell death (Rivera et al., 1997).
Likewise, TIMP1 knockout mice demonstrate resistance to cell death (Jourquin et
al., 2005). TIMP1 expression is also strongly correlated with malignancy in
particular forms of glioma (Groft et al., 2001), and is also elevated in the brains
and CSF of patients with Parkinson’s disease (Lorenzl et al., 2003) and multiple
sclerosis (Kouwenhoven et al., 2001).
The upregulation of TIMP1 found in these and other disease states
suggests that an imbalance between MMP activity and associated TIMP
inhibition may be a direct cause of decreased cell viability in pathological
conditions. In the case of MMP7 and TIMP1, increased MMP7 levels have been
shown to be necessary for pro- to mature NGF conversion in vivo, and these
levels inversely corresponded to TIMP1 expression (Kendall et al., 2009).
Furthermore, MMP7 production and activity was found to be significantly
decreased in both rat and clinical patient models of diabetic retinopathy, leading
to proNGF accumulation and enhanced retinal neurodegeneration via p75NTR (Ali
15
et al., 2011). Together, these data suggest that the activity of MMP7 and its
inhibitor, TIMP1, is a system that directly influences the proapoptotic or
prosurvival effect of p75NTR signaling.
2.2 The tissue plasminogen activator (tPA)-plasmin cascade
Another enzyme system linked to the extracellular proteolysis of
proneurotrophins is the tPA-plasmin cascade (Lee et al., 2001a; Bruno and
Cuello, 2006). tPA, or tissue plasminogen activator, is a serine protease found in
various mammalian tissues, including the brain (Tsirka et al., 1995; Bruno and
Cuello, 2006). Once secreted from a cell, tPA can catalyze the conversion of the
single-chain protein plasminogen to the double-chain active protease plasmin.
TPA and plasmin are most commonly associated with the pharmacological lysis
of blood clots, particularly following embolic or thrombotic stroke (Papadopoulos
et al., 1987). However, tPA therapy has also demonstrated neuroprotective
effects in a mouse model of multiple sclerosis (East et al., 2005), on β-amyloid
neurotoxicity (Ledesma et al., 2000; Jacobsen et al., 2008), on remyelination and
axonal regeneration after sciatic nerve crush (Zou et al., 2006), and following
hypoxic injury (Echeverry et al., 2010).
tPA-plasmin cascade activity has been shown to be important in motor
learning in adults (Seeds et al., 1995), tissue remodeling (Liu et al., 1995),
responding to stress (Skrzypiec et al., 2008), and proper development of
binocular vision during the critical period (Mataga et al., 2004). These studies
16
and others emphasize the role of tPA in neuronal plasticity (Basham and Seeds,
2001; Nagappan et al., 2009) and in fact, plasmin cleavage of proBDNF to BDNF
is essential for hippocampal long-term potentiation (LTP) (Pang et al., 2004).
Conversely, proBDNF was found to produce a physiologically opposite effect, as
perfusion of uncleavable proBDNF induced p75NTR–dependent long-term
depression (LTD) in hippocampal slices (Woo et al., 2005). These physiologically
opposing effects between the pro- and mature forms of the protein demonstrate
that elucidating the mechanisms of plasmin activity on neurotrophin processing
will be crucial for understanding synaptic modulation.
The tPA-plasmin cascade is inhibited at the level of tPA by the serine
protease inhibitor (serpin) neuroserpin (Hastings et al., 1997; Osterwalder et al.,
1998). In the adult, neuroserpin has been detected in neurons throughout the
brain and spinal cord, particularly where tPA is also located (Hastings et al.,
1997), and neuronal depolarization results in the concomitant release of tPA and
neuroserpin (Gualandris et al., 1996; Berger et al., 1999). Furthermore, tPA and
neuroserpin also colocalize with plasminogen and proNGF (Bruno and Cuello,
2006), all of which are secreted in conjunction upon stimulation, indicating a
tightly regulated system between plasmin cleavage of proNGF, its activation by
tPA, and a closely corresponding inhibitory loop regulated by neuroserpin (Bruno
and Cuello, 2006). Thus, proNGF processing may also depend upon the balance
between tPA and neuroserpin activity.
Two other serpin family members are also capable of inhibiting tPA
activity: plamsinogen activator inhibitor (PAI) -1 and -2. PAI-2 is produced by the
17
placenta and thus only detectable in the blood during pregnancy (Kruithof et al.,
1987). PAI-1 is expressed throughout the body and binds directly to tPA in a 1:1
stoichiometric complex to suppress activity (Lindahl et al., 1990). PAI-1 mRNA
and protein levels have been found to be elevated twenty-four hours following
ischemic injury and kainic acid-induced seizures, corresponding with decreased
tPA activity (Salles and Strickland, 2002; Kim et al., 2011). Furthermore, tPA:PAI-
1 complexes are found throughout multiple sclerosis lesions, suggesting a role
for PAI-1 in exacerbating axonal injury (East et al., 2005). However, the
functional roles of PAI-1 extend beyond that of inhibiting plasminogen activation.
Independent of tPA expression, PAI-1 is rapidly increased in response to injury
and inflammation (Kawasaki et al., 2000; Del Signore et al., 2006). Also in
response to inflammation, PAI-1 can act as a chemoattractant, mediating cell
migration by inducing changes in cell morphology, receptor expression, and
cytoskeletal organization (Degryse et al., 2001). In opposition to the thrombolytic
effects of tPA, PAI-1 activates the coagulation pathway (Chambers, 2003), but
may also function in conjunction with tPA to coordinate macrophage migration
(Cao et al., 2006), an effect that is also seen in the invasive nature of certain
types of cancer (Caccamo et al., 1994; Chazaud et al., 2002). In a mouse model
of Alzheimer’s disease, elevated proNGF and p75NTR levels (Cuello et al., 2007;
Capsoni et al., 2010), as well as decreased tPA axis activity (Melchor et al.,
2003), suggest that PAI-1 upregulation contributes to cell death in this disease.
Although heightened levels of PAI-1 have been found in the CSF of Alzheimer’s
patients (Sutton et al., 1994), conflicting studies suggest that PAI-1 is not
18
consistently associated with (Hino et al., 2001), or involved at all (Fabbro and
Seeds, 2009), in the pathological phenotype. Considering that all of the
components of the tPA-plasmin cascade, including its inhibitors, PAI-1 and
neuroserpin, display both protective and detrimental effects in the brain, it will be
relevant to examine the relationship between the tPA axis and neurotrophic
activity following injury, clarifying the mechanisms of plasmin-dependent
neuronal survival.
Unlike with proBDNF, the relationship between plasmin and proNGF
processing has remained relatively unexplored. However, this common
processing pathway suggests that improper proNGF cleavage by the tPA-
plasmin cascade may also significantly affect several aspects of cellular function.
We suggest that decreases in tPA activity following injury may prompt a
reduction in proNGF processing and thus an increase in the pro- to mature NGF
ratio, negatively impacting cell viability via increased p75NTR signaling.
3. CNS Injury and Kainic Acid-Induced Seizures: The Role of Neurotrophins
and p75NTR
NGF protein expression is increased after various forms of neuronal injury,
including corticospinal axotomy (Harrington et al., 2004), seizure (Ballarin et al.,
1991; Katoh-Semba et al., 1999) and Alzheimer’s Disease (Fahnestock et al.,
2001; Cuello and Bruno, 2007). Although NGF is known to promote cell survival
in neurons expressing trkA, pro- and mature NGF release is still increased after
19
injury in areas where this receptor is lacking (Ernfors et al., 1989; Roux et al.,
1999; Fahnestock et al., 2001). Additionally, p75NTR expression is also increased
after injury in these same areas that lack trk expression (Roux et al., 1999; Oh et
al., 2000), implicating a role for p75NTR-mediated signaling in the injury response.
Seizure models are commonly used to study injury-induced damage in the
brain (Turski et al., 1985; Vincent and Mulle, 2009), and cell death following
seizures has been strongly tied to p75NTR signaling. For example, pilocarpine-
induced seizures have been shown to increase p75NTR expression in apoptotic
hippocampal neurons (Roux et al., 1999), and mice lacking p75NTR demonstrate
significantly reduced neuronal death compared to wild-type mice (Troy et al.,
2002).
Kainic acid (KA), a glutamatergic agonist, is another widely used
neurotoxin for studying seizure-induced cell death. To induce seizures, kainic
acid can be injected intraperitoneally (IP) or intracerebrally (IC), including into the
ventricles (ICV) or amygdala (ICA). IC injection results in the most rapid and
severe seizure effects, including full body tremors, foaming at the mouth, bulging
eyes, necrosis at the injection site and in the pyriform cortex, and extensive
degeneration, gliosis and atrophy throughout the hippocampal region (Schwarcz
et al., 1978). IP injection of kainic acid produces similar motor and EEG
symptoms, but culminates in more mild sequelae, particularly reduced neuronal
damage that is also more symmetrical compared to IC injections (Ben-Ari et al.,
1980). The in vivo experiments in this thesis utilized IP injections to examine the
20
mechanisms that regulate apoptosis following seizure, as IC injections obscure
the difference between seizure-induced cell death and direct KA toxicity.
Kainic acid acts through AMPA/kainate receptors, and continued
stimulation of these receptors can lead to extended kindling and epileptic
behavior (Ernfors et al., 1991). KA-induced seizures evoke upregulation of
neurotrophin mRNA and protein, as well as cell death, in the dentate and CA1
areas of the hippocampus (Gall and Isackson, 1989; Frade et al., 1996; Rao et
al., 2006). Moreover, studies from our lab have confirmed that kainic acid-
induced seizures elicit the production of proNGF, leading to p75NTR-mediated
signaling and, consequently, increased cell death (Volosin et al., 2006).
Although increased mRNA and protein levels in tissue are indicative of
neurotrophin production, they do not necessarily evince what form of the
neurotrophin is released from cells following seizure. Examination of the CSF of
rats that had undergone seizure treatment revealed increased levels of proNGF
compared to rats that received vehicle treatment, indicating that proNGF is
produced and secreted under pathological conditions in vivo (Volosin et al.,
2008). More interesting, infusion of a blocking antibody to proNGF into the
hippocampus for three days following seizure induction dramatically reduced cell
death, further illustrating that endogenously secreted proNGF promotes apoptotic
signaling after seizures (Volosin et al., 2008).
Once released from a cell, proneurotrophins can be further processed to
their mature forms. What remains unclear, however, is what ultimately
21
determines the form of the neurotrophin available for signaling. Because the
production and secretion of proneurotrophins and their cleaving enzymes are
regulated after injury, the extracellular proteolysis of proneurotrophins to their
mature forms represents a critical checkpoint in determining whether a cell lives
or dies.
4. Significance
Since their discovery over sixty years ago, the subject of neurotrophins
has been recognized as a matter of great importance, particularly for their roles
in growth, differentiation and survival in the developing nervous system. Their
wide functionality throughout the nervous system has made neurotrophins
targets of particular interest for their potential therapeutic functions in numerous
disorders including ischemia (Han and Holtzman, 2000), spinal cord trauma (Ye
and Houle, 1997), Alzheimer’s Disease (Lad et al., 2003), and depression
(Shirayama et al., 2002). Recent findings indicating that NGF induces cell death
via the p75NTR, especially in its pro- form, tell us that the consequences of
neurotrophin signaling also depend on a number of other factors, including the
ratio of pro- and mature neurotrophins and the enzymes that regulate that
balance.
Although enhancing proneurotrophin cleavage in injury or disease
conditions appears to be a potential avenue for the restoration of cell viability, the
pleiotropic properties of proneurotrophin processing enzymes complicate this
22
idea. For example, MMP7 is a biomarker for the invasive phenotype of several
types of cancer, such as ovarian (Tanimoto et al., 1999), breast (Barrett et al.,
2002), prostate (Pajouh et al., 1991), pancreatic (Ohnami et al., 1999), colorectal
(Brabletz et al., 1999), hepatocellular (Ozaki et al., 2000), astro- and
oligodendrogliomas (Thorns et al., 2003; Rome et al., 2007), and others
(Amalinei et al., 2010). However, cancers are different from brain injury, and the
role of MMP7 in neuropathological conditions remains to be determined. tPA
contributes to blood-brain barrier damage following ischemic stroke (Yepes et al.,
2003) and cell death following spinal cord injury (Abe et al., 2003). More
interesting, tPA has been shown to be both neuroprotective (Kim et al., 1999)
and necessary for cell death following kainic acid-induced seizures (Tsirka et al.,
1995). This range of effects purports that if altered enzyme activity contributes to
improper trophic support, and if these enzyme systems are to be targeted in
clinical treatments, it will be crucial to understand the influence of enzymatic
regulation and the potential outcomes of shifting enzyme levels on neurotrophin
processing in the central and peripheral nervous systems.
23
VI. Research Aims
1. Specific Aim 1: Determine the mechanisms of cell death following kainic
acid treatment.
Previous results from our lab and others have demonstrated increased
p75NTR expression and cell death after seizure in vivo (Roux et al., 1999; Troy et
al., 2002; Yi et al., 2003; Volosin et al., 2008). To examine more thoroughly the
mechanisms of injury-induced cell death, a reduced hippocampal slice culture
system was utilized. This method allows for the manipulation of cellular
conditions and provides a simple way to measure protein secretion into the
media, all while maintaining cellular network structure. Furthermore, kainic acid
was used to induce cell death as it can be applied both in vitro and in vivo to
simulate epileptiform activity and injury (Routbort et al., 1999; Jourquin et al.,
2003). The experiments in this aim defined the mechanisms of p75NTR activation
and their effects on cell death after kainic acid treatment.
2. Specific Aim 2: Examine the expression and activity of MMP7 and TIMP1,
and their effects on proNGF processing, in the hippocampus following
kainic acid-induced injury.
proNGF has been shown in various systems to stimulate apoptotic
signaling via p75NTR (Beattie et al., 2002; Harrington et al., 2004; Nykjaer et al.,
2004; Domeniconi et al., 2007; Volosin et al., 2008; Rogers et al., 2010; Song et
al., 2010). The experiments in this aim investigated the expression and activity of
24
the proNGF extracellular processing enzyme, MMP7, in the hippocampus
following kainic acid treatment to determine if an association existed between the
levels of enzyme available for proNGF processing and proNGF-induced cell
death. The expression of the inhibitor to MMP7, TIMP1, was also examined to
determine whether inhibitor levels affected changes in MMP7 activity. Levels of
proNGF, p75NTR and cell death were measured to determine whether enhancing
enzyme activity would lead to augmented proNGF processing and reduced cell
death following kainic acid treatment.
3. Specific Aim 3: Determine the expression and activity of the tPA-plasmin
cascade in the hippocampus following kainic acid-induced injury.
The third aim evaluated the connection between proNGF/p75NTR-mediated
cell death and another proNGF processing enzyme, plasmin, following kainic
acid-induced injury. Both in vitro and in vivo, we examined whether levels of
tPA/plasmin were altered following kainic acid treatment, and the relationship
between enzyme and inhibitor expression. Similar to Aim 2, we examined the
consequences of artificially increasing proNGF processing using exogenous tPA
treatment. Finally, we examined whether altering the levels of proNGF
processing by increasing tPA after kainic acid treatment would reduce p75NTR-
mediated cell death.
25
VII. Materials and Methods
1. Animals and Kainic Acid-Induced Seizures.
Male Sprague Dawley rats (250–275 g) were injected with kainic acid (KA)
(10 mg/kg, i.p.) for induction of status epilepticus (SE). After 1 hour of
demonstrating SE behavior, rats were treated with diazepam (10 mg/kg, i.p.;
Henry Schein, Melville, NY) and phenytoin (50 mg/kg, i.p.; Sigma) to stop seizure
activity. Control rats received all the same treatments except they were injected
with saline instead of KA. Animals not displaying this seizure behavior were not
included in this study. During recovery, the animals were injected subcutaneously
with Hartmann’s solution (130 mM NaCl, 4 mM KCl, 3 mM CaCl, 28 mM lactate; 1
ml/100 g) twice daily until they were capable of eating and drinking freely.
2. Animal Surgery and Microinjection
In some cases, rats were cannulated 1 week before the induction of
seizures. Rats were anesthetized with ketamine hydrochloride (50 mg/kg) and
xylazine (10 mg/kg) and placed in a stereotaxic apparatus for cannula
implantation into the dorsal hippocampus with the following coordinates: bregma,
−3.1 mm; lateral, 2.0 mm; and depth, 3.1 mm (Paxinos and Watson, 1994).
Cannulas were fixed to the skull with a screw and dental cement. MMP7 (1 µg)
or tPA (1 µg) was infused on one side of the brain and saline on the other side at
a rate of 0.5 µl/minute immediately following the seizure, and twice daily
thereafter until the rats were sacrificed, for a total of 3 infusions over the course
of 24h. Animals found to have an incorrectly placed cannula were excluded.
26
All animal studies were conducted using the National Institutes of Health
guidelines for the ethical treatment of animals with approval of the Rutgers
Animal Care and Facilities Committee.
3. Analysis of CSF.
Twenty-four hours after KA-induced seizure, animals were anesthetized
with ketamine/xylazine. CSF (40–100 µl per animal) was collected from the
cerebello-medullarcisterna using a 25 gauge needle into tubes
containing protease inhibitors, snap frozen, and stored at –80°C until analysis.
Only CSF samples that did not contain blood contamination were used for
Western blot analysis.
4. Brain Preparation for Immunohistochemistry.
Animals were anesthetized with ketamine/xylazine and
perfused transcardially with saline followed by 4% paraformaldehyde. The brains
were removed and postfixed in 4% paraformaldehyde for 2 h, and cryoprotected
in 30% sucrose overnight. Sections (12 µm) were cut on a cryostat (Leica) and
mounted onto charged slides. Sections were blocked in PBS/10% goat
serum and permeabilized with PBS/0.3% Triton X-100, and then exposed to
primary antibodies overnight at 4°C in PBS/0.3% Triton X-100. Slides were then
washed three times in PBS. Sections for fluorescent microscopy were exposed
for 1h at room temperature to secondary antibodies coupled to appropriate
fluorophores (Alexa Fluor, Invitrogen; 1:1000 dilution), and washed again in PBS
in the presence of 4',6'-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich; 1:1000
27
dilution) to identify apoptotic neurons. Sections were coverslipped with anti-fading
medium (ProLong Gold; Invitrogen) and analyzed by fluorescence microscopy
(Nikon). Sections were alternatively prepared for immunohistochemical staining
using the Vectastain ABC kit (Vector Labs, Burlingame, CA) followed by
incubation in Sigmafast 3,3’Diaminobenzidine (DAB) substrate (Sigma Aldrich,
St. Louis, MO) according to the manufacturers’ instructions. Endogenous
peroxidase was quenched by incubating tissue in 0.3% H2O2 and MeOH for 30
minutes prior to staining. Sections were cleared in xylene and coverslipped with
Permount mounting media (Daigger, Vernon Hills, IL). Images were
captured digitally (ORCA-R2, Hamamatsu Photonics, Japan) and assembled in
Adobe Photoshop CS4 (Adobe Systems Inc., San Jose, CA).
