I I CYTOPROTECTIVE ACTIONS PF NICOTINE: THE INCREASED EXPRESSION OF a.7 NICOTINIC RECEPTORS AND NGF/TrkA RECEPTORS
' .
'
f !'b y' Ramamohima R. Jonnala ' '
' I' Submitted to the Faculty o~the School of Graduate Studies of Medical College of Georgia in Partial Fulfillment of the Requirements of the Degree of Doctor of Philosophy Juiy, 2001 I' l Cytoprotective Actions of Nicotine: The Increased Expression of a.7
Nicotinic Receptors a-!ld NGF/TrkA Receptors
This dissertation is submitted by Ramamohana R. Jonnala and has been examined and approved by an appointed committee of the faculty of the school of Graduate Studies of the Medical College of Georgia.
The signatures which appear below verify the fact that all required changes have been incorporated and that the dissertation has received final approval with reference to content, form and accuracy of presentation. ' This dissertation is therefore in partial fulfillment of the requirements of the degree of Doctor of Philosophy. ACKNOWLEDGMENTS
I would like to extend my appreciation to my major advisor, Dr. Jerry J.
Buccafusco, for his guidance, encouragement and support. I would also like to thank my committee members, Dr. Alvin V. Terry Jr., Dr. William D. Hill, Dr. Nevin A. Lambert and Dr. Clare M. Bergson and also my thesis readers Dr. Debra Gearhart and Dr. Dale W.
Sickles for their time and valuable suggestions.
My sincere thanks to Dr. Deborah L. Lewis, Dr. Robert W. Caldwell and Dr. Gary
C. Bond for encouraging me to enter the graduate program in the Department of
Pharmacology & Toxicology, Medical College of Georgia.
My sincere thanks to members of my laboratory Laura, Vanessa, Daniel, Cat,
Mark, Nandu, Lu, and Shyamala for their assistance and suggestions. TABLE OF; CONTENTS
Page
ABSTACT ...... : ...... lll
LIST OF FIGURES ...... vii
LIST OF TABLES ...... ix
LIST OF ABBREVATIONS ...... X
I. INTRODUCTION A. Significance of study ...... ,...... 1 B. Literature review ...... 3 I. Alzheierm's disease pathophysiology ...... 3 2. Genetics of AD ...... 6 3. The selective vulnerability ofBFC neurons ...... 7 4. NGF and NGF receptors ...... 9 5. Oxidative damage ...... 10 6. Cholinomimetic therapy ...... 12 7. N1cotmic receptors ...... 14 8. The PC12 cell model system ...... 18
II. MATERIALS AND METHODS Cell culture ...... 21 Compounds ...... 21 Strucutres of choline and choline analogs ...... 22 Cell vi;;tbility assay ...... 23 125 [ I]a-BGT binding assay ...... : ...... 23 Elisa assay ...... 24 Animals ...... 25 Surgical procedure ...... 25 Westemblot...... 25 Exposure of PC12 cells peroxynitrite ...... 26 Cytosensor Microphysiometer ...... 27 Cell lysate preparation ...... 27 Immunoprec1p1 . ·1at1· on s tudi es ...... 28 MAPKassay...... 29 Page I.RESULTS I. Stimulation of cx7nAChR protects differentiated PC12 cells from trophic factor withdrawal toxicity...... 3 0 2. Neuroprotective actions of cholii1e·and choline analogs...... 31 3. The abilities of different nAChR agonists to indµce neuroprotection...... 32 4. The abilities of different nAChR agonists to induce cx7nAChR upregulation...... 33 5. MLA induced increase in cx7nAChR expression and neuroprotection...... 34 6. Nicotine up-regulates TrkA receptors...... 35 7. Relationship between nicotine's neuroprotection and its ability to increase TrkA receptors ...... :...... 37 8. Metabolic response of PC12 cells to NGF...... 38 9. Effect of SIN-I on cell viability...... 39 10. Inhibition ofNGF mediated metabolic responses by SIN-1...... 40 11. Effects of SIN-I on TrkA receptor autophosphorylation...... 40 12. Effects of SIN-1 on MAPK activity...... 41
IV. DISCUSSION
I. Neuroprotective actions of nicotine...... 42 2. Choline and Choline analogs...... 45 3. Relationship between the ability to produce neuroprotection and the upregulation of cx7nAChRs...... 47 4. Relationship between increased TrkA receptors and neuroprotection.. 51 5. Importance ofNGF signaling...... 54
V. SUMMARY...... 61
VI. REFERENCES...... 89 Abstract: Certain epidemiological studies have reported a negative correlation between smoking and neurodegenerative diseases such as Alzheimer's disease (AD) and
Parkinson's disease, reflecting perhaps the neurotrophic actions of nicotine. In recent years there has been intense interest in the development° of new nicotinic acetylcholine receptor (nAChR) agonists. These agents have the potential to be used in the treatment of patients with AD. However, the mechanism for the neuroprotective action of the nicotine is not yet known, indeed, it is not yet clear as to which subtype of nAChR mediates the response. In neuronal cell lines, the induction of cytoprotection often requires exposure to nicotine for up to 24 hr to produce a full neuroprotective effect and this chronic exposure of nicotine is also known to increase nAChR receptors and cell surface nerve growth factor (NGF) receptors. The purpose of this study is to determine which subtype of nAChRs are involved in nicotine's neuroprotective actions and also to determine whether nicotine's neuroprotective actions are related to its ability to increase cell surface nAChRs and NGF receptors.
Preincubation of differentiated PC 12 _cells with nicotine for 24 hr protected the cells from growth factor withdrawal-induced toxicity in a time and concentration-dependent manner. Nicotine's cytoprotective actions were completely blocked oy non-selective nAChR antagonist mecarnylarnine, and the a7nAChR preferring antagonist methyllcaconitine (MLA) indicating that the response was primarily mediated by the subtype of a7 nAChR receptors. The acetylcholine precursor, choline is a very selective
111 and full agonist at a.7 receptors. Among five choline analogs tested for neuroprotection potential, acetylpyrrolidinecholine and pyrrolidinecholine were found to be more potent and more efficacious than their parent co_mpound, choline. The rank order of the six compounds tested for their cytoprotective ability is as follows: acetylpyrrolidinecholine = pyrrolidinecholine · > choline = monoethylcholine = diethylcholine = triethylcholine.
Further, to confirm the above structure activity relationships with respect to their binding affinities at u7nAChR, [1 25I]u-bungarotoxin (BGT) displacement binding studies were performed using differentiated PC12 cells. Choline was only 50 fold less potent than nicotine in displacing [125I]u-BGT binding. Pyrrolidinecholine, the most active analog, fully displaced [125I]u-BGT binding and it exhibited a slightly greater affinity for the site than did choline.
Next we compared the ability of seven different nAChR agonists with varying activities at a.7 receptors for their ability to produce cytoprotection. Among the seven compounds tested, nicotine was the most effective and the most potent followed in order of potency by
4OH-GTS21, epibatidine, methylcarbamylcholine, 1, 1-Dimethyl-4-phenyl-piperazinum, cytisine and tetraethylarnmonium. Since, epibatidine and cytisine were less efficacious than nicotine despite their greater affinity for a.7 receptors and because short-term exposure of cells to nicotine did not produce cytoprotective actions, we next compared the ability of these compounds to upregulate cell surface a.7 receptors. After, incubation of cells for 2 hr with either nicotine or cytisine, the number of [1 25I]u-BGT binding sites on differentiated
PC12 cells were measured. Nicotine, the most efficacious compound increased the [125I]u
BGT binding sites by ~40% over the untreated control cells. In contrast, cytisine, the least effective compound failed to do so, indicating that the ability to upregulate a.7 receptors
iv may provide one possible mechanism for neuroprotective actions of nicotine. Further, we confirmed that these additional populations of receptors were functional and that they mediate an enhanced neuroprotective response to subsequent nicotine stimulation.
Earlier studies had shown that prolonged exposure to nicotine increases NGF receptors on differentiated PC12 cells. In the present study, high affinity NGF receptors
(TrkA) were shown to increase with nicotine treatment in a time-and concentration dependent manner. This effect was blocked by co-treatment with mecamylamine and with MLA, but not a low concentration of dihydro-p-erythroidine (selective for P2 and
P4 containing receptors), indicating that the response was mediated by predominantly a7 nAChRs. Next, we measured the TrkA protein levels in rat hippocampal and cortical tissues after nicotine treatment. Chronic -nicotine infusion increased TrkA protein levels I within the hippocampus, and this effect was blocked by co-treatment with mecamyliunine. In contrast, TrkA protein levels in cortical tissues were not altered.
Since the majority of nAChRs in hippoc,ampus are of the a7 subtype and whereas in cortex consist largely of the a4P2 subtype, it is reasonable to conclude that the differences observed in TrkA receptor expression in hippocampus and cortex were due to presence of a7nAChR and non-a7nAChR. To determine whether the neuroprotective actions of nicotine were due to enhanced NGF trophic activity during the drug incubation period, the ability of nicotine to protect cells from trophic factor withdrawal in the presence and in the absence ofNGF were compared. Nicotine was found to be effective in both experimental paradigms. However, nicotine ·was found to be 10 fold more effective when it was incubated with cells in the presence of NGF. When cells were treated with nicotine and k252a or nicotine and anti-TrkA antibody, nicotine was only
V partially effective as a neuroprotective agent, indicating that mechanisms apart from enhanced NGF mediated trophic activity during drug incubation period were involved in nicotine's cytoprotective actions.
One consistent finding with regard to in AD pathology is the selective loss of basal forebrain cholinergic neurons (BFC). The survival and maintenance of these neurons depended on the availability ofNGF from target tissues. Evidence from previous studies suggests that impairment in NGF support could be an initial insult in AD pathology.
Previous studies have shown that much of the oxidative damage in AD tissue was mediated by peroxynitrite. Breif exposure of undifferentiated PCl2 cells to 3- morpholinosydnonmine (SIN-I, peroxynitrite generator) was sufficient to inhibit an NGF mediated cellular response by 67% of that measured in control cells. This inhibition of the NGF cellular response by SIN-I was not related to generalized cellular toxicity. In fact, the peroxynitrite scavenger uric acid significantly attenuated the inhibitory actions of SIN-I. Pretreatment with SIN-I also resulted in a decrease in the NGF-induced phosphorylation of TrkA protein. Furthermore, SIN-I treatment reduced the activity of mitogen activated protein kinase, a downstream kinase activated by TrkA receptor stimulation: These data suggest that SIN-I treatment inhibits NGF signaling by inactivating TrkA receptors through the formation of nitrotyrosine residues on the receptor. The inactivation of TrkA receptors may contribute to the initial insult that eventually leads to neuronal cell death.
vi List of Figures
Figure Page
1. The ability of nicotine to protect differentiated PC12 cells from the trophic factor withdrawal...... 65
2. The ability of choline to protect differentiated PC12 cells from trophic factor withdrawal...... 67
3. The relative cytoprotective ability of different choline analogs...... 68
4. Characterization of the pyrollidinecholine's cytoprotective actions... 69
5. Displacement of high-affinity [125I]a-Bungarotoxin binding to differentiated PC12 cells by nicotine and choline...... 70
6. Displacement of high-affinity [125I]a~Bungarotoxin binding to differentiated PC12 cells by pyrrolidinecholine,...... 71
7. The relative ability of different nAChR agonists to protect differentiated PC12 cells from the trophic facotoi; withdrawal...... 72
8. The effect of chronic nicotine and cytisine on the upregulation of
a7nAChR subtype...... 73
9. The effect of chronic methyllcaconitine on the upregulation of a7nAChR subtype and subsequent stimulation with nicotine...... 75
10. The ability of nicotine to increas~ cell surface TrkA receptors on differentiated PC12 cells...... 76
11. Blockade of nicotine induced increase in cell surface TrkA receptors by nAChR antagonists...... 77
12. TrkA protein levels in rat hippocampus and cortex tissues after
continuous infusion with nicotine...... 78
vii Page 13. Cytprotective.ability of nicotine in the presence and in the absence ofNGF...... 79
14. Cytprotective ability of nicotine in the presence and in the absence ofNGF...... 80
15. Cytoprotective ability of nicotine in the presence of anti-TrkA antibody .. ,...... 81
16. The rate of media acidification in response to exposure of PC12 cells
with different concentrations of nerve growth factor (NGF) for 12 min.... 82
17. The effect of SIN-I on cell viability ...... 83
18. The rate of media acidification in response to exposure of PC12 cells
With 25 ng/ml NGF after SIN-I treatment...... 84
19. The effect of uric acid treatment on SIN-I induced inhibitory responses to NGF stimulation...... 85
20. The extent ofTrkA receptor phosphorylation to NGF stimulation after SIN-I treatment...... 86
21. MAPKinase activity in immunoprecipitates of PC12 cells treated with NGF or SIN-! plus NGF...... 87
22. Schematic representation of the possible relationship between the upregulation of et7nAChR, the increased expression ofNGF/TrkA receptors and the neuroprotective actions of nicotine...... 8 8
. viii List of Tables
Table
Page
1. Effect of nicotinic receptor antagonists on the neuroprotective action produced by nicotine in differentiated PC12 cells...... 66
2. Effect of prolonged exposure of differentiated PC 12 cells to nicotinic ligands on the cell surface saturation binding of [125I]a.-bungarotoxin... 74
ix List of Abbreviations
A~ amyloid beta Ach acetylcholine AD Alzheimer's disease BDNF brain derived neurotrophic factor BFC basal forebrain cholinergic BGT bungarotoxin Bmax maximum binding ChAT choline acetyltransferase CNS central nervous system DH~E dihydro-~-erythroidine DMPP 1, 1-dimethyl-4-phenyl-piperaziniurn ECso effective concentration (50%) EGF epidermal growth factor FGF fibroblast growth factor :final femto moles i.v. intravenous Kd dissociation constant Ki inhibitory affinity concentration MAPK mitogen activated protein kinase MCC methylcabamylcholine Mee mecamylamine MLA methyllcaconitine MTT 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliurn bromide nAChR nicotinic acetylcholine receptor NbM neucleus basalis of Meynert NFTs neurofibrilary tangles NGF nerve growth factor NMDA N-methyl-D-aspartate PC12 Pheochromocytoma SIN-I 3-morpholinosydnonmine TEA tetraethyl ammonium TrkA tyrosine kinase receptor A 4OH-GTS21 3-(4-hydroxy, 2-methoxy benzylidine)-anabaseine
X I. Introduction:
A. Significance of study:
Alzheimer's disease (AD) is the most.common cause of progressive cognitive decline in the aged population. In one community-based study it was reported that approximately
4 million Americans are inflicted with AD (Evans et al., 1989). The disease is characterized by a progressive loss of memory, difficulties with language, decision making and visual perception. The decline in intellectual function progresses slowly, but unavoidably, and leads to severe debilitation and death between 4 and 12 years after onset.
The post mortem AD brain is often characterized by a relatively selective loss of basal forebrain cholinergi~ (BFC) neurons, the presence of arnyloid protein (A~), neurofibrillary tangles (NFTs), and an, abnormally phosphorylated form of the tau protein. Since BFC neurons play an important role in human cognition, restoring the integrity and functional ability of these neurons is of a particular interest in AD res~arch.
However, the mechanisms underlying the selective degeneration of BFC neurons have not been elucidated. Several attempts have been made to produce an animal model for
AD. Transgenic approaches have provided the presenilin-based transgenic mouse (Duff et al., 1996), the arnyloid precursor-protein based transgenic mouse (Nalbantoglu et al.,
1997), and the A~ peptide-based transgenic mouse (LaFerla et al., 1995) were found to be of limited utility. None of these models were able to reproduce all of the pathological changes that occur in the AD brain. Recently, Capsoni and co-workers (2000) have
1 2 reported an AD-like neurodegenerative process in aged transgenic mice that over-express antibodies against nerve growth factor (NGF). Strikingly, these mice developed an age dependent neurodegenerative pathology including AP plaques, insoluble and hyperphosphorylated i:, and NFTs in cortical and hippocampal neurons. In addition the mice exhibited certain cognitive deficits. However, in humans the process by which
' NGF trophic activity might become impaired in AD has never been addressed. Evidence from several studies using AD brains suggests, that a decrease in NGF receptor levels occurs in areas that are selectively damaged in AD (Boissiere et al., 1997; Mufson et al.,
1997). One of the approaches that has been proposed for AD therapeutics is the use of small molecules that could mimic NGF-induced trophic activity. Reports from this and other laboratories have indicated that nicotine and other nicotinic receptor agonists, can increase the levels of trophic factors and their receptors and evoke a neuroprotective response (Terry and Clarke, 1994; French et al., 1999; Bellurdo et al., 1998). However, it is not clear which of the several nicotinic acetylcholine receptor (nAChR) subtype(s) are involved in mediating these responses. Since nAChR agonists elicit many unwanted peripheral and central effects, it is imperative to determine the subtype(s) that are responsible for their trophic actions. On the basis of previous literature and on the preliminary work performed in our laboratory, the following specific aims were investigated.
1. To characterize the pharmacological properties of nicotine that play a role in the ability of the drug to produce cytoprotection in differentiated PC12 cells. 3
Corollary: To determine which subtype of nAChR primarily mediates nicotine's neuroprotective actions.
2. To determine whether the cytoprotective action of nicotine is also related to its ability to increase the expression of nicotinic receptors.
3. To determine whether nicotine's cytoprotective actions are related to the ability of the drug to increase the expression of cell surface NGF/TrkA receptors.
4. To_ determine whether TrkA cell signaling pathways are important for mediating the cellular response to NGF in PC12 cells.
