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Regulation of amyloid precursor protein catabolism in vitro : the role of mitogen-activited protein kinase… Mills, Julia 1998

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R E G U L A T I O N O F A M Y L O I D P R E C U R S O R P R O T E I N C A T A B O L I S M IN VITRO: T H E R O L E O F M I T O G E N - A C T I V A T E D P R O T E I N K I N A S E A N D P R O T E I N K I N A S E C by Julia Mills B.Sc.H., Queen's University, 1987 M.Sc, Queen's University, 1992 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES The Graduate Program in Neuroscience We accept this thesis as conforming tojttye required stymdard UNIVERSITY OF BRITISH COLUMBIA April 1998 © Julia Mills, 1998 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) Abstract Secretory processing of the amyloid precursor protein (APP) generating soluble APP fragments occurs via two routes broadly categorized as non-amyloidogenic (yielding a truncated APP derivative known as APPS) or potentially amyloidogenic (yielding intact AP). These catabolic pathways are mutually exclusive and subject to regulation by various first messengers. In continuous cell lines, nonamyloidogenic processing of APP is increased by cholinergic receptor stimulation and its downstream effector protein kinase C (PKC). However, at the time our first set of experiments were initiated, mechanisms underlying APP catabolism in central neurons were largely unknown. Therefore, we examined whether or not first or second messengers of cholinergic neurotransmission increase APPS in primary cultures of rat cortical neurons. Using western blot analysis to measure secreted proteins, we determined that although activation of PKC by phorbol esters increased production of APPS, activation of muscarinic receptors by oxotremorine-M or carbachol did not. One explanation for this apparent discrepancy is that cholinergic agonists and phorbol esters activate downstream effectors differentially. One such effector may be mitogen-activated protein kinase (MAPK) also known as extracellular signal-regulated protein kinase (ERK). APP has been shown to be regulated by a variety of first messengers which also regulate this signaling pathway. Specifically, we hypothesized that regulation of APP processing may involve the sequential activation of the enzymes MAPK kinase (MEK) and ERK. We provide evidence that the MAPK pathway is critically involved in regulation of APP processing by nerve growth factor (NGF), phorbol esters and A -^methyl-D-aspartate (NMDA). Western blot analysis of APPS demonstrated that the MEK inhibitor PD 98059 antagonized NGF stimulation of APPS ii production in a neuronal cell line. Moreover, inhibition of MEK blocked phorbol ester regulation of APPS in cortical neurons and AP release in cell lines. Finally, overexpression of a kinase-inactive MEK mutant inhibited both phorbol ester and NMDA receptor stimulation of APPS. Taken together, these data indicate that the MAPK pathway may be critically involved in regulating APP processing. As signal transduction is intimately associated with amyloid burden in Alzheimer's disease, understanding the critical effector systems responsible for this mismetabolism of APP is necessary in order for effective treatment strategies to be generated. TABLE OF CONTENTS Abstract ii Table of Contents iv List of Tables vi List of Figures vii Acknowledgments viii Preface ix List of Abbreviations x I Introduction 1 II Cholinergic Agonists do not Regulate the Release of APPS in Cultured Rat Cortical Neurons Introduction 31 Materials and Methods 33 Results 37 Discussion 49 III Regulation of APP Catabolism Involves the MAPK Signal Transduction Pathway Introduction 55 Materials and Methods 57 Results 60 Discussion 70 IV Requirement for MAPK Signaling in NMDA Regulation of APPS Secretion Introduction 75 Materials and Methods 77 Results 80 Discussion 84 V Ap-Induced Tau Hyperphosphorylation: A role for c-Jun 88 N-Terminal Kinase Stress-Activated Protein Kinase VI General Discussion 90 VII References 102 Appendix 1 Infection of rat cortical neurons with a mutant herpes 124 simplex virus iv Appendix 2 Effects of dexamethasone on basal APPS release. 125 Appendix 3 SAPK/INK phosphorylation in mixed cortical cultures is 126 increased by A0. v L I S T O F T A B L E S Table 1. A Summary of Second Messengers and Effectors Known to 21 Regulate APP Catabolism Table 2. PI Turnover Associated With Cholinergic Receptor Stimulation 44 vi L I S T O F F I G U R E S Figure 1. Secretory processing pathways of APP 6 Figure 2. MAPK and APP catabolism 29 Figure 3 Schematic of APP with antibodies specific for different epitopes 37 of APP or A p Figure 4. PKC stimulation increases APPS release. 