Primary antisera were as follows: anti-cleaved caspase-3 (pAb;
1:500; Cell Signaling Technology), anti-NGF (pAb; Sigma-Aldrich; 1:1000
dilution), anti-MMP7 (pAb; GeneTex; 1:1000 dilution), anti-TIMP1 (mAb; Abcam;
1:500 dilution), anti-p75 (pAb; 9651; 1:500 dilution) (Huber and Chao, 1995) or
192 IgG (mAb or pAb; Millipore Bioscience Research Reagents; 1:1000 dilution),
anti-proNGF (kindly provided by Dr. Barbara Hempstead; 1:1000 dilution).
Specificity of each antibody was determined by immunoblot analysis of whole
tissue lysates; antibodies demonstrating non-specific binding were not used for
histochemistry. No immunostaining was seen in controls with omission of the
primary antibodies or secondary antibodies.
28
5. In Situ Zymography.
24h following KA treatment, rats were anesthetized with
ketamine/zylazine, decapitated and their brains removed, flash frozen, and
cryosectioned (12µm). To assess MMP7 activity, sections were coated with an in
situ zymogram assay buffer (50mM Hepes, 200 mM NaCl, 1mM CaCl2, 0.01%
Brij-35, pH 7.5) containing 1% low-melting point agarose and 10µg/ml MMP7
fluorogenic substrate (Calbiochem, San Diego, CA) specific to MMP7 activity,
then incubated overnight in a humidified chamber at 37˚C. MMP7 activity was
observed using fluorescence microscopy (Nikon) as fluorescent holes on a black
background and calculated as a percentage of the entire hippocampal surface
area using Adobe Photoshop CS4 Adobe Systems Inc., San Jose, CA). Ethylene
diamine tetraacetic acid (EDTA), a chelating agent that inhibits MMP activity, was
added to the assay solution as a negative control and abolished enzymatic
activity. To determine substrate specificity, 10µg/ml MMP7 fluorogenic substrate
was incubated with varying concentrations (1-100ng/µl) of active MMP2, MMP3
and MMP7 in in situ zymogram assay buffer at 37˚C overnight, placed on a
coverslip, and imaged using fluorescence microscopy. Activity was only observed
in MMP7-incubated samples.
tPA activity was measured by overlaying fresh-frozen sections with an
assay solution containing 1% low-melting point agarose (BioRad, Hercules, CA),
0.1 M Tris, pH 7.5, 2.5% milk and 25 µg/mL plasminogen. Activity was observed
as dark areas on a light background using Bright field microscopy. Aprotinin was
added to the assay solution as a negative control.
29
6. Fluoro-Jade B Labeling
The number of dying neurons in rats after KA-induced seizures was
assessed by labeling with Fluoro-Jade B according to published protocol
(Schmued and Hopkins, 2000). Briefly, sections were air-dried on a slide warmer
at 50°C for 30 minutes following histochemical labeling. Sections were then
rehydrated for 2 minutes in distilled water and transferred to 0.06% potassium
permanganate for 10 minutes. The sections were then rinsed in distilled water
for 2 minutes and transferred to Fluoro-Jade B working solution (0.0001% Fluoro-
Jade B in 0.1% acetic acid) for 10 minutes, followed by three rinses of two
minutes each in distilled water. The sections were air-dried on a slide warmer at
50°C for 5 minutes, then cleared in xylene and coverslipped with Permount
mounting media (Daigger, Vernon Hills, IL). Images were captured digitally
(ORCA-R2, Hamamatsu Photonics, Japan) and assembled in Adobe Photoshop
CS4 (Adobe Systems Inc., San Jose, CA).
7. Organotypic hippocampal slice cultures
Organotypic hippocampal cultures were prepared as previously described
(Stoppini et al., 1991). Postnatal day 7 rat pups were sacrificed by exposure to
CO2 and soaked in 80% ethanol for ten minutes. Their brains were removed
under sterile conditions and placed into Hanks Balanced Salt Solution plus
glucose (100ml HBSS with 0.6g glucose). Hippocampi were removed in HBSS,
set on sterile Whatman chromatography paper and sectioned to 300 µm using a
McIlwain tissue chopper. Slices that retained the cytoarchitecture of the
hippocampus were placed onto Millipore filters suspended in 1.5 ml of serum-
30
containing media (25% horse serum, 75% MEM, 0.6g glucose, 1 uM penicillin-
streptomyocin). Cultures were incubated at 37 C in 95% O2 and 5% CO2 for 7
days to eliminate the initial inflammatory response. Afterwards, the media was
changed to Serum Free Media (SFM) containing 1:1 Minimum Essential Medium
(MEM):F12+ (6 mg/ml d-glucose, 100 µg/ml transferrin, 25 µg/ml insulin, 20 nM
progesterone, 60 uM putrescine, 30 nM sodium selenide) and incubated for 24
hours. Slices were treated with MK-801 (10 µM; Tocris Bioscience) for 30 min to
block NMDA receptor activity, then 5 µM KA. After 1h, KA was washed out and
slices returned to fresh SFM. Slices were lysed for Western analysis 2, 8 and 24h
following their return to SFM. Some slices were fixed in 4% PFA at room
temperature, followed by preparation for immunofluorescent staining as
described for brain sections. Control slices were prepared similarly, but treated
with vehicle instead of kainic acid. Additional controls included treating slices with
CNQX (10 µM; Tocris Bioscience) to block AMPA/kainite receptor activity.
8. Propidium Iodide Assay.
Propidium iodide (PI) nucleic acid stain (2.5 µl/ml) was added to SFM to
label cells with compromised membrane structure, an indication of cell death.
The amount of cell death was measured as the difference between PI
fluorescence intensity at each timepoint, subtracted from the intensity of the slice
after saturation with KA for 24h (max intensity). Fluorescence intensity was
measured using Adobe Photoshop CS4 (Adobe Systems Inc., San Jose, CA,
USA). For all samples at all timepoints, images were taken of the whole
31
hippocampus at equal magnification, exposure times and camera parameters
(Hammamatsu Orca).
9. Co-immunoprecipitation
Protease inhibitor tablets (Roche Diagnostics, Indianapolis, IN) were
added to slice culture media. Media was pre-cleared for 1h using Protein A/G
magnetic beads (New England Biolabs), then immunoprecipitated for 2h with
anti-BDNF (N-20) (Santa Cruz Biotechnology), anti-NT-3 (gift from David
Kaplan), or anti-TIMP1 (Abcam) at 4˚C. 25µl of magnetic beads were added to
media and incubated 1h at 4˚C. The supernatant was removed, and the beads
were rinsed three times with TNE buffer (10 mM Tris; 0.2 M NaCl; 1 mM EDTA;
pH 7.4). The beads were incubated for 5 min at 70˚C in 3x SDS sample loading
buffer (187.5 mM Tris-HCl (pH 6.8), 6% (w/v) SDS, 30% glycerol, 150 mM DTT,
0.03% (w/v) bromophenol blue, 2% β-mercaptoethanol), then the proteins were
subjected to electrophoresis using SDS polyacrylamide gels, followed by
immunoblot analysis.
10. Western Blot Analysis
Hippocampal tissue or slice tissue was lysed in RIPA buffer (50 mM
Tris/HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% Nonidet P40, 0.5%
deoxycholic acid and 0.5% SDS) supplemented with a protease inhibitor mixture
(Roche). Protease inhibitors were also added to slice culture medium and CSF.
Medium was concentrated in Pierce Protein Concentrators, 9K MWCO (Thermo
Scientific) to 30% of its original volume. Total protein was quantified by the
32
Bradford assay (Bio-Rad, Hercules, CA). Twenty µg of protein from lysates and
culture medium were subjected to Western blot analysis and probed with
antibodies to MMP7 (Abcam; 1:500), TIMP1 (Abcam; 1:500), p75NTR (Upstate;
1:1000), cleaved caspase 3 (Cell Signaling; 1:1000), NGF (Sigma; 1:1000), anti-
proBDNF (gift from Dr. Carlos Ibanez) and anti-NT-3 (gifts from Dr. David
Kaplan; 1:1000). Positive controls utilized were NGF (gift from Genentech),
BDNF (gift from Dr. Carlos Ibanez) and NT-3 (Genentech). To ensure equal
protein loading, blots were stained with Ponceau and stripped/reprobed for actin.
All Western blot analyses were performed at least three times with samples from
independent experiments.
11. Statistical Analysis
All data were expressed as mean ±SEM from three or more independent
experiments. Student’s t-test was used to analyze two comparison groups. One-
way analysis of variance (ANOVA) followed by Tukey post-hoc test were used to
analysis of three or more groups. For all statistical analyses, a value of p<0.05
was considered significant.
33
VIII. Results
Overview
Previous work has identified increased p75NTR-mediated cell death
following various forms of seizure in vivo (Roux et al., 1999; Troy et al., 2002; Yi
et al., 2003; Volosin et al., 2008). However, the mechanisms of p75NTR activation
following this type of injury-induced cell death remain unclear. Using
hippocampal slice cultures to assess the role of p75NTR in kainic acid-induced cell
death, activation of p75NTR by proNGF was found to be necessary for kainic acid-
induced cell death, independent of NMDA-mediated glutamate toxicity (Chapter
1). As the upregulation of extracellular proNGF was observed to be a part of the
injury response, the processing of proNGF to NGF represented a critical step in
determining the extent of cell death after injury. The expression and activity of the
proNGF processing enzyme MMP7 was found to inversely correspond with the
expression levels of proNGF, indicating that the reduction of proNGF processing
by MMP7 is associated with increased proNGF levels after injury. Furthermore,
restoring MMP7 activity rescued hippocampal cells from kainic acid-induced
death, both in vitro and in vivo, demonstrating a crucial connection between the
processing of proNGF by MMP7 and cell survival (Chapter 2). The effects of the
tPA-plasmin cascade were also examined with regard to their effects on proNGF
processing after injury. Like MMP7, tPA activity decreased following kainic acid
treatment, and expression of its inhibitors, PAI-1 and neuroserpin, increased. In
vitro, exogenous tPA completely prevented kainic acid-induced cell death in
hippocampal slices. However, exogenous application of tPA following seizure in
34
vivo did not result in as dramatic a rescue from cell death. tPA infusion into the
hippocampus following kainic acid treatment resulted in reduced cell death in the
CA1, but not the dentate. Thus, tPA-induced proNGF processing is region-
specific and ultimately influenced by the prevalence of its inhibitor (Chapter 3).
These findings are consistent with the idea that enhanced proNGF levels are due
in part to a reduction in the availability of the enzymes that cleave it. Together,
the two proNGF processing enzymes examined here demonstrated similar, but
not identical effects on cell viability after seizure.
Chapter 1. Mechanisms of p75NTR-mediated cell death in the hippocampus
following kainic acid-induced injury
1.1 Kainic acid treatment induces p75NTR mediated cell death in organotypic
hippocampal slice cultures
To examine the mechanisms by which hippocampal neurons undergo cell
death following kainic acid-induced injury, an organotypic slice culture
preparation was employed. Cultured hippocampal slices were pretreated with
MK-801 to block NMDA-mediated toxicity, then treated with vehicle or 5 µM
kainic acid, a glutamate agonist that can be used both in vivo and in vitro to
stimulate injury, for one hour and monitored 24 hours later for cell death by
measuring propidium iodide (PI) uptake. KA treatment induced cell death,
especially throughout the CA1, dentate gyrus and hilus, as demonstrated by PI
uptake that was absent in vehicle-treated slices (figure 3a). Kainic acid treatment
35
also induced p75NTR expression in the same areas, particularly in the CA1 (figure
3b).
To further confirm whether kainic acid treatment caused p75NTR induced
cell death in hippocampal slices, cultured tissue was lysed and analyzed by
Western blot for p75NTR and cleaved caspase-3, a downstream effecter of
p75NTR-mediated apoptosis (Troy et al., 2002). p75NTR expression levels as well
as caspase-3 activation were increased two-fold by 8h following KA treatment,
and this level was sustained through 24h (figure 4a). To ascertain whether the
increased cell death was due to p75NTR-mediated signaling, a blocking antibody
to p75NTR was applied to hippocampal slices prior to KA treatment. Cleaved
caspase-3 (cc3) was not significantly different from control in anti-p75-treated
cultures (figure 4b), suggesting that kainic acid-induced death in hippocampal
slice cultures is p75NTR-dependent.
1.2 proNGF increases after kainic acid treatment and causes cell death via
p75NTR
Numerous reports have observed seizure-induced upregulation of NGF
mRNA in the hippocampus (Gall and Isackson, 1989; Rocamora et al., 1992;
Morimoto et al., 1998; Katoh-Semba et al., 1999). However, these expression
studies did not indicate which form of the protein was secreted, or whether
proNGF directly functioned as a death-inducing ligand for p75NTR after seizure.
To assess whether proNGF might induce cell death following KA treatment,
hippocampal slices from KA-treated cultures were fixed and immunostained with
36
an antibody to the pro-region of proNGF. ProNGF immunodetection was
upregulated after kainic acid treatment, particularly in the CA1 and dentate, areas
that also demonstrated high levels of PI-positive cells (figure 5a-b). Previous
studies have demonstrated that proNGF is induced and secreted following
corticospinal axotomy (Harrington et al., 2004) and pilocarpine-induced seizure
(Volosin et al., 2008). To determine if proNGF is also released from cells in
hippocampal slices, tissue lysates and medium from kainic acid-treated slice
cultures were subjected to Western analysis and probed with an antibody to NGF
in order to detect both the mature and pro-forms. ProNGF was detected in both
the tissue and culture medium, reaching a 1.5-fold increase by 8h that was
maintained through 24h, indicating that proNGF is produced and secreted from
hippocampal cells following kainic acid treatment (figure 5c-d). Mature NGF was
detected only in the tissue, but not the media, indicating that following KA-
treatment, proNGF is the preferentially secreted form of the protein.
In other injury systems, proBDNF and proNT-3 have also been shown to
act as death-inducing ligands via p75NTR (Teng et al., 2005; Koshimizu et al.,
2009; Tauris et al., 2011). To ascertain that the increased cell death observed in
KA-treated slice cultures was due to proNGF activity, a proNGF blocking
antibody was applied to culture media prior to kainic acid treatment. Slices
treated with anti-proNGF together with KA demonstrated a 60% decrease in PI
uptake, compared to cultures treated with KA alone (figure 6a-b). Western
analysis also demonstrated that anti-proNGF treatment lead a reduction of
caspase-3 activation to levels comparable to control (figure 6c-d). To determine
37
whether proBDNF or proNT-3 is secreted from the hippocampus following KA
treatment, slice culture media was analyzed for the presence of either
proneurotrophin. A conventional Western analysis was unable to detect BDNF or
NT-3 in culture media (data not shown). To enhance neurotrophin detection,
media was immunoprecipitated with antibodies to BDNF and NT-3, followed by
Western blot. Bands were detected at ~30 kDa, which correlate to proBDNF and
proNT-3 (Lee et al., 2001b; Matsumoto et al., 2008), but demonstrated no
regulated changes following KA treatment (figure 7). Interestingly, the mature
form of either neurotrophin was not detected, possibly due to the limitations of
detection. Taken together, these results suggest that, in response to KA
treatment, proNGF is the primary proneurotrophin to be secreted and act as a
death-inducing ligand for p75NTR in hippocampal slice culture.
1.3 Kainic acid induces proNGF and p75-mediated apoptosis in the
hippocampus in vivo
We previously demonstrated that pilocarpine-induced seizures increased
proNGF expression and p75NTR-mediated apoptosis in the hippocampus (Volosin
et al., 2008). To determine if seizures induced by KA also elicited these effects,
adult rats were injected IP with KA (10 mg/kg) to evoke status epilepticus.
Twenty-four hours following treatment, KA-treated rats showed increased p75NTR
staining that colocalized with cleaved caspase 3, suggesting that KA-induced
seizures stimulate p75-mediated apoptosis, similar to pilocarpine-induced
seizures (figure 8a). In accordance with previous findings (Volosin et al., 2008),
38
proNGF levels also increased (figure 8b). Taken together, these data suggest
that, following seizure, proNGF is upregulated and stimulates apoptotic signaling
via p75NTR.
Chapter 2. Expression and activity of MMP7 and TIMP1 in the hippocampus
after kainic acid-induced injury
2.1 MMP7 and TIMP1 expression and activity following kainic acid
treatment in vitro
Increased levels of proNGF have been detected in the CSF after injury
(Harrington et al., 2004; Volosin et al., 2006). To determine the mechanisms that
stabilize proNGF and prevent its cleavage to NGF, we examined the expression
and release of MMP7, an enzyme known to cleave proNGF, and its inhibitor
TIMP1, in hippocampal slice cultures.
To assess whether MMP7 and TIMP1 expression patterns were affected
in a manner that could facilitate increased proNGF and cell death following KA
treatment in slice culture, PI-labeled, KA-treated slices were fixed and
immunostained with antibodies to MMP7 or TIMP1. MMP7 expression was
observed throughout the hippocampus in normal conditions, but demonstrated a
global decrease 24h following kainic acid treatment (figure 9a). Additionally,
TIMP1 levels increased following kainic acid treatment, and this expression
colocalized with PI-positive cells (figure 9b).