B. Literature review:
1. Alzheimer's disease pathophysiology:
Alzheimer's disease (AD) is one of the most common causes of mental deterioration in elderly people, accounting for 50%-60% of the overall cases of dementia among persons over 65 years of age (Evans et al., 1989). AD affects an estimated 15 million people worldwide. With the proportion of elderly in the population steadily increasing, the emotional burden of the disease on patients and caregivers, as well as the national economic burden is expected to substantially increase over next 2 to 3 decades. AD is a progressive neurodegenerative disorder with a mean duration of about 8.5 years between onset of clinical symptoms and death. Brain regions that are associated with higher mental functions, particularly the neocortex and hippocampus, are the most affected by the characteristic pathology of AD. The past two decades have witnessed a considerable research effort directed towards discovering the cause of AD, with the anticipation of developing safe and effective pharmacological treatments. 4
The major neuropathological features of AD were first described by Alois Alzheimer
(Alzheimer, 1907) in 1907. He reported the presence of intraneuronal deposits of proteinaceous material termed neurofibrillary tangles (NFTs ), as well as extracellular deposits termed amyloid plaques (A/3). More recently, the results from studies at the molecular level indicate the presence of extracellular deposits of amyloid peptides
(derived from amyloid precursor protein) in senile plaques, whereas the neurofibrillary tangles were shown to contain an abnormally phosphorylated form of the microtubule associated protein tau (Brion et al., 2001 ). Anatomically there was the loss of neuronal synapses and cortical pyramidal neurons. The appearance of these pathological features, which are correlated with the development of characteristic symptoms associated with
AD, are characterized by gross and progressive impairments of cognitive function, often accompanied by behavioral disturbances such as aggression, depression, and wandering
(Tariot et al., 2001). Caregivers find these features the most difficult to cope with and their prevalence often leads to the institutionalization of the patient (Esiri, 1996).
The systematic biochemical investigation of autopsied brain tissue derived from AD patients began in the late 1960s to early 1970s. · It was anticipated that a clearly defined neuroche~cal abnormality would be identified, providing the basis for the development of rational therapeutic interventions analogous to levodopa in the treatment of
Parkinson's disease. Support for this possibility came in the mid 1970s with the reports of substantial neocortical deficits in the enzyme responsible for the synthesis of acetylcholine (ACh), choline acetyltransferase (ChA T) (Davies and Maloney, 1976).
Subsequent discoveries of reduced choline uptake (Rylett et al., 1983), ACh release
(Nilsson et al., 1986) and the loss of cholinergic perikarciya within the nucleus basalis of 5
Meynert (NbM) (Whitehouse, 1982) confirmed the presence of a substantial presynaptic cholinergic deficit. These studies, together with the emerging role of ACh in learning and memory (Drachman and Leavitt, 1974), led to the "cholinergic hypothesis of
Alzheimer's disease" (Bartus et al., 1981). Thus it was proposed that degeneration of cholinergic neurons in the basal forebrain and the associated loss of cholinergic neurotransmission in the cerebral cortex and other areas contributed significantly to the deterioration in cognitive function observed in patients.
Anatomically and pathologically, AD is characterized by relatively selective loss of basal forebrain cholinergic (BFC) neurons, neurofibrillary tangle formation, and the presence of A~ plaques in circumscribed regions of the neocortex and hippocampus, primarily affecting pyramidal neurons and their synapses (Mann, 1996; DeKosky et al.,
1996). Neurotransmitter specific subcortical nuclei that project to the cortex are also affected by neurodegenerative process, including the cholinergic NbM and medial septum, the serotonergic raphe nuclei, and noradrenergic locus ceruleus. Biochemical investigations of biqpsy tissue taken from patients with AD 3.5 years (on average) after the onset of symptoms indicate that a selective neurotransmitter pathology occurs early in the course of the disease (Francis et al., 1993). Specifically, presynaptic markers of the cholinergic system appear uniformly reduced. This is exemplified by reductions in
ChAT activity and ACh synthesis, which are strongly correlated with the degree of cognitive impairment in patients with AD (Francis et al., 1993). Whereas serotonergic and certain noradrenergic markers are affected, markers for dopamine, y-aminobutyric acid (GABA), or somatostatin do not appear to be affected (Francis et al., 1993). In later stages of the disease these other (non-cholinergic) neurotransmitter systems are affected 6 to a greater extent, including GABA (Rossor, 1982) and somatostatin-releasing neurons.
Also, the neurochemcial markers attributed to cortical intemeurons are affected later in the disease process (Roser and Iversen, 1986). The fact that basal forebrain projection neurons to the neocortex are lost at an early stage of the disease process is substantiated by the findings of such neuronal changes in patients that have displayed clinical symptoms for less than one year. Although, diminished ChAT activity is a necessary correlate of AD, other components of cholinergic neural cells likely participate in the decline in cognitive function these may include the decline in nicotinic (Whitehouse et al., 1988) and muscarinic (M2) ACh receptors. The loss of these receptors may reflect their presynaptic location on cholinergic basal forebrain projection neurons, whereas other cholinergic receptors that are considered to be located on cells postsynaptic to these neurons (e.g., muscarinic (Ml, M3) receptors are largely spared (Nordberg et al., 1992).
2. Genetics of AD:
In a number of families AD is inherited as an autosomal dominant disorder, although in most cases inheritance appears to be multifactorial. Monozygotic twin studies indicate a variable concordance of between 18% (Raiha eta!., 1996) and 41% (Nee et al., 1987), showing that AD cannot be explained completely by a single autosomal dominant gene.
A relative risk of 3.5 has been demonstrated for those with at least one first-degree relative suffering from dementia (VanDuijn et al., 1991).
Three genes have been identified to date in which mutations result in early onset familial AD, inherited in an autosomal fashion. These are the APP, Presenilin 1 (PS-I), and PS-2 genes. The APP gene maps to chromosome 2lq21.1, and mutations in this gene are estimated to account for up to 5% of familial AD cases. A number of mutations have 7 been identified, at codons 717 (Chartier-Harlin et al.; 1991), 670/671 (Murrell et al.,
1991 ), 692 (Hendriks et al., 1992), and 693 (Levy et al., 1990). Mutations in the APP gene might result in altered metabolism of APP, leading to increased production of the
AP protein, or an increased production of more toxic form of AP (AP 1-42). This is the predominant form in senile plaques, and is thought to be more amyloidogenic, forming amyloid fibrils more rapidly than the shorter forms of AP (Jarrett et al., 1993).
A second familial AD locus was identified by genetic linkage analysis at chromosome
14q24 (Van Broeckhoven et al., 1992; Schellenberg et al., 1992). The gene responsible was subsequently identified as PS-I, and mutations in this gene are _thought to cause up to
80% of familial Alzheimer's disease cases, with onset as early as 29 years of age
(Campion et al., 1996), ranging to 62 years of age (Goate, 1997). To date, more than 35 missense mutations, and one in frame deletion· have been identified in the PS-I gene in affected families. The fact that most PS mutations are missense ones suggests that
Alzheimer's disease pathology is a result, not of the absence of functioning as a result of the mutation. Although mutations in the PS-I gene account for most cases of familial
Alzheimer's disease, there are some affected families in which both APP and PS-I mutations have been excluded. A third locus was identified at chromosome lq31-42
(Levy-Lahad et al., 1995a), and the mutated gene was identified subsequently as PS-2
(Levy-lahad et al., 1995b ). However, the functions of PS-! and PS-2 proteins are largely unknown, but it has been demonstrated that mutations in both the PS-I and PS-2 gene cause over production of the amyloidogenic AP1-42 form in both transfected cells and transgenic mice (Duff et al., 1996; Citrone al., 1997). 8
3. The selective vulnerability ofBFC neurons:
One of the consistent findings .in AD pathology is the selective degeneration of BFC neurons. Clinicopathological studies of the BFC neurons in-patients with AD indicate that this system plays a pivotal role in human cognition (Wilcock et al., 1982). However, the mechanisms underlying the selective degeneration of BFC neurons are not yet understood. Several attempts have been made to produce animal models for AD. These include presenilin based transgenic mice (Duff et al., 1996), amyloid precursor protein based transgenic mice (Nalbantoglu et al., 1997), and A~ peptide based transgenic mice
(LaFerala et al., 1995). However, none of these animals were consistently shown to produce all the pathological changes that are characteristic of the AD brain. Recently,
Capsoni and co-workers (2000) have reported an AD-like neurodegenerative process in aged transgenic mice that over-express antibodies against nerve growth factor (NGF). In the CNS, NGF mRNA and protein concentrations are found in highest concentration within sites of termination of BFC neurons such as the cerebral cortex and hippocampus
(Koh et al., 1989). Through interaction with its specific receptor, NGF affects the survival, fiber outgrowth, and expression of transmitter-specific enzymes associated with cholinergic neurons as studied both in vitro (Haritikka and Hefti, 1988; Knusel and Hefti,
1988) and in vivo (Fisher et al., 1987; Kromer, 1987). NGF, recognizes two classes of cell surface receptors. These two classes can be identified by their distinct binding affinities to NGF. A low-affinity binding site (Kd = 10·9 M), is termed the p75 neurotrophin receptor (p75NTR), whereas the high-affinity binding sites (Kd = 10· 11 M) is termed the TrkA receptor. The survival and maintenance of these BFC neurons are dependent on the availability of target derived NGF (Barde, 1989). It has been proposed 9 that one factor contributing to the deg~nerative changes associated with AD is the withdrawal of this NGF trophic support (Hefti and Weiner, 1986).
4. NGF and NGF receptors:
Studies on the development of the central nervous system in chick embryos during
1950's by Viktor Hamburger and Rita Levi-Montalcini have led to the discovery ofNGF.
NGF was initially isolated and purified from murine sarcomas 180 and 37 (Cohen et al.,
1954). During 1960's, studies by Levi-Montalcini on the development and maintenance of mice peripheral sympathetic system have provided the basic knowledge about NGF in the neuronal development and survival (Levi-Montalcini and Booker., 1960). NGF is synthesized in target neurons (Korsching et al., 1985; Shelton and Reichard, 1986; Ayer
!eLievre et al., 1988; Whittemore· et. al., 1986), taken up by terminals of septohippocampal cholinergic projection neurons through the high-affinity NGF receptor
TrkA (Richardson et al., 1986; Raivich and Kreutzberg; 1987; Batchelor et al., 1989;
Weskamp and Reichardt, 1991; Steininger et al., 1993; Gibbs and Pfaff, 1994; Chao and
Hempstead, 1995), and growth factor is retrogradely transported to the parent cell bodies in the medial septum-diagonal band complex (Johnson et al., 1987; Loy et al., 1994).
The neurotrophic actions of NGF on BFC neurons are required to maintain choline acetyltransferace (ChAT) activity (Kojima, 1995), cell size, cell number, the activity of antioxidant enzymes (Pan and Perez-Polo, 1993) and the regulation of its own tyrosine kinase (TrkA) receptor (Figueiredo et al., 1995; Zhou et al., 1995). Studies using AD brain tissue have revealed decreases in ChAT activity (Boissiere, 1997), cell size, and
TrkA receptor levels (Boissiere, 1997; Mufson et al., 1997), with no loss in NGF protein levels. Rather, Scott and co-workers (1995) reported that NGF protein levels were 10 increased in all regions of the AD brain except for the nucleus basalis of Meynert (NbM).
Since NGF reaches the NbM from nerve terminals through retrograde transport
(DiStefano, 1992), AD pathology may include impairment ofNGF retrograde transport.
It is possible therefore, that in the AD brain BFC neurons may receive reduced NGF
signaling. Recently, Capsoni and co-workers (2000) reported finding an Alzheimer-like
neurodegenerative process in aged transgenic mice that over-express antibodies against
NGF. Tuey demonstrated that these mice acquire an age-dependent neurodegenerative
pathology including amyloid plaques, insoluble and hyperphosphorylated tau, and
neurofibrillary tangles in cortical and hippocampal neurons. These mice also display
extensive cholinergic neuronal deficits in basal forebrain, as well as cognitive behavioral
abnormalities. These findings suggest that an impairment in NGF retrograde transport
may contribute to the initial insult in the pathogenesis of AD. However, the mechanism ' behind the loss in NGF retrograde transport has not been adequately addressed.
5. Oxidative damage:
Emerging evidence suggests that oxidative stress may be a common mechanism for
neurodegeneration in disorders such as amyotrophic lateral sclerosis (Rosen et al., 1993),
Parkinson's disease (Jenner and Olanow, 1996) and AD (Benzi and Moretti, 1995), as
well as in normal aging (Beal, 1995; Benzi and Moretti, 1995; Bains and Shaw, 1997).
During the past few years, a number of oxidative modifications have been found to
associate with the pathological lesions of AD. For example, advanced glycation end
products, lipid peroxidation, and free carbonyls have been detected in both NFTs and
senile plaques (Vitek et al., 1994). Such oxidative modifications of proteins may define
of the unique properties of the lesions, in particular their association with degenerating 11 neurons (Vitek et al., 1994; yan et al., 1994). Nonetheless, despite this evidence, the
source of oxygen radicals in vivo has not been determined.
Recent evidence from in vitro studies suggests that A~1-42, which is deposited in the
AD brain, can induce nitric oxide production (Vitek et al., 1997) as well as generate
oxidative radicals such as hydrogen peroxide and superoxide anion, both of which are
extremely cytotoxic chemical species (Huang et al., 1999). In humans, an antioxidant
system is present which protects cells from these reactive species. The cellular
antioxidant system consists of the enzymes catalase, glutathione peroxidase, and
superoxide dismutase. A decrease in the activity of any of these enzymes could lead to
cell death. Studies with AD brain tissue have shown that the enzyme activity of catalase
and superoxide dismutase is significantly decreased in frontal and temporal cortex
compared to age-matched control brains (Marcus et al., 1997) thus indicating the
involvement of oxidative stress in the pathogenesis of the disease.
Recently, there has been increased interest in the possibility of peroxynitrite-mediated
oxidative damage in AD. In fact, the results of one report suggest that most of the
oxidative damage measured in AD is mediated by peroxinitrite (Smith et al., 1997).
Peroxynitrite is a powerful reactive oxygen species produced as a result of the diffusion
limited reaction of superoxide with nitric oxide (Beckman et al., 1990). Superoxide and
nitric oxide each separately can cause oxidative damage to the neurons. However, since
the reaction between nitric oxide and superoxide is kinetically favored, any condition that
leads to the overproduction of either component might result in the production of
peroxynitrite. Peroxynitrite causes oxidation of proteins and other macromolecules with
resultant carbonyl formation from side-chain and peptide-bond cleavage, and these free 12
carbonyls also have been shown to occur in the AD brain (Smith et al., 1996). Although
carbonyl formation represents a major oxidative modification, peroxynitrite also causes
the nitration of tyrosine residues (Beckman, 1996) thus preventing their phosphorylation.
Peroxynitrite nitrates tyrosine residues on the ortho-position to form 3-nitrotyrosines and
this modification ultimately prevents phosphorylation at this position, a process which is
essential for many cellular responses. Nitrotyrosine immunoreactivity, a marker for
peroxinitrite-mediated damage, has been measured in AD brains in regions selectively
involved in AD pathology (Good et al., 1996; Smith et al., 1997). Moreover, this
nitrotyrosine immunoreactivity is found predominantly in the brain regions that are
selectively damaged in AD. Although the identity of specific proteins affected in this
way is currently unknown, one possibility is that tyrosine kinase receptors may become nitrated. If so, it is possible that peroxynitrite may decrease NGF/TrkA signaling by nitrating tyrosine residues on TrkA receptors, thus preventing them from becoming phosphorylated by NGF, a proximate step in the NGF/TrkA signaling pathway.
6. Cholinomimetic th.erapy:
A prediction of the cholinergic hypothesis is that drugs that potentiate central cholinergic function should improve cognition and perhaps they may resolve the behavioral problems experienced by AD patients. There are a number of approaches to the treatment of the cholinergic deficits in AD, most of which have initially focused on the replacement of ACh precursors (choline, lecithin). However, these agents did not prove to be effective treatments (Smith et al., 1978; Higgins and Flicker, 2000). Other studies have investigated the use of cholinesterase (ChE) inhibitors such as physostigmine that reduce the hydrolysis of ACh allowing it to available for longer 13 action. Currently, CbE inhibitors donepezil, rivastigmine, and galanthamine are the only drugs available for the treatment of AD. More recent investigational compounds include specific Ml muscarinic or nicotinic agonists, and M2 muscarinic antagonists. Additional potential symptomatic therapeutic avenues relevant to the cholinergic hypothesis of AD have resulted from the rapid development in the understanding of the molecular pathology of the disease. For example, during the development of cholinergic neurons in the basal forebrain, they express functional nerve growth factor (NGF) receptors. In adult life, these neurons seem to remain responsive to NGF. Numerous studies have confirmed that in AD patients, the number of neurons that expressed NGF receptors were markedly decreased in the NbM very likely as a consequence of cholinergic neuronal loss
(Boissiere, 1997; Mufson et al., 1997). It has been argued that an increase in NGF tonus might be beneficial in AD. A wealth of animal data, generated mainly in rodents, has shown that axotomized cholinergic neurons can be rescued by treatment with NGF.
Thus, when cholinergic projections are lesioned in animals, infusion of NGF can rescue the cholinergic neurons, stimulate axonal growth, and improve cholinergic function
(Hefti, 1986; Tuszynski et al.,· 1990). Furthermore, NGF and NGF receptor mRNA and protein levels are decreased in aged rodents (Larkfors, 1987), and decreased levels of
NGF in hippocampus correlate with deficits in spatial learning (Henriksson et al., 1992).
Treatment with NGF has been shown to counteract cholinergic atrophy and memory deficits in aged rats (Fischer et al., 1987). However, one of the main drawbacks in this approach is the route of administration of NGF (Eriksdotter et al., I 998; Olson et al.,
1992). Since NGF cannot cross the blood brain barrier, an intracerebral route of administration is required. Several alternative strategies to increase either endogenous 14
NGF or NGF receptor levels in AD patients have been proposed. These include slow release biodegradable implants, direct gene transfer, development of NGF receptor agonists and development of small molecular substances that enhance the NGF trophic support (Olson, 1993). Among all the strategies, only the development of small molecules such as nicotine analogs that may mimic NGF trophic effects has met with
moderate success.
