Characterizing the Interaction of NGF with p75m in Chick

Retinal Cells

BY Christopher AHington

A thesis submitted to the Department of Physiology, in conformity with the requirements for the degree of Master of Science

Queen's University

Kingston, Ontario, Canada

Copyright O Christopher Allington, 2000

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The completion of this thesis was made possible with the help of several

organizations and individuals. 1 would like to thank the Medicd Research Counsel of

Canada for funding this projecf and the School of Graduate Studies, the Department of

Physiology and the Department of Pharmacology for financiai assistance.

1 am grateful for the help of the research associates in our laboratory. First, to

Suzanne Dostaler for ber help in setting up the retinal ce11 culture system and counting

cells. To Mark Solc, for his help in leaming binding techniques and support throughout

this project. To Joan Post, for her help in editing this thesis and in counting cells. 1

would like especially like to thank Gai1 Lawrance, who was encouraging and supportive

in the lab, and was always willing to lend her technical expertise whenever needed.

Many of the conformational figures shown in this thesis were generated and provided by

Dr. 1. L. Sharnovsky. 1 would like to thank him sincerely for providing these figures and

for his usefiil discussions about the theoretical aspects of this thesis. 1 also thank other

students, post docs and staffwho helped out dong the way.

To Dr. R.J. Riopelle, my CO-supervisor, for sharing bis knowledge and thoughtfùl

insight, as well as helping immensely in the preparation of this thesis and other

manuscripts. Finally, to Dr. G.M. Ross, who helped in al1 stages of this project, and

who's unbound optimism and enthusiasm inspires everyone who works with him.

The radio-iodination of NGF was perforrned by Dr. G.M. Ross. Cells were

counted by myself and another lab member. Ml other work was done by myself unless

otherwise stated.

ii

A bsfract :

Neurotrophins have long been recognized to play a role in supporting the sumival

and outgrowth of developing neurons. However, recent evidence demonstrates that dong

with these effects, nerve growth factor (NGF; the prototypic neurotrophin) can potentiate

apoptotic ce11 death through the common neurotrophia receptor p7sNTR. in this thesis, we

characterize the interaction of NGF with p75m in embryonic day (ED) 5.5 chick retinal

cells. These cells express p75N"< but not TrkA. Binding of NGF to p 7 ~ m in retinal cells

displayed higher affinity (Kd = 0.4 QM) than when this receptor is expressed with TrkA

in pheochromocytoma (PC 12) cells. Further, exogenous NGF induced apoptosis in

dissociated retinal cultures (2.5 fold higher then control cultures), while BDNF and N T 4

did not. This characteristic allowed us to use retinal cells to evaiuate how protection

against ce11 death mediated by the interaction of NGF and p75mR might be achieved.

The effects of 2n2+ on NGF confomatiori and TrkA mediated functions have

been documented. To evaluate the ability of Zn2' to attenuate the biologicd activities

mediated by p75m in chick retinal cells, we characterized the effects of 2n2+ on the

binding of NGF to p75m, and evaluated the ability of this transition metal cation to

inhibit NGF induced apoptosis. The addition of 100 p M 2n2+ inhibited binding and

cross-linking of ' 2 S ~ - ~ ~ ~ to p75NTR and attenuated NGF induced ce11 death. Taken

together with previous findings, this study leads to the conclusion that zn2+ antagonizes

NGF's biological activity. Furthemore, the biological outcornes of the influence of zn2'

on neurotrophin activity are related to cellular context and receptor expression. When

neurons express p75NTR Hithout TrkA, zn2+ protects against the proapoptotic effects of

. . . 111

NGF.

To further investigate the intemction of NGF and p75m, we evaluated the

neuroprotective effects of a small organic molecule fïrst described by Parke-Davis, PD

90780. We show that 40 pM of this compound is sufficient to attenuate NGF induced

apoptosis in cultures of chick retina. This concentration inhibited receptor cross-linking

of to p75m in these ceiis, suggesting that PD 90780 exerts its protective

effects by antagonizing this receptor ligand interaction.

Table of Contents

Title.. ............................................................................ .i . . Acknowledgements.. .......................................................... .il ... Abstract. ......................................................................... -111

Table of Contents.. ............................................................. .v List of Figures.. ................................................................. .vi List of Tables.. .................................................................. .vi . . . . Abbrewations. .................................................................... vil

Introduction 1 Nerve Growth Factor. ................................................. .1 p75m: A Biological Paradox.. ...................................... -6 Apo tosis - Self Instructed Demise.. ............................... .10 L p75 Antagonists ..................................................... .15 Zinc and the Nervous System.. ... .. .................................. -16

Hypothesis and Objectives 21

Me thods 22 Chick Embryo Dissection.. .............................................. -22 '%NGF Preparation. .................................................... .22 Receptor Binding and Cross-Linking.. ............................... ..22 Retinal Cell Cultures.. ..................................................... 23 SurvivaI Assay ............................................................ .24 NGF Adsorbed Beads.. ................................................. ..24 Apoptosis Detennination.. .............................................. .24 Statistical Analysis.. ..................................................... ..25

Resul ts 27 .......................... Receptor Expression and Afnnity of p75m.. 27

.......... NGF Promotes Apoptosis in EDSS Chick Retinal Cells.. .3 1 .............. Changes in ' 2 5 ~ - ~ ~ ~ Binding in the Presence of zn2+. .33

Survival of ED5.5 Chick Retinal Cells.. ............................... -3 8 2n2+ ~nhibits p75m ~ed ia t ed ~ p ~ t o s i s . . ............................ -38

........................... PD 90780 Inhibits '%NGF Cross-Linking.. 42 PD 90780 Miibits p75m Mediated Apoptosis.. ....................... 42

Discussion 47

Conclusions 57

Re ferences 58

Vitae 67

List of Fi~ures

1-1. NGF 1-2- PD 90780

R- 1. Receptor Expression R-2. Scatchard Anal ysis R-3. Receptor Cross-Linking ICso R-4. Neurotrophin Mediated Apoptosis EDS.5 Chick Retinal Cells R-5. NGF Effect in ED IO Chick Retinal Cells R-6. zn2+ inhibits 1 2 5 ~ - ~ ~ ~ Receptor Cross-linking R-7. 2n2+ Inhibits Protomer Cross-linking R-8. Metd Ion Survival R-9. zn2+ Inhibits ~ 7 5 ~ ~ e d i a t e d TUNEL Staining R- 1 0. 2n2+ Inhibits p75- Mediated ssDNA Staining R- 1 1 . PD 90780 Inhi bits ' U ~ - ~ ~ ~ Receptor Cross-linking R- 12. PD 90780 Effect on '*'I-NGF Protomer Cross-Linking R- 1 3. PD 90780 Mubits p75m Mediated Apoptosis.

D- 1. Proposed Interaction of 2n2+ with NGF D-2. Proposed Interaction of PD 90780 with NGF

List o f Tables

Fig-1 . Effect of Transition Metals on Binding.

Abbreviations

ABC - Avidin biotinylated home radish peroxidase complex ANOVA - Anaiysis of variance BDNF - brain derived neurotropbîc factor B S ~ - Bis[sulfosucciaimidyl] substrate CMF - Calcium Magnesium Free Gey' buffered salt solution DM3 - Diaminobendine DD - death domain DRG - dorsal root ganglia ED- embryonic day EDC - 1 -Ethyl-3<3-dimethyIafninopropyno1) carbodihide FADD - fas associated death domain HKR - Hepes Krebs Ringer solution JNK - c-jun-kinase MAb - Monoclonal antibody MTï - 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazodium bromide NF& - nuclear factor kappa B NGF - nerve growth factor NT-3 - neurotrophin 3 N T 4 5 - neurotrophin 4/5 NRIF - Neurotrophin receptor interacting factor p 7 ~ m - common low atfinity neurotrophin receptor p75 PBS - Phosphate buffered d i n e PC 12 - pheochromocytoma PCD - programmed ce11 death PD - postnatal day IUP - receptor interacting factor S D S-P AGE - sodium dodec y1 sulfate pol y acry lamide gel electrophoresis SNHS - Sul fo-N-hydroxysul fosuccinimide ssDNA - single stranded DNA TRADD - TNF receptor associated death domain Trk - tyrosine kinase receptor TUNEL - Tramferase mediated dUTP nick end labeling

vii

Introduction

The focus of this thesis is on the interaction of nerve growth factor (NGF) with

the common neurotrophin receptor p75 @7sMR). Recent work suggests that this receptor

ligand interaction contributes to the death of specific ce11 populations. Zinc (zn23, dong

with copper (cu23, have previously been shown to inhibit NGF effects mediated by the

tyrosine kinase receptor (Trk; Wang et al., 1999; Ross et al., 1 997). Theoretical studies

predict that these transition metal cations should also inhibit NGF's effects mediated by

p75m, including p75NTR mediated apoptosis. Coosidenng that free 2n2' is found in the

nervous system, it may exert physiological activity by inhibiting the effects of NGF. If

this transition metal cation attenuates p75m mediated apoptosis, it would also be

interesting to determine if pharmacological compounds could have a similar effect.

Therefore, the antiapoptotic effect of a small organic NGF antagonist was also tested.

PD 90780 has been shown to block the binding of NGF to p75m (Spiegel et al., 1995).

If this compound inhibits NGF induced apoptosis, it could have pharmaceutical

implications, and possibly serve as a basis for future design of antagonists that block the

interaction of NGF and p75m with more efficacy. These compounds may be beneficial

in treating conditions where NGF mediated apoptosis occurs.