Western analysis of culture media was also performed to measure the
amount of protein released from cells following KA treatment. Twenty-four hours
39
after KA treatment, extracellular TIMP1 and proMMP7 levels were both detected,
and mature MMP7 was undetected (figure 10a). To determine whether secreted
TIMP1 interacted with pro- or mature MMP7, co-immunoprecipitation of culture
media was performed. A pull-down for TIMP1 and western analysis for MMP7
revealed increased proMMP7 bound to TIMP1 through 24h. Western analysis of
the supernatant was unable to detect either form of MMP7, suggesting that
although proMMP7 was released, it was inhibited by TIMP1 binding (figure 10b).
Mature MMP7 was not detected following TIMP1 immunoprecipitation, indicating
that TIMP1 bound only to the pro-form and inhibited MMP7 activation. These
data suggest that following kainic acid-induced injury, proMMP7 is released from
cells, but is bound to TIMP1, reducing the enzyme activity available for
neurotrophin processing.
If the endogenous inhibition of MMP7 contributes to increased proNGF
and cell death, then enhancing MMP7 activity should manifest opposite effects.
To determine if exogenously supplied MMP7 could prevent neuronal loss,
hippocampal culture medium was supplemented with 2.5µg MMP7 or the
equivalent volume of saline (5µl), 30 minutes prior to kainic acid treatment. After
24h, KA-treated slices in MMP7-supplemented medium demonstrated
comparable PI uptake to non-treated controls, indicating that exogenous MMP7
can protect cells from KA-induced death in slice culture (figure 11a).
Furthermore, Western analysis of culture medium from MMP7-supplemented
slices demonstrated proNGF levels similar to control (figure 11b), suggesting that
40
exogenously supplied MMP7 increases proNGF cleavage and simultaneously
decreases cell death.
2.2 MMP7 and TIMP1 expression and activity following kainic acid
treatment in vivo
Post-seizure rats were examined for changes in TIMP1 and MMP7
expression to determine whether these proteins’ levels were affected in a manner
that could lead to increased proNGF in vivo. TIMP1 was found to be upregulated
while MMP7 was downregulated in the hippocampal tissue 24h after seizure
(figure 12a), suggesting that inhibition of MMP7 may lead to increased proNGF.
Interestingly, TIMP1 and proNGF were found to be tightly colocalized throughout
the CA1 following seizure (figure 12b), indicating a relationship that may
correspond with cell death after injury (arrows).
To assess whether MMP7 activity, as well as its expression, decreased
following seizure, in situ zymography was performed on fresh frozen
hippocampal sections taken from adult rats with and without kainic acid
treatment. 24h following seizure, MMP7 activity was reduced throughout the
hippocampus, particularly in the CA1 and dentate, areas that coincide with
seizure-induced cell death (figure 13a). As a negative control, zymograms from
each animal were incubated in the presence of EDTA, a metal chelator that
inhibits MMP catalytic activity. No activity was detected in the presence of EDTA
(figure 13b).
41
2.3 MMP7 protects hippocampal neurons from death following seizure in
vivo
To determine whether the seizure-induced reduction of MMP7 activity
contributes to neuronal death after seizures, rats were given a direct infusion of
MMP7 into one hippocampal hemisphere immediately following seizure
treatment. The opposite hemisphere received an equal volume of saline. In situ
zymography confirmed MMP7 activity on the infused side, which showed almost
complete rescue from cell death compared with the vehicle-infused hemisphere,
as demonstrated by FluoroJade-B (FJ) labeling (figure 14a-b). Consistent with
increased cell death, both p75NTR staining and caspase 3 activation were found
throughout the saline-treated hemisphere of the hippocampus. Interestingly,
detection of p75NTR and cleaved caspase 3 was nearly abolished in the MMP7-
injected hemisphere (figure 15).
To ascertain whether the neuroprotective effect of MMP7 was due to
reducing proNGF, hippocampal sections from post-seizure rats were also
analyzed for proNGF expression. As with p75NTR, proNGF was only detected in
the saline-treated hemisphere of KA-treated rats, and was almost completely
eliminated on the MMP7-injected side (figure 16a). To determine whether
extracellular proNGF levels were affected, proNGF secretion was measured by
Western analysis of the CSF from cannulated, KA-treated rats, with and without
MMP7 infusions. ProNGF was not detected in rats that were not subjected to
seizures. ProNGF was strongly detected in the CSF of rats that underwent KA-
induced seizures, but levels were reduced by 54% when KA-treated rats also
42
received MMP7 infusions following seizure treatment (figure 16b). Mature NGF
was undetected in MMP7-injected rats, compared to both seizure and non-
seizure treated rats.
Because MMP7 can also cleave proBDNF to its mature form, CSF from
vehicle and KA-treated rats was also analyzed for BDNF expression.
Interestingly, mature BDNF, but not proBDNF, was detected in the CSF (figure
17a). However, there was no detectable difference between the level of BDNF in
KA or vehicle-treated rats.
proNT-3 also acts as an apoptosis-inducing ligand through p75NTR
activation and has recently been shown to be released from cerebellar granular
neurons following membrane depolarization (Yano et al., 2009). To determine
whether proNT-3 was released following seizure treatment, CSF from KA-treated
rats was immunoblotted for pro- and mature NT-3 expression. As with BDNF,
only the mature form of NT-3 was detected in CSF samples, and the level of
expression between KA and vehicle-treated rats was not significantly different
(figure 17b). The size of mature neurotrophins detected was lower than that of
the purified positive controls, possibly due to processing at alternate cleavage
sites.
Together, these data suggest that proNGF is the primary apoptotic
proneurotrophin produced following seizure. Furthermore, providing MMP7 leads
to the extracellular proteolysis of proNGF and reduction in cell death, indicating
that following seizure, the absence of available MMP7 directly enhances
proNGF, creating a cellular environment more vulnerable to apoptosis.
43
Chapter 3. Expression and activity of the tPA-plasmin cascade following
kainic acid-induced injury
3.1 tPA treatment reduces proNGF production following kainic acid
treatment in vitro
The tPA-plasmin cascade was also analyzed with regard to its role in
proNGF production and p75-mediated apoptosis following injury (Lee et al.,
2001b; Bruno and Cuello, 2006). Cultured hippocampal slices were pretreated
with MK-801 to block NMDA mediated toxicity, then treated with 5 µM kainic acid
or vehicle for one hour and monitored for 24 hr and assessed for cell death by
measuring propidium iodide (PI) uptake. Some slices received recombinant tPA
in culture media 30 min prior to KA treatment. As shown previously, slices treated
with kainic acid demonstrated increased cell death compared to vehicle-treated
slices, especially throughout the CA1, dentate gyrus and hilus, whereas slices
that received additional tPA treatment did not show a significant increase in cell
death compared to control (figure 18a). Culture medium was also analyzed to
determine whether proNGF release was affected by KA-induced injury and tPA
activity. proNGF levels were found to be increased following KA treatment, but
were not detected in samples collected from tPA + KA treated slices, indicating
that increased tPA activity can reduce extracellular proNGF and protect cells
from KA-induced death in slice culture (figure 17b).
tPA activity is endogenously regulated by the serine protease inhibitors
neuroserpin and PAI-1. To examine whether neuroserpin or PAI-1 expression
were affected in a manner that could influence proNGF levels following KA
44
treatment, tissue and medium from slice cultures was collected and examined for
neuroserpin and PAI-1 expression. Cultured slices demonstrated increased
neuroserpin immunodetection that colocalized with PI-positive cells following KA
treatment, particularly in the dentate (figure 19a-b). Secreted neuroserpin was
found to be increased in the media as early as 2h following KA treatment (figure
19c). Similarly, PAI-1 also increased in hippocampal tissue and culture media,
but reached maximum levels by 2h that were sustained through 24h, suggesting
different regulatory systems for each inhibitor (figure 20a-b).
Taken together, these data suggest that decreased tPA levels, as well as
increased levels of their inhibitors, PAI-1 and neuroserpin, may contribute to
decreased proNGF processing and thus, increased proNGF-induced cell death
following kainic acid treatment.
3.2 tPA activity decreases and proNGF and cell death increase following
seizure in vivo
tPA activity was measured using in situ zymography on fresh frozen
hippocampal sections taken from adult rats with and without kainic acid
treatment. 24h following seizure, tPA activity was reduced throughout the
hippocampus, particularly in the CA1-3 and hilus, areas that coincide with
increased proNGF and cell death after seizure (figure 21a). As a negative
control, zymograms from each animal were incubated in the presence of
aprotinin, a competitive serine protease inhibitor, to prevent tPA activity. No
activity was detected in the presence of aprotinin (figure 21b). Sections taken
45
from the same brains were subjected to immunostaining to examine levels of
neuroserpin and PAI-1, endogenous inhibitors of tPA activity. Neuroserpin
staining was most strongly detected in the dentate, with very little
immunostaining appearing in the CA1 (figure 22a-b). PAI-1 was similarly
upregulated after seizure, with additional immunostaining detected in the CA3
(figure 23a-b).
To determine whether the seizure-induced reduction of tPA activity
contributes to neuronal death after seizures, rats were given a direct infusion of
tPA into one hippocampal hemisphere immediately following seizure treatment.
The opposite hemisphere received an equal volume of saline. In situ zymography
confirmed increased tPA activity only on the infused side (figure 24a).
Additionally, the tPA-injected hemisphere showed an almost complete rescue
from cell death in the CA1, but not the dentate, when compared to the saline-
injected hemisphere (figure 24b-c), in accordance with increased neuroserpin
and PAI-1. Increased p75NTR was detected in both the CA1 and dentate of both
hemispheres. The CA1 area of the tPA-injected hemisphere, however, showed
an almost complete rescue from cell death compared to the vehicle-injected side,
and despite increased p75NTR staining in both hemispheres. Conversely, the hilus
did not show a significant difference in cell death compared to control (figure
24c), a finding in accordance with the increase in neuroserpin and PAI-1 staining
as shown in figures 22-23. These data suggest that endogenous tPA inhibitor
expression is sufficient to inhibit tPA activity, but regional differences lead to
differential vulnerability.
46
To verify whether the neuroprotective effect of tPA was through a
reduction of extracellular proNGF, proNGF secretion was measured by Western
blot analysis on CSF from cannulated, kainic acid-treated rats, with and without
tPA infusions. Increased proNGF was found in the CSF of seizure-injured rats
that had not received exogenous tPA, but was reduced in tPA-injected rats
(figure 25). These data suggest that decreased tPA activity, along with increased
neuroserpin following seizure lead to reduced proNGF processing and thus,
more available proNGF, creating a more vulnerable environment susceptible to
cell death.
47
IX. Discussion
Neurotrophins have long been recognized for their crucial roles in growth,
differentiation and survival in the developing nervous system. Recent findings
demonstrating that the pro- form of NGF induces cell death via the p75NTR
suggest that the consequences of neurotrophin signaling depend on various
factors, including the ratio of pro- and mature neurotrophins and the regulation of
that balance. Thus, understanding neurotrophin processing is an important step
towards discerning the pathway that a cell takes towards survival or death.
Proneurotrophin processing by MMP7after seizure is neuroprotective
This study investigated components of the extracellular environment that
can regulate whether the apoptosis-inducing ligand proNGF is processed to its
mature form. Once released from a cell, proNGF can be cleaved to mature NGF
by its processing enzyme MMP7 (Smith et al., 1995; Lee et al., 2001b). However,
after injury, we found that proNGF was not only upregulated in the extracellular
environment but MMP7 decreased in both expression and activity. Moreover,
TIMP1, which inhibits MMP7, was increased in expression and colocalized with
both proNGF and cell death following kainic acid treatment, indicative of a
functional association between TIMP1, proNGF and cell death after injury. In
slice cultures, release of TIMP1 and proNGF into the media were both
dramatically increased following kainic acid treatment, suggesting that proNGF
and TIMP1 are not only upregulated in cells, but also released from them as a
response to injury. Furthermore, restoring MMP7 by supplementing culture
48
medium or by direct infusion into the hippocampus resulted in a loss of proNGF
in the CSF and an almost complete rescue of hippocampal neurons from cell
death, suggesting that changes to the enzyme-inhibitor ratio may directly affect
the balance between pro- and mature NGF after injury, contributing to increased
cell death. More specifically, an increase of TIMP1 after injury inhibits MMP7
activity, leading to reduced proteolytic processing of proNGF and thus more
available ligand for the p75NTR-mediated apoptotic pathway.
It is worth noting that when post-seizure rats were treated with exogenous
MMP7, not only did proNGF and dying cells decrease, but p75NTR expression
decreased as well. These changes are in accordance with previous studies from
our lab showing that blocking proNGF activity in the hippocampus after seizure
reduced p75NTR expression (Volosin et al., 2008), suggesting that the presence
of proNGF may be necessary for the upregulation of its receptor.
We also observed that when recombinant MMP7 was infused into the
hippocampus following seizure, not only did the levels of proNGF decrease, but
so did mature NGF. A possible explanation for this lies in the intertwined network
of matrix metalloproteases. In inflammatory conditions such as seizure, a number
of MMPs are released from cells, including MMP9 (Nico et al., 2009; Girgenti et
al., 2010), an enzyme linked to the degradation of mature NGF (Bruno and
Cuello, 2006; Cuello and Bruno, 2007). However, MMP9 is released as a pro-
enzyme, requiring further processing for activation; MMP7 is one of the many
enzymes capable of cleaving the prodomain from MMP9 (Imai et al., 1995). The
influx of recombinant MMP7 may have also activated endogenous proMMP9,
49
increasing the degradation of mature NGF in the extracellular space, resulting in
lower levels of detection.
Because proNGF and mature NGF have differential effects on neuronal
survival, and because proNGF levels increase in parallel with neuronal apoptosis
following various forms of injury or pathological conditions, the processing of pro-
to mature NGF is critical in determining neuronal fate. Our findings on the MMP7-
proNGF relationship following excitotoxic injury demonstrate that the enzymatic
conversion of proNGF to NGF by MMP7 is important for cell survival and suggest
a novel mechanism by which MMP7 may function in the injury process. Changes
in MMP7 expression have been detected in an animal model of multiple sclerosis
(Buhler et al., 2009) and in the brains of Alzheimer’s patients (Ethell et al., 2002),
and the use of MMP inhibitors has been proposed as a treatment for diseases
including Alzheimer’s (Backstrom et al., 1996) and ALS (Lorenzl et al., 2003).
Elevated proneurotrophin levels have also been found during these conditions,
suggesting the MMP7-proNGF relationship as a possible therapeutic target for
disease or injury. But the precise roles of MMP7 and TIMP1 in these and other
disorders have not been elucidated. If alterations to the MMP7/TIMP1 balance
contribute to the increased proNGF and improper trophic support seen in
Alzheimer’s and other neurodegenerative disorders, it will be crucial to continue
examining the influence of enzymatic regulation and the potential outcomes of
altering enzyme activity on neurotrophin cleavage and the consequences for
neuronal survival or death.
50
Proneurotrophin processing by plasmin
MMP7 is not the only enzyme involved in the extracellular proteolysis of
proNGF. This study also examined the role of the tPA-plasmin cascade in the
regulation of proNGF processing after injury. tPA is a serine protease that
catalyzes the conversion of the inactive zymogen plasminogen to plasmin. The
active enzyme plasmin can then cleave proNGF to its mature form (Lee et al.,
2001b; Bruno and Cuello, 2006; Larsson et al., 2009). Plasminogen, tPA, and its
inhibitor, neuroserpin, are released from neurons in conjunction with proNGF
upon stimulation, indicating a relationship between the activation of plasmin, the
subsequent cleavage of proNGF, and a closely associated inhibitory loop
regulated by neuroserpin (Bruno and Cuello, 2006). Additionally, an increase in
p75NTR expression following sciatic nerve injury has been shown to suppress tPA
activity by upregulating another tPA inhibitor, PAI-1 (Sachs et al., 2007). Taken
together, these studies suggest that a reduction in tPA-plasmin activity following
injury leads to decreased proneurotrophin processing and subsequently,
increased p75NTR expression, creating an environment more susceptible to cell
death.
The work in this thesis found that tPA activity was downregulated following
seizures, particularly in areas that coincided with cell death. The expression of
neuroserpin and PAI-1 following injury also corresponded with dying cells, both in
vitro and in vivo. However, restoring tPA activity by direct infusion following
seizure did not result in a complete rescue of cell death as seen with MMP7.
Cells in the hilus of kainic acid-treated rats demonstrated no difference in the
51
amount of cell death between the hemisphere injected with tPA or vehicle. CA1
area cells, however, demonstrated a dramatic decrease in cell death compared
with vehicle-injected hippocampi. Neuroserpin expression was found to be
strongly increased throughout the hilus, but not the CA1, following seizure,
supporting the finding that only CA1 cells demonstrated increased survival
following tPA infusion. PAI-1 also increased following seizure in the dentate but
not the CA1, suggesting neuroserpin and PAI-1 play similar roles in mediating
plasminogen activation (Siconolfi and Seeds, 2001; Fabbro and Seeds, 2009).
Furthermore, proNGF levels were also sustained in the hilus, but not detected in
the CA1, after tPA treatment. Taken together, these results indicate that
differential expression of the enzymes and their inhibitors lead to differential
vulnerability of specific populations of neurons after injury, and that restored
tPA/plasmin activity could rescue cells from apoptosis by decreasing proNGF.
However, the protective effect of proNGF processing by the tPA-plasmin cascade
is restricted to regions expressing lower levels of inhibitor.
Similar to our findings, pharmacological upregulation of tPA activity has
also demonstrated beneficial effects following peripheral nerve injury (Zou et al.,
2006), and endogenous tPA following ischemic insult protects hippocampal
neurons from cell death (Echeverry et al., 2010). Furthermore, the progression of
cell death in mild cognitive impairment and Alzheimer’s disease has been
connected to downregulated proNGF processing by plasmin and subsequently
increased p75NTR-mediated cell death (Cuello and Bruno, 2007; Fabbro and
Seeds, 2009).
52
Based on established time courses from our lab and others (Roux et al.,
1999; Troy et al., 2002; Yi et al., 2003; Volosin et al., 2006), post-KA treated rats
were examined 24 hours after seizure, corresponding with elevated levels of
proNGF, p75NTR expression and cell death. Similar to other groups, we found that
24 hours after KA-induced seizure, tPA activity falls to nearly undetectable levels,
an effect complemented by the simultaneous upregulation of endogenous PAI-1
and neuroserpin (Salles and Strickland, 2002; Yepes et al., 2002; Siao et al.,
2003). More interesting, the cell death observed at this time point can be rescued
by application of exogenous tPA, an effect also observed by others (Kim et al.,
1999). Additionally, the activity timecourse of tPA is biphasic: tPA protein
expression sharply rises one hour following seizure induction, but falls to basal
levels within 8 hours (Salles and Strickland, 2002), in accordance with when we
were first able to detect significantly increased extracellular proNGF (figure 3)
and possibly accounting for lower levels of detected extracellular proNGF from 2-
6 hours following seizure induction. Although exogenous tPA resulted in
significantly reduced neuronal death, both in vitro and in vivo, we cannot
eliminate the possibility that supplying tPA may have only delayed cell death.