39 Figure 5. Oxotremorine-M does not increase the formation of APPS. 41 Figure 6. Carbachol does not increase the release of APPS. 42 Figure 7. KCl-induced depolarization does not increase the formation of 45 APPS. Figure 8. PKC stimulation but not cholinergic receptor stimulation 48 increases release of APPS and APLP. Figure 9. The MEK inhibitor PD 98059 antagonizes NGF receptor 63 stimulation of APPS secretion and ERK activation in PC 12 cells. Figure 10. The MEK inhibitor PD 98059 antagonizes phorbol ester 65 stimulation of APPS release and ERK activation in HEK 293 cells. Figure 11. The MEK inhibitor PD 98059 antagonizes PKC stimulation of 66 APPS secretion and ERK activation in cortical neurons. Figure 12. The kinase-dead MEK mutant K97A inhibits phorbol ester 68 stimulation of APPS secretion and ERK activation in HEK 293 cells. Figure 13. The MEK inhibitor PD 98059 antagonizes phorbol ester 69 inhibition of A P secretion in K695sw cells. Figure 14. The kinase-dead MEK mutant K97A inhibits NMDA receptor 83 stimulation of APPS secretion and ERK activation in HEK 293 cells. Figure 15. Regulation of APP catabolism via a MAPK-dependent or - 93 independent pathway. Aknowledgements TO FAMILY, FRIENDS AND COLLEAGUES I am indebted to my supervisor Peter Reiner who has dragged me (kicking and screaming) into an academic mindset. He is, and hopefully always will be, my scholastic mentor. I would like to thank those who both contributed directly and indirectly to this thesis. These include Andy Laycock, David Charest, Steven Pelech and Lynn Raymond. I would especially like to thank Fred Lam who was the first student to join the Alzheimer's research team, and who helped to make the latter years of research even more enjoyable than the former. For their helpful discussions and advice that can only come from the vantage point of post-docs, I would like to thank Bruce Connup and Kristi Kolmyer. For his very helpful suggestions at designing stronger experimental paradigms, I would like to thank Tim Murphy, who in our department sets the gold standard of experimental excellence. For their technical assistance, I would especially like to thank Monica Grunert and her successor Rouzbeh Shooshtarian who although had 'large shoes to fill', managed to do so. I would also like to thank my parents for their support and for hushing the chorus of disbelieving cries from friends and relatives when told that 'yes, I was still in school'. I feel both blessed and honored to be the first Doctor of Philosophy to represent our family. TO MY HUSBAND This work was initiated at a psychiatry research journal club as a follow up to an article sent to me by my then fiancee Don MacDougall from a local newspaper in Kelowna. A direct fallout of this journal club discussion has been the publication of two research articles, the submission a third research article and a review article. Since this time the Alzheimer's research team has grown from one to six, a paten has been submitted on the work carried out by Fred Lam and the company Activepass has been founded. Such growth has been highlighted by my now husband Don MacDougall, who has taken great pride in all of his achievements. Much more than the posting of that article however, I would like to thank Don for his unending support and personal sacrifices which have helped carry me through to the completion of these studies. Preface The contents of this thesis contain material taken from two published articles. These are cited in the references as Mills and Reiner (1996) and Mills et al. (1997). Contributions made by middle authors of the latter paper are as follows: (1) AP gels were run by Fred Lam from Dr. Peter Reiner's laboratory; (2) the dominant negative MEK mutant was provided by David Charest from Dr. Steven Pelech's laboratory , and (3) the WO-2 antibody was provided by Nobuo Ida from Dr. Konrad Beyreuther's laboratory. IX List of Abbreviations: A0 P-amyloid Api.40 P-amyloid terminating at amino acid 40 A0L42 P-amyloid terminating at amino acid 42 AD Alzheimer's disease APLP amyloid precursor-like protein APP amyloid precusor protein APPS soluble truncated APP derivative [3H]CDP-DAG [3H]CDP-Diacylglycerol ERK extracellular signal-regulated protein kinase JNK/SAPK c-Jun N-terminal kinase stress-activated protein kinases K97A kinase-inactive mitogen-activated protein kinase kinase LTP long term potentiation MAPK mitogen-activated protein kinase MEK mitogen-activated protein kinase kinase NFT neurofibrillary tangles NGF nerve growth factor [3H]NMS N-[3H]methylscopolamine NMDA 7V-methyl-D-aspartate p3 3 kDa truncated P-amyloid fragment plO 10 kDa C-terminal fragment of amyloid precursor protein PDBu phorbol 12, 13-dibutyrate PHF paired helical filaments PI phosphatidylinositol PKA cyclic AMP-dependent protein kinase PKC protein kinase C PLA 2 phospholipase A 2 PLC phospholipase C PMA phorbol 12-myristate 13-acetate SREBPs