Nerve Growth Factor

Neurotrophins are dirnenc proteins, and the mammalian family includes NGF,

brain-denved neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and neurotrophin 4/5

(NT-4/5). These proteins have k e n shown to play a vital role throughùut developrnent

of the nervous system (Segal & Greenburg, 1996; Oppenhiemer, 199 1 ; Levi-Montalchini,

1987). Neurotmphins are target derived and provide neurotrophic (survival) and

neurotropic (growth guidance) support to specific neuronal populations during

development (Lewin & Barde, 1996). Recentiy, they have been demonstrated to mediate

synaptic activity and be important in maintahhg specific neuronal populations in the

adult nervous system (Schuman et al., 1999; Cowen & Gavazzi, 1998).

The earliest suggestion that there was a target secreted substance that influenced

neuronal development came at the tum of the century fiom neurobiologist S. Ramon Y

Cajal. He hypothesized that neurons migrate toward a target by following a gradient of

molecular cues (Cajd, 1928). This hypothesis was mostly intuitive, as biochemical

techniques needed to support this theory were years fiom being developed.

Rita Levi-Montalcini and Victor Hamburger presented the first evidence

supporting Cajal's theory (1949). In an innovative experiment, they removed a limb bud

fiom a developing chick embryo. These embryos display ed hy perplasic nerve centers and

the neuronal loss could be attributed to the death of differentiated neurons (Hamburger &

Levi-Montalcini, 1949). Thjs showed that the absence of a target prompted the death of

neurons that would normally innervate it. Evidence supporting the hypothesis that this

effect was mediated by the lack of an unknown growth factor was provided in subsequent

experiments where pieces of rnouse turnor were grafied into chick embryos. In these

embryos, sympathetic and sensory neurites grew into the tumor (Levi-Montalcini &

Hamburger, 195 1). These investigators realized that not only did neurons grow into the

tumor, but also to visceral areas which would not normally be innervated (Levi-

Montalcini, 1952). This suggested that although derived from the tumor tissue, this

growth factor, temed nerve growth promoting factor (later shortened to nerve growth

factor; fig 1-l), was diffusable and could influence nerve growth in other areas.

Fig. 1-1. Three-dimensionai structure or the NGF dimer. (provided by LL.

Shamovsky) .

initial biochemical studies were performed in extraçts isolated fiom snake venom.

In an attempt to degrade any nucleic acids, snake venom (containing phosphodiesterase,

an enzyme that breaks down nucleic acids) was added to cultures of chick sensory

neurons and smail pieces of mouse tumor. Surprisingly, the venom increased neurite

outgrowth and was subsequentiy found to have a much higher concentration of the

growth factor than the hunor. Through a series of biochernical purification processes, the

nerve growth factor protein was isolated and found to have a molecular weight in the

order of 20 000 Da (Cohen & Levi-Montaicini, 1956).

The first in vivo evidence of NGFs neurotrophic activity in normal development

was seen following injection of antisenim to NGF (AS-NGF) into neonatal rnice (Levi-

Montalcini & Booker, 1960). This inhibited NGF activity, and prompted the

disappearance of the sympathetic ganglia, showing that the survival of these neurons was

NGF dependent. Later experirnents demonstrated that in addition to the above trophic

effects, NGF played a neurùtropic role as injections of NGF into the venûicle floor of

newborn mice resulted in the growth and penetration of nerve bundles (Levi-Montaicini.

1 976).

The discovery of the next neurotrophin did not happen for several decades. Using

homogenates of fetal pig brain, Y.A. Barde isolated BDNF using classical biochemical

techniques (Barde et al., 1982). The finding that this new neurotrophin had many

conserved amino acid regions with NGF, coupled with advancements in molecular

bioIogy techniques, quickiy led to the discovery of NT-3 and N T 4 5 (Hallbrook et al.,

1991 ; Hohn, et al., 1990). Together, neurotrophins are classified as a family due to their

similar structural motifs. These dimeric proteins are s ynthesized as precursors and

dimerized througb hydrophobic interactions. There are conserved regions in the primary

amino acid sequences, with regularly interspersed variations both within the sequence

and at the amino and carboxyl terminai ends. These variations are thought to mediate the

receptor specificity of each neurottophin. Considering the variation in receptor

interaction, it is not surprishg that individual neurotrophins can support different

neuronal populations (reviewed in Lewin & Barde, 1996).

Although neurotrophins were first shown to provide trophic and tropic neuronal

support, it is now recognized that they rnay intluence the development and maintenance

of the nervous system in other ways. NGF can influence the phenotype of developing

precursors and is responsible for increasing the size, dendritic complexity and synapse

formation of several neuronal populations (Ruit et al, 1990; Purves, 1988).

Neurotrophins have been implicated in augmenthg pain perception by promoting the

development of nociceptive neurons, and by increasing their neurotransrnitter activity and

ion charnel function (reviewed in Mendel1 et ai., 1999). However, the most perplexing

biological event mediated by neurotrophins is in direct contrast to their classical

supportive role. In limited neuronal populations, NGF can actually potentiate ce11 death

(reviewed in Barker, 1998).

This diversity can be explained in part by the existence of the two receptors with

which neurotrophins interact. It is well established that Trk receptors mediate many of

the survival, differentiation and growth promoting activities of neurotrophins. Each

neurotrophin specifically binds a Trk receptor. NGF associates with TrkA, BDNF and

N T 4 5 associate with TrkB, and NT-3 associates with TrkC (reviewed in Barbacid,

1994). Trk receptors associate with neurotrophins with picomolar afhity. The other

neurotrophin receptor, p 7 ~ m associates with ail neurotrophins with similar nanomola.

f i t i e s (Chao, 1 994). Although p75NTR was the first neurotrophin receptor identified

(Johnson et al., 1986), its biological role has been diflicult to elucidate. It was fïrst

perceived that the role of p75m was to help potentiate the astivity of Trk receptors.

However, recent experirnental data indicate that when expressed alone, p75m can signal

and mediate apoptosis.

p75m: A Biological Pamdox

The p75m receptor was the fïrst neurotrophin receptor discovered and belongs to

the tumor necrosis factor (TNF)/fas receptor family (Johnson et al., 1986). These

membrane-spanning proteins share common cysteine rich repeats. Upon ligand binding,

most TNF receptors initiate a signaling cascade leading to ce11 death. However, this

effect is rarely caused by p75m. The ambiguous effect on apoptosis is not the only

difference between p75m and the other TNF receptors. TNF ligands are membrane

bound trimers and their corresponding receptors trimerise upon specific ligand binding

(reviewed in Wallach et al., L 999). p75m binds al1 neurotrophins which are soluble

dimeric proteins, and forms a dimer on the membrane surface following ligand binding

(Ross et al., 1998). Another major difference is that most family members have

intracelluIar sequences that form self-associating death domains @D) which bind

intracellurlar death mediator protein. p75NTR has a DD which shares some conserved

regions with other TNF receptors, but does not self-associate or bind TNF intracellular

adapters (Liepinsh et al., 1997; Chapman, 1995). The DD in p75m is not essential for

apoptosis to occur, as tnincated p75m, which does not contain this domain, can still

potentiate apoptosis in a shilar fashion to the full-length receptor (Coulsen et al., 1999).

p75m does interact with the intracelluiar TRAF6 protein, and blocking this interaction

inhibits p75m signalhg (Khursigara et al., 1999). Other features unique to p75- are

its ability to signal in the absence of ligand and modulate the activity of other receptors

(reviewed in Barrett, 2000). Therefore, to understand the a m y of roles of p75m, one

must consider the three circurnstances in which it is active. First, p75m fonns a

complex with TrkA and promotes the activity of this receptor. Second, this receptor can

signal independently of ligand activation. Finaily, p75- can signal when activated by

neurotrophins. These plurimodal forms of activation make the biological tlnctions of

p75m dificuit to elucidate.

It first appeared that the function of p75- was primarily to augment Trk

mediated activity. Several studies have demonstrated that p75m acts as a facilitator of

Trk. p75m has k e n shown to increase the binding affinity of TrkA for NGF (Ross et

al., 1998), and the expression of p75- enhances NGF induced TrkA signalling (Verdi et

al., 1994). Also, the amount of NGF required for ce11 survivai is less when p75- is CO-

expressed with TrkA than when there is no p75NTR (L,ee et al., 1994). There are two

proposed mechanisms of how p75- interacts with TrkA. The k t , mechanism

postulates that p75- presents NGF to TrkA and facilitates binding though this

mechanisrn (Barker & Shooter, 1994). The second, mechanism suggests that p7~NTR and

T r U fonn a hetero complex independent of NGF which has a higher affinity for NGF

than TrkA on its own (Ross et al, 1998). In support of the latter theory, a complex which

c m be immunoprecipitated with antibodies to both p75- or TrkA has been found in

PC 12 cells crossed-linked prior to ligand binding (Ross et al., 1998).

Signalling via p75m was fwst observed in the absence of ligand. Apoptosis

following the withdrawal of survival maintainhg senun is a well-recognized

phenomenon. In PC 12 cells, the over expression of p75m leads to a substantial increase

in ce11 death when serum is removed (Rabizdeh et al, 1993). Further, primary cultures of

dorsal root ganglia @Re) sensory neurons treated with antisense oligonucleotides to

p75m survived better than untreated cells following the same conditions (Barret &

Bartlett, 1994). In vivo evidence of p75m signalhg cornes fiom the production of

p75m knockout mice which had increased numbers of sympathetic, spinal cord, and

retinal neurons compared to wild type mice (Barnji et al., 1998), suggesting that p75m is

prominent in the elimination of these populations. Transgenic mice with overproduction

of the intracellular domain of p75m demonstrated that certain (but not d l ) neuronal

populations expressing p75m underwent increased neuronal death throughout

development (Majdan et al, 1997). These observations established that p75m plays a

role in mediating ce11 elirnination, but that the expression of this receptor does not always

promote ce11 death. Further, there must be other (yet to be elucidated) cellular or

environmental factors that contribute to this elimination.