Examination of proNGF, p75NTR and cell death at later time points will be
necessary to elucidate the role of tPA in neuroprotection after seizure. Taken
together, this suggests a complex deregulation of the plasminogen system
following pathological conditions that can result in, among other events, a
reduction in proneurotrophin processing and subsequent cell death.
53
It is interesting to note that tPA injection also resulted in reduced mature
NGF detected in the CSF. As mentioned previously, proMMP9 is known to be
upregulated following various forms of stimulation and injury (Nico et al., 2009;
Girgenti et al., 2010). In addition, plasmin is a known activator of MMP9
(Baramova et al., 1997). Thus, the influx of recombinant tPA may activate
plasmin, which could in turn, activate MMP9 to facilitate the degradation of NGF.
This system illustrates the intricacy of extracellular matrix enzyme systems and
emphasizes the complexity in elucidating the consequences of altering any step
of the enzyme process.
The roles of proneurotrophin processing extend beyond regulating apoptosis
The basic tenet of the neurotrophic hypothesis states that, during
development, neurons that receive sufficient trophic support from target tissue
will form functional synapses with those targets, and those that do not will
eventually undergo programmed cell death. Thus, cells must receive a threshold
level of trophic support to counteract their innate apoptotic signaling. With the
discovery of proneurotrophin functionality, the neurotrophic hypothesis has
gained another dimension, one in which proneurotrophins bind to p75NTR to
initiate an active programmed cell death mechanism, both during development
and in response to pathological conditions (Frade et al., 1996; Majdan et al.,
1997; Bamji et al., 1998).
54
However, the functional significance of proneurotrophin signaling extends
beyond a simple decision between life and death. BDNF and the tPA axis are
known to play important roles in synaptic modulation, and TrkB stimulation by
BDNF is essential for various forms of synaptic plasticity (Seeds et al., 1995; Xu
et al., 2000). Moreover, because proBDNF is released from cells in a regulated
manner, the proteolysis of proBDNF to mature BDNF represents an important
step in proper synaptic transmission and in fact, extracellular proBDNF
conversion by plasmin is required for LTP (Pang et al., 2004). These data
suggest that the regulation of neurotrophin processing directly influences the
synaptic efficacy of proneurotrophins. However, a role for proNGF in synaptic
function remains to be determined.
Currently, it is unclear how p75NTR can distinguish between proNGF and
proBDNF to execute distinct downstream events, but it appears as though
proNGF binding favors apoptotic signaling and proBDNF stimulates processes
related to synaptic transmission. Along with apoptosis and synaptic
communication, p75NTR signaling can also function in mediating neuronal
outgrowth. p75NTR has been shown to bind RhoA to negatively modulate neurite
expansion, which is reversed by mature neurotrophin binding to p75NTR, resulting
in RhoA dissociation and subsequent neuronal growth (Yamashita et al., 1999).
Overexpression of p75NTR leads to stunted dendritic arborization and reduced
spine density in the adult mouse, and blocking p75NTR signaling manifests the
opposite results (Zagrebelsky et al., 2005). Additionally, p75NTR–mediated axonal
retraction can be overcome with concurrent TrkA activation by NGF (Singh and
55
Miller, 2005). Although these studies did not address whether p75NTR requires
proneurotrophin binding to execute these effects, proBDNF has been shown to
be a critical ligand for inducing presynaptic terminal retraction (Yang et al.,
2009a). Moreover, p75NTR is not limited to neurons, and its activation on other
cell types facilitates a variety of functions that do not affect apoptosis, including
enhancing Schwann cell myelination (Cosgaya et al., 2002) or regulating glial cell
activation in response to injury (Gschwendtner et al., 2003). Indeed, NGF
activation of p75NTR on astrocytes displayed no effect on cell death, but rather
attenuated mitogen-induced proliferation (Cragnolini et al., 2009). Taken
together, these data suggest that neurotrophin signaling through p75NTR
functions in shaping several aspects of the cellular network, that these functions
are highly dependent upon cellular context, and that death is not the only
potential consequence of p75NTR signaling.
56
X. Conclusion and Future Directions
The work in this thesis was designed to clarify some of the circumstances
that affect the stimulation of p75NTR-mediated apoptosis under pathological
conditions. Neurotrophins have long been targeted for their possible therapeutic
properties in numerous disorders including multiple sclerosis (Caggiula et al.,
2006), spinal cord trauma (Ye and Houle, 1997), and Alzheimer’s disease (Lad et
al., 2003). ProNGF in particular has been found to accumulate in the brains of
Alzheimer’s patients (Fahnestock et al., 2004; Peng et al., 2004; Pedraza et al.,
2005), while levels of the mature NGF receptor, TrkA, diminish and p75NTR levels
remain (Ginsberg et al., 2006). The regulation of proNGF processing in the
progression of this and other nervous system disorders awaits further
investigation.
Although the role of proNGF in mediating cell death following injury has
been well-characterized, we cannot rule out the involvement of other
neurotrophins as well, and future studies would examine the regulation of
proBDNF and proNT-3 processing, elucidating their roles in cellular pathology
and the injury response. To date, the majority of proneurotrophin research has
focused on the role of proNGF in pathology and proBDNF in physiology. Indeed,
the experiments in this thesis were unable to detect regulated release of
proBDNF in vitro or in vivo in response to injury. These results were surprising
because BDNF mRNA has been shown to be dramatically upregulated following
seizure activity (Gall and Isackson, 1989; Ernfors et al., 1991; Kokaia et al.,
1996). Possible explanations for these discrepancies include the time points at
57
which protein levels were examined, increased breakdown of proBDNF and/or
BDNF by various proteases, or perhaps that changes in BDNF regulation were
too small to detect using Western analysis. Furthermore, although proBDNF has
been shown to be an apoptotic ligand in an overexpression study (Teng et al.,
2005), examination in physiologically relevant conditions revealed that proBDNF
is released following low and high frequency stimulation to induce LTD and LTP,
respectively (Nagappan et al., 2009). More specifically, low frequency stimulation
resulted in proBDNF secretion and LTD, whereas high frequency stimulated
release of proBDNF and the components of the tPA axis, resulting in plasmin-
dependent pro- to mature BDNF conversion and LTP (Pang et al., 2004;
Nagappan et al., 2009). These results, as well as our findings that proBDNF
release was not detected in the CSF following seizure, suggest that it is unlikely
that MMP7 acts upon proBDNF in vivo to mitigate apoptotic signaling. This
hypothesis is supported by previous work that found BDNF and NGF to be
differentially regulated following certain types of nerve injury (Matsuoka et al.,
1991; Meyer et al., 1992; Harrington et al., 2004). Future experiments will
examine whether endogenous proBDNF plays a role in post-injury and disease
pathology.
ProNGF contains three possible sites of cleavage: a site recognized by
plasmin and furin that releases a 13.5 kDa product, and a site on each side of
that site, both recognized by MMP7, that releases a smaller or larger product,
depending on the target (Lee et al., 2001b). Interestingly, Western analysis of
CSF taken from sham-treated rats often detected smaller forms of NGF,
58
suggesting that the majority of secreted NGF in vivo is the pro-form (Reinshagen
et al., 2000; Fahnestock et al., 2001; Hasan et al., 2003; Bierl et al., 2005), which
is extracellularly processed by MMP7. The experiments in this thesis examined
the extracellular processing of proNGF, as the pro-form is reported to be the
predominant isoform in sympathetic neurons (Bierl et al., 2005), cortical cells
(Fahnestock et al., 2001), dorsal root ganglion neurons (Reinshagen et al.,
2000), and other neuronal and non-neuronal cell populations (Dicou et al., 1986;
Yamamoto et al., 1992; Skaper et al., 2001) under basal conditions. However, it
is important to note that proNGF may also be processed intracellularly by furin
and other proconvertases (Seidah et al., 1996). Thus, the cleavage of proNGF by
intracellular proconvertases also reflects a step in proNGF processing that may
affect the final form of the protein available for signaling. Examination of any
changes to the intracellular biosynthesis of NGF after injury awaits further
investigation.
To date, the cells responsible for the synthesis and secretion of
proneurotrophins have not been ascertained. Under different physiological and
pathophysiological conditions, studies from our lab and others have observed an
upregulation of higher neurotrophin isoforms in glia as well as neurons (Yune et
al., 2007; Volosin et al., 2008; Yano et al., 2009; Marler et al., 2010). Moreover,
the intracellular processing system in glia is not as active as in neurons (Mowla
et al., 1999), suggesting that glial cells may be a rich source of secreted
proneurotrophins. Further investigation would determine which form of the
59
protein is released under basal conditions, which cells make and secrete
proNGF, and which enzymes are most actively involved in its processing.
The data presented in this thesis strongly support the idea that the
regulation of proneurotrophin processing activity is an important means of
mediating cellular responses to injury. Exogenous application of MMP7 and tPA
were shown to alter the amount of proNGF processing, illustrating two separate
but overlapping mechanisms involved in regulating NGF activation of p75NTR.
The existence of redundant systems represents a complex, buffered network that
functions to perpetuate efficient synaptic communication. Thus, a downregulation
of proneurotrophin processing following injury may function as part of a complex
system to stimulate the retraction and pruning of injured cells, as well as to rewire
the parts of the cellular network that remain, via increased p75NTR signaling.
Figure 1
Lee et al., 2001
60
Figure 1. Schematic illustrating putative, highly conserved proteolytic cleavage
sites in proneurotrophins for serine proteinases and MMPs. Note that although
a processing site for MMP3 is indicated, only BDNF has been shown to
cleaveable by this enzyme. “A” and “B” designate previously characterized
cleavage sites by unknown proteases.
Adapted from Lee et al., 2001
61
Figure 2
Nykjaer et al., 2004
62
Figure 2. Schematic model of receptor-complex formation. Sortilin is a
necessary co-receptor for p75NTR to create a high-affinity binding site for
proNGF and to signal cell death. Regulated activity of extracellular proteases
may cleave proNGF to mature NGF to instead promote trk-mediated survival
signaling.
Adapted from Nykjaer et al., 2004
63
Pretreatment 24hV
eh
icle
24
h K
AA
B
Ve
hic
le2
4h
KA
Figure 3
p75 PI merge
64
Figure 3. Organotypic hippocampal slice cultures show increased cell death in
the presence of kainic acid (KA). A) Slices for p7 rat pups were cultured for 7
days in serum with 50% media changes every 3 days. On day 7, slices were
placed into serum free media (SFM) containing 2.5 µl/ml propidium iodide (PI).
24h later, pretreatment photos of slices were taken (left column). Slices were
treated 24h later with 10µl MK-801 and 1.5 µl vehicle or 5 µM KA. After 1h,
slices were placed into fresh SFM. Photos were again taken 24h following
return to SFM. Only slices that received KA treatment demonstrated propidium
iodide (PI) uptake 24h after kainic acid treatment, particularly in the CA1. B)
Magnification of CA1 showing p75NTR immunodetection and PI staining only
after kainic acid treatment. Note the colocalization between p75NTR and PI
positive staining, indicating dying cells are also positive for p75NTR . Photos are
representative staining from 5 independent experiments. Scale bar = 25 µm.
65
p75
cc3
actin
C 2 4 6 8 24hA
BC KA
KA+
αp75 αp75
Figure 4
cc3 -18 kDa
- 75 kDa
- 18 kDa
actin
Hours following KA
66
Figure 4. Cell death following kainic acid treatment is p75NTR -dependent. (A)
Following KA treatment, slice culture tissue was harvested at 2, 4, 6, 8 and
24h timepoints, lysed in buffer containing protease inhibitors and subjected to
Western analysis to examine levels of p75NTR and cleaved caspase-3. By 8h
post-KA treatment, levels of both p75NTR and activated caspase had doubled
compared to basal levels. (B) Slice cultures treated with a blocking antibody to
p75NTR in conjunction with KA treatment show levels of caspase-3 activation
comparable to control, indicating p75NTR is involved in KA-induced caspase
activation. Each graph represents the normalized densitometric index of
immunoblots from 3 independent experiments. Error bars represent SEM.
Data were analysed using ANOVA, followed by Tukey post-hoc test. *P<0.05
relative to control.
67
A
B
NGF
C 2 8 2437kDa-
15kDa-
Tissue Media
proNGF
ponceauactin
C 2 8 24
Figure 5
proNGF PI merge
Ve
hK
A
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
c 2h 8h 24h
Rel
ativ
e D
ensi
tom
etri
c U
nit
s
proNGF
NGF* *
Culture Tissue
0
0.5
1
1.5
2
2.5
3
3.5
c 2h 8h 24h
Rel
ativ
e D
ensi
tom
etri
cU
nit
s proNGF
NGF
*
*
Culture Media
C D
68
Figure 5. proNGF is upregulated and released after kainic acid treatment in
slice culture. (A) proNGF is undetected in CA1 area cells under control
conditions (top row). In response to KA treatment, proNGF is highly
upregulated throughout the CA1, near areas of PI-positive staining. Western
analysis (B) and quantification (C) of tissue from slice cultures detects very low
levels of proNGF in control conditions. However, KA induces a 1.5-fold
increase in proNGF detection by 8h following treatment. Mature NGF is
strongly detected in both control and treated conditions, but does not manifest
significant differences in expression levels in response to KA. (D) In culture
medium, proNGF is found in control conditions, but is nearly doubled by 8h
following KA stimulation, reaching 2.5-fold increase by 24h. Extracellular
mature NGF was not detected. Each graph represents the normalized
densitometric index of immunoblots from 3 independent experiments. Error
bars represent SEM. Data were analyzed using ANOVA, followed by Tukey
post-hoc test. *P<0.05 relative to control.
69
Veh KA
KA+
αproNGF αproNGF
cc3
actin
B
Veh
KA
KA+αproNGF
A
Figure 6
-18 kDa
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Re
lati
ve F
luo
resc
en
ce In
ten
sity
*
C
D
00.20.40.60.8
11.21.41.61.8
2
Re
lati
ve D
en
sito
me
tric
Un
its *
70
Figure 6. Blocking proNGF activity results in reduced cell death and
caspase 3 activation. (A) Slices treated with KA demonstrate a 68%
increase in PI uptake as measured by fluorescence intensity of the whole
slice. Slices treated with a blocking antibody to proNGF 30 min prior to KA
demonstrated decreased cell death that was insignificant from control.
Results are the normalized fluorescence index of slice images from 3
independent experiments. (B) Quantification of (A). Error bars represent
SEM. Data were analyzed using one-way ANOVA, followed by Tukey post-
hoc test. *P<0.01 relative to control. (C) Western analysis of slice culture
tissue demonstrating that blocked proNGF activity results in decreased KA-
induced caspase 3 activation, comparable to control. Activated caspase 3
was 67% higher than control levels. (D) Quantification of (C). bars
represent SEM. Data were analyzed using one-way ANOVA, followed by
Tukey post-hoc test. *P<0.01 relative to control. Images and blots are each
representative of 3 independent experiments.
71
Figure 7
C 2 8 24 C 2 8 24
- 25 -
kDa
- 15 -
IP: BDNF mAb
IB: BDNF pAb
IP: NT-3 pAB
IB: NT-3 pAb
0
0.2
0.4
0.6
0.8
1
1.2
1.4
c 2h 8h 24h
Re
lati
ve D
en
sito
me
tric
U
nit
s
0
0.2
0.4
0.6
0.8
1
1.2
1.4
c 2h 8h 24h
Re
lati
ve D
en
sito
me
tric
U
nit
s
A
B C
72
Figure 7. Secretion of proBDNF and proNT-3 is not regulated following kainic
acid treatment in vitro. Western analysis was unable to detect BDNF or NT-3
from hippocampal slice culture media (data not shown). To enhance signal
detection, media was immunoprecipitated and immunoblotted with antibodies
to BDNF (left) and NT-3 (right) as indicated. The pro-forms of each protein was
detected, but no significant difference was detected at any timepoint. Note the
mature forms were not detected even following immunoprecipitation. N=3,
p>0.05. Graphs represent the normalized densitometric index of blots from 3
independent experiments. Error bars represent SEM. Data were analyzed
using one way ANOVA, followed by Tukey post-hoc test.
73
A p75 cc3
Ctr
l2
4h
KA
B p75 proNGF
Ctr
l2
4h
KA
Figure 874
Figure 8. In vivo expression of p75NTR and cleaved caspase 3 increase
following kainic acid-induced seizures. Male Sprague Dawley rats (250–275 g)
were injected with kainic acid (KA) (10 mg/kg, i.p.) for induction of status
epilepticus (SE). After 1 hour of SE behavior, rats were treated with diazepam
and phenytoin to stop seizure activity. Control rats received the same
treatments but were injected with saline instead of KA. Twenty-four hours after
stopping seizure activity, the rats were anesthetized and brains prepared for
histochemistry. (A) Hippocampal sections were co-immunolabeled with anti-
p75NTR (green) and anti-cleaved caspase 3 (cc3, red). Arrows indicate
colocalization of p75NTR and cleaved caspase-3 in response to KA-induced
seizure. (B) Histochemical analysis for expression of proNGF shows proNGF
induction 24h after KA-induced seizure. Arrows indicate colocalization of
proNGF with p75NTR. All images are taken from the CA1 area of the
hippocampus and are representative images from 5 independent experiments.
Scale bars = 50um.
75
MMP7 PI mergeC
trl
24
h K
A
A
B
Figure 9
PI merge
Ctr
l2
4h
KA
TIMP1
76
Figure 9. Expression of MMP7 and TIMP1 in the CA1 following KA
treatment in vitro. Following 7 days in culture, slices were placed into fresh
serum-free medium (SFM) containing 2.5 µl/ml propidium iodide (PI). The
next day, slices were treated with 5µm KA for 1 hour, then placed back into
SFM. Twenty-four hours following treatment, slices were fixed in 4% PFA,
rinsed in PBS, and subjected to immunolabeling with anti-MMP7 or anti-
TIMP1. (A) MMP7 expression is found throughout cell bodies and
processes in the hippocampus under basal conditions, but is barely
detectable in tissue 24h after KA treatment. (B) TIMP1 expression is
detected at relatively low levels under basal conditions, but is strongly
upregulated throughout hippocampal tissue in response to KA treatment.