7. Nicotinic receptors:
The concept of nicotinic receptors was first developed by Langley during early
1900's, who first reported that nicotine could block neuronal transmission in the superior
cervical ganglion. Until only a few years ago, knowledge of neuronal nicotinic receptors
remained confined to the ganglia. The structure, function, pharmacology and distribution
of brain receptors have more recently begun to be systematically investigated, and these
studies have shown that nicotinic receptors are also relevant to our understanding of a
number of complex brain functions and the pathogenic mechanisms of some brain
pathologies. Nicotinic receptors are ligand gated ion channels that permit the passage of
cations through the post-synaptic membrane in response to the binding of ACh. Much of
the early evidence of biochemical and: pharmacological chara~terization of nicotinic
receptors comes from the study of these receptors in the electric organ Torpedo
califomica and the fresh water teleost Electrophorous electricus (Popat and Changeux,
1984). Subsequently, molecular approaches have clearly demonstrated that nicotinic
receptors are oligomeric proteins composed of five subunits (Changeux, 1990; Cooper et
al., 1991). In muscle, nicotinic acetylcholine receptors are composed of four subunits
according to the mole ratio, a:2~yo. The subunits are oriented like barrel staves around a 15 central pore and all subunits contribute to the lining of the channel (Brisson and Unwin,
1985). All subunits are glycosylated and they span the membrane four times, resulting in four hydrophobic regions (Unwin, 1993). The individual subunits exhibit about 40% amino acid sequence homology, and the a. subunit can be distinguished by the presence of cytisines at the 192 and 193 positions in second hydrophobic region. The ACh binding ' . sites are located on the extracellular surface of a. subunits. Thus far, eleven members of the nicotinic receptor subunit gene family are known to exist in the CNS. These genes and their products are termed a2 - a9, p2 - P4 (Boulter et al., 1987; Boulter et al., 1986;
Boulter et al., 1990; Deneris et al., 1987; Lindstrom et al., 1987). These subunits are known to form two classes of receptors, homooligomers and heterooligomers.
Association of a.7, a.8, and a.9 subunits c_an form homooligomeric receptors, whereas the combination of a.2/P2, a.3/P2, a.2/p4, a.4/P2, a.3/P4, and a4/ P4 subunits can form heterooligomeric receptors.
It has been known since the early days of pharmacology that nicotinic receptors are important in ganglionic transmission and control the function -of the peripheral autonomic system (Asher et al., 1979), but their functions in the brain are still unclear, They are known to be involved in various complex cognitive functions, such as attention, learning, memory consolidation, arousal, sensory perception and in the control of locomotor activity, pain perception, body temperature regulation (Gotti et al., 1997). Although every potential subtype of the central nicotinic receptor has not yet been fully ' pharmacologically characterized, using radioligand binding techniques, at least two major classes have been identified in rodent brain. One major class of receptor binds agonists such as nicotine, cytisine, methyl carbamylcholine, ABT-418 and epibatidine with high 16 affinity (Marks et al., 1986; Pauly et al. 1989, Whiting and Lindstrom, 1988). These receptors are localized throughout the rat brain in discrete regions which include the medial hebenula, interpenduncular nudei, thalamic nuclei, substantia nigra pars compacta, cortical areas and striatum. Mice lacking these high affinity receptors (P2 knockout) was shown to have an impairment in spatial learning and also an increased neurodegeneration during ageing (Zoli et al., 1999). One other major class of receptors exhibits a high affinity for a.-bungarotoxin and very low affinity for nicotine or cytisine.
Moreover, a.-bungarotoxin binding sites exhibit strikingly different anatomical localization compared with cytisine binding sites. The a-bungarotoxin sites are mainly located in deep layers of the cerebral cortex, hippocampus, hypothalamus, and superior and inferior colliculli (Marks et al., 1986; Pauly et al., 1989). The results of ligand binding or immunoaffinity and/or immunoprecipitation studies indicate that the class of high affinity nicotinic receptor is composed primarily of a.4/P2 subunits (Whiting and lindstrom, 1986; Whiting and lindstrom, 1987), whereas the a.-bungarotoxin subtype is a.7 homooligomer (Schoepfer et al., 1988; Segula et al., 1992).
In general, continued stimulation of most neurotransmitter receptors with agonists results in a decrease in the number of binding sites (down-regulation), and the development of tolerance to the effects that follows subsequent exposure to the same concentration of drug. However, chronic treatment with nicotine and its analogs has been shown to upregulate nicotine binding sitys both in vivo and in vitro (Marks et al., 1992;
Pauly et al., 1991; Schwartz and Kellar, 1985; Wonnacott, 1990). One possible explanation for the upregulation of nicotinic receptors is that receptor number is increased as a regulatory response to continued receptor desensitization. It has also been 17 shown that this increased population of receptors is functional (Molinari et al., 1998;
Buisson et al., 2001).
In order to be able to control different nicotinic brain functions pharmacologically, it is very important to have drugs that selectively affect the different receptor subtypes in such a way as to maximize the desired effect and minimize the unwanted effects. The possible clinical uses of nicotinic receptor agonists and antagonists are for controlling smoking addiction, improving brain ·function in dementia, due to ageing or degenerative diseases, and controlling pain. The pharmacology of nicotinic receptors has always been selective, but this has previously been confined to the discrimination of muscle and ganglion receptors with the aim of obtaining curare-like drugs without ganglionic activity and ganglionic agents without any activity at neuromuscular junction. The problem of finding drugs that selectively discriminate neuronal receptor subtypes is more complex since the differences are more subtle and the number of receptor subtypes is much larger.
Although a number of compounds have been synthesized and some are now in the clinical testing, very few of them are subtype selective (Holladay et al., 1997).
The results from early epidemiological studies suggest that an inverse relationship between smoking and the prevalence or rate of progression of neurodegenerative diseases like Parkinson's disease (Baron, 1986) and AD (Brenner et al., 1993) may exist, although more recent studies have disputed a beneficial action of smoking as related to AD (Ott et al., 1998). It has also been reported from PET studies using 11 C-nicotine that there is a
43% deficit of nicotinic receptors in temporal cortex of AD patients compared to aged matched controls (Nordberg et al., 1997). The deficit of these nicotinic receptors was also shown to correlate with the decrease in cognitive performance. Moreover, evidence 18 from recent studies indicates that there is a huge reduction (-40%) in nicotinic receptor density in patients with Lewy body disease (dementia) compared to aged matched controls (Rei et al., 2000). Over the past several years it has been demonstrated that nicotine and certain nicotinic acetylcholine receptor (nAChR) agonists can increase the levels of neurotrophic factors such as NGF and fibroblast growth factor-2 and their receptors both in vivo and in vitro (French et al., 1999; Terry and Clarke, 1994; Bellurdo ' et al., 1998). Moreover, nicotinic ligands also have been shown to protect neural cells against the cytotoxic effects of glutamate (Akaike et al., 1994; Marin et al., 1994), ~ amyloid (Kihara et al., 1997; Zarnani et al., 1997), trophic factor-deprivation (Martin et al., 1994), and the neurotoxic destruction of the basal forebrain (Owaman et al., 1989).
However, the specific subtype(s) of nAChR involved and the mechanisms underlying the neuroprotective actions of nicotine have not been determined. In addition, stimulation of nAChR has been shown to improve memory related tasks in rodents (Levin, 1992; Levin et al., 1995; Terry et al., 1996) and non-human primates (Elrod et al., 1988; Buccafusco et al., 1995). These findings should support the hypothesis that the use of nAChR agonists in the treatment of AD patients will have an indirect neuroprotective action along with their ability to improve working memory.
8. The PC12 cell as model neuronal system:
PC12 cells originally derived from, a rat medullary pheochromocytoma cell line
(Greene and Tischer, 1976), have been extensively used as a model system to study the effects of NGF. The long-term effect' of NGF on PC12 cells is to cause cellular differentiation into neurite-bearing cells resembling mature sympathetic neurons. The cells then stop dividing, they attach more strongly to the substratum, and they extend long 19
neuritic process (Greene, 1978). NGF exerts its effects via specific low-affinity
receptors (p75NTR) and high affinity receptors (TrkA). Dissociation of NGF from high
affinity receptors is considerably slower than from low-affinity receptors; therefore, they
· are also referred to as slow receptors and fast receptors respectively. Several lines of
evidence have demonstrated that activation of TrkA receptors is necessary and sufficient
to elicit NGF responses (Bernd and Greene, 1984; Meakin and Shooter 1991). The role
of p75NTR in NGF mediated actions is not clearly understood. However, it has been
suggested that p75NTR increases the association of NGF to TrkA receptors (Mahadeo et
al., 1994). NGF initiates its actions by binding to the TrkA receptor on the cell surface,
which in turn induces autophosphorylation of receptor tyrosine residues (Jing et al.,
1992). There are two cellular consequences of receptor atuophosphorylation of tyrosine
residues. Tyrosine phosphorylation is coµsidered to be required for both the initiation of
receptor activity and to provide recognition or docking sites for cellular signaling
proteins. Four proteins have been shown to form complexes with NGF activated Trk:
Phospholipase C (PLC)-yl, SHC, phosphoinosidine (PI)-3 kinase, and Erk!. Acting in a
signal transduction pathway downstream of these proteins are the small GTPase Ras,
regulators of Ras, and mitogen activated protein kinase (MAPK) activated directly or
indirectly by Ras activity (Szeberenyi et al., 1990; Kremer e al., 1991; Thomas et al.,
1992; Wood e al., 1992; Li e al., 19.92). Thus, a signal transduction pathway is
estrablished whereby the activation of TrkA stimulates the activities of PLC-y I and other
TrkA associated proteins that in tum sequentially activate Ras and MAPK. The
activation of TrkA receptor tyrosine kinase has been shown to participate in ligand
internalization (Meakin and Shooter, 1992), axonal growth (Sinder, 1994), neuronal cell 20 survival (Sinder, 1994; Chao, 1992; Barbacid, 1994), and cell proliferation (Chao, 1992;
Barbacid, I 994).
Differentiation of PC12 cells in the presence ofNGF results in neurite-bearing cells, which can express cholinergic markers. For example, NGF treatment has been shown to increase ChAT activity, increase the relative levels cholinergic receptors (nAChR, muscarinic), and also to increase the responses to cholinergic agonists (Furukawa et al.,
1994). In a manner similar to BFC neurons, differentiated PC12 cells also depend upon the availability ofNGF for their survival and maintenance. The deprivation ofNGF from differentiated PC12 cells results in neurite retraction and eventually apoptosis in a similar fashion to that which occurs in AD. · In fact, it also has been reported that NGF deprivation causes aberrant tau phosphorylation in differentiated PC12 cells (Nuydens et al., 1997). Evidence from recent studies strongly suggest that nicotine and nicotinic receptor agonists protect differentiated PC12 cells from NGF deprivation (Martin et al.,
1994). However, the specific subtype(s) of nAChR involved and the mechanisms underlying the neuroprotective actions o( nicotine have not been determined. PC12 cells are known to express nAChR subunits u3, u5, u7, ~2, ~3, and ~4. Differentiation of
PC12 cells with NGF has been shown t~ increase the expression of u3, u5, u7, and ~4
(Takahashi et al., 1999) and as well as to increase the responses to nicotinic agonists
(Furukawa et al., 1994). Thus, growth factor withdrawal from differentiated PC12 cells has been employed extensively as a useful model system in AD literature to determine the mechanisms underlying neuroprotective actions of nicotine as well as to evaluate potential novel molecules for their ability to exhibit neuroprotection. II. Materials and Methods:
Cell Culture: The rat pheochromocytoma (PC12) cell line was purchased from
American Type Culture Collection (Cat No# CRL 1721). PC12 cells were maintained in
150-cm2 tissue-culture flasks in Dulbecco's modified Eagles medium containing
(DMEM) (Gibco BRL) 7% horse serum (VN), 7% fetal calf serum (VN), 1% non essential amino-acids and 1% streptomycin. The cells were incubated at 3 7 °c in a 5%
COi-enriched, humidified atmosphere. To attain maximum differentiation, the cells were maintained in DMEM.NGF medium for 7 days, with the medium being changed every 2 or 3 days.
Compounds: Nicotine hydrogen tartarate, mecamylamine hydrochloride, methyllcaconitine citrate (MLA), (-)-cytisine, tetraehtylarnmonium chloride (TEA), 1,1- dimethyl-4-phenyl-piperazinum iodide (DMPP) choline iodie, a.BGT, and 7s NGF were obtained from Sigma Chemicals, St. Louis, MO. Biochemicals International, Natick,
MA. (±)-Epibatidine chloride was obtained from ICN Biomedials lnc, Aurora,, OH.
Dihydro-~-erythroidine was obtained from Merch Sharp & Dohme Research Labs.
Methylcarbamylcholine was purchased from Arcos organics. K252a was purchased from
Cal Biochem Labs, CA. SIN-I (3-morpholinosydnonmine) was purchased from Cayman
Chemicals. Pyrrolidinecholine iodide, acetylpyrrolidinecholine iodide, monoehtylchoilne iodide, diethylcholine iodide, and triethylcholine iodide were synthesized as described previously (Arenstam et al., 1988), benzylchoilne iodide was synthesized by Dr. Warren
Beach, University of Georgia, Athens, Georgia. All the drug concentrations refer to the
21 22 salt. [1251]- a.BGT was obtained from NEN Lifescience, Boston, MA. 3-( 4-hydroxy, 2- methoxy benzylidene)-anabaseine (40HGTS-21) was generously supplied by Dr. Edwin
M. Meyer, University of Florida, Gainesville, Florida.
Structures of Choline and Choline anal!)gs:
Nicotine Choline
3 CHrCH2 /H3 CH3-CH2- :"cH2-CH2-0H ,"!;!"-CHrCHi-OH1 £H3 CHrCH2 MonoEthylcholine Diethylcholine CH3 I CH2 I CH3-CH2· N -CH2-CH2-0H fH3 I + CH2 c~CH2CH20H I CH3
Triethylcholine Pyrrolidinecholine
fH3 ~ c~CH2CH20 OH
Acetyl Pyrrolidinecholine 23
Cell Viability Assay: Cells were dissociated by trituration and plated at lx104 cells per
. well in 96 well plates. For the experiments that involving nAChR ligands, cells were dissociated by trituration and plated at lxl04 cells per well on poly-L-lysine coated 96 well plates containing DMEM.NGF media. Next, the differentiated cells were incubated with the test drug at different concentrations for the specified period of time. Cell viability (cytotoxicity) was determined using the Cell Titer 96 non-radioactive cell proliferation/cytotoxicity assay kit (Promega), which is based on the cellular conversion of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) into a formazan product that can be detected spectrophotometrically (Mosmann, 1983). After deprivation of cells from NGF and serum, the medium was aspirated and 15 µl of dye solution dissolved in DMEM was added. After an additional 4 hr incubation at 37 °c,
100 µl of solubilization/stop solution was added and the absorbance of solubilized MTT formazan products was measured at 570 nm. The data represent an average of at least three experiments. Each experiment is done in quadruplicate. For the experiments that t involved SIN-1, the cells were exposed to different concentrations ofSIN-1 for 20 min at
37 °c. After SIN-1 exposure, the buffer was removed and replaced with DMEM for the specified time intervals.
25 [1 I]a.-BGT Binding assay: Cell surface a.-bungarotoxin sites were caluculated as mentioned previously (Blumenthal et al.; 1997). PC12 cells were plated (100,000 cells per well) on poly-L-lysine coated 24 well plates containing DMEM.NGF media. The culture medium was removed from differentiated cells and replaced with identical medium, but containing [125I]a.-bungarotoxin ([125I]a.BGT) with or without I µM unlabeled a.BGT (non-specific binding). The cells were incubated for 2 hr at 37 °c and 24 rinsed four times with 2 ml aliquots ofHEPES solution (137 mM NaCl, 5.4 mM KC!, 0.8 mM MgSO4, 0.9 mM Na3PO4, 0.4 mM K2PO4, 1.8 mM CaCiz, 2 mg/ml BSA, and 1.0 mM HEPES, pH 7.4). Bound radioactiv!ty was quantified by gamma counting. Levels of specific binding were normalized to total protein, which was assayed by scraping cells in 2% SDS and O. lN NaOH and conducting the BCA protein assay with BSA as a standard.
ELISA Assay: Cell surface TrkA receptors were measured as described previously
(Terry and Clarke, 1994). After incubation, the cells were washed with warm D-PBS
(Dulbecco's Phosphate-Buffered Saline) buffer and fixed with D-PBS containing 0.1 % glutaraldehyde for 10 min at room temperature to preserve cell surface antigenicity. The fixed cells were incubated in D-PBS containing 10 mM ammonium sulfate for 10 min to neutralize reactive aldehyde groups and then washed with D-PBS. Next the cells were treated with D-PBS containing 1% H2O2 and 0.02% sodium azide to block endogenous peroxidase activity and then washed with D-PBS. Non-specific protein binding was blocked by incubating the wells with D-PBS containing 2% BSA (Sigma Chemicals) for
90 min at 37 °c. Cells were incubated with a mouse monoclonal antibody raised against the extracellular epitope of TrkA receptor (Cal Biochem) at a concentration of 1 µg/ml in
1% BSA.D-PBS for 60 min at room temperature. Cells were then washed with D-PBS three times to remove non-specifically bound primary antibody. This was followed by incubation with anti-mouse IgG conjugat~d with horseradish peroxide (Sigma Chemicals) at a dilution of 1:2000 in 1% BSA.D-PBS containing 0.01% goat serum for 40 min at room temperature. Cells were then washed with D-PBS 3 times to remove non specifically bound secondary _antibody. Secondary antibody binding was quantified by 25 incubating the wells with 0.4 mg/ml o-phenylenediamine in 0.05 M phosphate-citrate buffer at pH 5.0 (Sigma Chemicals) for 30 min at room temperature. The reaction was stopped by adding 3M sulphuric acid and the optical density was read at 490 nm.