It has also proven difficult to determine the biological role of p 7 ~ m following

activation by neurotrophins. Most TNF receptors potentiate ce11 death following ligand

binding. For a long time, it was thought that p75m did not share this role. However,

recent experiments have s h o w that under certain conditions, specific cell types die

through apoptosis following the interaction of NGF with p75m. In order for this to

occur, cells must express p75m and not TrkA, afthough this is not the only requirement.

NGF was first shown to promote apoptosis in embryonic chick retinal neurons and

postnatal day 1 (PD 1) rat oligodendrocytes (Frade et al, 1996; Casaccia-Bonnefil et al,

19%). In developrnent, chick retinal neurons go through a phase of ce11 death between

embryonic days (ED) 4-7. Frade et al., (1996), showed that this stage is dependent on

NGF binding to p75-. Casaccia-Bonnefil et al, (1996) showed that when PD 1 rat

oligodendrocytes are cultured for 14 days and treated with NGF ceil death will occur.

Interestingly, if cultured oligodendrocytes are assayed earlier, no ce11 death is seen- Mso,

oligodendrocytes cultureci from adult rats do not undergo ce11 death when treated the

sarne way (Ladiwala et al., 1998). Since these discovenes, published work has shown

that several immortalized ce11 lines genetically aitered to express p7sm, as well as

embryonic rat motoneurons, die when treated with exogenous NGF (Sedel et al., 1999;

Bunone et al., 1997).

The signal transduction pathways of p75N+R mediated apoptosis have yet to be

fully characterized, but are thought to involve the production of ceramide (Dobrowski et

al., 1994), activation of c-jun kinase (JNK; Casaccia-Bonnefil et ai., 1996), translocation

o f nuciear factor KB (NFKB; Carter et al., 1 996), accumulation of p53 (Aloyz et al, 1 W8),

and activation of various caspases (Gu et al., 1999). Recent data suggests the

involvement of an intracellular zinc finger adapter protein, neurotrophin receptor

interacting factor (NRIF; Casademunt et al., 1999). Knockout mice missing this

intI-ace1lula.r protein show similar increases in spinal cord and retinai neurons as p75m

knockout rnice. However, how this adapter might mediate p75MR signahg is still

llnknown.

Apoptosis-Self Instructed Demise

Throughout development, most neurons go through many neurogenic events

including stages of proliferation, migration, axon formation, and synaptic connection.

These stages are al1 vitai for the formation of a fùnctional, intact nervous system.

However, at some point in development, most populations aIso go through a stage of

elimination where many developing neurons die. Vertebrates have adapted an organized.

controlled mechanism to perforxn this elimination; termed programmed ce11 death (PCD),

or apoptosis (Oppenheim, 199 1). The sumival of developing neurons depends primarily

on target derived neurotrophin support, although interactions with various molecular cues

from other neurons or neighboring glial cells are also vital (Raffet al., 1993). A balance

between many apoptotic and antiapoptotic factors ensures that although produced in

excess, the proper number of developing neurons innervate a target tissue. Experimental

data indicate that as much as 50% of a developing neuronal population is elirninated

before maturity (Johnson et al., 1980). In development, this elimination is beneficial to

the nervous system, as it ensures functional cornpliance between many neural networks.

However, when homeostasis is altered in the mature nervous system, undesirable

neuronal death can occur. This ce11 death is often apoptotic and may potentiate

neurodegeneration seen in conditions such as Parkinson's disease, Alzheimer's disease,

Arnyotrophic lateral sclerosis, spinal multiple sclerosis, epilepsy and stroke (reviewed in

Thompson, 1995).

Regardless of how a ce11 dies, it goes through many changes. Kerr et al., (1 972)

descnbed two types of ceil death. The first, necrosis, results fiom injury, the second,

apoptosis, occurs in normal development. Apoptosis is now recognized to also occur in

the mature nervous system, and is often referred to as a fonn of ce11 suicide because in

development, or after insult, tbis type of ce11 death is delayed and requires ongoing

cellular activity. This is in contrast to necrosis, whereby a ce11 will deplete its energy

store, Ioose membrane structure, cease cellular activities and die following an insult. In

necrosis, there is ~ a + and ca2+ influx and K' efflux. Cytoplasmic and membrane

compartments swell and many organelles are destroyed. Chromatin disperses, there is a

loss of energy (ATP), and celluiar production of macromoiecules stops.

In apoptotic ce11 deatb, there are few initial changes and ionic homeostasis is

sustained. Chromatin condensation occurs and macromolecule synthesis proceeds.

There is no initiai depletion of energy and cellular organelles are left intact. For

apoptotic ce11 death to occur there must be an active initiation of transcription. Much of

the work to detennine which genes are tumed on in PCD has been done using

Canhabdites elegans. The first gene discovered that was active in causing apoptosis was

CED- 1. CED- 1 was f o n d to be homologous to marnrnalian interleukin converting

enzyme (ICE). Since this fïnding, many similar genes have been found. These genes

encode proteins that are referred to as caspases, and there are currently 13 members in the

mammalian family. Caspases are cysteine proteases and work by cleaving proteins after

Asp residues in an amho acid c h a h They are activated through self-cleavage

mechanisms (reviewed in Thornberry & Lazebnik, 1998).

Although there are defined morphological differences between apoptotic and

necrotic ce11 death, recent reports suggest that in the adult nervous system, cells which

were originally though to die through necrosis show some apoptotic ce11 death markers

(Roy & Sapolsicy, 1999). This makes distinguishing the difference between the two

forms of ce11 death difficuit, and suggests that apoptosis in the mature nervous system

differs fiom ceIl death seen in development. Further, this presents the possibility that

there is a continuum between apoptosis and necrosis, as cells can show characteristics of

each.

Throughout development and in the mature nervous system, there are many

factors that may promote apoptosis. Generally, apoptosis can occur under two different

sets of circumstances. First, cells have a means of self-directed apoptosis, which is

activated by a ligand binding to specific death receptors. Second, apoptosis can be

propagated by a loss of homeostatic balance between a nurnber of parameters in a cell's

environment. A prirnary cause of apoptosis in development is the loss of neurotrophin

support. In vivo knockout studies show that loss of specific neurotrophins leads to

increased apoptosis in specific neuron populations (Crowley et al., 1994). In vitro,

several neuronal populations undergo apoptosis following neurotrophin withdrawal

(Garcia et al., 1992; Milligan et al., 1 995). Other known changes which can lead to

apo ptosis include:

- Loss of ion homeostasis (KC or ca23.

- Exposure to high concentration of excitatory neurotransrnitters.

- Accumulation of toxic particles such as AP.

- Oxidative stress caused by ischemic conditions, hydrogen peroxide, or loss of

functions of antioxidant systems such as superoxide dismutase.

- Lipids, such as ceramides.

- Ultraviolet irradiation.

- BuiId up of nitric oxide.

- Neurotoxins such as MPTP and cocaine.

(reviewed in Sastry & Rao)

When a ce11 dies due to loss of homeostasis there are severai events wbich occur.

The initiator phase can involve several factors. There is a generation of the intracellular

lipid ceramide. This lipid has many biological effects and can be is generated through

sphingomyelinase or via ceramide synthase (Bose et al, 1995). Apoptosis is also initiated

by an influx of ca2+ through glutamatergic receptor channels or voltage gated ca2+

channels. This leads to the activation of P-1-3 kinase, skews mitochondria membrane

potential, and the activation of nitrk oxide synthase (NOS), leading to increases in nitric

oxide (NO). High ca2+ also increases the expression of Bax protein, which can inhibit

mitochondrid function and lead to the formation of fiee radicais by attenuating the cells

antioxidant system. When mitochondria loose membrane potential, cytochrome C is

released into the cytoplasm. This intermediate step, dong with JNK, ceramide, and Bax

lead to the activation of caspases which dong with AP-1, ROS and p53, cause DNA

fragmentation (reviewed in Sastry & Roa, 2000).

Apoptosis can also be caused by receptor-ligand interactions. This instructive

apoptosis plays an important role in the immune system and in regenerating tissue, and

also occurs in neurons. Apoptosis mediated by changes to a cell's environment or

through receptor activation differs not only in the way in which it is initiated, but also

through the signaling events which propagate ce11 death. This has contnbuted to the

difficulty of d e m g the signal transduction events of apoptosis. Ligand mediated

apoptosis is initiated by receptor oligomerization leading to the recruitment and

activation of independent intracellular protein associated death domains. These proteins

Vary slightly fiom receptor to receptor, but commonly serve the sarne purpose of

signding apoptosis. The best characterized death receptor is TNFRl. Upon eimerising,

intracellular adapters, TRADD and RIP bind to the TNFRl trimer through their own

death domains. Following ligand binding, ceramide is formed fkom sphyngom yelin by

the activation of sphyngomyelinase. Ceramide activates JNK through MEKKl

phosphorylation, and can activate initiator caspases. The association of RIP leads to the

activation of JNK and the transcription factor NFKB. JM( can activate the transcription

factor AP-1 through a phosphorylation reaction. Activation of AP- t promotes

proapoptotic gene expression, and NF& is thought to mediate survival signais.

Interaction between these two transcription factors coordinate gene expression to

influence cell death (reviewed in Ashkenazi & Dixit, 1998).