Photos are from CA1 and are representative of 3 independent
experiments. Scale bar = 50µm.
77
A
B
ponceau
TIMP1
Figure 10
proMMP7
MMP7
-28.5 kDa
-20 kDa
ponceau
C 2 8 24
C 2 8 24
proMMP7
TIMP1
IP: TIMP1
IB: MMP7, TIMP1
MMP7-20 kDa
Precipitate Supernatant
C 2 8 24 C 2 8 24
78
Figure 10. MMP7 and TIMP1 secretion following KA treatment. (A) Western
analysis of culture medium shows increased secretion of TIMP1 and
proMMP7 2-24h following KA treatment. Note mature MMP7 was undetected.
(B) Medium from slice cultures was co-immunoprecipitated for TIMP1 and
immunoblotted for MMP7 and TIMP1. Extracellular proMMP7 was detected in
TIMP1 precipitate, but not in the supernatant, and mature MMP7 was not
detected in either sample set, indicating that secreted proMMP7 is bound to
TIMP1. All experiments were repeated three times. Images are representative
from 4 independent experiments.
79
Pretreatment 8 hr 24 hr
Ctrl
KA
MMP7
+ KA
A
B
proNGF
ponceau
Veh KAKA+
MMP7 MMP7
Figure 11
-36 kDa
*
80
Figure 11. Exogenously supplied MMP7 reduces the presence of
extracellular proNGF and cell death in slice culture. (A) After 7 days in
culture, hippocampal slice cultures were treated with KA or with 1 µg/ml active
MMP7 in conjunction with KA. Slices that received KA treatment alone
demonstrated 70% increase in PI uptake as calculated by fluorescence
intensity. Slices that received exogenous MMP7 demonstrated significant
rescue from cell death, as depicted by diminished PI uptake, comparable to
control levels. Graph represents are the normalized fluorescence index of
slice images from 3 independent experiments. Error bars represent SEM.
Data were analyzed using one-way ANOVA, followed by Tukey post-hoc test.
*P<0.05 relative to control. (B) Western analysis of culture media from slices
treated with vehicle, KA, or KA+MMP7 and probed with anti-proNGF to
determine the effect of exogenous MMP7 on extracellular proNGF levels.
ProNGF was nearly abolished in medium from slices that received 1 µg/ml
active MMP7 in conjunction with KA compared with those that received KA
alone. Images and blot are representative of three independent experiments.
81
A
Figure 12
MM
P7
Ctrl 24h KA
B
pro
NG
FT
IMP
1
Ctrl 24h KA
82
Figure 12. Expression of MMP7, TIMP1 and proNGF 24h following KA-
induced seizure in vivo. (A) Hippocampal tissue from post-seizure rats
immunolabeled with anti-MMP7. Vehicle-treated rats demonstrated wide
MMP7 expression throughout cell bodies and processes, but is greatly
diminished throughout the hippocampus after seizure. (B) Expression of
TIMP1 and proNGF are strongly induced and are found in the same cells
throughout the CA1 following KA-induced seizure. Images are from the CA1
area of the hippocampus and are representative of 5 independent
experiments. Scale bars = 50um.
83
ACtrl KA
0
0.2
0.4
0.6
0.8
1
1.2
Ctrl KA
Rel
ati
ve
Flu
ore
scen
ce I
nte
nsi
ty
B+EDTA
Figure 13
84
Figure 13. MMP7 activity is reduced in response to KA-induced seizure.
Twenty-four hours following seizure, rats were anesthetized with, decapitated
and their brains removed, flash frozen, and cryosectioned (12µm). To assess
MMP7 activity, sections were coated with an in situ zymogram assay buffer
containing10µg/ml MMP7 fluorogenic substrate that is specific to MMP7
activity. Following overnight incubation in a humidified chamber at 37˚C,
MMP7 activity can be observed using fluorescence microscopy, appearing as
fluorescent areas on a black background. (A) In situ zymogram
demonstrating 60% reduction in MMP7 activity in the hippocampus,
particularly in the CA1 and dentate, areas that coincide with seizure-induced
cell death. and calculated as a percentage of the entire hippocampal surface
area Relative fluorescence intensity, normalized to background fluorescence,
is quantified below. N=5. Error bar represents SEM. Data were analyzed using
Student’s t-test. P<0.01. (B) In situ zymogram performed in the presence of
EDTA, a metal chelator that inhibits MMPs, demonstrating abolished activity.
Scale bar = 100um.
85
Vehicle MMP7
A
B Vehicle MMP7
Figure 14
86
Figure 14. Exogenous MMP7 protects hippocampal neurons from death
following seizure in vivo. (A) Panoramic montage of photos illustrating both
hippocampal hemispheres from a single rat following seizure and unilateral
infusion of active MMP7 (1µg) at a rate of 0.5 µl/min. In situ zymography was
performed on sections to confirm MMP7 activity in the infused hemisphere, as
observed by bright, fluorescent areas on a dark background. (B) FluoroJade
B staining showing decreased cell death in the hippocampal hemisphere
ipsilateral to MMP7 injection (arrows).
87
Figure 15
p75
cc3
merge
Vehicle MMP7
88
Figure 15. Sections from a single, bilaterally cannulated, seizure-induced rat
that received infusions of active MMP7 (1µg) into one hippocampal
hemisphere, as well as vehicle infusions (0.5µl) into the opposite hemisphere,
immediately following seizure treatment and every 12 hours thereafter. The left
column represents the vehicle-infused side, and the right column represents
the MMP7-infused side of the same rat. Twenty-four hours following seizure,
immunostaining for cleaved caspase 3 and p75NTR is diminished on the MMP7-
infused side compared to the vehicle-infused hemisphere, indicating MMP7
protects against KA-induced, p75NTR–mediated cell death. Images are from the
CA1 area of the hippocampus and are representative of 6 independent
experiments. Scale bar = 50um.
89
Vehicle MMP7
pro
NG
FF
JM
erg
e
A
B37-
15-
25-
Veh MMP7 Veh
KA
Figure 16
NGF
proNGF
NGF
0
0.5
1
1.5
2
2.5
3
3.5
Veh KA + MMP7
KA + Veh
Re
lati
ve D
en
sito
me
tric
Un
its
proNGF
*****
90
Figure 16. MMP7 infusion reduces proNGF protein levels after seizure. (A)
Image from opposite hippocampal hemispheres of a single, bilaterally
cannulated rat that had undergone seizure treatment, followed by unilateral
infusions of MMP7 and vehicle. Sections were immunolabled for proNGF
detection as well as subjected to Fluoro-Jade B staining (FJ) to label dying
cells. MMP7 infusions resulted in diminished proNGF as well as FJ-postiive
cells compared to the contralateral, vehicle infused hemisphere. Images are
representative of 6 independent experiments. (B) Western analysis of CSF
from cannulated, seizure-treated rats that received MMP7 or vehicle
infusions. Rats that received seizure treatment and MMP7 infusions
demonstrated a 54% reduction in extracellular proNGF compared with those
that received vehicle infusions. Graph represent the normalized densitometric
index of blots from 3 independent experiments. Error bars represent SEM.
Data were analyzed using one way ANOVA, followed by Tukey post-hoc test.
**p<0.05, ***p<.001. Scale bar = 50µm.
91
KA KABDNF NT-3Veh Veh
Probe: BDNF Probe: NT-3
- 25 -
- 15 -
- 37 -
CSF CSF
A B
Figure 17
92
Figure 17. Secreted proBDNF and proNT-3 are not detected after KA-induced
seizures. Twenty-four hours following seizure, rats were anesthetized and their
CSF collected for Western analysis. Each lane represents 15µg of CSF. Only
rats from the same experimental group were used for comparisons. (A)
Western analysis of CSF (15µg) from vehicle and KA-treated rats did not
reveal the presence of proBDNF. (B) proNT-3 was also not detected following
seizure. Purified BDNF and NT-3 were loaded as positive controls. Blots are
representative of 3 independent experiments.
93
Pretreatment 24h KA
Ctr
lK
AtP
A
+ K
A
A
B
Figure 18
C KA KA+tPA tPA
proNGF
ponceau
-36kDa
0
0.5
1
1.5
2
Veh KA KA+tPA tPA
*
94
Figure 18. tPA reduces PI uptake and cell death in slice culture. (A)
Hippocampal slices were cultured for one week, then subjected to KA
treatment, with and without a pretreatment of exogenous tPA. Only slices that
received KA treatment alone demonstrated PI uptake. (B) Western analysis of
culture medium from slices that received vehicle, KA, KA+tPA or tPA. Medium
that was supplemented with recombinant tPA did not demonstrate a significant
difference in proNGF levels compared to control, indicating exogenous tPA
reduces extracellular proNGF. Graph represents the normalized densitometric
index of blots from 3 independent experiments. Error bars represent SEM.
*p<0.001 Data were analyzed using one way ANOVA, followed by Tukey post-
hoc test.
95
A
B
Nsp PI merge
Nsp PI merge
CA1
Dentate
Ctr
l24h K
AC
trl
24h K
A
CNsp
ponceau
C 2 8 24h
Figure 19
-55 kDa
96
Figure 19. Neuroserpin expression is induced in hippocampal cells in
response to KA treatment in vitro. (A) Confocal image of CA1 cells displaying
increased neuroserpin immunostaining and PI uptake 24h after KA treatment.
(B) Neuroserpin increase is particularly strong throughout the hilus in
response to KA. (C) Time course demonstrating increasing neuroserpin
release into culture medium 2-24h following KA treatment. Scale bar = 50um.
97
A
Figure 20
C 2 8 24
PAI-1
ponceau
B
PAI-1 PI mergeV
eh
24h K
A
-46kDa
98
Figure 20. Expression and release of PAI-1 in the dentate. (A) Twenty-four
hours after KA treatment, slices demonstrated moderate induction of PAI-1.
Images are representative of 3 independent experiments. Scale bar = 50um.
(B) Western analysis of culture medium was performed to examine secretion
of PAI-1. PAI-1 was not detected in culture medium of vehicle-treated slices,
but was induced by 2h following KA treatment and sustained through 24h.
99
Ctrl 24hA
Figure 21
B +aprotinin
100
Figure 21. tPA activity is downregulated following seizure. Twenty-four hours
following seizure, rats were anesthetized with, decapitated and their brains
removed, flash frozen, and cryosectioned (12µm). To assess endogenous tPA
activity, sections were subjected to in situ zymography. Fresh-frozen sections
were coated with an in situ zymogram assay buffer containing 1% casein and
10µg/ml plasminogen and incubated overnight in a humidified chamber at
37˚C. Endogenous tPA activates the plasminogen, which then digests the
casein, creating dark areas in the overlayed substrate buffer and visualized
using dark field microscopy. (A) In situ zymogram demonstrating tPA activity
in the hippocampus with and without seizure treatment. Note the decrease in
areas of activity particularly in the CA1-CA3 and dentate. (B) In situ
zymogram in the presence of aprontinin, a serine protease inhibitor,
demonstrating abolished tPA activity.
101
CA1 Dentate
24h post-seizure
A
B
Ctrl 24h
Figure 22
CCtrl KA
nsp
actin
102
Figure 22. Neuroserpin expression is induced by seizures in vivo. (A) DAB
staining for neuroserpin shows upregulated expression in the dentate and CA3
areas of the hippocampus 24h following KA-induced seizures. (B) High
magnification of CA1 and dentate areas from (A), showing differential
neuroserpin expression following KA treatment. (C) Western blot of whole
hippocampal lysates demonstrating upregulated neuroserpin in response to KA-
induced seizures.
103
A
Figure 23
Ctrl KA
BCtrl KA
PAI-1
actin
104
Figure 23. Neuroserpin expression is induced by seizures in vivo. (A) DAB
staining demonstrating PAI-1 expression is upregulated 24h following KA
treatment. Note that expression is particularly localized to the dentate and
CA3, similar to the pattern of neuroserpin upregulation as seen in figure 22.
(B) Western blot showing differential PAI-1 levels from whole hippocampal
lysates 24h following KA-induced seizures.
105
Figure 24
B
A Veh tPA
p75
FJ
Merge
Veh tPA
106
p75
FJ
Merge
CVeh tPA
107
Figure 24. Exogenous infusion of recombinant tPA (1µg every 12 hours)
leads to decreased cell death in the CA1 24h following KA-induced seizure.
(A) In situ zymogram demonstrating increased tPA activity in the injected
hemisphere and confirming tPA infusion. (B) Co-staining for p75NTR and FJ
demonstrates decreased p75NTR immunoreactivity and cell death in the CA1
ipsilateral to the tPA infusion. (C) FJ and p75NTR staining reveal no difference
in the dentate gyrus compared between vehicle or tPA injections, suggesting
regional differences in the rescue effect of tPA. All images are taken bilaterally
from the same rat and representative of images from 5 independent
experiments. Scale bar = 50um.
108
Figure 25
proNGF
NGF
-37 kDa
-15 kDa
Veh tPA Veh NGF
KA
109
Figure 25. Exogenous tPA infusions (1µg every 12 hours) reduce extracellular
proNGF 24h following KA-induced seizures. Western analysis of CSF from
tPA-injected rats demonstrate decreased proNGF detection compared with
non-seizured or vehicle-injected rats. Only rats from the same experimental
group were used for comparisons. Blot is representative of 3 independent
experiments.
110
111
XII. References
Abe Y, Nakamura H, Yoshino O, Oya T, Kimura T (2003) Decreased neural damage after spinal cord injury in tPA-deficient mice. J Neurotrauma 20:43-57.
Adler JE, Kessler JA, Black IB (1984) Development and regulation of substance P in sensory neurons in vitro. Dev Biol 102:417-425.
Ali TK, Al-Gayyar MM, Matragoon S, Pillai BA, Abdelsaid MA, Nussbaum JJ, El-Remessy AB (2011) Diabetes-induced peroxynitrite impairs the balance of pro-nerve growth factor and nerve growth factor, and causes neurovascular injury. Diabetologia 54:657-668.
Amalinei C, Caruntu ID, Giusca SE, Balan RA (2010) Matrix metalloproteinases involvement in pathologic conditions. Rom J Morphol Embryol 51:215-228.
Backstrom JR, Lim GP, Cullen MJ, Tokes ZA (1996) Matrix metalloproteinase-9 (MMP-9) is synthesized in neurons of the human hippocampus and is capable of degrading the amyloid-beta peptide (1-40). J Neurosci 16:7910-7919.
Ballarin M, Ernfors P, Lindefors N, Persson H (1991) Hippocampal damage and kainic acid injection induce a rapid increase in mRNA for BDNF and NGF in the rat brain. Exp Neurol 114:35-43.
Bamji SX, Majdan M, Pozniak CD, Belliveau DJ, Aloyz R, Kohn J, Causing CG, Miller FD (1998) The p75 neurotrophin receptor mediates neuronal apoptosis and is essential for naturally occurring sympathetic neuron death. J Cell Biol 140:911-923.
Baramova EN, Bajou K, Remacle A, L'Hoir C, Krell HW, Weidle UH, Noel A, Foidart JM (1997) Involvement of PA/plasmin system in the processing of pro-MMP-9 and in the second step of pro-MMP-2 activation. FEBS Lett 405:157-162.
Barrett GL, Bartlett PF (1994) The p75 nerve growth factor receptor mediates survival or death depending on the stage of sensory neuron development. Proc Natl Acad Sci U S A 91:6501-6505.
Barrett JM, Puglia MA, Singh G, Tozer RG (2002) Expression of Ets-related transcription factors and matrix metalloproteinase genes in human breast cancer cells. Breast Cancer Res Treat 72:227-232.
Basham ME, Seeds NW (2001) Plasminogen expression in the neonatal and adult mouse brain. J Neurochem 77:318-325.
112
Beattie MS, Harrington AW, Lee R, Kim JY, Boyce SL, Longo FM, Bresnahan JC, Hempstead BL, Yoon SO (2002) ProNGF induces p75-mediated death of oligodendrocytes following spinal cord injury. Neuron 36:375-386.
Ben-Ari Y, Tremblay E, Ottersen OP, Meldrum BS (1980) The role of epileptic activity in hippocampal and "remote" cerebral lesions induced by kainic acid. Brain Res 191:79-97.
Benedetti M, Levi A, Chao MV (1993) Differential expression of nerve growth factor receptors leads to altered binding affinity and neurotrophin responsiveness. Proc Natl Acad Sci U S A 90:7859-7863.
Berger P, Kozlov SV, Cinelli P, Kruger SR, Vogt L, Sonderegger P (1999) Neuronal depolarization enhances the transcription of the neuronal serine protease inhibitor neuroserpin. Mol Cell Neurosci 14:455-467.
Bierl MA, Jones EE, Crutcher KA, Isaacson LG (2005) 'Mature' nerve growth factor is a minor species in most peripheral tissues. Neurosci Lett 380:133-137.
Birkedal-Hansen H (1993) Role of matrix metalloproteinases in human periodontal diseases. J Periodontol 64:474-484.
Boutilier J, Ceni C, Pagdala PC, Forgie A, Neet KE, Barker PA (2008) Proneurotrophins require endocytosis and intracellular proteolysis to induce TrkA activation. J Biol Chem 283:12709-12716.
Brabletz T, Jung A, Dag S, Hlubek F, Kirchner T (1999) beta-catenin regulates the expression of the matrix metalloproteinase-7 in human colorectal cancer. Am J Pathol 155:1033-1038.
Bresnahan PA, Leduc R, Thomas L, Thorner J, Gibson HL, Brake AJ, Barr PJ, Thomas G (1990) Human fur gene encodes a yeast KEX2-like endoprotease that cleaves pro-beta-NGF in vivo. J Cell Biol 111:2851-2859.
Bruno MA, Cuello AC (2006) Activity-dependent release of precursor nerve growth factor, conversion to mature nerve growth factor, and its degradation by a protease cascade. Proc Natl Acad Sci U S A 103:6735-6740.