sterol regulating element binding proteins I. INTRODUCTION The progressive deposition of P-amyloid (A3) in the cerebral and limbic cortices and in the walls of the cerebral microvasculature is a hallmark of Alzheimer's disease (AD) pathology (Selkoe, 1991). Evidence that A P deposition plays a pivitol role in the etiology of AD comes from several lines of inquiry. Perhaps the most convincing is genetic analysis indicating that three genetic alterations underlying familial AD increase the production of A P in the brain (Selkoe, 1997; Roses, 1996). These include mutations in the genes encoding the amyloid precursor protein (APP, the precursor to AP) (Goate et al., 1991; Mullan et al., 1992) and presenilin-1 and -2, (Sherrington et al., 1995; Rogaev et al., 1995). Further support for the amyloid hypothesis is the fact that individuals with trisomy 21 develop AD in their 4th or 5th decade of life (Tanzi et al., 1987). As the APP gene is located on chromosome 21, it has been suggested that this is due to a gene-dosage effect. Finally, allelic variation of apolipoprotein E is a significant risk factor for sporadic AD (Corder et al., 1993; Brousseau et al., 1994; Rebeck et al., 1993): as apolipoprotein E binds to Ap (Strittmatter et al., 1993a; Strittmatter et al., 1993b) it may be involved in the formation of senile plaques (Ma et al., 1994). Animal and cell culture studies characterizing the effects of familial AD mutations also support the amyloid hypothesis. Expression of these mutants in cell lines results in increased production of A p (Citron et al., 1992; Cai et al., 1993; Citron et al., 1997). Transgenic mice expressing APP genes bearing the familial AD mutations exhibit some characteristics of the classic AD phenotype including neuritic plaques, age-dependent memory deficits and hyperphosphorylated tau (Games et al., 1995; Masliah et al., 1996; Hsiao et al., 1996; Nalbantoglu et al., 1997; Sturchler-Pierrat et al., 1997). AMYLOID PLAQUES, Ap FIBRILLOGENESIS AND CYTOXICITY Two distinct forms of A P plaques are found in the A D brain (Selkoe 1993). The classic senile or neuritic plaques are a critical diagnostic feature of AD. These plaques consist of fibrillar A P deposits that are associated with abnormal neuronal processes known as dystrophic neurites. Due to their P-pleated nature, these AP aggregates stain with dyes such as Congo Red or thioflavin. In contrast, diffuse plaques, commonly found in the aged brain, consist of nonfibrillar A p deposits that are not associated with neuronal pathology. The formation of these A P fibrils is thought to relate directly to A P cytotoxicity. The spontaneous formation of full-length synthetic A P i . 4 0 and A P i . 4 2 fibrils has been observed during incubation and this conformation has been shown to be neurotoxic (Mattson 1997). Moreover, A p secreted from cultured cells are able to form sodium dodecyl sulfate resistant oligomeric aggregates. The amino acids 35 and 41-42 appear to be especially critical to fibril formation (Mattson 1997). Specifically, fibrils are thought to consist of antiparallel strands in which amino acids 41-42 in one peptide strand interact with amino acids 34-35 of the second peptide monomer. A P i . 4 2 is much more amyloidogenic than its shorter counterpart, as it forms fibrils much more readily and can act as a 'seed' for A P i . 4 0 fibril formation. In this regard the process of A p fibril formation and neurotoxicity are strikingly similar to those of 2 other diseases where an insoluble protein in an aggregating state can cause the normal endogenous peptide to adopt an abnormal conformation (Welch and Gambetti 1998). In addition to peptide length, A(3 fibril formation is also dependent on species, amyloidogenic factors and local concentration. The amino acid sequence of human and rodent A3 differ by three amino acids near the N-terminal. Species-specific differences in A3's amino acid sequence may be a significant variable in the species specificity of A3 deposition and neurodegeneration (Mattson 1997). For example, several independent studies have shown that synthetic A3 having the rodent amino acid sequence does not readily form fibrillar aggregates and is not as neurotoxic as human A3- Enviromental factors promoting amyloid formation include metal cations, pH and lipoproteins (Nitsch and Growdon, 1994; Selkoe 1993; Mattson 1997). Finally, local concentrations of A 3 are governed by the rate of A3 formation versus the rate of its clearance. Clearance of A3 is achieved either by the proteolytical activity of soluble serine proteases or through receptor-dependent endocytosis (Qui et al., 1997; Paresce et al., 1996; Boland et al., 1996; Narita et al., 1997). Factors affecting A3 production will by reviewed extensively in the following paragraphs as these cellular processing decisions