Study of the morphological changes and biochemical events that occur in the

apoptotic process has revealed several markers that can be used to determine if ce11 death

occurs through this mechanism. One hallmark of apoptosis is changes in a cell's DNA.

DNA Iaddering is seen in apoptotic cells and can be tested for by running DNA on an

agarose gel, and looking for fiagmentation patterns (McConkey et al., 1998). Although

this event occurs in apoptosis, it is not exclusive to this fonn of ce11 death, as cells which

die through necrosis may also show DNA Iaddering (Fukuda et al., 1993). Termina1

deoxynucleotide transferase mediated dUTP nick end labeling (TUNEL) is a prominent

and often used histochemicd marker for apoptosis (Neguescu et al., 1996). This assay

looks only for DNA fiagmentation, regardless of how the hgmentation occurs.

Therefore, like DNA laddering, it is not always specific for apoptotic cells (Frankfûrt et

al., 1 996). Recentl y, a speci fic immunohistochernical method for detecting apoptosis was

developed based on the binding of a monoclonai antibody to single stranded DNA

(ssDNA). This offers a distinct advantage over previous immunohistochemical assays

because it is specific for apoptotic cells and recognizes earlier stages of apoptosis then

TUNEL staining or DNA laddering (Ffankfilrt et ai., 1996). Other means of

distinguishing apoptotic cells includes assaying for caspase, activity and identieing

pyknotic nuclei with propidium iodide or Hoechst dye.

p75MR Antagonists

With recent reports showing that p75m can promote apoptosis following NGF

binding, there is an interest in finding p75m antagonists. Previously, our lab has shown

that transition metal cations, particularly 2n2+ and cu2+, can uncompetitively antagonize

binding of NGF to its receptors. Further, they are able to inhibit protomer cross-linking

of BDNF and NT-3, as well as NGF, thus, it is likely that 2n2+ and cu2+ can also block

binding of other neurotrophins to the receptors with which they interact. In biological

assays, 2nZC has been shown to attenuate NGF mediated chick DRG neurite outgrowth,

and the ability of NGF to rescue PC12 cells fiom oxidative stress (Wang et al., 1999;

Ross et al., 1997). Both effects are mediated by the TrkA receptor. Theoretical and

binding studies have predicted that 2nZf will influence NGF conformation and

compromises the ability of this protein to induce p75m activity (Shamovsky et al.,

1999). Considering that there is a significant overlap in brain regions where neurons are

susceptible to 2n2' and where NGF levels are increased following insult (Harrison &

Gibbons 1994; Lindvall et al., 1994), the interaction of zn2' and NGF could have

biological significance.

Since discoverhg that p75m play a deleterious role in specific ce11

populations, there has been much effort to fhd compounds which inhibit binding to this

receptor. A specific p75m antagonist would be usefd not oniy to inhibit the

proapoptotic effects of this receptor, but also to M e r elucidate other biological

functions that this receptor mediates. One compound which was shown to

block binding of NGF to p75m, PD 90780, was discovered by Spiegel et al., (1995; Fig

1-2). This compound blocks binding of 1 2 S ~ - ~ ~ ~ to PC 12 cells and in an immortalized

ce11 line with transfected full length human p75NTR and no TrkA expression. Like 2n2',

PD 90780 binds the NGF protein directly and does not interaft with p75m in the

absence of NGF. Supporting this suggestion is the fact that an analogue of PD 90780

radiolabeled with 12'1 could inhibit NGF binding, but did not bind to PC12 cells or

isolated membranes. However, this analogue did specifically bind to sepharose-linked

NGF. Considering PD 90780's ability to antagonize NGF binding to p75m, it Hill be

interesting to determine how this compound effects NGF's biological activity.

Zinc and the Nervous System

Zinc is the second most abundant trace element in the body, and thus is the focus

of much research interest. This transition metal has a high binding affinity for many

macrornolecules and is an important structural and catalytic element of many proteins

(reviewed in Choi & Koh, 1998). 2n2+ binds to electronegative nitrogen, oxygen and

sulfùr moieties. Under physiological conditions, zn2+ does not gain or lose electrons,

therefore is not redox reactive or prornote the formation of toxic f k e radicais (Berg and

Shi, 1996).

Fig. 1-2. Structure of p75m antagonist PD 90780.

CH,

In the nervous system, 2n2+ can be found in four components; in synaptic vesicles,

membrane or receptor bound, bound to intracellular proteins, or in fkee ionic pools. The

release of 2n2+ nom synaptic stores can be stimulated chemicaily, electricaily, with

increased extracellular K' concentrations, or with the administration of kainic acid

(Aniksztejn et al., 1987; Assaf and Chung, 1984). 2n2+ release has k e n shown to occur

in a caZf dependent manner (Howell et al., 1984). Upon release, free 2n2+ may reach

concentrations greater than 300pM (As&& Chung, 1984; Xie & Srnart, 1991). With

even a brief exposure, this concentration of zn2' c m be toxic to specific cell populations.

Once in the synaptic clefi, there are a number of mechanisms through which zn2'

is regdated. With its high affinity for many proteins, it is not surprising that this

transition metal binds to, and fhctionaily alters, the activity of many membrane

receptors. There is a binding site for 2n2' on the NMDA, AMPA and GABA receptors

(Peters et al, 1987; Westbrook & Mayer 1987). While 2n2+ inhibits NMDA and GABA,

it potentiates AMPA rnediated currents. The result is that Zn2' influence on AMPA are

thought to be protective by decreasing ca2+ influx, while the activity of this transition

metal on NMDA and GABA is toxic by increasing ca2+ influx, and decreasing CI- influx

into the cell. Other membrane proteins which this transition metal alters include glycine

and opiate receptors as well as transporters of dopamine, glutamate, and GABA.

However, the effects of these interactions on neuronal death is not clear (reviewed in

Choi & Koh, 1998).

Considering the multitude of effects of 2n2+ on cellular function, it is not

surprising that the nervous system has adapted many mechanisms to regulate its

extracelluluar concentrations. Three 2nZf transporters (T) have been identified. 2n2+ T-l

and 2n2+ T-3 have been found in the CNS. zn2+ T-1 is a non-energy dependent

membrane bound protein that facilitates the re-uptake of 2n2' in presy~pt ic neurons

(Palmiter & Findley, 1995). 2n2+ T-3 is involved in intracellular synaptic uptake of this

transition metal (Palmiter et al., 1996). 2n2' can enter post synaptic neurons through

voltage gated ca2+ channels, NMDA or caZf sensitive AMPA receptor channels, or via a

zn2+/Na+ exchanger (Sensi et al., 1997; Koh & Choi, 1994; Simons et al., 1991).

The study of the homeostatic mechanisms through which zn2' is regulated once

inside the ce11 becomes quite complicated. This is due to the powerful affinity this

transition metai has for many macromolecules. The most important regulator of zn2+ in

the cytoplasm is binding to metailothionins (MTs). There are four isoforms of MT

recognized. These are al1 single amino acid chahs and act as 2n2+ buffers. When

intracellular 2n2' levels become too high, the buffering effects of MT are exceeded and

this transition metal can alter numerous cellular processes (reviewed in Cuajungco &

Lees' 1997).

The simplest explanation of intracellular zn2' toxicity is that because zn2' influx

is often accompanied by ca2+, 2n2+ affects the sarne cellular processes as this metal (the

cytotoxic role of ca2' is well docurnented; reviewed in Leist & Nicotera, 1998).

However, this has proved to be not true, because these two metals have been shown to

perform different bctions. Unlike caZC, 2n2+ does not activate NOS, a key event in

ca2+ cytotoxicity (Snider et al., 1987). Increased concentrations of 2n2' can initiate the

production of ceramide through sphyngomyelinase (Spence et ai., 1989). Further, 2n2'

can interact with calcium binding proteins, including calmoduiin, thereby inhibithg their

ability to bind fke caZC, and increase the toxic actions of ca2' influx. At high

concenaations, Zn 2' decreases energy production both by altering glycolysis and

inhibiting electron transport in the mitochondria (Skulachev et al., 1967; Krotkiewska &

Banas, 1992). Aithough the above effects are for the most part neurotoxic, 2n2+ also has

some newoprotective effects, such as inhibiting endonucleases, enzymes which are

essential for DNA fragmentation (Cohen & Duke, 1984). The pluripotent influences of

2n2'influences in the nervous system make the study of this transition metal an area of

great scientific interest.

Hvmtlieses

This thesis will test the following hypotheses:

1. NGF will promote apoptosis in dissociated cultures of EDS.5 chick retinal cells.

Previous work in explants of EDS.5 chick retina demonstrates that NGF causes

apoptosis. We will use dissociated cultures of chick retinal cells and evaluate the

proapoptotic effect of NGF, BDNF, and NT-3.

2. In cells that undergo p75- mediated apoptosis, p75- will have a higher

binding afnnity for NGF than in PC12 cells which express p75m and TrkA.

We will test binding affinity through equilibrium binding of 1 2 S ~ - ~ ~ ~ to retinal

cells, and by the ability of various concentrations of unlabeled NGF to inhibit

cross-linking of 1 2 5 1 - ~ ~ ~ to p75* in both PC 12 and retinal cells.

3. 2n2+ and PD 90780 wül attenuate p75- mediated apoptosis.

To evaluate the antiapoptotic effects of 2n2+ and PD 90780, we will perfonn

TUNEL and ssDNA staining in chick retinal ce11 cultures following incubation

with NGF, in the presence and absence of these antagonists. To show that the

effect of these antagonists is mediated by their ability to inhibit binding, we will

evaluate their ability to inhibit '''1-NGF fkom binding and cross-linking to

p75-.

Muterials and Methods

Chick Embryo Dissection.