Buhler LA, Samara R, Guzman E, Wilson CL, Krizanac-Bengez L, Janigro D, Ethell DW (2009) Matrix metalloproteinase-7 facilitates immune access to the CNS in experimental autoimmune encephalomyelitis. BMC Neurosci 10:17.
113
Caccamo DV, Keohane ME, McKeever PE (1994) Plasminogen activators and inhibitors in gliomas: an immunohistochemical study. Mod Pathol 7:99-104.
Caggiula M, Batocchi AP, Frisullo G, Angelucci F, Patanella AK, Sancricca C, Nociti V, Tonali PA, Mirabella M (2006) Neurotrophic factors in relapsing remitting and secondary progressive multiple sclerosis patients during interferon beta therapy. Clin Immunol 118:77-82.
Cao C, Lawrence DA, Li Y, Von Arnim CA, Herz J, Su EJ, Makarova A, Hyman BT, Strickland DK, Zhang L (2006) Endocytic receptor LRP together with tPA and PAI-1 coordinates Mac-1-dependent macrophage migration. EMBO J 25:1860-1870.
Capsoni S, Tiveron C, Vignone D, Amato G, Cattaneo A (2010) Dissecting the involvement of tropomyosin-related kinase A and p75 neurotrophin receptor signaling in NGF deficit-induced neurodegeneration. Proc Natl Acad Sci U S A 107:12299-12304.
Carmichael DF, Sommer A, Thompson RC, Anderson DC, Smith CG, Welgus HG, Stricklin GP (1986) Primary structure and cDNA cloning of human fibroblast collagenase inhibitor. Proc Natl Acad Sci U S A 83:2407-2411.
Casaccia-Bonnefil P, Carter BD, Dobrowsky RT, Chao MV (1996) Death of oligodendrocytes mediated by the interaction of nerve growth factor with its receptor p75. Nature 383:716-719.
Casademunt E, Carter BD, Benzel I, Frade JM, Dechant G, Barde YA (1999) The zinc finger protein NRIF interacts with the neurotrophin receptor p75(NTR) and participates in programmed cell death. EMBO J 18:6050-6061.
Chambers RC (2003) Role of coagulation cascade proteases in lung repair and fibrosis. Eur Respir J Suppl 44:33s-35s.
Chang A, Nishiyama A, Peterson J, Prineas J, Trapp BD (2000) NG2-positive oligodendrocyte progenitor cells in adult human brain and multiple sclerosis lesions. J Neurosci 20:6404-6412.
Chao MV (1992) Neurotrophin receptors: a window into neuronal differentiation. Neuron 9:583-593.
Chao MV, Bothwell M (2002) Neurotrophins: to cleave or not to cleave. Neuron 33:9-12.
Chao MV, Bothwell MA, Ross AH, Koprowski H, Lanahan AA, Buck CR, Sehgal A (1986) Gene transfer and molecular cloning of the human NGF receptor. Science 232:518-521.
114
Chazaud B, Ricoux R, Christov C, Plonquet A, Gherardi RK, Barlovatz-Meimon G (2002) Promigratory effect of plasminogen activator inhibitor-1 on invasive breast cancer cell populations. Am J Pathol 160:237-246.
Chen KS, Nishimura MC, Armanini MP, Crowley C, Spencer SD, Phillips HS (1997) Disruption of a single allele of the nerve growth factor gene results in atrophy of basal forebrain cholinergic neurons and memory deficits. J Neurosci 17:7288-7296.
Cosgaya JM, Chan JR, Shooter EM (2002) The neurotrophin receptor p75NTR as a positive modulator of myelination. Science 298:1245-1248.
Cragnolini AB, Huang Y, Gokina P, Friedman WJ (2009) Nerve growth factor attenuates proliferation of astrocytes via the p75 neurotrophin receptor. Glia 57:1386-1392.
Crocker SJ, Milner R, Pham-Mitchell N, Campbell IL (2006) Cell and agonist-specific regulation of genes for matrix metalloproteinases and their tissue inhibitors by primary glial cells. J Neurochem 98:812-823.
Cuello AC, Bruno MA (2007) The failure in NGF maturation and its increased degradation as the probable cause for the vulnerability of cholinergic neurons in Alzheimer's disease. Neurochem Res 32:1041-1045.
Cuello AC, Bruno MA, Bell KF (2007) NGF-cholinergic dependency in brain aging, MCI and Alzheimer's disease. Curr Alzheimer Res 4:351-358.
Degryse B, Sier CF, Resnati M, Conese M, Blasi F (2001) PAI-1 inhibits urokinase-induced chemotaxis by internalizing the urokinase receptor. FEBS Lett 505:249-254.
Del Signore A, De Sanctis V, Di Mauro E, Negri R, Perrone-Capano C, Paggi P (2006) Gene expression pathways induced by axotomy and decentralization of rat superior cervical ganglion neurons. Eur J Neurosci 23:65-74.
Deppmann CD, Mihalas S, Sharma N, Lonze BE, Niebur E, Ginty DD (2008) A model for neuronal competition during development. Science 320:369-373.
Dicou E, Lee J, Brachet P (1986) Synthesis of nerve growth factor mRNA and precursor protein in the thyroid and parathyroid glands of the rat. Proc Natl Acad Sci U S A 83:7084-7088.
Domeniconi M, Hempstead BL, Chao MV (2007) Pro-NGF secreted by astrocytes promotes motor neuron cell death. Mol Cell Neurosci 34:271-279.
115
East E, Baker D, Pryce G, Lijnen HR, Cuzner ML, Gveric D (2005) A role for the plasminogen activator system in inflammation and neurodegeneration in the central nervous system during experimental allergic encephalomyelitis. Am J Pathol 167:545-554.
Echeverry R, Wu J, Haile WB, Guzman J, Yepes M (2010) Tissue-type plasminogen activator is a neuroprotectant in the mouse hippocampus. J Clin Invest 120:2194-2205.
Edwards RH, Selby MJ, Mobley WC, Weinrich SL, Hruby DE, Rutter WJ (1988) Processing and secretion of nerve growth factor: expression in mammalian cells with a vaccinia virus vector. Mol Cell Biol 8:2456-2464.
Ernfors P, Merlio JP, Persson H (1992) Cells Expressing mRNA for Neurotrophins and their Receptors During Embryonic Rat Development. Eur J Neurosci 4:1140-1158.
Ernfors P, Henschen A, Olson L, Persson H (1989) Expression of nerve growth factor receptor mRNA is developmentally regulated and increased after axotomy in rat spinal cord motoneurons. Neuron 2:1605-1613.
Ernfors P, Ibanez CF, Ebendal T, Olson L, Persson H (1990) Molecular cloning and neurotrophic activities of a protein with structural similarities to nerve growth factor: developmental and topographical expression in the brain. Proc Natl Acad Sci U S A 87:5454-5458.
Ernfors P, Bengzon J, Kokaia Z, Persson H, Lindvall O (1991) Increased levels of messenger RNAs for neurotrophic factors in the brain during kindling epileptogenesis. Neuron 7:165-176.
Esposito D, Patel P, Stephens RM, Perez P, Chao MV, Kaplan DR, Hempstead BL (2001) The cytoplasmic and transmembrane domains of the p75 and Trk A receptors regulate high affinity binding to nerve growth factor. J Biol Chem 276:32687-32695.
Ethell DW, Kinloch R, Green DR (2002) Metalloproteinase shedding of Fas ligand regulates beta-amyloid neurotoxicity. Curr Biol 12:1595-1600.
Fabbro S, Seeds NW (2009) Plasminogen activator activity is inhibited while neuroserpin is up-regulated in the Alzheimer disease brain. J Neurochem 109:303-315.
Fahnestock M, Michalski B, Xu B, Coughlin MD (2001) The precursor pro-nerve growth factor is the predominant form of nerve growth factor in brain and is increased in Alzheimer's disease. Mol Cell Neurosci 18:210-220.
Fahnestock M, Yu G, Michalski B, Mathew S, Colquhoun A, Ross GM, Coughlin MD (2004) The nerve growth factor precursor proNGF exhibits
116
neurotrophic activity but is less active than mature nerve growth factor. J Neurochem 89:581-592.
Farhadi HF, Mowla SJ, Petrecca K, Morris SJ, Seidah NG, Murphy RA (2000) Neurotrophin-3 sorts to the constitutive secretory pathway of hippocampal neurons and is diverted to the regulated secretory pathway by coexpression with brain-derived neurotrophic factor. J Neurosci 20:4059-4068.
Fiore M, Talamini L, Angelucci F, Koch T, Aloe L, Korf J (1999) Prenatal methylazoxymethanol acetate alters behavior and brain NGF levels in young rats: a possible correlation with the development of schizophrenia-like deficits. Neuropharmacology 38:857-869.
Fischer W, Bjorklund A, Chen K, Gage FH (1991) NGF improves spatial memory in aged rodents as a function of age. J Neurosci 11:1889-1906.
Fischer W, Sirevaag A, Wiegand SJ, Lindsay RM, Bjorklund A (1994) Reversal of spatial memory impairments in aged rats by nerve growth factor and neurotrophins 3 and 4/5 but not by brain-derived neurotrophic factor. Proc Natl Acad Sci U S A 91:8607-8611.
Fosang AJ, Neame PJ, Last K, Hardingham TE, Murphy G, Hamilton JA (1992) The interglobular domain of cartilage aggrecan is cleaved by PUMP, gelatinases, and cathepsin B. J Biol Chem 267:19470-19474.
Frade JM, Barde YA (1999) Genetic evidence for cell death mediated by nerve growth factor and the neurotrophin receptor p75 in the developing mouse retina and spinal cord. Development 126:683-690.
Frade JM, Rodriguez-Tebar A, Barde YA (1996) Induction of cell death by endogenous nerve growth factor through its p75 receptor. Nature 383:166-168.
Frade JM, Bovolenta P, Rodriguez-Tebar A (1999) Neurotrophins and other growth factors in the generation of retinal neurons. Microsc Res Tech 45:243-251.
Friedman WJ (2000) Neurotrophins induce death of hippocampal neurons via the p75 receptor. J Neurosci 20:6340-6346.
Friedman WJ, Greene LA (1999) Neurotrophin signaling via Trks and p75. Exp Cell Res 253:131-142.
Gall CM, Isackson PJ (1989) Limbic seizures increase neuronal production of messenger RNA for nerve growth factor. Science 245:758-761.
117
Giehl KM, Rohrig S, Bonatz H, Gutjahr M, Leiner B, Bartke I, Yan Q, Reichardt LF, Backus C, Welcher AA, Dethleffsen K, Mestres P, Meyer M (2001) Endogenous brain-derived neurotrophic factor and neurotrophin-3 antagonistically regulate survival of axotomized corticospinal neurons in vivo. J Neurosci 21:3492-3502.
Ginsberg SD, Che S, Wuu J, Counts SE, Mufson EJ (2006) Down regulation of trk but not p75NTR gene expression in single cholinergic basal forebrain neurons mark the progression of Alzheimer's disease. J Neurochem 97:475-487.
Girgenti MJ, Collier E, Sathyanesan M, Su XW, Newton SS (2010) Characterization of electroconvulsive seizure-induced TIMP-1 and MMP-9 in hippocampal vasculature. Int J Neuropsychopharmacol:1-10.
Glass DJ, Nye SH, Hantzopoulos P, Macchi MJ, Squinto SP, Goldfarb M, Yancopoulos GD (1991) TrkB mediates BDNF/NT-3-dependent survival and proliferation in fibroblasts lacking the low affinity NGF receptor. Cell 66:405-413.
Gogly B, Fournier B, Couty L, Naveau A, Brasselet C, Durand E, Coulomb B, Lafont A (2009) Gingival fibroblast inhibits MMP-7: evaluation in an ex vivo aorta model. J Mol Cell Cardiol 47:296-303.
Gomez DE, Alonso DF, Yoshiji H, Thorgeirsson UP (1997) Tissue inhibitors of metalloproteinases: structure, regulation and biological functions. Eur J Cell Biol 74:111-122.
Gomis-Ruth FX, Maskos K, Betz M, Bergner A, Huber R, Suzuki K, Yoshida N, Nagase H, Brew K, Bourenkov GP, Bartunik H, Bode W (1997) Mechanism of inhibition of the human matrix metalloproteinase stromelysin-1 by TIMP-1. Nature 389:77-81.
Gotz R, Koster R, Winkler C, Raulf F, Lottspeich F, Schartl M, Thoenen H (1994) Neurotrophin-6 is a new member of the nerve growth factor family. Nature 372:266-269.
Groft LL, Muzik H, Rewcastle NB, Johnston RN, Knauper V, Lafleur MA, Forsyth PA, Edwards DR (2001) Differential expression and localization of TIMP-1 and TIMP-4 in human gliomas. Br J Cancer 85:55-63.
Gschwendtner A, Liu Z, Hucho T, Bohatschek M, Kalla R, Dechant G, Raivich G (2003) Regulation, cellular localization, and function of the p75 neurotrophin receptor (p75NTR) during the regeneration of facial motoneurons. Mol Cell Neurosci 24:307-322.
118
Gualandris A, Jones TE, Strickland S, Tsirka SE (1996) Membrane depolarization induces calcium-dependent secretion of tissue plasminogen activator. J Neurosci 16:2220-2225.
Hallbook F, Ibanez CF, Persson H (1991) Evolutionary studies of the nerve growth factor family reveal a novel member abundantly expressed in Xenopus ovary. Neuron 6:845-858.
Hamburger V, Levi-Montalcini R (1949) Proliferation, differentiation and degeneration in the spinal ganglia of the chick embryo under normal and experimental conditions. J Exp Zool 111:457-501.
Han BH, Holtzman DM (2000) BDNF protects the neonatal brain from hypoxic-ischemic injury in vivo via the ERK pathway. J Neurosci 20:5775-5781.
Hardingham TE, Fosang AJ (1992) Proteoglycans: many forms and many functions. FASEB J 6:861-870.
Harrington AW, Leiner B, Blechschmitt C, Arevalo JC, Lee R, Morl K, Meyer M, Hempstead BL, Yoon SO, Giehl KM (2004) Secreted proNGF is a pathophysiological death-inducing ligand after adult CNS injury. Proc Natl Acad Sci U S A 101:6226-6230.
Harvey MB, Leco KJ, Arcellana-Panlilio MY, Zhang X, Edwards DR, Schultz GA (1995) Proteinase expression in early mouse embryos is regulated by leukaemia inhibitory factor and epidermal growth factor. Development 121:1005-1014.
Hasan W, Pedchenko T, Krizsan-Agbas D, Baum L, Smith PG (2003) Sympathetic neurons synthesize and secrete pro-nerve growth factor protein. J Neurobiol 57:38-53.
Hastings GA, Coleman TA, Haudenschild CC, Stefansson S, Smith EP, Barthlow R, Cherry S, Sandkvist M, Lawrence DA (1997) Neuroserpin, a brain-associated inhibitor of tissue plasminogen activator is localized primarily in neurons. Implications for the regulation of motor learning and neuronal survival. J Biol Chem 272:33062-33067.
Heinrich G, Lum T (2000) Fish neurotrophins and Trk receptors. Int J Dev Neurosci 18:1-27.
Hempstead BL, Martin-Zanca D, Kaplan DR, Parada LF, Chao MV (1991) High-affinity NGF binding requires coexpression of the trk proto-oncogene and the low-affinity NGF receptor. Nature 350:678-683.
Henriksson BG, Soderstrom S, Gower AJ, Ebendal T, Winblad B, Mohammed AH (1992) Hippocampal nerve growth factor levels are related to spatial learning ability in aged rats. Behav Brain Res 48:15-20.
119
Heymach JV, Jr., Shooter EM (1995) The biosynthesis of neurotrophin heterodimers by transfected mammalian cells. J Biol Chem 270:12297-12304.
Heymach JV, Jr., Kruttgen A, Suter U, Shooter EM (1996) The regulated secretion and vectorial targeting of neurotrophins in neuroendocrine and epithelial cells. J Biol Chem 271:25430-25437.
Hino H, Akiyama H, Iseki E, Kato M, Kondo H, Ikeda K, Kosaka K (2001) Immunohistochemical localization of plasminogen activator inhibitor-1 in rat and human brain tissues. Neurosci Lett 297:105-108.
Hohn A, Leibrock J, Bailey K, Barde YA (1990) Identification and characterization of a novel member of the nerve growth factor/brain-derived neurotrophic factor family. Nature 344:339-341.
Imai K, Yokohama Y, Nakanishi I, Ohuchi E, Fujii Y, Nakai N, Okada Y (1995) Matrix metalloproteinase 7 (matrilysin) from human rectal carcinoma cells. Activation of the precursor, interaction with other matrix metalloproteinases and enzymic properties. J Biol Chem 270:6691-6697.
Ip NY, Ibanez CF, Nye SH, McClain J, Jones PF, Gies DR, Belluscio L, Le Beau MM, Espinosa R, 3rd, Squinto SP, et al. (1992) Mammalian neurotrophin-4: structure, chromosomal localization, tissue distribution, and receptor specificity. Proc Natl Acad Sci U S A 89:3060-3064.
Jacobsen JS et al. (2008) Enhanced clearance of Abeta in brain by sustaining the plasmin proteolysis cascade. Proc Natl Acad Sci U S A 105:8754-8759.
Jansen P, Giehl K, Nyengaard JR, Teng K, Lioubinski O, Sjoegaard SS, Breiderhoff T, Gotthardt M, Lin F, Eilers A, Petersen CM, Lewin GR, Hempstead BL, Willnow TE, Nykjaer A (2007) Roles for the pro-neurotrophin receptor sortilin in neuronal development, aging and brain injury. Nat Neurosci 10:1449-1457.
Jee BK, Park KM, Surendran S, Lee WK, Han CW, Kim YS, Lim Y (2006) KAI1/CD82 suppresses tumor invasion by MMP9 inactivation via TIMP1 up-regulation in the H1299 human lung carcinoma cell line. Biochem Biophys Res Commun 342:655-661.
Jourquin J, Tremblay E, Decanis N, Charton G, Hanessian S, Chollet AM, Le Diguardher T, Khrestchatisky M, Rivera S (2003) Neuronal activity-dependent increase of net matrix metalloproteinase activity is associated with MMP-9 neurotoxicity after kainate. Eur J Neurosci 18:1507-1517.