Animals: Male Wistar rats, weighing 300-350 g at the time of the experiment were obtained from Harlan, Sprague-Dawley, (Indianapolis, IN). The animals were housed in our animal care facilities for at least 5 days prior to experiments. Food (Harlan Teklad ' rodent diet, Madison, WI) and tap water were supplied on an unlimited basis. A 12 h light/dark cycle was maintained. Procedures involving animals were approved by the
Institutional Committee for Animal Use in Research and Education.
Surgical Procedure: Rats were anesthetized with methohexital (65 mg/kg), and a midsagittal incision was made in the neck. The jugular vein was then exposed, and a polyethylene cather (PE5o) filled with heparinized saline (20 units/ml) was inserted. The distal end of the catheter was directed subcutaneously to emerge at the back of the neck.
The catheter was passed through a spring support and connected to a watertight swivel mounted 300 mm above the cage floor. This surgical procedure allowed the chronically catheterized rat unrestricted movement to' all areas of the cage for the duration of the experiment while receiving a constant infusion ofheparinized saline (8 ml/day). Each rat was allowed 24 hr recovery period after the surgery. After the recovery, the rats were continuously infused with nicotine (12 mg/kg/day) and/or mecamylamine (24 mg/kg/day) for24 hr.
Western blot: After the drug infusion period, rats were sacrificed and the tissue from frontal cortex and hippocampus was collected. The tissue was snap frozen in liquid nitrogen prior to protein extraction. TrkA protein in these tissues was quantified as 26 described previously (Mufson et al., 1997). The frozen samples were solubilized in ' buffer (150 mM NaCl, 50 mM Tris-Cl, pH 7.8, 0.01% sodium-azide, 10 mM NaF, 30 mM sodium-pyrophospp.ate; 2% (VN) NP-40), and supplemented with protease and phosphate inhibitors (1 mM Na3 V04, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 20 mM iodoacetamide, 1 mM PMSF) for 30 min at 4 °c. Following lysis, organelles and cell ' debris were removed by centrifugation (16;000g, 15 min at 4 °c). Proteins in the lysates were measured with the Bio-Rad protein assay kit (Bio-Rad Labs). Equal amounts of ' protein (20 µg) were resolved by 7.5% SOS-PAGE, and electrotransferred to PVDF membranes. Each membrane was immunoblotted with the monoclonal anti-TrkA antibody (Upstate biotechnology) raised against the extracellular domain of TrkA receptor. Membranes were subsequently incubated with the appropriate horseradish peroxidase-conjugated secondary antibody;for 1 hr. After a final wash in TBS, detection was performed (Storm system, Molecular Devices) using an enhanced chemiluminescence system.
Exposure of PC12 cells to peroxynitrite:: Undifferentiated PC12 cells were treated with
SIN-I solution (in Earle's balanced salt s~lution, pH 7.4) at 37 °c for 20 min. Under these conditions the decomposition of SIN-I has been shown to generate peroxyrtitrite
(Ischiropoulous et al., 1995). For cytosensor microphysiometer experiments, PC12 cells were plated at a density of 5xl 05 cells/dish in 100 mm-diameter dishes coated with poly
L-lysine. Twenty-four hours after the plating, cells were washed with PBS and exposed to freshly prepared SIN-I solution for 20 min. After the SIN-I treatment, cells were plated into sterile capsule cups as described in the next section. For experiments involving the measurement of phosphotyrosine levels, cells were treated with NGF in 27
DMEM media for 10 min after SIN-I exposure.
Cytosensor Microphysiometer: The metabolic actions of PC12 cells stabilized in the flow chambers of a Cytosensor microphysiometer were measured as described previously
(Pitchford et al., 1995). Briefly, PC 12 cells were centrifuged and resuspended in low buffered serum-free RPMI 1640 modified medium (Molecular Devices) containing 3 %
BSA. The cells were plated into sterile cell capsule cups (Molecular Devices) at a density of 100,000 cells/capsule. Cells were immobilized using a cell entrapment medium (Molecular Devices). The cups were then placed into the chambers of the microphysiometer and medium was pumped across the cells at a rate of 100 µI/min. To measure the extracellular acidification rate, the flow of the medium was periodically interrupted, allowing the accumulation of excreted acid metabolites. In these experiments, the flow was interrupted for 30 s, in every 2 min cycle.
Basal acidification rates were monitored for at least 20 min. For those experiments that involved SIN-1 treatment, cells were pretreated as mentioned above. Cells were exposed to NGF for a total of 12 min at various concentrations. Different concentrations of NGF were added to separate chambers of cells. For each experiment, acidification rates were normalized to 100% prior to the addition ofNGF. Responses to NGF.were monitored for at least 60 min after initial treatment. It has been previously demonstrated that the change in metabolic rate of PC12 cells upon NGF exposure, is mediated specifically by the activation of NGF receptors and it is sustained for at least 2 hr I (Pitchford et al., 1995).
Cell lysate preparation: PC12 cells we~e seeded in 10 cm plates at a density of lx107 cells/dish. Cells were allowed to grow in DMEM medium.for 24 - 48 hr before lysis. 28
' Cells were rinsed with cold PBS containing 1 mM sodium orthovanidate (phosphatase inhibitor). Cell lysates were prepared by ;adding 1 ml of lysis buffer A (50 mM Tris, pH ' 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM sodium' orthovanidate, 0.1% 2-mercaptoethanol,
1% Triton X-100, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 1 mM PMSF, I
µg/ml of aportonin, pepstatin, leupeptin and 1 µM rnicrocystine (Sigma Chemicals)) to each dish, and ·lysed on ice for 40 i min. The lysate was then transferred to microcentrifuge tubes and centrifuged fqr 10 min at 12,000 x g. Proteins in the cell lysates were measured with the Bio-Rad DC protein assay kit (Bio-Rad Labs). lmmunoprecipitation studies: PC12 :cells were washed twice in ice-cold PBS containing 1 mM sodium orthovanidate aµd lysed as described above. Equal amounts of protein were incubated with anti-phosphotyrosine (Santa Cruz Biotechnology) antibody I overnight (4 °C) and subsequently incubated with protein A agarose beads· (Upstate
Biotechnology) for 3 - 4 hr at 4 °c. Immunoprecipitates were washed three times with i washing buffer (TBS, 2 mM sodium orthcivanidate, 0.1% triton X-100, and I mM PMSF) and they were boiled in Laemmli buffer! for 5 min before electrophoresis using 7.5 % I i SDS-polyacrylamide gel (Bio-Rad). The protein was electroblotted onto PVDF I membranes (Bio-Rad). Each membrane :was immunoblotted with the monoclonal. anti- , . TrkA antibody (Upstate Biotechnology) ~aised against the extracellular domain of TrkA receptor. Membranes were subsequently incubated with the appropriate horseradish I peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) for I hr. After a I final wash in TBS, detection was performed' (Strom system, Molecular Devices) using an enhanced chemiluminescence (ECL) system. 29
MAPK assay: The mitogen activated protein kinase (MAPK) assays were performed
using a MAPK assay kit (Upstate Biotechnology). Anti-rabit IgG MAPK-1/2 (Erk-1/2) ' was incubated in a centrifuge tube contaicing 100 µI of 50% protein A agarose beads and
500 µI of lysis buffer A for 1 hr at 4 °c. The antibody-protein A agarose complex was washed twice with lysis buffer A by collecting the pellet at 14,000 rpm for 1 min. The
pellet was resuspended in 100 µI of buffer A. I mg protein of cell lysate was added to this suspension and incubated for 2 hr on a rotator at 4°C to immunoprecipitate MAPK-
1/2. The protein A agarose/enzyme immunocomplex was washed twice with 500 µI of lysis buffer A and 100 µI of buffer B ,(20 mM MOPS, pH 7.2, 25 mM p-glycerol I phosphate, 5 mM EGTA, 1 mM sodium orthovanidate, 1 mM dithiothreitol). The enzyme immunocomplex was incubated with 10 µI of buffer B, 10 µI of inhibitor cocktail
(20 µM PKC inhibitory peptide, 2 µM protein kinase A inhibitor peptide, and 20 µM compound R24571 in buffer B), 10 µI ofMAPK substrate cocktail (2 mg/ml mylein basic protein in buffer B), and 10 µI of [y-32P] ATP mixture (75 mM magnesium chloride, 500
µM ATP, and lµci/µl of [y-32P] ATP) fo~ 20 min at 30 °c in a shaking incubator. The ' supernatant (3 0 µI) of the reaction mixtur~ was spotted on to a P81 paper square. MAPK activity was quantified by measuring the y-radioactivity on the P8 I paper square. Non specific activity was measured from the cells treated neither with SIN-I nor with NGF. III. Results:
1. Stimulation of a.7nAChR protects differentiated PC12 cells from trophic factor
withdrawal:
Withdrawal ofNGF and serum from differentiated PC12 cells for 24 hr reduced cell viability to 66.6 ± 5.4% of non depriv~d control cells (Fig. 1). Even though serum contains many trophic· factors, the contr;ol experiments revealed that NGF deprivation alone can reduce cell viability to 66.8 ±• 1.1 % of non deprived control cells (Table 1).
Pre-incubation of differentiated PC12 cells with nicotine (10 µM) for 24 hr increased the cell viability to 95.6 ± 2.1 % from the cytotoxicity induced by NGF and serum
' ' withdrawal. This protective effect of nic?tine was inhibited in a concentration-dependent ' manner by co-treatment with the a.7nAChR-preferring antagonist MLA. In fact, MLA
' was particularly effective in this regard, as 10 nM of the a.7 antagonist almost completely prevented the neuroprotective action to ,10 µM nicotine (Table 1). A similar level of inhibition was afforded by 5 µM mecamylamine and 100 nM bungarotoxin.
Upon exposure to agonist, a7nAG:hRs rapidly desensitize. To determine whether this initial activation and the desensitization of the receptor is sufficient to protect the cells from trophic factor withdrawal, th~ cells were incubated with nicotine for a short ' period of time (10 min). After the 10 min nicotine treatment the media was replaced with I DMEM.NGF media for next 24 hr, which was followed by NGF and serum deprivation.
Whereas the continuous presence of nicotine for 24 hr completely protected the cells
30 31 from trophic factor deprivation, 10 min exposure to nicotine failed to produce significant protection from growth factor withdrawal0 induced cytotoxicity (Table 1).
2. Neuroprotective actions of choline and choline analogs:
Since choline may serve as natural ligand for the a:7nAChR (Mandelzys et al., 1995;
Papke et al., 1996), and since stimulation of a:7nAChR by nicotine produced a neuroprotective effect, we next sought to determine whether there was a relationship between the neuroprotective actions and. the affinity for a:7nAChR for choline and six I other choline analogs. The selectivity of choline for a:7nAChR has provided a template for the synthesis of novel a:7nAChR selective compounds. In this series, differentiated
PC12 cells were deprived of neurotrophic factors for 24 hr to promote cytotoxicity. Cell viability was measured by the MIT assay under control conditions, and in cultures that I were pre-incubated for 24 hr with a drug., Pre-treatment of differentiated PC 12 cells with choline produced a dose dependent cytopwtective effect offering a protective action up to
-40% of control deprived cells at the 10 :mM concentration. (Fig 2). The cytoprotective actions to choline were completely blocked by co-treatment with the non-selective I nAChR antagonist mecamylamine (10 µM) and a:7nAChR selective antagonist MLA (10 nM), supporting the contention that the protective actions to choline were largely mediated by a:7nAChR stimulation.
The relative levels of effectiveness as neuroprotective agents for 6 choline analogs was as follows: acetyl pyrrolidinecholine = pyrrolidinecholine > choline = I monoethylcholine = diethylcholine = triethylcholine. Among the 5 analogs studied (Fig
3), Mono, di, and triethyl analogs produced concentration dependent cytoprotective actions (with the maximum protection from cytotoxicity -40%) being identical to that for 32 choline. However, the analogs pyrroli1inecholine and acetyl pyrrolidinecholine were most effective, improving cell viability to over 60% from control. In fact, examination of the full dose response curve for pyrrolidinecholine revealed that this analog is more potent than choline, with significant protection observed at only JOO µM (Fig 4A).
Further, we characterized the pyrrolidinecholine cytoprotective actions with respective to nAChRs. Co-treatment with mecamylamine (10 µM) or MLA (10 nM) each completely prevented the cytoprotective actions 0£ pyrrolidinecholine (Fig 4B), indicating that response was mediated primarily through u7nAChR.
To confirm the above structure activity profile with respective to their binding affinities at u7nAChR, we performed [1r5I]u-BGT displacement binding studies using differentiated PC12 cells. These data are presented in Fig 5 along with the displacement curve for nicotine as a comparison. Choline (Ki = 86.2 µM) was only 50 fold less potent than nicotine (Ki = 1.67 µM) in displacing [125I]u-BGT binding. We next measured pyrrolidinecholine, the most active choline analog, for its ability to displace the cell surface binding of [125I]u-BGT to PC12 cells. As indicated in Fig 6, pyrrolidinecholine fully displaced [125I]u-BGT binding (Ki = 33 µM), and it exhibited a slightly greater affinity for the site than choline - which correlates with pyrrolidinecholine's greater activity in the neuroprotection assay.
3. The abilities of classical nAChR agoni~ts to induce neuroprotection:
The nAChR agonists epibatidine, 4OHGTS-21, DMPP, cytisine, methylcabamylcholine and TEA were evaluated for their ability to protect differentiated
PC12 from cytotoxicity induced by NGF and serum withdrawal (Fig. 7). Each drug was pre-incubated with cells using a 4 - 5 fold range of concentrations for 24 hr prior to 33
I trophic factor deprivation. Among the ·7 compounds tested, nicotine was the most efficacious neuroprotective agent and the 9nly compound to completely prevent toxicity
(near 100% cell viability) associated with tr9phic factor withdrawal. The next most effective group included epibatidine, 4OHGTS-21, MCC, and DMPP. These least effective group included cytisine and TEA. In terms of potency, nicotine, epibatidine,
4OHGTS-21, and MCC represented a group exhibiting a higher degree of potency than the group represented by cytisine, DMPP, and TEA. Thus, although the drug shared potency with nicotine, the effectiveness pf 4OHGTS-21 (the principal metabolite of
(2,4)-dimethyl phenyl piperazinium also known as GTS-21), a selective agonist at a.7 receptors (Meyer et al., 1998b ), exhibited 1a significant decrement in response at the 10
µM concentration. A similar profile of action was previously noted for GTS-21 (Li et al., ' 1999). Epibatidine, an extremely potent nAChR agonist (including the a.7 subtype), also failed to fully protect the cells. DMPP, an a.3-preferring (ganglionic nAChR) agonist, exhibited low potency, but effectiveness that was equivalent to epibatidine. Even the acetylcholine analog MCC failed to achieve levels of protection equivalent to nicotine.
Cytisine (preference for ~2 subunit-containing receptors) and TEA ( classical agonist at peripheral ganglionic nAChRs) each ~xhibited low potency and weak or no efficacy.
4. The abilities of different nAChR agonists to induce a.7nAChR upregulation:
The next series of experiments was designed to determine whether neuroprotective effectiveness could be related to the ability to induce upregulation of cell surface
125 [ I]a.BGT binding sites. Saturation binding experiments were performed_ using
[1 25I]a.BGT to measure the extent of cell surface a.7 nAChR upregulation associated with the preincubation of nicotinic drugs as determined for the neuroprotection studies (Table 34
2). Consistent with earlier studies (Takahashi et al., 1999), differentiation of PC12 cells with NGF increased the maximum binding (Bmax) of [1251]-aBGT binding sites from
16.4 ± 1.21 to 82.6 ± 3.67 fi:nol/mg. In ,the next series, nicotine, the most efficacious compound as a neuroprotectant, was compared with cytisine, one of the least efficacious, for their ability to up-regulate a7nAChRs. Incubation of differentiated PC 12 cells with nicotine (10 µM) for 24 hr, significantly .increased the Bmax for [1 251]-aBGT by 41%
(Fig. 8). Cytisine, however, failed to significantly increase the Bmax for [1251]-aBGT.
Nicotine and cytisine produced nd significant effect on the calculated Kds for the ligand.
5. MLA induced increase in a7nAChR expression and neuroprotection:
This experimental series was designed to determine whether the cytoprotective action produced by chronic exposure of the cells to nicotine was related to the agonist' s ability to induce a7nAChR up-regulation during the incubation period. The a7nAChR antagonist MLA can dramatically increase the number of cell surface [125I]aBGT binding sites (Molinari et al., 1998). We determined in preliminary experiments that incubation of differentiated PC-I 2 cells with MLA (100 µM) for 96 hr induced a maximal increase in cell surface [1 25I]aBGT binding (Table 2). Since MLA itself had no intrinsic neuroprotective activity (Fig. 9B) we used this drug to obtain cells expressing increased populations of a7nAChR receptors. In order to determine whether increasing the expression of a7nAChRs could enhance the neuroprotective action of nicotine, we first needed to employ a paradigm that provided a lower degree of neuroprotection to the agonist (since our standard regimen already resulted in near 100% cell viability). This was accomplished by decreasing the nicotine incubation time to only 4 hr (see Fig. 9B).
Therefore, the protocol for this series involved exposure of the cells to MLA ( I 00 µM) 35
for 96 hr to evoke a maximal increase in cell surface [125I]aBGT binding sites. This was
followed by exhaustive washing to remove traces of the antagonist, and subsequent
incubation of the cells with 10 µM nicotine for an additional 4 hr (the shorter time
interval) prior to trophic factor withdrawal. Treatment of cells with MLA (100 µM) for
96 hr significantly decreased the Bmax for [125I]aBGT by-100% (control value for 96 hr
treatment is 87.2 fmol/mg) (Table 2). The ligand binding affinity measured in these cells
was significantly increased as compared with control cells (Table. 2). ' Trophic factor withdrawal in control (non-drug treated) differentiated PC 12 cells under the conditions of this experiment reduced the cell viability to 46% of trophic factor-intact cells (Fig. 9B). This slight increase in cytotoxicity relative to our previous controls was most likely a consequence of the additional 4 days of pre-trophic factor withdrawal maintenance (required in the present experiment for the MLA incubation).