Fertilized white hom hen eggs were incubated at 38OC in 70% humidity, and

staged as previously described (Hamburger & Hamilton, 195 1). The dissection was

performed by excising both eyes fiom the optic cups and removing the retinal layer.

Single ce11 suspensions of retinal cells were prepared by digesting explants in 0.05

mg/mL DNase, 0.01 mg/mL trypsin in calcium magnesium-fkee Gey's balanced salt

soulution (CMF; pH 8.1) for 15 min at 37OC. Cells were then suspended in RPMI 1640

with 10% heat inactivated home senun (Gibco), spun through fetal calf senun (Gibco),

and washed three times in WMI 1 640.

'=I-NGF Preparation.

Full-length (1 - 11 8) mouse NGF was obtained fiom Cedarlane Laboratories. The

radioiodination of NGF was performed as previously described (Sutter et al., 1979) with

modification (Ross et al., 1997).

Receptor Binding & Cross-linking.

Both equilibrium binding and cross-linking were used to determine the affinity

state of p75m in retinal and PC12 cells. PC l2 cells were grown in RPMI with 10% fetal

calf s e m . Cells were harvested by replacing medium with CMF and incubating for 1 h

at 37OC, collecting in centrifuge tubes, and washing in HKR buffer ([pH 7.351; 10 mM

Hepes, 125 m M NaCl, 4.8 rnM KCl, 1.3 m M CaClt, 1.2 mM MgSO1, 1.2 mM KHzP04, 1

g/L glucose, 1 g/L bovine semm albumin PSA]). For binding, cells were suspended at 1

x 1 06/rnI, at 4OC. Each sample contained 5 x 1 o5 cells and '=I-NGF (at specified

concentrations). To detennine non-specific binding, a replicate of each tube were made

with 40 ph4 unlabeled NGF. Samples were incubated for 2 h at 4°C with continuous

mixing. Aliquots of 100 pL were transferred to 400 pL microcentrifuge tubes containing

200 pL of 10% glycerol in HKR b d e r and spun at 5,000 RPM for 2 mui. Tips

containing the ce11 pellet were cut and radioactivity bound to the celb counted. For

receptor cross-linking studies, cells were placed in HKR bufTer with 1 2 S ~ - ~ ~ ~ (at

concentration of 0.1 nM) and incubated for 2 hours at 4 O C . Bis[suLfosuccinimidyl]-

suberate (BS'; 0.4 mM) or 1 -Ethyl-3-(3-dimethyfaminopropyl) carbodiimide (EDC; 5.0

mM)) and N-hydroxysuifosuccinimide (SNHS; 2.0 mM) were added at room temperature

for 30 min. Cross-linked ceils were washed 3 times in TBS, and the reaction was

quenched with reducing SDS-PAGE sample buffer. Electrophoresis was performed

using a 6% acrylamide gel and followed by autoradiography. For immunoprecipitations,

cross-linking was performed as above, but d e r incubations with cross-linker, cells were

placed in ice cold TBS lysis bufTer ([pH 7.51 10% glycerol, 1 % Triton X-100,2 mM

PMSO, 200 KlU/mL aproptinin, 1 pg/mL leupeptin), incubated at 4 ' ~ for 1 hr, and spun

at 10,000 RPM for 15 min. The supernate was removed, and placed in a fiesh tube where

a specific antibody was added. Following ovemight incubation at OC, ultraiink

immobilized protein G (Pierce) was added to collect bound antibody. The beads were

w h e d 3 times in 1 ysis buffer, and the antigen released £kom the beads in reducing SDS-

PAGE sample buffer.

Retinal Ce11 Culture.

To culture cells for death determination, they were washed three times in RPMI

and suspended in RPMI + 2% B27 supplement (Gibco) and plated on 16 well chamber

slides coated with poly-d-lysine (PDL; O. 1 m m ) . Wells were individually treated and

incubated overnight at 3 7 ' ~ with 5% COt and 95% air.

Cell Survival Assay.

The 3-(4,5-dimethylthiazo1-2-y1)-2,5diphenyl tetrazoliurn bromide ( M m assay

was performed. Briefly, 25,000 neurons were plated in 100 pL of RPMI + 2% B27

(Gibco) in a 96 well plate coated with PDL. Metals were added at designated

concentration, and plates were incubated overnight at 37'~ with 5% CO* and 95% air.

MTT (Sigma) was dissolved at 5 rng/ml in PBS and 10 pL was added to each well. After

incubating for 2 h at 37'~, the reaction was stopped and 50 PL 100% DMSO was added

to solubilise the MTT-formazan product. After mixing absorbance was measured at 540

nM on a microplate reader-

NGF Adsorbed Beads.

Full-length mouse NGF was adsorbed on controlled pore glass beads as

previously described (Gotz et al., 1992). Bnefly, glass powder was incubated overnight

in tnfluoroacetic acid at room temperature, then beads were washed with distilled water

and PBS six times each. Neurotrophins or cytochrome C were added to the activated

beads at concentrations of 0.01 m g h l and placed on ice for 1 h. Beads were washed

with PBS six times and applied immediately on top of the plated neurons.

Apoptosis Determination.

To determine the number of apoptotic cells, staining for TUNEL and ssDNA

MAb were performed. For TUNEL, cells were fixed in PBS with 4% formaldehyde and

4% sucrose, solubilized in 0.25% Triton X- 100 for 5 min, then suspended in 3% Hz02

for 5 min. PBS with 2% BSA was used as a blocker (30 min). DNA nick translations

24

were marked with biotin-16-dUTP as described in manufacmers instructions

(Boehringer Mannheim). Adding 300 mM sodium chloride and 30 mM sodium citrate

for 45 min stopped the reaction. Biotin labeled DNA was marked using an ABC kit as

per the manufacturer's insmsctions (Vector). ssDNA staining was performed as

described (Frankfùrt et d.,1996). Briefly, neurons were fixed with methanoVPBS (6: 1) at

-2ooc, then resuspended with 4.5 rnM MgCi* in PBS and heated to 100'~ for 5 min.

Following a wash with PBS, plated neurons were exposed to MAb F-27 (Alexis) for 30

min, washed and exposed to biotin conjugated goat anti mouse IgM (Sigma) for 15 min,

then washed again and exposed to Extra Avidin peroxidase (Sigma) for 15 min. In

TUNEL and ssDNA assays, neurons were stained with DAI3 (Vectastain) solution as

described by the manufacturer. Slides were then washed with water and dehydrated in

ethanol(75- 100%) and xylenes for 5 min each. Permount was used to fix the coverslips

for microscopy. Apoptotic cells were scored blindly by the investigator and confirmed

with blind counting by another lab member.

S tatistical Analysis.

Statistical analyses were chosen using Modem Elementary Statistics (Freund &

Simon) and performed using GraphPad Pnsm (GraphPad Software). In apoptosis assays,

two wells were used for each culture group and four fields were counted with an average

of 100 cells per field. Averages of the cytochrome C treated cultures were normalized to

100% and al1 other culture averages were taken as a percentage of this value. In receptor

cross-linking studies, bands with incorporated radioactivity were excised and counted in a

gamma counter. For each lane, a background area of equal size was also counted and

subtracted from receptor bands. Control untreated lanes were normalized to 1000/0 and al1

other lanes were taken as percentage of this value. Protomer cross-linking studies were

analyzed by counting total radioactivity added and radioactivity incorporated into the

NGF dimer. Statistical analysis performed include t-tests, ANOVA, and Dunnett's poc

hoc tests, with particuiar tests king stated in the results. Al1 values are expressed plus or

minus the standard error of the mean (SEM).

Resufts

Receptor Expression and AtTîty of p75m

To confirm that homogeneous binding occurred between 1 2 5 ~ - ~ ~ ~ and p75m in

dissociated cultures of EDS.5 chick retinal cells, we performed receptor cross-linking

with EDUSNHS and BS) to evaluate the presence of receptors which interacted with

1 2 ' 1 - ~ ~ ~ . Receptor cross-linking followed by SDS-PAGE resolution and

autoradiography demonstrated that both the monorner and homodimer of p75NTR are

present and bind '*'I-NGF, while no Trk receptor was detectable at this time (Fig. R- 1 ).

The presence of p75m was confirmed with an immunoprecipitation using the rabbit

polyclonal Ab 9992 against p75m. TrkA was detected in PC 12 cells with rabbit

polyclonal Ab 545.

Equiiibriurn binding was performed using dissociated retinal cells and '**I-NGF

concentrations between 0.005 and 1 nM. Linear transformation of the steady state

sahuation binding data of I 2 ' 1 - ~ G ~ to retinal cells demonstrated homogeneous binding

affinity parameters consistent with one binding site. Cross-linking studies showed that

p75- is the only receptor available for interacting with NGF in these cells. The

rneasured dissociation equilibriurn constant (Kd) was 0.42 +- 0.12 n M (Fig. R-2). To

confirm this observation we also determined the ICso value using various concentrations

of unlabelled NGF (0.04 - 40 nM) to inhibit 1 2 5 ~ - ~ ~ ~ (0.1 5 nM) from chemically cross-

linking p75- in retinal and PC 12 cells. Results were evaluated with SDS-PAGE and

autoradiography (Fig. R-3A&B) followed by excision of bands and counting of

radioactivity incorporated into both monomers and dirners of p75mR (Fig. R-3C&D). In

Fig. R-1. Receptor cross-linking and immunoprecipitation of p75m and TrkA

following incubation with '%NGF in retinal cells (lanes 1 4 ) and PC 12 cells (lanes 5-8).