appear to be critical for production of either the potentially neurotoxic A3 or neurotrophic APPS. 3 FUNCTIONS OF APP S AND Ap The initial observation that neurons release APPS in response to neurotransmitters and electrical stimulation suggested that APPS may function in activity-dependent processes. In fact, many of the physiological roles of APPS in neurons appear to fall within two main categories: (1) neuroprotective functions owing to the ionic effects of APPS and (2) neurotrophic activities attributed to various functional domains located along its sequence (Fraser et al., 1997; Mattson 1997; Mattson et al., 1997). The neuroprotective function of APPS has been observed in vitro as hippocampal cultures pretreated with APPS were resistant to excitotoxicity and glucose deprivation (Mattson 1997; Mattson et al., 1997). This protective function may be due in part to its ionic effects. APPS has been shown to activate high-conductance charybdotoxin-sensitive potassium channels and supress Ca 2 + influx through both ligand-gated and voltage-dependent channels (Fraser et al., 1997; Mattson 1997; Mattson et al., 1997). The neurotrophic functions of APPS are now well established and many of its survival and neurite outgrowth-promoting properties have been localized to various domains along its sequence (Mattson 1997). For example, a heparin-binding domain near the N-terminus of APPS is involved in cell adhesion and regulation of neurite outgrowth. Moreover, a 5-amino acid sequence within APPS (RERMS) produced neurite elongation in vitro and increased both cortical synaptic density and memory retention in vivo. Although considerable data indicate that APPS subserves neurotrophic function, evidence that A P plays a survival-promoting role is not convincing. Rather, many of AP's cellular functions appear to be neurotoxic and its ionic effects directly oppose those of APPS (Mattson et al., 4 1997; Fraser et al., 1997). For example, APi_ 4 0 selectively inhibits K + channels, increases Ca 2 + conductance and impairs Na+-K+-ATPase thereby disrupting ion homeostasis (Mattson et al., 1997; Fraser et al., 1997). Moreover, AP increases neuronal vulnerability to excitotoxicity by promoting membrane depolarization, Ca 2 + influx and glutamate release (Mattson et al., 1997; Fraser et al., 1997). Mechanisms underlying this toxicity include membrane lipid peroxidation, reduced glutamate uptake and inhibition of glucose transport. Cholinergic neurons may be especially vulnerable to AP-induced toxicity as AP has been shown to supress acetylcholine synthesis and result in uncoupling of muscarinic receptor subtypes from their heterotrimeric G-protein (Gq) (Mattson et al., 1997). SECRETORY PROCESSING PATHWAYS APP is so named because it contains the AP peptide (39-42 amino acids in length) within its sequence. APP comprises a family of type 1 (spanning the membrane once and having the C-terminus intracellular) membrane spanning glycoproteins (Kang et al., 1987; Tanzi et al., 1987): nine APP gene transcripts generated by alternative splicing have been identified giving rise to proteins ranging from 365-770 amino acid residues. The three major APP isoforms expressed in the brain are APP 6 9 5, APP751 and APP770. Of these, APP695 is the only isoform lacking a 57-residue domain homologous to the family of kunitz serine protease inhibitors and is the isoform most highly expressed in neurons (Goedert, 1987; LeBlanc et al., 1991). APP matures while being transported through the secretory pathway, becoming N - and O-glycosylated and tyrosine-sulfated while moving through the tram-Go\g\ network (Weidemann et al., 1989). Immature APP may be cleaved before being trafficked through the 5 Golgi while the mature APP holoform is degraded rapidly as it moves through the cell and following arrival at the cell surface (Weidemann et al., 1989). Processing of APP is complex and can occur via several different routes (Checler, 1995; Selkoe et al., 1996; Nitsch et al., 1994). For the purposes of this review we will focus only on secretory pathways generating soluble APP fragments via two routes broadly catagorized as non-amyloidogenic (yielding a truncated APP derivative known as APPS and a truncated A P fragment known as p3) or amyloidogenic (yielding intact A P ) (Fig. 1) N 8=-° _ - o 0— 0— 0—. o— - 0 - 0 N APP. a Y P3 NON-AMYLOIDOGENIC P N Truncated APPC Ap AMYLOIDOGENIC Figure 1. Secretory Processing Pathways of APP. Processing of APP occurs via a nonamyloidogenic pathway (yielding soluble APPS and p3) or potentially amyloidogenic pathway (yielding soluble A P and a truncated APPS fragment 16 residues shorted than the a-secretase product). 