Pretreatment of cells with MLA (100 µM, 96 hr) had no significant effect on the cytotoxicity induced by trophic factor withdrawal in comparison with untreated cells maintained under similar conditions. Pretreatment of control (trophic factor deprived, but non-MLA treated) cells with nicotine for 4'. hr improved cell viability from 46% to 68%.
However, in the cells previously exposed to MLA that exhibited the increased expression
25 of [1 I]aBGT binding sites, the improvement in cell viability produced by nicotine was significantly increased to 79% of control.
6. Nicotine up-regulates TrkA receptors:
Differentiated PC12 cells were incubated with different concentrations of nicotine
(0.01 -10 µM) for various time intervals (Fig 10). After the incubation period, the media was removed and the relative levels of cell surface TrkA receptors were determined by 36 cell based ELISA as described in methods section. Nicotine increased the expression of cell surface TrkA receptors, an effect that was time- and concentration- dependent.
Nicotine increased the expression of TrkA receptors by 2 - 12% and 14 - 36%, respectively, for the 4 and 24 hr incubatidn periods. These effects of nicotine apparently I were not due to an increase in overall cell growth because nicotine did not change the total protein levels (39.5 ± 1.07 µg/ml) from control (40.2 ± 0.87 µg/rnl) (N = 27). The nicotinic effects were completely blockeq by co-treatment with non-competitive nAChR antagonist mecarnylarnine (5 µM) and with the a7nAChR selective antagonist methyllcaconitine (10 nM) (Fig 11) indicating that the response may be largely mediated by a7nAChR stimulation. In addition, co-treatment with the competitive antagonist dihydro-beta-erythroidine (10 nM), at c.oncentrations selective for the a4~2 nAChR subtype did not block the nicotine induced TrkA receptor upregulation. The next series of experiments was performed to determine whether in vivo administration of nicotine increases the expression of brain NGF/TrkA receptor protein. Test drugs were administered intravenously as indicated in the Methods section. After the drug infusion period, rats were sacrificed and the levels of TrkA protein in cortex and hippocarnpal tissues were quantified by westembloting technique. Since the continuous presence of nicotine is essential for neuroprotection and TrkA receptor upregulatin, in order reflect
the in vitro we continuously infused the 1 rats with nicotine at 12 mg/kg/day for 24 hr.
Nicotine administration to the rats resu\ted a 43% of increase in hippocarnpal TrkA I protein levels above that determined in c.ontrol' (saline) animals (Fig 12A). This effect was completely blocked by concurrent administration of non-selective nAChR antagonist mecarnylarnine (24 mg/kg/day), indicating that this effect was resulted from the 37
stimulation of nAChRs. Interestingly, when these animals were given mecamylamine
alone, the TrkA receptor protein levels in hippocampus were moderately increased (by
-20% over that of control animals). However, the mecamylamine-induced increase in
I TrkA protein levels was not statistically significant (One way ANOV A). In contrast, in
cortex neither nicotine nor mecamylamine were able to produce any changes in TrkA
protein levels (Fig 12B).
7. Relationship between ,nicotine-induced neuroprotection aud its ability to increase
TrkA receptors:
The proposed hypothesis is that the neuroprotective actions of nicotine are partly due
to the increased NGF-mediated trophic support that occurs during the incubation period
through the increased expression of TrkA receptors. To test this . hypothesis the
neuroprotective ability of nicotine incubated in the presence and absence of NGF was
compared. According to the hypothesis, if the neuroprotective actions of nicotine were
mediated through the increased expression of TrkA receptors, nicotine should not be protective when incubated with cells in the absence of NGF. However, nicotine was i protective in both experimental paradigms.' Examination of the dose-response relationships for nicotine in the presency and in the absence of NGF revealed· that nicotine is much more potent in its ability to protect cells than it is to rescue cells from trophic factor deprivation (Fig 13). Nicotine, when incubated in the presence of NGF
completely protected the cells from trophic' factor withdrawal at the 10 µM dose.
Whereas in the absence ofNGF, a 10 fold higher dose of nicotine (I 00 µM) was required to achieve the same level of cell viability. Further, this effect was completely blocked by 38 cotreatment with bungarotoxin (100 nM, 66.3±4.94 % cell viability) indicating that this effect was mainly resulted from a.7 nicotimc receptor stimulation.
In the next series of experiments a selective TrkA receptor inhibitor K252a was used to block the NGF trophic support during nicotine incubation period. K252a is a non selective protein tyrosine kinase inhibitor. However, when used at low concentrations it has been shown to selectively inhibit TrkA receptor activity (Pitchford et al., 1995).
When the differentiated PC 12 cells were incubated with K252a alone prior to the deprivation, K252a enhanced the cytotoxicity induced by trophic factor withdrawal
(-60% viability). The treatment of cells with nicotine (10 and 100 µM) plus K252a (I
µM) for 24 hr partially protected the cells from trophic factor withdrawal (-72% viability) (Fig 14).
Next, a selective TrkA receptor antibody raised against the entire extra cellular domain of TrkA receptor was used to inhibit NGF trophic support during nicotine incubation (Clary et al., 1994). The cells incubated with anti-TrkA exhibited an enhanced cytotoxicity to growth factor withdrawal (-63%). Preincubation of differentiated PC 12 cells with the maximum effective dose of nicotine (IO µM) and with anti-TrkA (1 µg/ml) resulted in a partial protection from trophic factor withdrawal
(-81%) (Fig 15). Since anti-TrkA completely inhibits NGF support during drug incubation period, this effect might have ~esulted due to the extended deprived state. (i.e. drug incubation period plus trophic factor withdrawal period). ' 8. Metabolic response of PC12 cells to NGF:
The activation of TrkA receptors by NGF is known to cause a rapid metabolic response in PC12 cells (Pitchford et al.; 1995). In fact, this response observed in a 39
Cytosensor microphysiometer previously has been shown to correlate with neurite
. outgrowth in these cells. Characteristic pyrfusate acidification responses to the addition of
NGF to PC12 cells are shown in Fig. 16A. This figure represents the averaged metabolic responses induced in PC12 cells to control (no NGF added) and four different concentrations ofNGF ranging from I - 100 ng/rnl infused over 12 min. Upon exposure to NGF, the acidification rates for PC12 cells began to increase rapidly. At higher concentrations, the response peak was attained IO - 12 min after initial exposure. After the withdrawal of NGF, the response ;emained at the elevated levels throughout the experimental period (1 hr). The ECso for the NGF response under the present conditions was 15.2 ng/rnl (Fig I 6B). For subsequent experiments, the 25 ng/rnl concentration, which resided on the linear portion ofthe_dose response curve, was used.
9. Effect of SIN-1 on cell viability:
Since peroxynitrite can induce cellular damage to PC12 cells (Estevez et al., 1995), it was first necessary to determine whether .any decrease in the metabolic response to NGF did not result indirectly from generalized cytotoxicity. MTT reduction assays were performed to measure the percentage of c,ell viability after SIN- I treatment. This assay is I • used as a marker of cell viability becaus~ MTT can only be reduced if oxidases, and cell metabolism in general, are unimpaired. After SIN-I treatment, the buffer was removed and replaced with DMEM media containing serum for the specified time intervals. ' Exposure to SIN-I did not cause immediate cell death (Fig 17). Consistent with earlier studies (Estevez et al., 1995), cell death was a time and concentration-dependent process.
In fact, a significant decrease in cell viability was observed only after 12 hr of treatment, and at aconcentration of 2 mM. In order to preclude the non-specific cytotoxic actions 40 of SIN-1, exposure was limited to concentrations up to 500 µM, and for 20 min in duration for the subsequent experiments.
10. Inhibition ofNGF mediated metabolic responses by SIN-1:
PC12 cells were pre-treated for 20 min with various concentrations of SIN-1 (I - 500
µM). After the treatment, the cells were removed and plated into the capsule cups and challenged with NGF (25 ng/rnl) in the microphysiometer. As indicated in Fig. 18A, exposure to .SIN-I inhibited the metapolic responses in PCl2 cells to NGF m a concentration-dependent manner. At 'the highest concentration (500 µM), SIN-I decreased the maximum response to NQF from 124.4% to 107.8%, i.e., a decrease of
67% from the control response (Fig 18B).
Uric acid was previously shown tq suppress peroxynitrite accumulation in cells exposed to variety of inducers of nitric oxide and superoxide (Szabo et al., 1996).
Pretreatment of PCl2 cells with uric acid significantly attenuated the inhibitory actions of
SIN-I on NGF mediated metabolic responses (Fig 19A). Cells were treated with uric acid (1 mM) for 1 hr prior to SIN-1 (500 µM) exposure and later these cells were challenged with NGF in the microphysio'meter. As indicated in Fig 19B, treatment with ' uric acid prior to SIN-I exposure significantly reversed SIN-I induced inhibition of the maximum response to NGF (from 107.8% to 118.6%). Uric acid treatment alone produced no significant effect on NGF mediated metabolic responses.
11. Effects of SIN-I on TrkA receptor :i,utophosphorylation:
The binding of NGF to TrkA recepto'rs results in autophosphorylation of the tyrosine residues on TrkA receptors. The extent of tyrosine phosphorylation on TrkA receptors was us'ed to provide an estimate of receptor activity to NGF. Western blots of 41 phosphotyrosine immunoprecipitates from cells treated with NGF (25 ng/ml) for 10 min ' after SIN-1 treatment were probed with an antibody raised against the extracellular domain of the TrkA receptor, a 140-kD~ transmembrane receptor tyrosine kinase (Zapf
Colby and Olefsky, 1998) (Fig. 20). As the concentration of SIN-1 increased, the relative extent of tyrosine phosphorylation on TrkA receptors decreased, indicating decreased autophosphorylation in response to NGF. At the highest concentration (500 µM), SIN-I I inhibited the NGF-induced TrkA receptoi autophosphorylation by about 80%.
12. Effects of SIN-1 on MAPK activity:: ' Activation of the TrkA receptor by NGF leads to the activation of MAPK, an important downstream step in the signaling pathway leading to neuroprotection. The level of MAPK activation directly correlates with the extent of TrkA receptor activation. ' MAPK activity (measured by using myle1n basic protein as a substrate) was estimatea'in immunoprecipitates of the enzyme. Pretreatment of PC12 cells with SIN-I inhibited the increase in MAPK activity to NGF in a dose-dependent manner (Fig. 21). At the highest concentration (500 µM), SIN-1 inhibited the NGF-induced increase in MAPK activity by about 50%. IV. Discussion:
1. N europrotective actions of nicotine:
In patients with AD, the memory and attention deficits associated with the disease are considered to be largely due to the degeneration of the forebrain. choli_nergic neurons.
The first class of drugs that were developed for the treatment of AD act by preventing the breakdown of acetylcholine, e.g., donepezil and riviistigmine. One alternative approach under development is based upon the ability of nicotine to improve both the performance
of cognitively demanding tasks in normal healthy adult human subjects (Wesnes and
Warburton, 1984), and information processing and attention in patients with AD type
dementia (Sahakian et al., 1989). In fact, earlier results from this lab and others have
convincingly demonstrated that nicotine administration increases performance in memory
related tasks both in non-human primates as well as iri rodents (Buccafusco et al., 1995;
Buccafusco et al., 1995; Terry et al., 1996; Elrod et al., 1988Levin, 1992; Levin et al.,
1995). Moreover, in animal models of cholinergic defects such as those produced by
NbM lesions, chronic pretreatment with nicotine reduced the cell loss in the neocortex
(Sjak-Shie and Meyer, 1993). These findings suggest that nicotine not only exerts a
therapeutic effect with regard to cognitive function, but the drug also may produce
cytoprotective actions for cholinergic neurons.
Recent evidence suggests that neurodegeneration associated with certain brain
neuropathies may occur as a result of the lack of growth factor maintenance of the
neurons (Capsoni et al., 2000). During the past decade the results of a number of reports
42 \
43 support a role for central nAChRs in the regulation of growth factors and their receptors.
For example, administration of nicotine was shown to protect rodents from MPTP toxicity by increasing brain BDNF and FGF levels (Maggio et al., 1998; Maggio et al.,
1997). It was suggested that the increase in BDNF and FGF levels produced by nicotine would subsequently activate their respective trophic signaling pathways within the cells thus offering protection from toxicity. Moreover, it has been demonstrated that nicotine increased the expression of EGF receptor levels on cervical cancer cells in culture through the drug's ability to enhance EGF levels (Mathur et al., 2000). In contrast, the survival and maintenance of BFC neurons depended on the availability ofNGF. In mice a reduction in the endogenous levels ofNGF was shown to impair the animals' cognitive abilities and to induce pathology similar to that which occurs in the AD brain (Capsoni et al., 2000). Since it is not practical to administer NGF to humans, it was proposed (Olson,
1993) that restoring or enhancing the NGF support to degenerating BFC neurons by any means would prevent the further progression of the disease. Earlier findings from this laboratory and others have demonstrated an increase in NGF (Bellurdo et al., 1998) and
NGF receptors on differentiated PC12 cells after nicotine treatment (Terry and Clarke,
1994; French et al., 1999). Based on these findings we proposed that nicotine treatment would protect neurons from certain types of toxicity by enhancing NGF trophic support through the increased expression ofNGF/TrkA receptors.
To study the neuroprotective actions of nicotine in vitro we adopted the model of withdrawal of growth factor-induced cytotoxicity in differentiated PC-12 cells.
Withdrawal ofNGF and serum for 24 hr reduced cell viability to 66.6 + 5.4% of control cells (Table 1). However, when we deprived differentiated PC12 cells from NGF alone, 44 we observed similar decreases in cell viability. Since the reduced cell viability is directly proportional to the duration of trophic factor deprivation, it reasonable to speculate that the cells that are more NGF-dependent, and therefore more vulnerable, might represent the reduced cell viability during the short-term deprivation (24 hr). In that case it necessary to deprive cells for longer time periods in order to compare the toxic insults resulting from either NGF plus serum deprivation or NGF deprivation alone. The ability of nicotine to produce its neuroprotective action was blocked by co-treatment with mecamylarnine, a classical nAChR antagonist. Whereas differentiated PCl2 cells express several potential subtypes of nAChRs on their surface, we were able to block nicotine's neuroprotective action with only 10 nM MLA and 100 nM of Bgt, (Table. 1).
Moreover, concentrations of DHPE sufficient to block P2 and P4 containing nicotinic receptors was not effective in this regard. Thus, our data are consistent with a primary role for the a.7 subtype in nicotine's neuroprotective action, but as yet we cannot rule out contributions from all of the other subtypes expressed in these cells. Several mechanisms have been proposed to explain the neuroprotective actions of nicotine, including neurotrophic factor modulation (Freedman et al., 1993), inhibition of nitric oxide formation (Shimohama et al., 1996), and activation of protein kinase C (Li et al., 1999).
2 However, each of these processes is triggered by the initial Ca + influx that is mediated
2 by the activation of a.7 receptors. Moreover, it has been reported that Ca + influx through a.7 receptors increases the survival of spinal cord motor neurons (Messi et al., 1997).
The a.7 receptor functions as a homo-oligomeric ion channel that is as selective for Ca2+ as is the potentially excitotoxic NMDA receptor, but the former is much more rapidly desensitizing in the presence of agonist (Seguela et al., 1993). Since a.7 receptors 45 desensitize more rapidly than NMDA receptor, they may be more able to self-limit
2 intracellular Ca + to a level necessary and sufficient to produce neuroprotection rather than cytotoxicity. Further, the expression of a mutated, non-desensitizing form of a7 receptor was found to mediate a reduce cell viability, apparently due to intracellular calcium-overload (Treinin and Chalfie, 1995).
2. Choline and Choline analogs:
An important advance in understanding the function of nAChRs in the CNS was the demonstration that choline, a precursor and metabolite of ACh, is an effective and selective agonist of a7nAChRs (Mandelzys et al., 1995; Papke et al., 1996). It was also demonstrated in rat hippocampal neurons and in PC 12 cells that choline activates both postsynaptic and presynaptic a7 receptors, with the later action resulting in the release of other neurotransmitters (Alkondon et al., 1997). Although choline was approximately one order of magnitude less potent than ACh (ECSO 1.6 mM for choline and 0.13 mM for
ACh), the precursor acts as a full agonist at a7 receptors. In contrast, choline was shown to be ineffective at other nAChR subtypes supporting the selectivity at a7 receptors
(Papke e al., I 996). It recently has been demonstrated that human cortical intemeurons express a7 receptors that are also effectively activated by choline (Alkondon e al., 2000).
Similar to ACh, choline is an efficacious nicotinic receptor desensitizing agent, with significant effects observed at concentrations above IO µM (Alkondon et al., 1997). This concentration is close to the mean extracellular concentration of choline in the brain, which is generally maintained at 4 to 6 µM (Klein et al., I 988). After the activation of cholinergic neurons, the concentration of choline is expected to rise sharply in the areas near ACh release sites due to the rapid hydrolysis of ACh. This resultant elevation of 46 synaptic choline likely modifies the responsiveness of the nicotinic receptor to A Ch. It is also possible that choline is the only potential agonist to act at perisynaptic a7 receptors located apart from the acetylcholine release sites. The selectivity of choline for a7nAChR over other nAChR subtypes may provide a useful structural template for the design and synthesis of novel a7nAChR selective compounds. The results from epidemiological studies suggests a potential neuroprotective action in long-term smokers from neurodegenerative diseases. Since the blood nicotine concentrations (nM) in smokers are not usually sufficient to activate a7 receptors directly, it is possible that the a7 receptors might be activated by the high synaptic choline concentrations resulting from nicotine-induced ACh release. Thus the further development of novel choline analogs inducing neuroprotective actions·is warranted.