In retinal cells p75m is seen with cross-linking (lane 1) and confirmed with

immunoprecipitation with the Ab 9992 (lane 2). No TrkA cotdd be detected (lanes 3&4).

PC 12 cells express both recepton, as p75m can be seen in lanes 5&6 and TrkA in lanes

7&8 (Trk detected with Ab 545; lane 8).

Retinal Cells PC12 Cells

Fig. R-2. Scatchard aiiysis of specific bhding of '%NGF to p75N"< in chick retinal

cells verses bound over free ' 2 5 ~ - ~ ~ ~ in the incubation buffer. Bùiding displays

hornogeneous affinity consistent with single receptor binding to p 7 ~ m wïth a

dissociation constant of 0.42 + 0.12 n M ( ~ 7 ) .

0.15-

Q, 2 0.10- e O t

0.05-

0.00- I

O 1 I 1

10000 20000 30000 40000

Bound (cpm)

Fig. R-3. To compare the affuiity of the p75m monorner and homodimer in retinal and

PC 12 cells, various concentrations of unlabeled NGF were added to the incubation to

inhibit chernical cross-linking of 0.15 n M ' 2 S ~ - ~ ~ ~ to this receptor. in both ce11 types,

the displacement of 1 2 5 ~ - ~ ~ ~ in the monomer and homodimer did not difEer statistically.

The loss of cross-linking of ' 2 S ~ - ~ ~ ~ with increasing concentration of unlabeled NGF

was determined by autoradiography followed by excising bands and counting

incorporated radioactivity. Displacement analysis revealed that the p75m monomer in

retinal cells (A) had an ICso value of 0.23 + 0.03 nM and a value of 0.54 + 0.1 1 nM in PC

12 cells (B). The displacement profile of both monomer and dimer are shown in each

ce11 type (C & D). The two monomer means were compared with a student-t test (n=4)

-1 O 1 2

Log Unlabelled NGF (nM)

Log Unlabelled NGF (nM)

both ce11 types. the displacement of '*'I-NGF fiom monomers and dimen did not differ

statistically. The 1Cso values for the monomer of p75m in retinal and PC12 cells were

0.23 k 0.03 nM and 0.54 +, 0.1 1 nM respectively. The lower ICso vaiue in retinal neurons

demonstrates that in these cells, p75m has a higher affinity for NGF than in PC 12 cells.

NGF Promotes Apoptosis in Dissociateci Cultures of ED5.5 Chick Retuial CeUs

Recent reports have shown that NGF has the ability to kill different ce11

populations that express p7~m. NGF bound to glas beads has previously been shown

to kill chick retinal explants (Frade & Barde, 1998). Here we use dissociated cultures of

chick retinal cells and demonstrate the apoptotic effect of NGF using ssDNA staining. To

determine the effects of neurotrophins on apoptosis, dissociated neurons from ED5.5

chick retina were plated on 16 well chamber slides. Each well was treated independently

and incubated ovemight at 3 7 C with 5% COz and 95% air. Control apoptosis was

deterrnined using groups treated with g las beads coated in cytochrome C. Cytochrome

C was chosen as a control because of its similar physical characteristics to neurotrophins.

The percent of apoptotic cells ranged between 9.2-14.2% ssDNA positively stained cells

in these control cultures. Groups cultured with NGF bound to glass beads showed

increased nurnbers of apoptotic cells with ssDNA positive neurons ranging between 22.4-

28.9%. It is aiso known that other neurotrophins bind p75m, so we tested NT-3 and

BDNF (on g las beads) in the ssDNA apoptosis assay. These treatments did not

significantly increase cell death compared to the cytochrome C treated group as BDNF

and NT-3 showed average numbers of apoptotic neurons of 9.1 t 2.3% and 12.7 + 1.1 %

respectively (Fig. R-4). We also tested to determine if NGF caused apoptosis at later

Fig. R-4. To test the ef%ects of neurotrophins adsorbed to on glass beads, ED5.5 chick

retinai cells were cultured with individual neurotrophins overnight, futed the next day and

stained for ssDNA. NGF treated cultures showed a significant increase in apoptotic cells.

Results are shown as percentage of positively stained cells. A one way ANOVA

followed by a Dunnett's post hoc test were performed to determine which values differ

fiom the cytochrome C treated cultures (n=3; * denotes groups that are significantiy

different then cytochrome C treated group at p < 0.01 confidence).

stages of retinal development. Here, EDlO retina were used. These cells still express

p75NTR and not TrkA (Frade et ai., 1996). Again a ssDNA assay was used. The number

of positive cells in cultures treated with cytochrome C coated beads was 12.02 +_ 1.2%.

Cultures treated with NGF beads showed no significant difference and had an average of

12.9 f 1 .O% (Fig. R-5). This resuit shows that at this stage of development NGF, even

bound to glass beads, does not cause apoptosis in these ceils.

Changes in %NGF Bindîng in the Presence of 2n2+

Previous work has shown that 2n2+ alters the conformation of NGF (Ross et al.,

1 997; Sharnovsky et al., 1 999), and inhibits bindhg to p75m (Ross et ai., 1 997). Here

we demonstrate that 2n2+ blocks the binding of ' 2 5 ~ - ~ ~ ~ to p75m in dissociated retinai

cells. As p75m is the only neurotrophin receptor that interacts with NGF in these cells,

we could use both total binding and chernical cross-linking analysis to evaluate the dose

response profile of zn2+. At concentrations of 100 FM, 2n2+ (and cu23, effectively block

' 2 5 ~ - ~ ~ ~ binding (Table 1). This finding was selective for these metais as equal

concentrations of MC did not influence binding. Dose response analysis revealed that

the addition of 50 and 25 p M 2n2+ partially blocked binding, while concentrations of 1 O

PM had no effect. The inhibitory effects of 2n2+ could be prevented by the addition of

equirnolar CaEDTA into the incubation buffer. 2n2+ inhibited EDC/SNHS cross-finking

of 1 2 S ~ - ~ ~ ~ to p75m with a similar dose response profile (Fig. R-6a). Results were

quantified by excising bands and counting incorporated radioactivity (Fig. R-6b). To

determine if 2n2+ inhibited the ability of BS' to cross-link the NGF dimer, 2n2' was

added to '*'I-NGF in HKR sample buffer. 100 pM of 2n2+ inhibited the ability of BS' to

covalently cross-link the NGF dimer (Fig. R-7).

Fig. R-5. To test the efEects of neurotrophins adsorbed to on glass beads, ED 1 O chick

retinal cells were cuitured with individuai neurotrophins ovemight, fixed the next day and

stained for ssDNA. Results are shown as percentage of positive1 y stained cells. NGF

treatrnent showed no significant increase in positively stained cells compared to

cytochrom C cultures. A one way ANOVA was performed to determine which vaiues

differ fiom the cytochrome C treated cultures (n=3; * denotes groups that are

significantly different then cytochrome C treated group at p < 0.01 confidence).

NGF Cytahrome C No Treatment

Table 1. Effect of Zn2+ on binding to retinal cells. Binding of "'1-NGF to p75m in

chick retinal cells is blocked by 100 pM 2n2+. The binding efficiency was restored was

with the addition of equal molar CaEDTA. Partial inhibition was achieved with the

addition of 50 and 25 pM 2n2', while 10 p M had no effect. With the addition of 100 pM

M ~ ~ ' , no change in binding was observed. Cu 2C aiso blocked '=I-NGF binding.

Table 1.

Binding of ' 2 5 ~ - ~ ~ ~ to ED 5.5 retinal cells

Treatment % Controf Bindïng SEM

No Treatment 1 100

Fig. Rd. Zinc inhibits EDCISNSH receptor cross-linking of '*Sr-NGF of p75m in

retinal cells effectively at concentrations of 100 FM, partially at 50 and 25 pM and has

no effect at 10 pM (n=4).

Fig. R-7. At 100 pM, Zn2+ alters the conformation of NGF as shown by the inhibitory

effect of this metal on the ability of B S ~ to covalently cross-link the two monomers of

' 2 5 ~ - ~ ~ ~ at this concentration.

Suwival of ED5.5 Chick Retinai Ceiis

To evaluate the toxic effects of metal ions on EDS.5 chick retinal ce11 survival, an M M

assay was performed to determine mitochondrial activity. Mitochonàriai activity is

a strong correlate with ce11 survival. To evaluate the results, control absorbance (540

nM) of untreated cultures were normalized ?O 100% (Fig. R-8). 2nzf does not show a

significant decrease in absorbance at concentrations up to 200 pM, suggesting that the

addition of 1 00 p M 2n2' alone is not sufficient to cause cell death. On the other hand,

CU'' is toxic at the lowest concentration assayed (50 CiM), thus was not tested in other

assays.

zn2+ Inhibits p7~hTR Mediated Apoptosis

To determine the effect of 2n2+on apoptosis, TUNEL (Fig. R-9) and ssDNA MAb

(Fig. R-1 0) staining were perfonned. Al1 slides had two control wells, one with no

treatment and one with control beads with cytochrome C. There was no significant

difference between these two control groups in either assay. The basal percentage of

apoptotic cells was taken fiom the cytochrome C treated group and ranged between 9.5-

12.7% in TUNEL stained cultures, and 1 1.1-13.9% in ssDNA stained cells. The control

cytochrome C groups were normalized to 100%. With the addition of NGF coated glass

beads, the number of TUNEL and ssDNA positively stained nuclei was significantly

increased to 208 + 7.5% and 258.9 6.2% of control level respectively. When co-

administered with NGF, 100 HM 2n2* proved to be neuroprotective as the number of

TUNEL and ssDNA MAb positive nuclei was decreased significantly to 107 + 4.5% and

1 10 _+ 2.3% of control respectively. This effect could be blocked with equimolar

CaEDTA (1 86 i: 14.2% and 209 f 3.2% positive apoptotic cells). As predicted through

38

Fig R-8. MTT analysis was used to determine the viability of retinal cells treated with

various concentrations of 2n2+ and Cu 2'. Results are expressed as percentage of

absorbence at 540 n M of control untreated cultures. 2n2' is not toxic until200 (IM, while

Cu 2' is toxic at 50 PM. A one way ANOVA and Dunnett's p s t hoc tests perfomied to

determine which value first differed fiom the control untreated cultures (n=3 ; p < 0.0 1 ).