Jourquin J, Tremblay E, Bernard A, Charton G, Chaillan FA, Marchetti E, Roman FS, Soloway PD, Dive V, Yiotakis A, Khrestchatisky M, Rivera S (2005)
120
Tissue inhibitor of metalloproteinases-1 (TIMP-1) modulates neuronal death, axonal plasticity, and learning and memory. Eur J Neurosci 22:2569-2578.
Kaplan DR, Miller FD (2000) Neurotrophin signal transduction in the nervous system. Curr Opin Neurobiol 10:381-391.
Kaplan DR, Martin-Zanca D, Parada LF (1991a) Tyrosine phosphorylation and tyrosine kinase activity of the trk proto-oncogene product induced by NGF. Nature 350:158-160.
Kaplan DR, Hempstead BL, Martin-Zanca D, Chao MV, Parada LF (1991b) The trk proto-oncogene product: a signal transducing receptor for nerve growth factor. Science 252:554-558.
Katoh-Semba R, Takeuchi IK, Inaguma Y, Ito H, Kato K (1999) Brain-derived neurotrophic factor, nerve growth and neurotrophin-3 selected regions of the rat brain following kainic acid-induced seizure activity. Neurosci Res 35:19-29.
Kawamata H, Kawai K, Kameyama S, Johnson MD, Stetler-Stevenson WG, Oyasu R (1995) Over-expression of tissue inhibitor of matrix metalloproteinases (TIMP1 and TIMP2) suppresses extravasation of pulmonary metastasis of a rat bladder carcinoma. Int J Cancer 63:680-687.
Kawasaki T, Dewerchin M, Lijnen HR, Vermylen J, Hoylaerts MF (2000) Vascular release of plasminogen activator inhibitor-1 impairs fibrinolysis during acute arterial thrombosis in mice. Blood 96:153-160.
Kenchappa RS, Zampieri N, Chao MV, Barker PA, Teng HK, Hempstead BL, Carter BD (2006) Ligand-dependent cleavage of the P75 neurotrophin receptor is necessary for NRIF nuclear translocation and apoptosis in sympathetic neurons. Neuron 50:219-232.
Kendall TJ, Hennedige S, Aucott RL, Hartland SN, Vernon MA, Benyon RC, Iredale JP (2009) p75 Neurotrophin receptor signaling regulates hepatic myofibroblast proliferation and apoptosis in recovery from rodent liver fibrosis. Hepatology 49:901-910.
Kerwin JM, Morris CM, Perry RH, Perry EK (1992) Hippocampal nerve growth factor receptor immunoreactivity in patients with Alzheimer's and Parkinson's disease. Neurosci Lett 143:101-104.
Kessenbrock K, Plaks V, Werb Z (2010) Matrix metalloproteinases: regulators of the tumor microenvironment. Cell 141:52-67.
121
Kim JW, Lee SH, Ko HM, Kwon KJ, Cho KS, Choi CS, Park JH, Kim HY, Lee J, Han SH, Ignarro LJ, Cheong JH, Kim WK, Shin CY (2011) Biphasic regulation of tissue plasminogen activator activity in ischemic rat brain and in cultured neural cells: Essential role of astrocyte-derived plasminogen activator inhibitor-1. Neurochem Int 58:423-433.
Kim YH, Park JH, Hong SH, Koh JY (1999) Nonproteolytic neuroprotection by human recombinant tissue plasminogen activator. Science 284:647-650.
Klein R, Nanduri V, Jing SA, Lamballe F, Tapley P, Bryant S, Cordon-Cardo C, Jones KR, Reichardt LF, Barbacid M (1991) The trkB tyrosine protein kinase is a receptor for brain-derived neurotrophic factor and neurotrophin-3. Cell 66:395-403.
Koh S, Chang P, Collier TJ, Loy R (1989) Loss of NGF receptor immunoreactivity in basal forebrain neurons of aged rats: correlation with spatial memory impairment. Brain Res 498:397-404.
Kohn J, Aloyz RS, Toma JG, Haak-Frendscho M, Miller FD (1999) Functionally antagonistic interactions between the TrkA and p75 neurotrophin receptors regulate sympathetic neuron growth and target innervation. J Neurosci 19:5393-5408.
Kokaia Z, Kelly ME, Elmer E, Kokaia M, McIntyre DC, Lindvall O (1996) Seizure-induced differential expression of messenger RNAs for neurotrophins and their receptors in genetically fast and slow kindling rats. Neuroscience 75:197-207.
Koshimizu H, Kiyosue K, Hara T, Hazama S, Suzuki S, Uegaki K, Nagappan G, Zaitsev E, Hirokawa T, Tatsu Y, Ogura A, Lu B, Kojima M (2009) Multiple functions of precursor BDNF to CNS neurons: negative regulation of neurite growth, spine formation and cell survival. Mol Brain 2:27.
Kouwenhoven M, Ozenci V, Gomes A, Yarilin D, Giedraitis V, Press R, Link H (2001) Multiple sclerosis: elevated expression of matrix metalloproteinases in blood monocytes. J Autoimmun 16:463-470.
Kruithof EK, Tran-Thang C, Gudinchet A, Hauert J, Nicoloso G, Genton C, Welti H, Bachmann F (1987) Fibrinolysis in pregnancy: a study of plasminogen activator inhibitors. Blood 69:460-466.
Lad SP, Neet KE, Mufson EJ (2003) Nerve growth factor: structure, function and therapeutic implications for Alzheimer's disease. Curr Drug Targets CNS Neurol Disord 2:315-334.
Lai KO, Fu WY, Ip FC, Ip NY (1998) Cloning and expression of a novel neurotrophin, NT-7, from carp. Mol Cell Neurosci 11:64-76.
122
Lamballe F, Klein R, Barbacid M (1991) trkC, a new member of the trk family of tyrosine protein kinases, is a receptor for neurotrophin-3. Cell 66:967-979.
Larsson E, Kuma R, Norberg A, Minde J, Holmberg M (2009) Nerve growth factor R221W responsible for insensitivity to pain is defectively processed and accumulates as proNGF. Neurobiol Dis 33:221-228.
Ledesma MD, Da Silva JS, Crassaerts K, Delacourte A, De Strooper B, Dotti CG (2000) Brain plasmin enhances APP alpha-cleavage and Abeta degradation and is reduced in Alzheimer's disease brains. EMBO Rep 1:530-535.
Lee FS, Kim AH, Khursigara G, Chao MV (2001a) The uniqueness of being a neurotrophin receptor. Curr Opin Neurobiol 11:281-286.
Lee KF, Li E, Huber LJ, Landis SC, Sharpe AH, Chao MV, Jaenisch R (1992) Targeted mutation of the gene encoding the low affinity NGF receptor p75 leads to deficits in the peripheral sensory nervous system. Cell 69:737-749.
Lee R, Kermani P, Teng KK, Hempstead BL (2001b) Regulation of cell survival by secreted proneurotrophins. Science 294:1945-1948.
Leibrock J, Lottspeich F, Hohn A, Hofer M, Hengerer B, Masiakowski P, Thoenen H, Barde YA (1989) Molecular cloning and expression of brain-derived neurotrophic factor. Nature 341:149-152.
Levi-Montalcini R (1964a) Growth Control of Nerve Cells by a Protein Factor and Its Antiserum: Discovery of This Factor May Provide New Leads to Understanding of Some Neurogenetic Processes. Science 143:105-110.
Levi-Montalcini R (1964b) The Nerve Growth Factor. Ann N Y Acad Sci 118:149-170.
Levi-Montalcini R (1987) The nerve growth factor 35 years later. Science 237:1154-1162.
Lindahl TL, Ohlsson PI, Wiman B (1990) The mechanism of the reaction between human plasminogen-activator inhibitor 1 and tissue plasminogen activator. Biochem J 265:109-113.
Linggi MS, Burke TL, Williams BB, Harrington A, Kraemer R, Hempstead BL, Yoon SO, Carter BD (2005) Neurotrophin receptor interacting factor (NRIF) is an essential mediator of apoptotic signaling by the p75 neurotrophin receptor. J Biol Chem 280:13801-13808.
123
Liotta LA, Steeg PS, Stetler-Stevenson WG (1991) Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation. Cell 64:327-336.
Liu YX, Chen YX, Shi FW, Feng Q (1995) Studies on the role of plasminogen activators and plasminogen activator inhibitor type-1 in rat corpus luteum of pregnancy. Biol Reprod 53:1131-1138.
London CA, Sekhon HS, Arora V, Stein DA, Iversen PL, Devi GR (2003) A novel antisense inhibitor of MMP-9 attenuates angiogenesis, human prostate cancer cell invasion and tumorigenicity. Cancer Gene Ther 10:823-832.
Lorenzl S, Albers DS, LeWitt PA, Chirichigno JW, Hilgenberg SL, Cudkowicz ME, Beal MF (2003) Tissue inhibitors of matrix metalloproteinases are elevated in cerebrospinal fluid of neurodegenerative diseases. J Neurol Sci 207:71-76.
Maisonpierre PC, Belluscio L, Squinto S, Ip NY, Furth ME, Lindsay RM, Yancopoulos GD (1990a) Neurotrophin-3: a neurotrophic factor related to NGF and BDNF. Science 247:1446-1451.
Maisonpierre PC, Belluscio L, Friedman B, Alderson RF, Wiegand SJ, Furth ME, Lindsay RM, Yancopoulos GD (1990b) NT-3, BDNF, and NGF in the developing rat nervous system: parallel as well as reciprocal patterns of expression. Neuron 5:501-509.
Majdan M, Miller FD (1999) Neuronal life and death decisions functional antagonism between the Trk and p75 neurotrophin receptors. Int J Dev Neurosci 17:153-161.
Majdan M, Lachance C, Gloster A, Aloyz R, Zeindler C, Bamji S, Bhakar A, Belliveau D, Fawcett J, Miller FD, Barker PA (1997) Transgenic mice expressing the intracellular domain of the p75 neurotrophin receptor undergo neuronal apoptosis. J Neurosci 17:6988-6998.
Malemud CJ (2006) Matrix metalloproteinases (MMPs) in health and disease: an overview. Front Biosci 11:1696-1701.
Marcinkiewicz M, Marcinkiewicz J, Chen A, Leclaire F, Chretien M, Richardson P (1999) Nerve growth factor and proprotein convertases furin and PC7 in transected sciatic nerves and in nerve segments cultured in conditioned media: their presence in Schwann cells, macrophages, and smooth muscle cells. J Comp Neurol 403:471-485.
Marler KJ, Poopalasundaram S, Broom ER, Wentzel C, Drescher U (2010) Pro-neurotrophins secreted from retinal ganglion cell axons are necessary for ephrinA-p75NTR-mediated axon guidance. Neural Dev 5:30.
124
Mataga N, Mizuguchi Y, Hensch TK (2004) Experience-dependent pruning of dendritic spines in visual cortex by tissue plasminogen activator. Neuron 44:1031-1041.
Matsumoto T, Rauskolb S, Polack M, Klose J, Kolbeck R, Korte M, Barde YA (2008) Biosynthesis and processing of endogenous BDNF: CNS neurons store and secrete BDNF, not pro-BDNF. Nat Neurosci 11:131-133.
Matsuoka I, Meyer M, Hofer M, Thoenen H (1991) Differential regulation of nerve growth factor and brain-derived neurotrophic factor expression in the peripheral nervous system. Ann N Y Acad Sci 633:550-552.
McDonald NQ, Lapatto R, Murray-Rust J, Gunning J, Wlodawer A, Blundell TL (1991) New protein fold revealed by a 2.3-A resolution crystal structure of nerve growth factor. Nature 354:411-414.
Melchor JP, Pawlak R, Strickland S (2003) The tissue plasminogen activator-plasminogen proteolytic cascade accelerates amyloid-beta (Abeta) degradation and inhibits Abeta-induced neurodegeneration. J Neurosci 23:8867-8871.
Meyer M, Matsuoka I, Wetmore C, Olson L, Thoenen H (1992) Enhanced synthesis of brain-derived neurotrophic factor in the lesioned peripheral nerve: different mechanisms are responsible for the regulation of BDNF and NGF mRNA. J Cell Biol 119:45-54.
Miyazaki K, Hattori Y, Umenishi F, Yasumitsu H, Umeda M (1990) Purification and characterization of extracellular matrix-degrading metalloproteinase, matrin (pump-1), secreted from human rectal carcinoma cell line. Cancer Res 50:7758-7764.
Morimoto K, Sato K, Sato S, Yamada N, Hayabara T (1998) Time-dependent changes in neurotrophic factor mRNA expression after kindling and long-term potentiation in rats. Brain Res Bull 45:599-605.
Mowla SJ, Farhadi HF, Pareek S, Atwal JK, Morris SJ, Seidah NG, Murphy RA (2001) Biosynthesis and post-translational processing of the precursor to brain-derived neurotrophic factor. J Biol Chem 276:12660-12666.
Mowla SJ, Pareek S, Farhadi HF, Petrecca K, Fawcett JP, Seidah NG, Morris SJ, Sossin WS, Murphy RA (1999) Differential sorting of nerve growth factor and brain-derived neurotrophic factor in hippocampal neurons. J Neurosci 19:2069-2080.
Mufson EJ, Presley LN, Kordower JH (1991) Nerve growth factor receptor immunoreactivity within the nucleus basalis (Ch4) in Parkinson's disease: reduced cell numbers and co-localization with cholinergic neurons. Brain Res 539:19-30.
125
Nagappan G, Zaitsev E, Senatorov VV, Jr., Yang J, Hempstead BL, Lu B (2009) Control of extracellular cleavage of ProBDNF by high frequency neuronal activity. Proc Natl Acad Sci U S A 106:1267-1272.
Nagase H (1997) Activation mechanisms of matrix metalloproteinases. Biol Chem 378:151-160.
Nagase H, Brew K (2003) Designing TIMP (tissue inhibitor of metalloproteinases) variants that are selective metalloproteinase inhibitors. Biochem Soc Symp:201-212.
Nagase H, Enghild JJ, Suzuki K, Salvesen G (1990) Stepwise activation mechanisms of the precursor of matrix metalloproteinase 3 (stromelysin) by proteinases and (4-aminophenyl)mercuric acetate. Biochemistry 29:5783-5789.
Nataf S, Naveilhan P, Sindji L, Darcy F, Brachet P, Montero-Menei CN (1998) Low affinity NGF receptor expression in the central nervous system during experimental allergic encephalomyelitis. J Neurosci Res 52:83-92.
Nico B, Mangieri D, De Luca A, Corsi P, Benagiano V, Tamma R, Annese T, Longo V, Crivellato E, Ribatti D (2009) Nerve growth factor and its receptors TrkA and p75 are upregulated in the brain of mdx dystrophic mouse. Neuroscience 161:1057-1066.
Nykjaer A, Lee R, Teng KK, Jansen P, Madsen P, Nielsen MS, Jacobsen C, Kliemannel M, Schwarz E, Willnow TE, Hempstead BL, Petersen CM (2004) Sortilin is essential for proNGF-induced neuronal cell death. Nature 427:843-848.
Oh JD, Chartisathian K, Chase TN, Butcher LL (2000) Overexpression of neurotrophin receptor p75 contributes to the excitotoxin-induced cholinergic neuronal death in rat basal forebrain. Brain Res 853:174-185.
Ohnami S, Matsumoto N, Nakano M, Aoki K, Nagasaki K, Sugimura T, Terada M, Yoshida T (1999) Identification of genes showing differential expression in antisense K-ras-transduced pancreatic cancer cells with suppressed tumorigenicity. Cancer Res 59:5565-5571.
Osterwalder T, Cinelli P, Baici A, Pennella A, Krueger SR, Schrimpf SP, Meins M, Sonderegger P (1998) The axonally secreted serine proteinase inhibitor, neuroserpin, inhibits plasminogen activators and plasmin but not thrombin. J Biol Chem 273:2312-2321.
Ozaki I, Mizuta T, Zhao G, Yotsumoto H, Hara T, Kajihara S, Hisatomi A, Sakai T, Yamamoto K (2000) Involvement of the Ets-1 gene in overexpression of matrilysin in human hepatocellular carcinoma. Cancer Res 60:6519-6525.
126
Pajouh MS, Nagle RB, Breathnach R, Finch JS, Brawer MK, Bowden GT (1991) Expression of metalloproteinase genes in human prostate cancer. J Cancer Res Clin Oncol 117:144-150.
Pang PT, Teng HK, Zaitsev E, Woo NT, Sakata K, Zhen S, Teng KK, Yung WH, Hempstead BL, Lu B (2004) Cleavage of proBDNF by tPA/plasmin is essential for long-term hippocampal plasticity. Science 306:487-491.
Papadopoulos SM, Chandler WF, Salamat MS, Topol EJ, Sackellares JC (1987) Recombinant human tissue-type plasminogen activator therapy in acute thromboembolic stroke. J Neurosurg 67:394-398.
Park JA, Lee JY, Sato TA, Koh JY (2000) Co-induction of p75NTR and p75NTR-associated death executor in neurons after zinc exposure in cortical culture or transient ischemia in the rat. J Neurosci 20:9096-9103.
Parks WC, Wilson CL, Lopez-Boado YS (2004) Matrix metalloproteinases as modulators of inflammation and innate immunity. Nat Rev Immunol 4:617-629.
Patapoutian A, Reichardt LF (2001) Trk receptors: mediators of neurotrophin action. Curr Opin Neurobiol 11:272-280.
Pedraza CE, Podlesniy P, Vidal N, Arevalo JC, Lee R, Hempstead B, Ferrer I, Iglesias M, Espinet C (2005) Pro-NGF isolated from the human brain affected by Alzheimer's disease induces neuronal apoptosis mediated by p75NTR. Am J Pathol 166:533-543.
Peng S, Wuu J, Mufson EJ, Fahnestock M (2004) Increased proNGF levels in subjects with mild cognitive impairment and mild Alzheimer disease. J Neuropathol Exp Neurol 63:641-649.
Ra HJ, Parks WC (2007) Control of matrix metalloproteinase catalytic activity. Matrix Biol 26:587-596.
Rao MS, Hattiangady B, Reddy DS, Shetty AK (2006) Hippocampal neurodegeneration, spontaneous seizures, and mossy fiber sprouting in the F344 rat model of temporal lobe epilepsy. J Neurosci Res 83:1088-1105.