We next sought to evaluate and compare the potential neuroprotective actions of choline and six choline analogs to identify the structural requirements for cytoprotective efficacy. Using our model of growth factor withdrawal in differentiated PC-12 cells we found that the cytoprotective actions of choline were completely blocked by co-treatment with the non-selective nAChR antagonist mecamylarnine, and also with the a.7nAChR selective antagonist MLA (Fig. 2), indicating the role of a.7nAChRs in mediating the response. However, a significant degree of protection was achieved only at concentrations ~I mM. The ranking in terms of relative cytoprotective potencies for the six compounds tested was as follows: acetyl pyrrolidinecholine = pyrrolidinecholine > choline = monoethylcholine = diethylcholine = triethylcholine. Among the six compounds, acetyl pyrrolidinecholine and pyrrolidinecholine exhibited the most efficacious responses (Fig. 3). In fact, the dose-response profile for pyrrolidinecholine 47 revealed that this compound was the most potent (significant effect at 100 µM) and the most efficacious among the six tested (Fig 4A). These findings were also confirmed by measuring each compound's affinity for cx.7nAChRs. According to their neuroprotective profile, choline was found to be SO fold less potent than nicotine in displacing [1 25I]cx.
BGT binding (Ki = 86.2 µM for choline and Ki = 1.67 µM for nicotine) (Fig. S).
Pyrrolidinecholine, the most effective compound as a neuroprotectant fully displaced
[1 25I]cx.-BGT binding (Ki = 33 µM) (Fig. 6), and it exhibited a slightly higher affinity for the site than did choline.
Choline is a charged· quaternary amine. For specific nAChR subtypes, other structural elements are either permissive (neither increasing nor decreasing activity) or non-permissive ( decreasing activity). Choline is a full agonist at a7 receptors, but a hydroxyl group is strongly non-permissive for other receptor subtypes including all3lyll, cx.3134, cx.3/32, and cx.4132 (Papke et al.,1996). Among the six analogs tested, monoethylcholine, diethylcholine, and triethylcholine proved equi-effective to choline, indicating that the additional N-ethyl groups are ineffective. In contrast, the modification of the pyrrolidine ring (an important structural feature of nicotine) was found to enhance the potency of choline approximately 3 fold and its efficacy at least ~2 fold.
3. Relationship between the ability to produce neuroprotection and the upregulation of a7nAChRs:
Upon stimulation, cx.7 receptors rapidly desensitize in the presence of agonist. Since in this study, incubation of the cells with nicotine for only 10 min failed to produce a significant degree of neuroprotection (Table. 1), it is reasonable to conclude that the initial rapid desensitization (initial Ca2+ influx) phase does not play an important role in 48 this response. It is well established that chronic nicotinic exposure results in the
increased expression of [3H]nicotine and [ 125I]-aBGT binding sites in brain (Marks et al.,
1992; Pauly et al., 1991; Schwartz and Kellar, 1985; Wonnacott, 1990). In fact, it has recently been reported that nicotine-induced stimulation of a.7 nAChR in primary hippocampal cultures first results in a rapid transient increase in Ca2+ influx with a return to baseline levels, followed by a prolonged and gradual increase in cytosolic Ca2+ levels
(Federico et al., 2000; Gueorguiev et al., 1999). This smaller more gradual elevation in
2 intracellular Ca + appears to be temporally related to the time course of development of nicotine's. neuroprotective action. It is possible that this secondary elevation in Ca2+ levels may have resulted as a consequence of the nicotine-induced expression of additional a.7 receptors. To test this hypothesis we compared the ability of chronic exposure of differentiated PC-12 cells to several nAChR agonists to induce a.7 receptor upregulation with their ability to produce neuroprotection.
For the nAChR agonists used in this study, their rank order of potencies (EC50) for activating (channel currents) a.7 receptors has been reported to be: epibatidine (3
µM)>4OHGTS-2l=cytisine (6-10 µM)>nicotine (49 µM)>DMPP (64 µM); which correlates well with their rank order of binding affinities (Ki) for the a.7 receptor: epibatidine (0.02 µM)>4OHGTS-21 (0.17 µM)>cytisine (1 µM)>nicotine (1.6
µM)>DMPP (7.6 µM). (Amar et al., 1993; Gopalakrishnan et al., 1995; Meyer et al.,
1998b). However, in our assay for neuroprotection, nicotine was the singularly most efficacious compound and cytisine and TEA proved to be virtually ineffective in this regard. This lack of correlation between the neuroprotection profile and the agonist potencies in activating a.7 receptors indicates that the mechanism involved in 49 neuroprotection requires more than simply activation of a.7 receptors. For example, a.7 agonists are known to have both acute agonist and protracted antagonist actions (Briggs and McKenna, 1998) and the kinetics of these two properties vary with different .a.7 agonists. This antagonistic activity was shown to interfere particularly with the neuroprotective effects of a.7 agonists (Meyer et al., 1998a), suggesting that analysis of both agonist and antagonist activities may be necessary for predicting the cytoprotective efficacy of a.7 agonists. In the next series of experiments we compared the extent of up regulation induced by chronic exposure with nicotine or cytisine, which were the most and one of the least efficacious compounds 1n our neuroprotection assay. After the period 24 hr of drug exposure, only cells treated with nicotine expressed significant increases in cell surface rt 25I]a.BGT binding sites, whereas cytisine, failed to do so.
Although, cytisine binds to a.7 receptors with greater affinity than nicotine, its inability to
upregulate a.7 receptors may underlie its poor cytoprotective efficacy. Moreover, the published rank order of these agonists for inducing maximal up-regulation of [ 125!]
a.BGT binding sites: nicotine> GTS-21 > epibatidine > DMPP (Molinari et al., 1998),
better correlates with their effectiveness as neuroprotective agents.
Chronic treatment of cells with the a.7 selective antagonist MLA, has been reported to
upregulate [125I]a.BGT binding sites, and this increased population of a.7 receptors have
been shown to be functional to subsequent nicotine application (Molinari et al., 1998). To
more directly test whether the additional population of a.7 receptors play a role in the
neuroprotective actions to nAChR agonists, MLA was used as means to increase the
expression of cell surface a.7 receptors (MLA has no intrinsic activity to induce a
neuroprotective response, and it was not cytotoxic under the conditions of the 50
experiment). Since MLA is a competitive antagonist at a.7 receptors, the newly
expressed receptors with MLA treatment are presumably in a non-desensitized state.
Exposure of differentiated PCl2 cells to MLA (100 µM) for 96 hr increased the cell
surface [125I]a.BGT binding sites by -100% (control value for 96 hr treatment is 87.2
fmol/mg) (Fig9A). The ligand affinity (Kd) measured in these cells was significantly
greater than the Kd value measured in control cells (Table. 2). Although the cells were
washed extensively prior to the assay, it is possible that potentially residual MLA may
have caused the increase in ligand affinity. Not withstanding the change in a.7 affinity,
under conditions that produced significant upregulation of a.7 receptors, MLA itself had
no effect on cell viability. However, after exhaustive washout of the antagonist and
subsequent incubation with nicotine, the protective effect of nicotine was significantly enhanced (Fig. 9B). Thus, the stimulation of MLA-induced additional a.7 receptor population likely mediated the enhanced level of neuroprotection to nicotine.
These results support those from previous reports in that nicotine protects differentiated PC12 cells from trophic factor withdrawal. We also report that this effect was mediated predominantly through the a.7 subtype of nAChRs and that chronic exposure to an agonist is essential in order to exhibit its protective actions. One explanation for the time-dependent effect of nAChR agonists is that neuroprotection is a consequence of the time-dependen~ induction of additional a.7nAChRs expressed during agonist incubation, possibly countering a state of agonist-induced receptor desensitization, and regulating "neuroprotective" levels of intracellular calcium. One puzzling finding was the observation that, of the 7 compounds examined, only nicotine was capable of evoking a near complete level of neuroprotection. Even the highly potent 51 epibatidine was not as effective as nicotine in this regard. Part of the explanation may reside in our unpublished observation that nicotine, at concentrations 10-100 fold higher than its EC50 for neuroprotection, can rescue differentiated PC~12 cells from trophic factor withdrawal. That is, at concentrations near or above 100 µM, nicotine can maintain cell viability when it is administered at the time of trophic factor withdrawal.
Although the mechanism for this latter action of nicotine is ·unknown, the fact that the other agonists tested in this study were incapable of producing optimal levels of protection even at their highest doses suggests that only nicotine may engage this alternate mechanism of cytoprotective action. Thus, there is still much to be learned regarding the mechanisms by which nicotine protects and maintains cell viability in the face of a toxic challenge, and there is the promise of new targets for drug discovery in this context.
4. Relationship between increased TrkA receptors and neuroprotection:
Earlier results from this lab and others have shown that nicotine treatment will increase NGF levels and NGF receptors both in vivo (French et al., 1999) and in vitro
(Terry and Clarke, 1994). The working hypothesis is that nicotine treatment will increase
NGF-mediated trophic actions by increasing cell surface NGF/frkA receptors thus protecting cells from toxic insults. In the present study, we demonstrated that nicotine increases the expression of cell surface TrkA receptor~ . in a time and concentration dependent manner (Fig. 10). This effect was blocked by co-treatment with the non selective nAChR anatagonist mecamylamine and the a7nAChR selective anatagonist
MLA (Fig 11). Together, these findings suggest that the stimulation of a7nAChRs results in increase in TrkA receptor expression in differentiated PC12 cells. Since the 52
nicotine-induced increase in TrkA receptors is also a time dependent process, it is
reasonable to speculate that this increased expression resulted from the gradual elevation
2 in intracellular Ca +. In rats, the chronic administration of nicotine significantly
increased hippocampal TrkA protein levels (Fig. 12A). This effect was completely
blocked by co-treatment with mecamylamine, indicating that this effect was mediated by
stimulation of nAChR. Under similar experimental conditions, chronic administration of
mecamylarnine alone resulted in a moderate, but non-significant increase in hippocampal
TrkA protein levels. However, neither nicotine nor mecamylamine administration altered
TrkA protein levels in cerebral cortical tissues (Fig 12B). In rat brain nAChR are
differentially and variably distributed. For example in hippocampus the majority of
nAChR belong to a.7 subtype, whereas in cortex the a.4~2 subtype predominates. Based
upon the results of our in vitro studies of receptor subtype selectivity it is reasonable to
conclude that the differences observed in TrkA receptor expression in hippocampus and
cortex were due to relative differences between these two regions in the abundance of
a.7nAChR and non-a.7nAChR. Moreover, it also has been reported that a.-bungarotoxin
nicotinic receptors located on certain hippocampal cells regulate the expression of local
growth factors (Freedman et al., 1993).
Since nicotine's neuroprotective ability as well as its ability to increase the expression
of TrkA receptors appear to be parallel events (in time and concentration), it may be
reasonable to consider that a relationship exists between these two events. Thus, nicotine's ability to increase TrkA receptors may constitute one possible mechanism for
drug's neuroprotective actions. To test this hypothesis we compared the neuroprotective ability of nicotine in differentiated 1PC-12 cells when the drug was included several hours 53 prior to, or simultaneous with, growth factor withdrawal. Surprisingly, nicotine was found to be effective in both experimental paradigms (Fig. 13). However, examination of the nicotine dose-response relationships revealed that nicotine is 10 fold more potent when incubated with cells prior to growth factor withdrawal. These results indicate that nicotine may activate at least two mechanisms contributing to its neuroprotective actions, one that is prevalent at lower doses and is capable of neuroprotection, with the other available at higher doses and able to rescue cells from the toxic insult. However, since nicotine is more efficient as a neuroprotectant when administered in the presence ofNGF, the agonist may enhance NGF-induced trophic actions contributing to the neuroprotection. To further confirm these results, we used K252a and anti-TrkA antibody (selective inhibitor ofTrkA receptors, Pitchford et al., 1995) to inhibit the NGF mediated trophic actions during nicotine treatment (Fig 14, 15). When the cells were co treated with nicotine and K252a or _nicotine and anti-TrkA, nicotine was unable to completely protect the cells from trophic factor withdrawal. However, in control experiments both K252a and anti-TrkA were shown to decrease cell viability on their own. Despite this enhanced cytotoxicity, in the presence of K252a or anti-TrkA nicotine treated cultures contained more viable cells than those exposed to the two TrkA inhibitors alone. These results support our previous findings suggesting that nicotine may activate two distinct mechanisms supporting its neuroprotective actions.
In this set of experiments we showed that nicotine could increases TrkA receptor expression both in vivo and in vitro. During the past few years it has been demonstrated that a.7nAChR selective agonists will enhance cognitive abilities in rodents (Meyer e al.,
1997) as well as in non-human primates (Buccafusco and Terry, 2000). Together these 54 findings suggest that the development of selective a7nAChR agonists in the treatment of
AD patients may have an indirect neuroprotective action by enhancing the NGF trophic actions along with their ability to improve working memory.
5. Importance of NGF signaling:
AD brain is typically characterized by a selective loss of BFC ·neurons. These neurons are continuously depended on the availability of NGF for their survival and maintenance. Recent evidence suggests that impairment in NGF signaling to BFC neurons may be an initial insult in AD pathology (Caposini et al., 2000). However, the mechanism behind the loss in NGF retrograde transport has not been adequately addressed. Emerging evidence from a number of studies suggests that oxidative stress may constitute a final common pathway in various neurodegenative disorders including
AD. The cellular antioxidant system consisting of the enzymes catalase, glutathione peroxidase, and superoxide dismutase protect cells from oxidative radicals. A decrease in the activity of any of these enzymes could lead to cell death. It has been suggested that neurotrophic factors play an important role in the synthesis of antioxidant enzymes.
Moreover, it has been shown, in vitro, that NGF increases the activity of glutathione and . . catalase in PC12 cells (Pan and Perez-polo, 1993; Jackson et al., 1990). Furthermore, several studies have documented the ability of NGF to protect cells against various oxidative insults (Estevez et al., 1995; Jackson et al., 1990). NGF has been shown to maintain ChAT activity and prevent cholinergic cell death induced by fimbria-fornix transection (Kojima et al., 1995; Wilcox et al., 1995). Furthermore, NGF deprivation has been shown to cause aberrant tau phosphorylation and neurite retraction in differentiated
PC12 cells (Nuydens et al., 1997), both of which occur in AD. The widespread 55 occurrence of nitrated tyrosine, an indicator of peroxynitrite involvement in postmortem
AD brains, has been documented (Good et al., 1996; Smith et al., 1997), suggesting that peroxynitrite could be implicated in mediating the neuronal damage in AD (Good et al.,
1996; Smith et al., 1997). Moreover, this nitrotyrosine immunoreactivity is found only in the brain regions that are selectively damaged in AD. Although the identity of specific proteins affected in this way is currently unknown, a possibility is that tyrosine kinase receptors may become nitrated. If so, it is possible that peroxynitrite may decrease
NGF/TrkA signaling by nitrating tyrosine residues on TrkA receptors, thus preventing them from becoming phosphorylated by NGF, a initial step in NGF/TrkA signaling pathway.
In our studies we used . a cytosensor microphysiometer to monitor the cellular responses in PC12 cells after NGF stimulation. Stimulation of NGF receptors rapidly increased cell metabolic activity and the extracelluar acidification of PC12 cells in a concentration-dependent manner (Fig. 15). Previously, PHchford and co-workers (1995) demonstrated that this increased metabolic response to NGF was mediated via TrkA
.receptor autophosphorylation. They also documented the specificity of these metabolic responses to the NGF signaling pathway. In our experiments, exposure to SIN-I
(peroxynitrite generator) decreased the metabolic responses in PC12 cells to NGF (Fig.
18). This decrease in NGF-activated metabolic responses to SIN-I treatment was
concentration dependent, up to a maximum level of inhibition of about 67% at 500 µM.
Pretreatment of PC12 cells with the peroxynitrite scavenger uric acid significantly
attenuated the inhibitory actions of SIN-1, indicating that the inhibitory effect to SIN-I treatment was mediated for the most part by peroxynitrite formation (Fig. 19). A small 56 contribution by nitric oxide or superoxide has not been ruled out. The exposure of PC I 2 cells to peroxynitrite has been shown to cause decreased cell viability and DNA fragmentation. However, the concentrations required to induce these changes were -1 mM (ECso = 850 µM), and the exposure time required to observe these effects was at least 8 hrs (Estevez et al., 1995). In order to preclude the possibility of these non-specific actions to SIN-I, we exposed the PC12 cells to SIN-I for only 20 min and at much lower concentrations (I - 500 µM). Under these conditions SIN-I failed to induce cell death or to produce obvious morphological changes (Fig 17). This obviates the possibility that the
SIN-I induced decreased metabolic responses in PC12 cells to NGF resulted indirectly from decreased cell viability. .
NGF initiates cellular responses leading to neuroprotection by causing the autophosphorylation of the tyrosine residues on TrkA receptors. Our experiments have shown that SIN-I interferes with NGF induced autophosphorylation of TrkA receptors thus leading to impairment of receptor function (Fig. 20). These results are consistent with those from the microphysiometer experiments, which demonstrated that exposure of ' the cells to SIN-I decreased the metabolic response to NGF in PC12 cells. This decrease in metabolic response to NGF was likely a direct result of the decreased TrkA autophosphorylation. Since nitrated tyrosine residues cannot be phosphorylated by tyrosine kinases (Martin et al., 1990), it is possible that SIN-I may interfere with the signal transduction initiated by NGF. However, our current data do not allow us to exclude the possibility that oxidation of the lipid membrane by SIN-I may have a similar effects on TrkA receptor autophosphorylation. 57
MAPK is one of the downstream kinases activated by NGF. In PC12 cells, the activation of MAPK by NGF has been implicated in several cellular processes ranging from differentiation to cellular apoptosis (Pang et al., 1995). NGF activates MAPK by recruiting the enzymes to phosphotyrosine proteins in signal transduction. Because the recruitment and activation of MAPK are conditional on binding to tyrosine phosphorylated proteins, the extent of activation of the enzyme has been used as a measure of cell surface receptor activation. In our experiments the exposure of cells to
SIN-I (500 µM) produced about a 50% decrease in NGF-mecliated increased MAPK activity (Fig. 21). This decrease in -MAPK activity was concentration dependent and significant even after only 10 µM. Recently, Perry and co-workers (1999) also presented evidence linking extracellular receptor kinase (ERK) dysregulation to abnormal phosphorylation of cytoskeletal proteins in AD brains. These findings probably reflect the consequences of chronic oxidative stress, which eventually leads to the activation of
ERK. The decrease in MAPK activity to NGF measured in the presence of SIN-I may be a consequence of the nitration of tyrosine residues, which inhibits tyrosine phosphorylation catalyzed by tyrosine kinases (Kong et al., 1996). Spear and co-workers
(1997) reported similar changes in PI-3 kinase activity (another downstream kinase activated by NGF stimulation).