Metai Concentration (PM)

Fig. R-9. Exogenous NGF on glass beads induces chick retinal cells to undergo

apoptosis show ushg TCMEL staining. The addition of 100 ph4 2n2' attenuates this

effect and NGF function c m be restored with the addition of equal molar CaEDTA.

This effect is independent of the g las beads as the number of positively stained cells

does not significantly differ between the group treated with glass beads loaded with

cytochrome C and untreated cultures. Zinc has no effect when administered without

NGF. A one way ANOVA followed by a Dunnett's post hoc test were perfomed to

determine which values differ fiom the cytochrome C treated cultures (n=5, *denotes

groups that are significantly different then cytochrorne C treated group at p < 0.01

confidence). Retinal cells are shown with TUNEL stainùig (B, arrows indicate apoptotic

cells).

Fig. R-10. Exogenous NGF on glass beads induces chick retinal cells to undergo

apoptosis shown using ssDNA staining. The addition of 100 pA4 zn2' attenuates this

effect and NGF iùnction can be restored with the addition of equaI molar CaEDTA.

This effect is independent of the glass beads as the number of positively stained cells

does not significantly differ between the group treated with glass beads loaded with

cytochrome C and untreated cultures. Zinc has no effect when administered without

NGF. A one way ANOVA followed by a Dunnett's p s t hoc test were performed to

determine which values differ from the cytochrome C treated cultures (n=3; * denotes

groups that are significantly different then cytochrome C treated group at p < 0.01

confidence). Retinal cells are shown with ssDNA staining (B, arro ws indicate apoptotic

cells).

binding aoalysis, this effect was specific for 2n2+, as 100 pM M ~ ~ + (which did not alter

binding) did not significantly alter NGF mediated cell death (195 t 9.2% T U W L

positive neurons).

PD 90780 Inhibits '3-NGF Receptor Cross-Linking

PD 90780 is a small cyclic compound onginally discovered by Parke-Davis.

Initially, it was reported that this compound blocked NGF binding to p75m through an

interaction with NGF (Spiegel et ai., 1995). To determine if PD 90780 could block

binding of p75- in retinal cells, we evaiuated the effect of this compound on receptor

cross-linking of '"1-NGF. This compound was able to block 1 2 * ~ - ~ ~ ~ receptor cross-

linking to both p75- monomer and dimer at 40 pM, while 4 ph4 partially inhibited

cross-linking and 0.4 p M had no effect (Fig. R-1 1). To determine if this molecule worked

by inhibiting dimer cross-linking with B S ~ , we perfonned protomer cross-linking in the

presence of PD 90780. This compound does not inhibit the ability of B S ~ to covaiently

cross-link NGF monomers as concentrations up to 100 pM had no effect on BS'

protomer cross-linking (Fig. R-12).

PD 90780 Inhibits p7~MR Mediated Apoptosis

To test if PD 90780 had the ability to block apoptosis mediated by the interaction

of p75m and NGF, we assayed dissociated cultures of ED5.5 chick retina. These cells

were incubated ovemight with NGF coated to g las beads in the presence and absence of

40 p M PD 90780. To analyze this apoptotic effect, staining for ssDNA was perfonned.

Compared to control cytochrome C treated groups that were normalized to 100%,

cultures treated with NGF coated to glas beads showed 258 f 6.2% ssDNA positive cells

Fig. R-11. PD 90780 inhibits EDCISNSH receptor cross-linking of '*'I-NGF to p75m

in retinal cells partially at 4 piid and effectively at 40 piid (n=3).

O 0.4 4 40

Concentration PD90780 (PM)

Fig. R-12. PD 90780 does not inhibit the ability of BS' to covalently cross-link the two

monomers of 1 2 5 ~ - ~ ~ ~ as 100 pM of this compound has no significant effect on

inhibiting the percentage of NGF dimer fomed.

NGF Dimer

NGF Monomer

No B S ~ O 0 . 1 0.4 1 4 40 100

Concentration PD 90780 (PM)

Fig . R-13). When 40 j.M PD 90780 was added with NGF apoptosis was inhibited

significantly to 109 + 3 -396, normalized against cytochrome C control. When applied

without NGF, groups treated with 40 pM PD 90780 showed no significant difference in

the number of ssDNA positive neurons than the control cytochrome C groups.

Fig R-13. The addition of 40 pM PD 90780 attenuates the apoptotic effect of NGF as the

number of ssDNA positive cells increases significantly with NGF. This increase is

reversed with the addition of PD 90780. A one way ANOVA followed by a Dunnea's

post hoc test were perforrned to determine which values differ fiom the cytochrome C

treated cultures (n=3; denotes groups that are significantly different then cytochrome C

treated group at p < 0.01 confidence).

Discussion

There are four principal observations emerging from these studies. First, the proapoptotic

effects of NGF on embryonic chick retina demonstrated initially by Frade et al., (1 996,

1998) have been extended to dissociated cultures. Second, in cells where NGF rnediates

apoptosis through p75m signaling, '"1-NGF binds to p75m with high affuiity. Third,

2n2', which has been shown to alter NGF conformation and influence TrkA mediated

survival fiinctions (Ross et. al, 1997, Wang et al., 1999), also attenuates the proapoptotic

activities mediated by the high aanity interaction beîween p75m and NGF. F o u . the

antiapoptotic effect demonstrated by zn2+ is also seen with PD 90780.

Early studies with NGF showed that throughout development this protein was

vital for the maintenance, survival, and neurite outgrowth of developing neurons. When

NGF was supplied in limiting amounts, neuronal death occurred. This led to the

neurotrophic hypothesis, stating that developing neurons compete for a limited amount of

neurotrophin secreted by target tissues. The limited amount of neurotrophins produced

ensures that the proper number of neurons innervate a target (Purves, 1988). However.

recent findings have shown that neurotrophins can play a paradoxical role and may

actually potentiate ce11 death through a receptor interaction.

There is an increasing body of evidence demonstrating that NGF c m promote

apoptosis in sorne cell populations which express p 7 ~ m and not TrkA (Sedel et ai.,

1 999; Frade et al., 1996; Casaccia-Bonnefil et ai., 1 996). A feature of this ceil death

unique to chick retina is that aithough NGF has been shown to promate cell death in

normal development, exogenous NGF in solution does not cause apoptosis, while NGF

bound to g las beads does (Frade & Barde, 1998). The reason for this is unknown but it

47

has been suggested that the way microglia present NGF to developing neurons in vivo

may be critical @rade & Barde, 1998). However, studies examining other potential

factors involved in presenting NGF to p75m expressing retinal cells in vivo have yet to

be explored.

in our cultures, NGF treated neurons demonstrated twice the number of TUNEL

positive nuclei, and two and a half times the number of ssDNA positive nuclei than seen

in control and non-treated cultures. The reason for the greater number of ssDNA positive

neurons may be attributed to the fact that this antibody is more sensitive to the early

stages of apoptosis than TUNEL labeling (Franktiut et al., 1996). The increased number

of apoptotic neurons can be attributed to the bound NGF, as groups treated with beads

covered with cytochrome C had a similar number of positive nuclei as untreated control

cultures.

Consistent with previous observations, the apoptotic effect in these cultures was

specific for NGF, as BDNF and NT-3 had no significant effect in killing these cells.

What is inûiguing is the hd ing that NGF does not kill these cells at ED 10. Previous

studies have reported that the apoptotic effect of NGF in cultured oligodendrocytes,

which express p75m, CM be attenuated when these cells are transfected with TrkA

(Yoon, et al., 1998). However, retinai newons have been s h o w not to express TrkA

until after ED 14 (Frade et al., 1996). Although it is possible that TrkA in these cells is

present yet undetectable, NGF's inability to cause apoptosis is not likely mediated by

TrkA expression.

The literature suggests that several factors rnay be important for p75m mediated

apoptosis to occur. Gu et ai., (1999), showed that p75m mediated ce11 death involved

several caspases (caspase-1, caspase-2, caspase-3). Barde's group showed that NRIF

interacts with p75m. In wild type mice' p75K"< mediates apoptosis in the developing

retina. However, both p75m and NRIF knockout mice show reduced numbers of

apoptotic cells in the retinal population (Casademunt et al., 1999). It is possible that

either a specific caspase or NRIF could be down regulated between ED5.5 and EDlO in

the chick retina. Considering that many retinal precursors undergo differentiation

between these days (Parda, et al., 1991), it is plausible that gene expression changes in

this time. Further, many other genes have been shown to inhibit apoptosis, the best

studied of which belonging to the bcl-2 family (Garcia et al., 1992). Therefore, it is also

possible that one of these antiapoptotic genes becomes active between these

developmental stages.