Rattenholl A, Ruoppolo M, Flagiello A, Monti M, Vinci F, Marino G, Lilie H, Schwarz E, Rudolph R (2001) Pro-sequence assisted folding and disulfide bond formation of human nerve growth factor. J Mol Biol 305:523-533.
Ray JM, Stetler-Stevenson WG (1995) Gelatinase A activity directly modulates melanoma cell adhesion and spreading. EMBO J 14:908-917.
127
Reinshagen M, Geerling I, Eysselein VE, Adler G, Huff KR, Moore GP, Lakshmanan J (2000) Commercial recombinant human beta-nerve growth factor and adult rat dorsal root ganglia contain an identical molecular species of nerve growth factor prohormone. J Neurochem 74:2127-2133.
Ringstedt T, Lagercrantz H, Persson H (1993) Expression of members of the trk family in the developing postnatal rat brain. Brain Res Dev Brain Res 72:119-131.
Rivera S, Tremblay E, Timsit S, Canals O, Ben-Ari Y, Khrestchatisky M (1997) Tissue inhibitor of metalloproteinases-1 (TIMP-1) is differentially induced in neurons and astrocytes after seizures: evidence for developmental, immediate early gene, and lesion response. J Neurosci 17:4223-4235.
Robinson RC, Radziejewski C, Stuart DI, Jones EY (1995) Structure of the brain-derived neurotrophic factor/neurotrophin 3 heterodimer. Biochemistry 34:4139-4146.
Rocamora N, Palacios JM, Mengod G (1992) Limbic seizures induce a differential regulation of the expression of nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3, in the rat hippocampus. Brain Res Mol Brain Res 13:27-33.
Rogers ML, Bailey S, Matusica D, Nicholson I, Muyderman H, Pagadala PC, Neet KE, Zola H, Macardle P, Rush RA (2010) ProNGF mediates death of Natural Killer cells through activation of the p75NTR-sortilin complex. J Neuroimmunol 226:93-103.
Rome C, Arsaut J, Taris C, Couillaud F, Loiseau H (2007) MMP-7 (matrilysin) expression in human brain tumors. Mol Carcinog 46:446-452.
Routbort MJ, Bausch SB, McNamara JO (1999) Seizures, cell death, and mossy fiber sprouting in kainic acid-treated organotypic hippocampal cultures. Neuroscience 94:755-765.
Roux PP, Barker PA (2002) Neurotrophin signaling through the p75 neurotrophin receptor. Prog Neurobiol 67:203-233.
Roux PP, Colicos MA, Barker PA, Kennedy TE (1999) p75 neurotrophin receptor expression is induced in apoptotic neurons after seizure. J Neurosci 19:6887-6896.
Sachs BD, Baillie GS, McCall JR, Passino MA, Schachtrup C, Wallace DA, Dunlop AJ, MacKenzie KF, Klussmann E, Lynch MJ, Sikorski SL, Nuriel T, Tsigelny I, Zhang J, Houslay MD, Chao MV, Akassoglou K (2007) p75 neurotrophin receptor regulates tissue fibrosis through inhibition of plasminogen activation via a PDE4/cAMP/PKA pathway. J Cell Biol 177:1119-1132.
128
Safieh-Garabedian B, Poole S, Allchorne A, Winter J, Woolf CJ (1995) Contribution of interleukin-1 beta to the inflammation-induced increase in nerve growth factor levels and inflammatory hyperalgesia. Br J Pharmacol 115:1265-1275.
Salehi AH, Roux PP, Kubu CJ, Zeindler C, Bhakar A, Tannis LL, Verdi JM, Barker PA (2000) NRAGE, a novel MAGE protein, interacts with the p75 neurotrophin receptor and facilitates nerve growth factor-dependent apoptosis. Neuron 27:279-288.
Salles FJ, Strickland S (2002) Localization and regulation of the tissue plasminogen activator-plasmin system in the hippocampus. J Neurosci 22:2125-2134.
Schmued LC, Hopkins KJ (2000) Fluoro-Jade B: a high affinity fluorescent marker for the localization of neuronal degeneration. Brain Res 874:123-130.
Schwarcz R, Zaczek R, Coyle JT (1978) Microinjection of kainic acid into the rat hippocampus. Eur J Pharmacol 50:209-220.
Seeds NW, Williams BL, Bickford PC (1995) Tissue plasminogen activator induction in Purkinje neurons after cerebellar motor learning. Science 270:1992-1994.
Seidah NG, Benjannet S, Pareek S, Savaria D, Hamelin J, Goulet B, Laliberte J, Lazure C, Chretien M, Murphy RA (1996) Cellular processing of the nerve growth factor precursor by the mammalian pro-protein convertases. Biochem J 314 ( Pt 3):951-960.
Shirayama Y, Chen AC, Nakagawa S, Russell DS, Duman RS (2002) Brain-derived neurotrophic factor produces antidepressant effects in behavioral models of depression. J Neurosci 22:3251-3261.
Siao CJ, Fernandez SR, Tsirka SE (2003) Cell type-specific roles for tissue plasminogen activator released by neurons or microglia after excitotoxic injury. J Neurosci 23:3234-3242.
Siconolfi LB, Seeds NW (2001) Induction of the plasminogen activator system accompanies peripheral nerve regeneration after sciatic nerve crush. J Neurosci 21:4336-4347.
Singh KK, Miller FD (2005) Activity regulates positive and negative neurotrophin-derived signals to determine axon competition. Neuron 45:837-845.
Singh KK, Park KJ, Hong EJ, Kramer BM, Greenberg ME, Kaplan DR, Miller FD (2008) Developmental axon pruning mediated by BDNF-p75NTR-dependent axon degeneration. Nat Neurosci 11:649-658.
129
Skaper SD, Pollock M, Facci L (2001) Mast cells differentially express and release active high molecular weight neurotrophins. Brain Res Mol Brain Res 97:177-185.
Skrzypiec AE, Buczko W, Pawlak R (2008) Tissue plasminogen activator in the amygdala: a new role for an old protease. J Physiol Pharmacol 59 Suppl 8:135-146.
Smith MM, Shi L, Navre M (1995) Rapid identification of highly active and selective substrates for stromelysin and matrilysin using bacteriophage peptide display libraries. J Biol Chem 270:6440-6449.
So T, Ito A, Sato T, Mori Y, Hirakawa S (1992) Tumor necrosis factor-alpha stimulates the biosynthesis of matrix metalloproteinases and plasminogen activator in cultured human chorionic cells. Biol Reprod 46:772-778.
Song W, Volosin M, Cragnolini AB, Hempstead BL, Friedman WJ (2010) ProNGF induces PTEN via p75NTR to suppress Trk-mediated survival signaling in brain neurons. J Neurosci 30:15608-15615.
Sposato V, Bucci MG, Coassin M, Russo MA, Lambiase A, Aloe L (2008) Reduced NGF level and TrkA protein and TrkA gene expression in the optic nerve of rats with experimentally induced glaucoma. Neurosci Lett 446:20-24.
Springman EB, Angleton EL, Birkedal-Hansen H, Van Wart HE (1990) Multiple modes of activation of latent human fibroblast collagenase: evidence for the role of a Cys73 active-site zinc complex in latency and a "cysteine switch" mechanism for activation. Proc Natl Acad Sci U S A 87:364-368.
Squinto SP, Stitt TN, Aldrich TH, Davis S, Bianco SM, Radziejewski C, Glass DJ, Masiakowski P, Furth ME, Valenzuela DM, et al. (1991) trkB encodes a functional receptor for brain-derived neurotrophic factor and neurotrophin-3 but not nerve growth factor. Cell 65:885-893.
Srinivasan B, Roque CH, Hempstead BL, Al-Ubaidi MR, Roque RS (2004) Microglia-derived pronerve growth factor promotes photoreceptor cell death via p75 neurotrophin receptor. J Biol Chem 279:41839-41845.
Stoppini L, Buchs PA, Muller D (1991) A simple method for organotypic cultures of nervous tissue. J Neurosci Methods 37:173-182.
Suter U, Heymach JV, Jr., Shooter EM (1991) Two conserved domains in the NGF propeptide are necessary and sufficient for the biosynthesis of correctly processed and biologically active NGF. EMBO J 10:2395-2400.
130
Sutton R, Keohane ME, VanderBerg SR, Gonias SL (1994) Plasminogen activator inhibitor-1 in the cerebrospinal fluid as an index of neurological disease. Blood Coagul Fibrinolysis 5:167-171.
Suzuki K, Enghild JJ, Morodomi T, Salvesen G, Nagase H (1990) Mechanisms of activation of tissue procollagenase by matrix metalloproteinase 3 (stromelysin). Biochemistry 29:10261-10270.
Tan HK, Heywood D, Ralph GS, Bienemann A, Baker AH, Uney JB (2003) Tissue inhibitor of metalloproteinase 1 inhibits excitotoxic cell death in neurons. Mol Cell Neurosci 22:98-106.
Tanimoto H, Underwood LJ, Shigemasa K, Parmley TH, Wang Y, Yan Y, Clarke J, O'Brien TJ (1999) The matrix metalloprotease pump-1 (MMP-7, Matrilysin): A candidate marker/target for ovarian cancer detection and treatment. Tumour Biol 20:88-98.
Tauris J, Gustafsen C, Christensen EI, Jansen P, Nykjaer A, Nyengaard JR, Teng KK, Schwarz E, Ovesen T, Madsen P, Petersen CM (2011) Proneurotrophin-3 may induce Sortilin-dependent death in inner ear neurons. Eur J Neurosci 33:622-631.
Teng HK, Teng KK, Lee R, Wright S, Tevar S, Almeida RD, Kermani P, Torkin R, Chen ZY, Lee FS, Kraemer RT, Nykjaer A, Hempstead BL (2005) ProBDNF induces neuronal apoptosis via activation of a receptor complex of p75NTR and sortilin. J Neurosci 25:5455-5463.
Thorns V, Walter GF, Thorns C (2003) Expression of MMP-2, MMP-7, MMP-9, MMP-10 and MMP-11 in human astrocytic and oligodendroglial gliomas. Anticancer Res 23:3937-3944.
Troy CM, Friedman JE, Friedman WJ (2002) Mechanisms of p75-mediated death of hippocampal neurons. Role of caspases. J Biol Chem 277:34295-34302.
Tsirka SE, Gualandris A, Amaral DG, Strickland S (1995) Excitotoxin-induced neuronal degeneration and seizure are mediated by tissue plasminogen activator. Nature 377:340-344.
Turski L, Ikonomidou C, Cavalheiro EA, Kleinrok Z, Czuczwar SJ, Turski WA (1985) Effects of morphine and naloxone on pilocarpine-induced convulsions in rats. Neuropeptides 5:315-318.
Van der Zee CE, Ross GM, Riopelle RJ, Hagg T (1996) Survival of cholinergic forebrain neurons in developing p75NGFR-deficient mice. Science 274:1729-1732.
131
Vincent P, Mulle C (2009) Kainate receptors in epilepsy and excitotoxicity. Neuroscience 158:309-323.
Volosin M, Song W, Almeida RD, Kaplan DR, Hempstead BL, Friedman WJ (2006) Interaction of survival and death signaling in basal forebrain neurons: roles of neurotrophins and proneurotrophins. J Neurosci 26:7756-7766.
Volosin M, Trotter C, Cragnolini A, Kenchappa RS, Light M, Hempstead BL, Carter BD, Friedman WJ (2008) Induction of proneurotrophins and activation of p75NTR-mediated apoptosis via neurotrophin receptor-interacting factor in hippocampal neurons after seizures. J Neurosci 28:9870-9879.
von Bredow DC, Cress AE, Howard EW, Bowden GT, Nagle RB (1998) Activation of gelatinase-tissue-inhibitors-of-metalloproteinase complexes by matrilysin. Biochem J 331 ( Pt 3):965-972.
von Schack D, Casademunt E, Schweigreiter R, Meyer M, Bibel M, Dechant G (2001) Complete ablation of the neurotrophin receptor p75NTR causes defects both in the nervous and the vascular system. Nat Neurosci 4:977-978.
Watanabe T, Ito T, Inoue G, Ohtori S, Kitajo K, Doya H, Takahashi K, Yamashita T (2008) The p75 receptor is associated with inflammatory thermal hypersensitivity. J Neurosci Res 86:3566-3574.
Wielockx B, Libert C, Wilson C (2004) Matrilysin (matrix metalloproteinase-7): a new promising drug target in cancer and inflammation? Cytokine Growth Factor Rev 15:111-115.
Woessner JF, Jr., Taplin CJ (1988) Purification and properties of a small latent matrix metalloproteinase of the rat uterus. J Biol Chem 263:16918-16925.
Wojtowicz-Praga SM, Dickson RB, Hawkins MJ (1997) Matrix metalloproteinase inhibitors. Invest New Drugs 15:61-75.
Wong V, Arriaga R, Ip NY, Lindsay RM (1993) The neurotrophins BDNF, NT-3 and NT-4/5, but not NGF, up-regulate the cholinergic phenotype of developing motor neurons. Eur J Neurosci 5:466-474.
Woo NH, Teng HK, Siao CJ, Chiaruttini C, Pang PT, Milner TA, Hempstead BL, Lu B (2005) Activation of p75NTR by proBDNF facilitates hippocampal long-term depression. Nat Neurosci 8:1069-1077.
Woolf CJ, Safieh-Garabedian B, Ma QP, Crilly P, Winter J (1994) Nerve growth factor contributes to the generation of inflammatory sensory hypersensitivity. Neuroscience 62:327-331.
132
Xu B, Gottschalk W, Chow A, Wilson RI, Schnell E, Zang K, Wang D, Nicoll RA, Lu B, Reichardt LF (2000) The role of brain-derived neurotrophic factor receptors in the mature hippocampus: modulation of long-term potentiation through a presynaptic mechanism involving TrkB. J Neurosci 20:6888-6897.
Yamamoto T, Yamakuni T, Okabe N, Amano T (1992) Production and secretion of nerve growth factor by clonal striated muscle cell line, G8-1. Neurochem Int 21:251-258.
Yamashita T, Tucker KL, Barde YA (1999) Neurotrophin binding to the p75 receptor modulates Rho activity and axonal outgrowth. Neuron 24:585-593.
Yang F, Je HS, Ji Y, Nagappan G, Hempstead B, Lu B (2009a) Pro-BDNF-induced synaptic depression and retraction at developing neuromuscular synapses. J Cell Biol 185:727-741.
Yang J, Siao CJ, Nagappan G, Marinic T, Jing D, McGrath K, Chen ZY, Mark W, Tessarollo L, Lee FS, Lu B, Hempstead BL (2009b) Neuronal release of proBDNF. Nat Neurosci 12:113-115.
Yano H, Torkin R, Martin LA, Chao MV, Teng KK (2009) Proneurotrophin-3 is a neuronal apoptotic ligand: evidence for retrograde-directed cell killing. J Neurosci 29:14790-14802.
Ye JH, Houle JD (1997) Treatment of the chronically injured spinal cord with neurotrophic factors can promote axonal regeneration from supraspinal neurons. Exp Neurol 143:70-81.
Yeo TT, Chua-Couzens J, Butcher LL, Bredesen DE, Cooper JD, Valletta JS, Mobley WC, Longo FM (1997) Absence of p75NTR causes increased basal forebrain cholinergic neuron size, choline acetyltransferase activity, and target innervation. J Neurosci 17:7594-7605.
Yepes M, Sandkvist M, Moore EG, Bugge TH, Strickland DK, Lawrence DA (2003) Tissue-type plasminogen activator induces opening of the blood-brain barrier via the LDL receptor-related protein. J Clin Invest 112:1533-1540.
Yepes M, Sandkvist M, Coleman TA, Moore E, Wu JY, Mitola D, Bugge TH, Lawrence DA (2002) Regulation of seizure spreading by neuroserpin and tissue-type plasminogen activator is plasminogen-independent. J Clin Invest 109:1571-1578.
Yi JS, Lee SK, Sato TA, Koh JY (2003) Co-induction of p75(NTR) and the associated death executor NADE in degenerating hippocampal neurons after kainate-induced seizures in the rat. Neurosci Lett 347:126-130.
133
Yoon S, Lee HW, Baek SY, Kim BS, Kim JB, Lee SA (2003) Upregulation of TrkA neurotrophin receptor expression in the thymic subcapsular, paraseptal, perivascular, and cortical epithelial cells during thymus regeneration. Histochem Cell Biol 119:55-68.
Yoon SO, Casaccia-Bonnefil P, Carter B, Chao MV (1998) Competitive signaling between TrkA and p75 nerve growth factor receptors determines cell survival. J Neurosci 18:3273-3281.
Yune TY, Lee JY, Jung GY, Kim SJ, Jiang MH, Kim YC, Oh YJ, Markelonis GJ, Oh TH (2007) Minocycline alleviates death of oligodendrocytes by inhibiting pro-nerve growth factor production in microglia after spinal cord injury. J Neurosci 27:7751-7761.
Zagrebelsky M, Holz A, Dechant G, Barde YA, Bonhoeffer T, Korte M (2005) The p75 neurotrophin receptor negatively modulates dendrite complexity and spine density in hippocampal neurons. J Neurosci 25:9989-9999.
Zeng J, Too HP, Ma Y, Luo ES, Wang S (2004) A synthetic peptide containing loop 4 of nerve growth factor for targeted gene delivery. J Gene Med 6:1247-1256.
Zou T, Ling C, Xiao Y, Tao X, Ma D, Chen ZL, Strickland S, Song H (2006) Exogenous tissue plasminogen activator enhances peripheral nerve regeneration and functional recovery after injury in mice. J Neuropathol Exp Neurol 65:78-86.
134
XI. Vita
Audrey Pham Le
1981 Born June 4 in La Mesa, California
1999 Graduated from Grossmont High School, La Mesa, California
1999-2003 Attended the University of California, Berkeley
2003 B.A. in Cognitive Science, minor in Education from the University of
California, Berkeley
2003-2004 Employed as a researcher and database programmer at RTZ Associates, Oakland, California 2004-2011 Graduate work in the joint Integrative Neurosciences program at the University of Medicine and Dentistry, New Jersey and Rutgers University, Newark, New Jersey 2011 Ph.D. in Integrative Neurosciences, joint with the University of Medicine and Dentistry, New Jersey and Rutgers University, Newark, New Jersey