In summary, SIN-I exposure inhibits the NGF signaling in PC12 cells. This may be due to the nitration of tyrosine residues on TrkA, thus preventing them from becoming phosphorylated, a process that is essential for many cellular responses. In this study, rnicromolar concentrations of SIN-I inhibited NGF signaling. Whereas it is difficult to estimate peroxynitrite concentrations achieved in vivo, oxidative stress in aging and in 58
diseased brains is thought to be a cumulative process. It is possible that these tissues are
under reduced NGF mediated trophic support, which in tum causes neurons to exhibit
increased vulnerability to further damage. In the case of BFC neurons, NGF signaling is
essential for maintaining ChAT activity and antioxidant machinery. A decrease in NGF
signaling subsequent to long-term peroxynitrite exposure may enhance the vulnerability
of forebrain cholinergic neurons to additional insults in AD.
The pharmacological and physiological properties of nAChRs are complex. The
overall problem regarding. the means by which chronic nicotine exposure results in
cytoprotection remains partially unresolved. However, from our studies it is clear that
stimulation of a:7nAChRs results in cytoprotection and modulation of NGF/TrkA
receptor expression and that these responses are directly related to the ability of the
agonist to upregulate a:7 receptors. One explanation is that neuroprotection is a
consequence of the time-dependent upregulation of a:7nAChRs during agonist
incubation, possibly countering a state of agonist-induced receptor desensitization, and
regulating "neuroprotective" levels of intracellular calcium. The rationale behind nAChR
upregulation is thought to lie in their rapid desensitization and consequent inactivation following chronic agonist exposure, putatively resulting in a deficit in cholinergic
function, which is then counteracted by an increase in receptor number (Schwartz and
Kellar, 1985). Upregulation, desensitization and the eventual inactivation ofnAChRs are
dependent upon the duration of agonist exposure and upon the nature of the agonist itself
(Rowell and Duggan, 1998; Reitstetter et al., 1999). For example, the duration of the
agonist occupancy drives the receptor molecule into deeper states of desensitization, each
requiring progressively longer times for recovery to the ground state after the removal of 59 agonist. Chronic nicotine results in upregulation of both intracellular as well as surface nAChR receptors (Lei et al., 1998). The functional role of these intracellular receptors is not known. Among the agonists tested in this study, only nicotine was able to completely protect the cells from trophic factor withdrawal. Even though the affinity of nicotine for a.7 receptors is reduced as compared with cytisine, its ability to cross lipid membranes and interact with the intracellular a.7 receptors may be responsible for its marked efficacy. In support of this concept is the reduced degree of efficacy exhibited by quaternary nitrogen-containing compounds DMPP, MCC, cytisine, TEA, choline and choline analogs, as a result of their inability to interact with intracellular nicotinic receptors. Like nicotine, 4OH-GTS21 and epibatidine are lipid soluble and they also are able to interact with the intracellular receptors. Since 4OH-GTS21 is a partial agonist at a.7 receptors and also exhibits an antagonistic activity at high concentrations, it may not be an ideal compound to reject the above possibility. However, epibatidine, being the most potent and also lipid soluble represents the only exception thus far tested. Further studies investigating the role of intracellular nAChR receptors are required. Evidence from recent studies indicates that Pl-3 kinase activity was responsible for as much as 80% of neurotrophin regulated cell survival, indicating PI-3 kinase is the major survival promoting protein in neurons (Crowder and Freeman, 1998). PI-3 kinase, stimulates the activities of many target proteins. Among is the serine/threonine kinase Akt, a target of
NGF induced PI-3 kinase activity (Dudek et al., 1997). Akt activity was shown using, dominant-inhibitory Akt, to be necessary for approximately 80% of NGF-induced survival of sympathetic neurons (Virdee et al., 1999; Dudek et al., 1997). The direct targets of Akt activity are the proteins that regulate cell survival in many cell systems: 60
these mclude Bad, an inhibitor of the Bcl-2 anti-apoptotic protein; pro-caspase-9, which
is cleaved into the pro-apoptotic caspase-9; and Forkhead, a transcription factor that
induces apoptosis by increasing levels of Fas ligand (Kaplan and Miller, 2000). Apart
from PI-3 kinase-Akt pathway, Ras-MEK-MAPK pathway was implicated in NGF
mediated neuronal survival. However, this pathway was shown to account for only
-20% ofNGF mediated neuronal survival (Klesse et al., 1999). Recent evidence from
studies aimed at molecular mechanisms indicates that nicotine through· stimulating a.7
nicotinic receptors increase the activities of PI-3/Akt (Kihara et al., 2001; Shimohama
and Kihara, 2001) and Ras!MAPK (Tang et al., 1998). However, it is not understood
whether this effect was directly resulted from the nicotinic receptors or indirectly through
stimulation of increased TrkA receptors. Further studies at the molecular level are
required to understand the neurotrophic actions of nicotinic receptors. There currently is
enormous interest in the development of selective nAChR agonists for the treatment of
neurodegenerative disease. Since the behavior of nAChR subtypes is unpredictable and varies with different agonists, knowledge of the selectivity and the affinity of the molecule may not predict outcome. Therefore, it is imperative to understand functional
drug-receptor interactions and examine each new compound in a relevant animal model. V. Summary:
I. Preincubation of differentiated PC12 cells with nicotine protected the cells from the cytotoxicity induced by NGF and serum deprivation. The high degree of sensitivity of this nicotine-induced response to the blocking actions of MLA suggests that nicotine's action was mediated through u7nAChR subtype.
2. Choline is a selective and full agonist at u7nAChRs. Among five choline analogs tested, pyrrolidinecholine and acetyl pyrrolidinecholine were found to be more potent and more efficacious than the parent compound choline. The rank order for producing cytoprotection among the six compounds was as follows: acetyl pyrrolidinecholine = pyrrolidinecholine > choline = monoethylcholine = diethylcholine = triethylchoilne.
Further, [125I]u-BGT displacement binding studies revealed that the pyrrolidinecholine exhibits -3 fold greater affinity for u7 receptors than choline.
3. Among the series of seven nAChR agonists, nicotine was the most efficacious and the most potent in its ability to protect cells from trophic factor withdrawal. Nicotine was followed by 4OH-GTS21, epibatidine, methylcarbamylcholine, DMPP, cytisine, and
TEA. neuroprotection. For the nAChR agonists used in this study, their rank order of potencies (ECS0) for activating (channel currents) a7 receptors has been reported to be: epibatidine>4OHGTS-2l=cytisine>nicotine >DMPP which failed to correlate with the rank order of their ability to produce cytoprotective actions. This relationship indicates that the mechanism involved in neuroprotection requires more than simply activation
61 62 of a7 receptors. However, the rank order nAChR agonists in producing cytoprotection correlates well with their ability to upregulate a7 receptors (nicotine > GTS-21 > epibatidine > DMPP). The extent of upregulation induced by chronic exposure to nicotine or cytisine, which were the most and one of the least efficacious compounds in our neuroprotection assay were compared. After 24 hr of drug exposure, cells treated with nicotine expressed significant increases in cell surface [125I]-aBGT binding sites, whereas cytisine, failed to do so. Although, cytisine binds to a7 receptors with greater affinity than nicotine, its inability to upregulate a7 receptors may underlie its poor cytoprotective efficacy.
4. Chronic treatment with MLA induced receptor upregulation. However, after exhaustive washout of the antagonist and subsequent incubation with nicotine, the protective effect of nicotine was significantly enhanced. Thus, the stimulation of MLA induced additional a7 receptors likely mediated the enhanced level of neuroprotection to nicotine. Since the prolonged exposure to nAChR agonist is essential to exhibit neuroprotective actions, one explanation for the time-dependent effect is that neuroprotection is a consequence of the time-dependent induction of additional a7 nAChRs expressed during agonist incubation, possibly countering a state of agonist induced receptor desensitization, and regulating "neuroprotective" levels of intracellular calcium.
5. Incubation of PC12 cells with nicotine produced a concentration and time dependent increase in· the expression of TrkA receptors. This effect was blocked by co treatment with the a7 nAChR antagonist MLA again indicating that the effect was largely mediated by the a7 nAChRs. The continuous infusion of nicotine in rats resulted 63
in an increase TrkA protein levels with in the hippocampus, and this effect was blocked
• by co-treatment with nAChR antagonist mecamylamine. In contrast, in cortical tissues
no changes were observed in TrkA protein levels after the drug administration. The
majority of nAChRs in hippocampus belong to the a.7 subtype whereas in the cortex the
predominant subtype is a.4~2. Based upon the results from in vitro studies of receptor
subtype selectivity it is reasonable to conclude that the differences observed in TrkA
receptor expression in hippocampus and cortex were due to specific subtypes located in
those brain regions.
6. Nicotine was 10 fold more efficient when incubated with cells in the presence of
NGF than in the absence ofNGF. Further, when cells were incubated with nicotine and k252a or nicotine and anti-TrkA antibody, nicotine was unable to completely protect the cells from trophic factor withdrawal. These results indicate that there are other mechanisms apart from enhanced NGF trophic support during drug incubation period involved in nicotine's neuroprotection.
7. Pretreatment of PCI2 cells with the peroxynitrite donor, SIN-I, produced a concentration dependent inhibition of the cellular response to NGF. The SIN-I effect was significantly reduced by pretreatment with peroxynitrite scavenger uric · acid indicating that response was primarily mediated by peroxynitrite generation.
8. NGF was shown to enhance the phosphorylation of tyrosine residues on TrkA receptors, and pretreatment with SIN-I produced a concentration dependent decrease in phosphorylation of tyrosine residues. SIN-I nitrates tyrosine residues in the ortho position to form 3-nitrotyrosines thus preventing the autophosphorylation induced by 64
NGF, an action that most likely accounts for the reduction in cellular response produced by SIN-1.
9. After TrkA autophosphorylation (in response to NGF) adapter proteins recognize and bind to the phosphotyrosine residues on the receptor. These adapter proteins initiate the activation of second messenger pathways. One of these is the pathway that leads to the activation of MAPK, which is known to be essential for gene expression and cell survival. Consistent with its effects on TrkA autophosphorylation, SIN-I also was shown to inhibit the increase in MAPK activity to NGF in these cells. Since BFC neurons depend upon endogenous NGF activation of TrkA receptors, oxidative damage with the production of peroxynitrite could lead to a loss of NGF-mediated responses, which may be the one of the initial insults in AD pathology.
10. The continuous exposure of the cells with nicotine results in the upregulation of a7nAChR receptors. Agonist activation of this additional population of receptors might results in, a gradual increase in intracellular calcium levels. The increase in intracellular
calcium initiates the transcription of genes responsible for neurotransmitter release,
neuroprotection, growth factors, and growth factor receptors. Further, the released
growth factors, along with endogenous growth factors, contribute to the enhanced trophic
activity leading to the neuroprotection. 65
Figure 1. Differentiated PC12 cells were deprived ofNGF and serum for 24 hr and cell viability was determined from the ability of mitochondria to reduce MTT. Preincubation of cells with nicotine for 24 hr protected the cells from trophic factor withdrawal cytotoxicity.
This effect was blocked by the nAChR antagonists mecamylamine (Mee), and methyllcaconitine (MLA). * significantly different from control (intact cells); ** significantly different from control (trophic factor deprived/no treatment); # significantly different from "nicotine 10 µM for 24 hr"; F4, 54 = 237.7, p<0.001, by one way ANOVA
(post hoc test: Tukey multiple comparision, p<0.05). 100 **
90 (Mee 5 µM) (MLA 10 nM) 80 -e C 70 -0 0 60 0~ ->, ·- 50 ·-.c- ra ·-> 40 -Cl) 0 30
20
10
0 Control nicotine 10 µM for 24 hr Deprived for 24 hr (-NGF, ~serum) 66
Table 1
Except for the Control (intact cells), in each experiment, cells were ·trophic factor-(NGF and serum) deprived for 24 hr. Nicotine was included in the media 24 hr (except for the one experiment where exposure time was only 10 min) prior to trophic factor withdrawal.
The nicotinic receptor antagonists mecamylamine (Mee) or methyllcaconitine (MLA) or dihydro-f3-erythroidine (DHf3E) or bungarotoxin (Bgt) were administered as a co-treatment with nicotine. * significantly different from Control (intact cells), t=19.3, p<0.001; ** significantly different from Control (trophic factor deprived/no treatment, -NGF, -serum), t=23.0, p<0.001; t significantly different from "nicotine 10 µM for 24 hr", F4,38=41.9, p<0.001, by one way ANOVA (post hoc test: Dunnett's, multiple comparison, p<0.05). Treatment % Cell Viability Control (intact cells) 100
NGF and serum deprivation 66.6±5.4 *
NGF deprivation alone 66.8±1.1 nicotine 10 µM for 24 hr 95.6±2.1 ** nicotine 10 µM + Mee 5 µM 72.2±I.3t nicotine 10 µM + MLA 0.1 nM 94.6±1.2 nicotine 10 µM + MLA 1 nM 79.4±2.8 t nicotine 10 µM + MLA 10 nM 71.6±1.2 t
~cotine 10 µM + MLA 100 nM 67.8±5.1 t nicotine 10 µM + Bgt 100 nM 71.0±3.9t nicotine 10 µM + DHl3E 10 nM 94.4±2.7 nicotine 100 µM for 10 min 72.9±2.0 67
Figure 2. Differentiated PC12 cells were deprived ofNGF and serum for 24 hr and cell viability was determined by MTT reduction assay. Preincubation of cells with choline for 24 hr protected the cells from trophic factor withdrawal cytotoxicity. This effect was blocked by the nAChR antagonists mecamylamine (Mee, 10 µM), and methyllcaconitine
(MLA, 10 nM). The cytoprotective ability of choline is expressed as the ability of choline to reverse the cell death induced by trophic factor withdrawal. * significantly different from untreated cells, F3, 64 = 10.3, p<0.001 one way ANOVA (post hoc test:
Tukey, p<0.05). # significantly different from cells treated with choline 10 mM, Fz, 33 =
7.9, p<0.002 one way ANOVA (post hoc test: Tukey, p<0.05). MTT reduction -e 45 C: * -0 ------Choline . ' 40 ....0 -0- Choline+ Mee 10 µM • 0 35 -T- Choline+ MLA .10 nM '$. 30 .c:- ca 25 -Cl) C 20 -Cl) 15 # 0 .... 10 0 C: 5 0 .:l 0 (.) :::s "C -5 Cl) 0:: 0.1 1 10 mM 68
Figure 3. The relative ability of different choline analogs to protect differentiated PC12 cells from the cytotoxicity induced by 24 hr of NGF and serum (trophic factor) deprivation. Various concentrations of mono ethyl choline, diethyl choline, triethyl choline, Pyrrolidine choline, Acetyl Pyrrolidine choline, were incubated for 24 hr prior to trophic factor withdrawal. The concentration-response curves for each compound exhibited significant increases in cell viability compared with untreated control values
(not shown) by ANOV A. F ratios ranged from 3.3 - 23.2, all at the p<0.001 level of significance. - Monoethyl choline -o- Diethyl choline ----.- Triethyl choline ----?-- Pyrrolidine choline - Acethylpyrrolidine choline
-e 70 C: -0 60 (.) '#,. 50 -.c: ca 40 -Cl) C 30 -Cl) (.) 20 --0 10 C: 0 :.:: 0 0 :I "C Cl) -10 0:: 0.1 1 10 mM 69
Figure 4. A) The dose response curve for the most effective compound
Pyrrolidinecholine. Cells were incubated with various concentrations of pyrrolidinecholine for 24 hr prior to the trophic factor withdrawal. * significantly different from untreated control cells, F6, 107 = 23.8, p<0.001, one way ANOVA (post hoc Tukey, p<0.05). B) Pyrrolidinecholine effect was blocked by concurrent treatment with the nAChR antagonists mecamylamine (Mee, 10 µM), and methyllcaconitine (MLA,
10 nM). * significantly different from Pyrrolidinecholine (1 mM) treated cells, F2, 49 =
65.5, p<0.001 one way ANOVA (post hoc test: Tukey, p<0.05). - 60..------e A * * -§ 50 * (.)