Previous work has shown that the binding characteristics of p75m for NGF are

dependent on the presence of TrkA (Ross et al., 1998). However, binding analysis of

' 2 5 1 - ~ ~ ~ to p75- has not been reported in cells that undergo ce11 death following this

receptor ligand interaction. in PC12 cells, p 7 ~ m is the low affinity receptor with a Kd =

4.0 nM (Ross et al., 1998). in these cells, p75m serves a secondary role of promoting

the binding of NGF to TrkA by influencing the allosteric properties of this receptor,

possibly through the formation of a heteroreceptor complex (Ross et al., 1998). When

p75m is expressed alone in ~ ~ 1 2 ~ " cells, the binding f i n i t y is much higher (Kd = 0.2

nM), suggesting that TrkA has an allosteric effect on p75m. In chick retinal cells, our

finding that p7sNTR has a Kd = 0.4 n M and ICso of 0.23 nM (compared to an ICso value of

0.55 n M in PC12 cells), corresponds to the high affinity binding state of this receptor.

nierefore, apoptosis mediated by the interaction between NGF and p75m occurs when

this receptor is in its hi& affinity conformation.

Through cornpetition bindiing and receptor cross-linking studies, we have shown

that 100 @A zn2+ is sufficient to inhibit binding of NGF to p75m in chick retïnal cells.

These findings extend previous observations that ~ n " attenuates binding to this receptor

in its low d h i t y conformation when CO-expressed with TrkA (Ross et al., 1997)-

Molecdar modeling and ab initio calculations have been used to determine the most

stable structure of the zn2+: NGF coordinated complex and optimal geometq of the

coordination site (Fig. D-2; Shamovsky et ai., 1999; Ross et al., 1997). When 2n2+

interacts with NGF, it is chelated by four residues fiorn the NGF dimer forming a 5-

coordinate state with donor atoms NE of His4, NE of His8, NS of His84 and both oxygens

of Asp 105. Transition metal cations split into three groups by their intrinsic

stereochemicai preference in the pentacoordinated environment, such that the most active

transition metal cations, zn2' and cu2+, are consistent with the square pyramidal

coordination environment within NGF (Shamovsky et al., 1999). The confonnational

transition within NGF induced by 2n2+, pulls the N-terminus of NGF, (the proposed

binding detennint of TrkA; Shamovsky et al., 1998), towards the loop 1 region (binding

determint for p75NTR (Shamovsky et ai., 1999b, Ibanez et al., 1992). With this

codonnational aiteration, an ionic bond is fonned between the N-terminal Serl and

Glu35 in loop 1. This interaction stabilizes the complex and creates a sterical hindrance

within the p75* binding determinant in both its high and low affinity states. These

stexicai considerations explain the inhibitory role of zn2+ for both TrkA and p75m

mediated activities of NGF.

Fig. D-1. Three-dimensional structure of the NGF conformation with 2n2' mediated

conformational changes (provided by I.L. Shamovsky). Conformation of the arnino

termini (TrkA receptor binding detemiuiant) is significantly altered and inhibits p75MR

binding through a covalent interaction with loop 1 @75- binding deteminant).

The addition of 100 p M 2n2+ to NGF treated cultures provided protection against

p75m mediated apoptosis, as the percentage of TUNEL and ssDNA MAb positive

nuclei were attenuated significantly. Previous studies have shown that chick retinal cells

can be protected against NGF induced apoptosis with the addition of the loop peptide

dc28-36 (Ross et al., 1994). This compound mimics loop 1 of NGF, the binding

determinant of p75m (Ibanez et al., 1992). The likely mode of action of this compound

is through direct competitive inhibition of p75m and not by altering ligand structure.

We propose that 2n2+ protection is mediated by the ability of this metal to alter the

conformation of NGF which leads to the inhibition of p75m binding. Consistent with

this hypothesis, we show that 2n2+ inhibits protomer cross-linking of the two NGF

monomers, suggesthg that a conformational alteration has taken place.

There is evidence that 2n2' is present in the developing rodent nervous system

(Sawashita et al., 1997). It is also likely that this transition metal is present in the

developing chick. After hatching, and throughout life, 2n2+ is an essential part of chicken

diet (Wedeking & Baker, 1990). In ovulating hens, 2n2+ and other transition metals are

transferred to the oocyte fiom hepatic stores. Egg white proteins mediate the transfer of

trace elements fiom the oviduct to the egg. The delivery of trace metals to the

developing embryo is mediated by embryonic membranes, and inuriorgan transport is

facilitated by specific metd binding proteins (reviewed in Richard & Steele, 1987).

Although no reference could be found to directly implicate 2n2+ in retinal neuron

development, the fact that this metai is present in the embryo suggests that it might play a

functional role. Considering this, and our findings that 2n2+ attenuates NGF induced

apoptosis, it is possible that this transition metal influences development by inhibiting

NGF's biological activity. Further, since BDNF and NT-3 have been shown to play a

role in retinai neuron development (Frade, et at., 1999), and that zn2+ inhibits protomer

cross-linking of these two neurotrophins (Ross et al., 1997), this transition metal may also

inhibit the efTects of these factors. However, the actual physiological role of zn2' on

neuroirophin function in chick retind development will have to be determined at a later

date.

The findings that 2n2' modulates neuronal îùnction have led to much interest into

the possible toxic roles of this transition metal in neurodegeneration. Under conditions

seen in Alzheimer's disease, 2n2' has been shown to promote the aggregation of P-

arnyloid and senile plaques (Bush et d., 1994). Fÿrther, expenmental data suggest that

2n2' is released fiom presynaptic vesicles in the hippocampus and cortex and contributes

to neuronal death following middle cerebral artery occlusion induced ischemia (Johanson

et al., 1993; Koh et al., 1996). 2n2' release has been observed in the hippocampus

following induced seizure activity, however, evidence that this release leads to ce11 death

is controversial (Lees et al, 1998). Following insult, presynaptic vesicular zn2+ release

c m reach concentrations between 100-300 pM (Xie & Smart, 199 1). Our results show

that this concentration would be sufficient to inhibit not oniy the protective effects of

NGF but also the p75m mediated proapoptotic properties of this molecule, suggesting a

possible neuroprotective role for this transition metal.

Although NGF has clsissically been associated with promoting neuronal swival .

it has recently been suggested that this protein may contribute to neurodegeneration under

conditions when p75m is expressed in the absence of TrkA. There have been several

studies suggesting that p75m contributes to the elimination of neurons in various

conditions. Xncreased p75M expression has been observed following middle cerebral

artery occlusion induced ischemia (Kokaia et al., 1998). Experimental data also suggest

that f3-amyloid protein binds p75m and this interaction can signal apoptosis (Yaar et al.,

1997). However, the most compelling evidence for the direct involvement of p75m in

neuronal degeneration under pathologie conditions may be the observations that ce11

death follows both pilocarpine induced seizure activity and unilateral kainic acid

administration. Both insults correlate strongly with the expression of p75m in

hippocampus, cortex and basal forebrain regions (Roux et al., 1999; Oh et ai., 2000).

Further, the chronic administration of anti rat p75m substantially decreases kainic acid

induced ce11 death of cholinergic neurons (Oh et al., 2000). The fmding that 2n2' inhibits

apoptosis mediated by p75m and NGF is significant, considering that vesicular zn2'

release following insult occurs in many areas of the nervous system where p75m has

been im,-licated in neuronal death (Harrison & Gibons, 1994). Therefore, this

mechanism for zn2' mediated neuroprotection offers novel insight into the potential

neuroprotective interaction of this transition metal with NGF and p75- under

conditions associated with ce11 death.

The increasing evidence that NGF can promote apoptosis through p75m has

prompted interest into finding small non-peptide compounds that antagonize this

interaction. One publicly disclosed compound shown to block this receptor ligand

binding is PD 90780. When tested at 40 pM, this compound was able to attenuate the

number of ssDNA positive cells following NGF treatment. Further, 40 PM PD 90780 did

not significantly inhibit or promote ce11 death in the absence of NGF. The same

concentration blocked receptor cross-linking, but at concentrations up to 100 pM, had no

54

effect on NGF protomer cross-linking, s ign img this compound does inhibit the ability

of B S ~ to covalently cross-link NGF monomers. However, since PD 90780 has

previously been shown to bind NGF directly, and not interact with p75NTR, it is likely that

the compound interferes with the p75m binding detenninant of NGF through another

mechanism. Molecular modeling studies predict that PD 90780 interacts directly with

Ioops 1 and 4 of the NGF dimer (I.L. Shamovsky personal communication). This area

encompasses the NGF binding determinant for p75m but does not compromise the

cross-linking site of the two monomers (Ross et al., 1997). It is hypothesized that the

electronegative moieties of PD 90780 form hydorgen bonds with Lys residues of NGF

(Lys 32, Lys 34, Lys 88, and Lys 95; Fig D-2). This interaction is sufficient to inhibit

NGF fiom binding p75NTR.

The ability of PD 90780 to inhibit the deleterious biological activity of NGF, and

non-toxicity on it's own, confirms that it is a good fust generation antagonist of the pro

apoptotic interaction between NGF and p75m. From this structure, more specific

compounds with higher efficacy can be generated through computer guided-structure-

based design and basic screening procedures. Such compounds may prove usefid in

treating conditions where the p75m NGF interaction contributes to the

neurodegenerative process.

Fig. D-2. Proposed interaction of PD 90780 with NGF (provided by I.L. Shamovsky).

Conciusion

This thesis was successfiil in determining that ED 5.5 chick retind cells die with

the administration of NGF bound to glas beads. This effect is mediated by p75m, as it

is the only NGF receptor present at this stage of deveiopment. Binding between NGF

and p75m OCCLUS with high atfinity in cells that undergo apoptosis through this

interaction. NGFYs apoptotic effect can be blocked with the administration of 2nZ' or PD

90780. These findings provide insight into how NGF mediated toxicity rnight be

attenuated in p75MR expressing cells.

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