0~ 40 -.c: 11' 30 -Cl) C -'a) 20 (.) 'o ·10 s::: 0 .. 0 (.) ::I "C ~ -10 '--....-----.----.-----.------_..I 1e-7 1e-6 1e-5 1e-4 1e-3 1e-2 [Pyrrolidinecholine], M :=- 60 e B -§ 50 (.) ';!. - 40 .s::: 11' -Cl> 30 C
-Cl> 20 .....(.) 0 Mee (10 µM) MLA (10 nM) s::: 10 0 .:; (.) 0 ➔- ::I ---...J "C a:Cl) * Pyrrolidinecholine (1 mM) 70
Figure 5. Displacement of high-affinity [125I]a.-Bungarotoxin binding to differentiated
PC12 cells by nicotine and choline. The [125I]a.-Bungarotoxin displacement studies were performed as mentioned in the methods section. The non-specific binding was determined in the presence of 1 µM unlabeled a.-Bungarotoxin. 125 [ 1]Bgt Displacemen~
• Choline (Ki = 86.2 µM) 100 • Nicotine (Ki = 1.67 µM)
o, 80 C: ·-"C C: iii 60 0, :::s c 4o 'cf:. 20
0
1e-7 1e-6 1e-5 1e-4 1e-3. [Drug], M 71
Figure 6. Displacement of high-affinity [125I]a-Bungarotoxin binding to differentiated
PC12 cells by pyrrolidinecholine. The [1 25I]a-Bungarotoxin displacement studies were performed as mentioned in the methods section. The non-specific binding was determined in the presence of I µM unlabeled a-Bungarotoxin. 120 ..,.....------, I • Pyrollidinecholine (Ki = 33 µM) I 100
C) 80 C: ·-'C C: ·-m 60 C) ::::s I,, C 40 ~ 20 t I 0
1e-5 1e-4 1e-3 [Drug], M 72
Figure 7. The relative ability of different nAChR agonists to protect differentiated PC12 cells from the cytotoxicity induced by 24 hr of NGF and serum (trophic factor) deprivation. Various concentrations of (A) nicotine, epibatidine, 4OHGTS-21, (B) methylcarbamylcholine (MCC), l,l-dimethyl-4-phenyl piperazinium (DMPP), cytisine, and tetraehtylarnmonium (TEA) were incubated for 24 hr prior to trophic factor withdrawal. The concentration-response curves for each compound (except for TEA which was not significant) exhibited significant increases in cell viability compared with untreated control values (not shown) by ANOV A. F ratios ranged from 8.7 - 25.8, all at the p<0.001 level of significance. 8 100 'A 100 ~ • DMPP :::::- j --te1-- nicotine --o-- Cytisine _g 95 --o-- epibatidine 95 ~ ... TEA 5 T 40H-GTS21 "ff-MCC O 90 90 '#. -_a, 85 85 ·-- ~ 80 80 ·-> "a;- 75 75 0 70 70
65 65 1e-9 1e-8 1e-7 1e-6 1e-5 1e-4 1e-9 1e-8 1e-7 1e-6 1e-5 1e-4 Cone [M] Cone [M] 73
Figure 8. The effect of chronic nicotine and cytisine on the up-regulation of a.7nAChR subtype. Differentiated PC12 cells were incubated with either nicotine (10 µM) or cytisine
(100 µM) for 24 hr. The ability of these ligands to up-regulate [125I]a.-bungarotoxin binding sites was quantified by saturation binding analysis. The results (Bmax values) derived from these binding curves are presented in Table 2. Undifferentiated PC12 Cells 100 • 0 Differentiated PC12 Cells T nicotine 100 µM 80 ■ Cytisine 100 µM C) -E -0 60 .s -C) C 40 ·-"C C ·-al 0 20 -C) al
0 0.1 1 10 [Bgt] Cone nM 74
Table2
Effect of prolonged exposure of differentiated· PC12 cells to nicotinic ligands, on the number of cell surface [125I]a.-bungarotoxin (Bgt) binding sites. Differentiated PC2 cells were incubated with one of the ligands nicotine (10 µM, 100 µM), cytisine (100 µM), methyllcaconitine (MLA) (100 µM) for specified time intervals. * significantly different from control, Bmax, F4,11=13.3, p<0.001; Kd, F3,s=5.00, p<0.031 by one way ANOVA
(post hoc test: Dunnett's, multiple comparison, p<0.05). t Since the MLA treated cells were maintained for extra 96 hr, it might be appropriate to compare the Bmax value derived from the control cells that were maintained for the similar duration of time period. After 15 months of the-initial experiments, we measured the Bgt binding sites on the control cells that were maintained for the same time period
(9 days) as that ofMLA treated cells. The Bmax values obtained from these experiments were control cells 87.2±3.65 final/mg, Kd 1.61±0.07 nM; MLA 100 µM treated cells
181.7±4.0 final/mg, Kd = 2.11±0.4 nM. In the similar experiments we also measured the
ability of mecamylarnine (nAChR channel blocker) to upregulate Bgt binding sites on
differentiated PC12 cells. Chronic treatment of cells with mecamylamine (100 µM, 96
hr) completely failed to upregulate cell surface a.7 nicotinic receptors (76.0±3.09
final/mg, Kd = 1.47±0.08nM). Treatment Bmax % increase (fmoUmg) from control Kd (nM)
Undifferentiated PC12 cells 16.4±1.21 0.54±0.105
Differentiated PC12 cells 82.6±3.67 0 1.47±0.129 nicotine 10 µM for 24 hr 117±10.3* 41 1.46±0.45 nicotine 100 µM for 24 hr 115±8.4* 39 1.77±0.113
Cytisine 100 µM for 24 hr 87.3±2.97 5 1.55±0.049
MLA 100 µM for 24 hr 116.8±13.5* 41 2.69±0.55*
MLA 100 µM for 96 hr 159.6±9.16* 93 2.6±0.424*
Differentiated PC12 cells 87.2+3.65t 1.61+0.07
MLA 100 mM for 96 hr 181.7+4.0t 107 2.11+0.4
Mee 100 mM for 96 hr 76.0+3.09t -12 1.47+0.08 75
Figure 9. The effect of chronic exposure to methyllcaconitine (MLA) and mecamylamine
(Mee) on the up-regulation of a7nAChRs on differentiated PC-12 cells. The cells were incubated with either with MLA (100 µM) or with Mee (100 µM) for 96 hr and the ability of the antagonist to up-regulate [125I]a-bungarotoxin binding sites was quantified by saturation binding analysis. The Bmax values derived from these binding curves are presented in Table 2. B) The effect of MLA-induced a7nAChR up-regulation on the ability of sub-maximal (4 hr) neuroprotective exposure of differentiated PC-12 cells to nicotine. The protocol for this series involved exposure of the cells to MLA (100 µM) for 96 hr to evoke a maximal increase in cell surface [125I]aBGT binding sites (see Table
2). This was followed by exhaustive washing to remove traces of the antagonist, and subsequent incubation of the cells with 10 µM nicotine for an additional 4 hr (the shorter time interval) prior to trophic factor withdrawal. All the values are derived from cells that were maintained for similar duration of time (9 days). Pretreatment of cells with
MLA (100 µM, 96 hr) had no significant effect on the cyto_toxicity induced by trophic factor withdrawal in comparison with untreated cells maintained under similar
conditions. Pretreatment of control (trophic factor deprived, but non-MLA treated) cells with nicotine for 4 hr improved cell viability from 46% to 68%. However, in the cells
previously exposed to MLA that exhibited the increased expression of [125I]aBGT
binding sites, the improvement in cell viability produced by nicotine was significantly
increased to 79% of control. * F3, 100=113.2, p<0.001 by one way ANOVA (post hoc test:
Dunnett's, multiple comparison, p<0.05). 1~:::,,,1;rz.1j Control Deprived for 24 hr 1111111111 MLA96hr I nicotine 10 µM for 4 hr MLA 96 hr+ nicotine 10 µM for 4 hr 180 100 B 11!'.:t:r A m!:1Pi * p<0.05 m 160 I 90 :~£~'.~i r<+t:~, E ~ ,,:.,; ,'/ 140 :::::- 0 80 ~::·) -0 ~'<'-~-,\ Control +-'... ;;:,., L-..• C: $·~:; (ti t 120 j •0 MEC 100 µM 0 70 (.) tn 100 MLA 100 µM Q) ... 'cf;. 60 ttl, ·- (' '"'-i tn 80 - ;,l-: '') - 50 !:. •{t~: 0, ~ r~•-Y• C: 60 ·-.c ·-. 40 f>.il,v ~~,;::1 cu 1 "C f#);{~·-~ C: ·-> 1L~,;,;-~ ·- 40 30 ~t\~,·; m - , • J I_.~• -Cl) ft~!.~ (.) }y,.;;::t 0, 20 20 fr:•J;f"'J - ~,}i/J.~; m fj:,1. 0 10 0 ~j 1e-10 1e-9 1e-8 Deprived for 24 hr [Bgtl, nM 76
Figure 10. Differentiated PC-12 cells were incubated with nicotine for 4 or 24 hr at different concentrations ranging from 10 nM to 10 µM. After the incubation period the cell surface TrkA receptor expression was measured by cell based ELISA. Nicotine increased the expression of cell surface TrkA receptors an effect that was time- and concentration- dependent. These effects of nicotine do not seem to be due to an increase in overall cell growth because nicotine did not change the total protein levels (39.5 ± 1.07
µg/ml) from control (40.2 ± 0.87 µg/ml) (N = 27). There is a significant interaction between time and concentration responses, F4 , 34 = 14.3, p<0.001. * significantly different from untreated control cells (pos hoc test: Tukey, multiple comparison, p<0.05). 40 Nicotine incubation period - 24hours * ji;;a;;:,'flliilll!I 4 ho u rs -0 35 I., +' C: 0 C.) 30 Cl) > * 0 25 .c ca ~ 20 C: * ca .c: * (.) 15
:::e!0 10
5
0 0.01 0.1 1 10 [Nicotine], µM 77
Fig 11. Blockade of nicotine induced increase in cell surface TrkA receptors by nAChR antagonists. Differentiated PC12 cells are incubated for 24 hr with nicotine plus one of the following nAChR antagonists mecamylamine (Mee), methyllcaconitine (MLA), dihydro-beta-erythroidine (DH~E). Each data point represents the mean ± SEM of at least
8 independent experiments. After the incubation period the relative levels of cell surface
TrkA receptors are determined. * significantly different from cells treated with nicotine alone F3, 56 = 27.1, p<0.001 one way ANOVA (post hoc test: Tukey, p<0.05) 30 -....0 25 C -0 (.).... 20 C1) > 0 15 C1) en C 10 cu .c (.) 5 ?!, * 0 Mee MLA DHPE (5 µM) (10 nM) (10 nM)
Nicotine 1 µM Present 78
Figure 12. TrkA protein levels in hippocampus and cortex tissues. Rats were chronically infused with indicated drugs for 24 hr as mentioned in the methods sections. After the treatment the amount of TrkA protein levels in A) hippocampus and B) cortex were quantified by using western bloting techniques. * significantly different from saline treated rats, F3, 16 = 8.6, p<0.001 one way ANOVA (post hoc test: Tukey, p<0.05). Hippocampus Cortex
120 -t B 1601 A * 140 100
120 80 e 100 C - 60 0 80 0 ';fe. 60 40 40 20 20 0 ...__ _.. 0 .
Control Nie Nie+ Mee Mee Control Nie Nie + Mee Mee
{-)-nicotine 12 mg/kg/day {I.V) mecamylamine 24 mg/kg/day {I.V) 79
Figure 13. Cytprotective ability of nicotine in the presence and in the absence ofNGF.
Differentiated PC12 cells were incubated with indicated concentrations of nicotine for 24 hr in the presence ofNGF prior to the trophic factor withdrawal(•) and in the absence of
NGF (during NGF withdrawal) (0). 105 ~------...... ,::..,___ , • Presence of NGF -o- Absence of NGF 100
-...0 95 C: -0 0 90 -~ ·-~ -.c 85 cu ·-> -Cl) 0 80
75
70 ..___ __,,__.....,. ______,....__-....-I 1e-9 1e-8 1e-7 1e-6 1e-5 1e-4 1e-3 nicotine[M] 80
Figure 14. Cytoprotective ability of nicotine in the presence of TrkA receptor inhibitor
K.252a. Differentiated PC12 cells were incubated with K.252a (1 µM) or nicotine (10
µM, 100 µM) plus K.252a for 24 hr prior to the withdrawal. # significantly different from deprived group; * significantly different from cells treated with k252a alone F2 35 = ' 14.0, p<0.001 one way ANOVA (post hoc test: Tukey, multiple comparision, p<0.05). 100 90 nic (10 µM)nic (100 µM) -0 .... 80 C * * -0 0 70 -~C, 60 ·-~ 50 ·--.c ctl 40 ·-> 30 -Q) 0 20 10 0 Control K252a (1 µM) Deprived for 24 hr 81
Figure 15. Cytoprotective ability of nicotine in the presence of anti-TrkA antibody.
Differentiated PC12 cells were incubated with anti-TrkA (1 µg/ml) or nicotine ( 10 µM) plus anti-TrkA for 24 br prior to the withdrawal. * significantly different from deprived alone;# significantly different from cells treated anti-TrkA alone, F3, 59 = 113.3, p<0.001 one way ANOV A, (post hoc: Tukey, multiple comparision, p<0.05) 110
100 nic ~o µM -0 I.. 90 # C -0 80 0 70 * - 60 ·-b ·-.c- 50 ca ·-> 40 30 -Cl) 0 20
Cl~ 10
0 ,!So' anti-TrkA 1 µg/ml § C, > Deprived 82
Figure 16. The rate of media acidification in response to exposure of PC12 cells with different concentrations of nerve growth factor (NGF) for 12 min. (A) The data represent mean values ± SD from 3 - 4 experiments for each of the following concentrations: 1 ( o ),
10 (T), 25 ('v), 100 ng/ml ( ■) and vehicle (0 ng/ml) (•). (B) Acidification response data calculated from area under acidification rate response curves for 32 min after addition ofNGF. The Half-maximal effective concentration (ECso) calculated from this graph is 15 .2 ng/ml. Time Course ...------~ Peak NGF Response 150 140 I -e- Vehicle A 1 I B -o- 1 ng/ml 0
140 T 0c: ---T- 10 ng/ml I I EC50=15.2 ng/ml --.- 25 ng/ml 130 ·-ca - 100ng/ml ....--~ 130 =a (.) 120 <( 120 cu ('ISU) 110 110 m 0 ~ 100 100 NGF (ng/ml) 90 -'-T----r-----r------r-----r---r---r---r___,J 0 5 10 15 20 25 30 35 1 10 100 minutes NGF (ng/ml) 83
Figure 17. The effect of SlN-1 on cell viability. PC12 cells were exposed to different concentrations of SlN-1 for 20 min and then maintained in DMEM for 0 (•), 12 (0), 24
(T) hr. The% cell viability was determined by using the MTT reduction assay. SlN-1 produced no significant decrement in cell viability under the conditions described for the experiments in Fig. 3, 4. Data are mean values ± SEM from at least three separate experiments performed in quadruplicates. * significantly different from control (p<0.05, one way ANOVA). 105
-0 b 100 r:: 0 0
~0 95 -r:: 0 -e-- 0 hr .:l 90 CJ -0- 12 hr ::I "C Q) ----T- 24 hr 0::: 85 I- I- :!!: 80
75 .L...-.------.------.------.------' 1 10 100 1000 SIN-1 µM 84
Figure 18. The rate of media acidification in response to exposure of PC 12 cells with 25 ng/ml NGF for 12 min. (A) The data represent mean values± SD from 3 - 4 experiments
in absence(•), and after 20 min exposure to SIN-11 (o), 10 (T), 100 ('v), 500 ( ■) µM.
(B) The peak NGF (25 ng/ml) response in the absence, and after 20 min exposure to SIN-
1 (1, 10, 100, 500 µM). * F4,13=14.7, p<0.001 by one way ANOVA (post hoc test:
Tukey, p<0.05). Time Course
....,.._OµM A 140 ~1µM C: ~10µM 0 """9-100µM ·-ns 130 - 500µM (.) ·-.;:: :E(.) 120 <( -cu 1/) 110 cu ca ~0 100 NGF 25 ng/ml
0 5 10 15 20 25 30 35 .minutes
140 -.------, Peak NGF Response 8 C: 0 130 ·-cu ·--(.) ·-- 120
-cu 110 cu1/) ca
0~ 100
0 1 10 100 500 [SIN] µM 85
Figure 19. The effect of uric acid treatment on SIN-1 induced inhibitory responses to
NGF stimulation. (A) PC12 cells were exposed to the peroxynitrite scavenger uric acid
(UA) (1 mM, 1hr) prior to SIN-I (500 µM, 20 min) exposure and subsequently the cells were placed in microphysiometer and challenged with NGF 25 ng/ml. The data represent mean values ± SD from 3 - 4 experiments. Cells pretreated with uric acid alone were used as the control. (B) The peak NGF (25 ng/ml) response to uric acid, SIN-I and uric acid plus SIN-I treatment. * significantly different from control ( cells treated with uric acid alone), t=l9.0, p<0.001; # significantly different from cells treated with SIN-I alone, t=5.8, p<0.004. 130 ..,...... ------, -+- Control (UA) A C 125 -- 135 ...... -'------, Peak NGF Response 8 C 130 0 +ica 125 0 !E 120 ·-"C ~ 115 -: 110 ca m 0~ 105 100 Uric Acid + + SIN-1 + + 86 Figure 20. Representative western blot showing tyrosine phosphorylation in TrkA receptors (140-kDa). PC12 cells were stimulated with 25 ng/ml NGF after a 20 min exposure to SIN-1 at the indicated concentrations. After the treatment, the cells were harvested and the lysates were immunoprecipitated with phosphotyrosine antibody and immunobloted with anti-TrkA antibody. IS IN -1 I, p M 0 I 10 100 500 0 --205 Kd -120 Kd -84Kd -52Kd -- 36 Kd 87 Figure 21. MAPKinase activity in immunoprecipitates of PC12 cells treated with NGF or SIN-I plus NGF. After the treatment, MAPKinase was immunoprecipitated with anti MAPK-1/2 antibody. The activity of MAPKinase is expressed as a percentage of the value determined in control cells that had been treated with NGF 25 ng/ml alone. Data indicate mean values± SD derived from 3 - 4 separate experiments. * F4,10=9.8, p=0.002 one way ANOV A (post hoc test: Tukey, p<0.05). 100 -0 ...... C: 80 0 (.) ?fl. 60 ->, ·s:.... +' (J 40 0 SIN-1 1 µM 10 µM 100 µM 500 µM 88 Figure 22. Schematic representation of the possible relationship between the upregulation of a.7nAChR, the increased expression of NGF/TrkA receptors and the neuroprotective actions of nicotine. 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