6 Secretion of APPS and p3 Nonamyloidogenic processing of APP was the first pathway to be characterized. This processing route involves cleavage of APP at Lys 16 within the Ap sequence by an unidentified enzyme designated a-secretase (Esch et al., 1990; Anderson et al., 1991; Wang et al., 1991). The principal determinants of cleavage by a-secretase appear to be an ct-helical conformation around the cleavage site and the distance of the hydrolyzed peptide bond from the membrane (which occurs most efficiently at a distance of 12 amino acid residues from the plasma membrane on the extracellular side) but is apparently independent of sequence (Sisodia, 1992). As would be expected of an extracellular cleavage event, the presence of the cytoplasmic domain of the holoprotein is not an absolute requirement (Haass et al., 1993; Efthimiopoulos et al., 1994; Sisodia, 1992). Cleavage by a-secretase results in the release of a soluble N-terminal truncated APP fragment designated APPS and the retention of a 10 kDa C-terminal fragment at the cell membrane (Weidemann et al., 1989). This 10 kDa C-terminal fragment (also called plO) may undergo additional cleavage by an enzyme known as y-secretase which cleaves APP at the C-terminus of AP (Haass et al., 1993). The resulting 3 kDa C-terminal fragment of Ap known as p3 appears to be stoichometrically coupled to APPS (Busciglio et al., 1993) and like APPS, is thought to be a product of the nonamyloidogenic pathway (Haass et al., 1993; Haass et al., 1992). Both APPS and p3 are secreted by a variety of cultured cells and are found in human brain and CSF (Weidemann et al., 1989; Palmert et al., 1989; Oltersdorf et al., 1990; Selkoe et al., 1988; Schubert et al., 1989a; Schubert et al., 1989b). 7 Production and Secretion of Afi A n alternative physiological processing pathway for APP results in the release of intact A P (Busciglio et al., 1993; Haass et al., 1993). The AP segment begins on the extracellular side of APP, 28 amino acids from the membrane and extends 11 to 15 residues into the transmembrane domain. A P production from the mature APP holoprotein occurs via the sequential action of two enzymes termed P- and y-secretase. The P-secretase cleavage occurs first (Citron et al., 1995; Paganetti et al., 1996) generating a truncated APPS fragment (16 residues shorter than the a-secretase product) that ends at the N-terminus of the A P domain and is released into the extracellular space (Seubert et al., 1993). The remaining 11.5 kDa membrane associated fragment (Gabuzda et al., 1994) yields intact soluble A0 following a second cleavage event occuring at the C-terminus of the AP domain by y-secretase (Anderson et al., 1992). P-secretase cleavage is sequence specific: the majority of A P production occurs by cleavage of APP at the met-asp bond preceeding the AP N-terminus; substitutions at either met or asp substantially decrease AP production (Citron et al., 1995). Soluble Ap is secreted by a variety of cells and has been found in human CSF (Sisodia et al., 1990; Haass et al., 1992; Shoji et al., 1992; Busciglio et al., 1993; Seubert et al., 1992). Secretion of Afi^o vs. Ap^2 Secretase cleavage at the C-terminus of A P varies considerably, liberating a series of peptides ranging from 39-43 amino acid residues. The majority of these fall into one of two populations of A P known as AP1-40, terminating at amino acid 40 and accounting for approximately 90% of secreted Ap, or A P i . 4 2 terminating at amino acid 42 and accounting for 8 only 10% of secreted A p (Haass et al., 1992; Busciglio et al., 1993). One explanation for the variability of A p length is that C-terminal cleavage by y-secretase occurs in at least two intracellular compartments. This has been most convincingly demonstrated in neuronal cells where an unusually high percentage of intracellular A p is produced from immature APP (Wild-Bode et al., 1997; De Strooper et al., 1995; Hartman et al., 1997). In neurons, the longer isoform, AP1.42, has been shown to be generated predominantly in the endoplasmic reticulum and nuclear envelope (Cook et al., 1997; Hartmann et al., 1997) while the shorter AP1.40 is produced in the toms-Golgi network (Hartmann et al., 1997). Moreover, differential sensitivity to cleavage at position 40 and 42 by various protease inhibitors has also been observed (Higaki et al., 1995; Klafki et al., 1996a). Specifically, the calpain inhibitor MDL 28170, calpain inhibitor I and MG 132 preferentially inhibited y-secretase cleavage at residue 40 in a variety of cell lines (Klafki et al., 1996a; Citron et al., 1996). As the activity of y-secretase varies in different compartments it has been suggested that two forms of y-secretase exist with different cleavage sites (Hartmann et al., 1997). Alternatively, cellular compartmentalization itself may dictate cleavage preference: cleavage at position 42 might be facilitated at the thinner endoplasmic reticulum membrane while the greater thickness of the trans-Go\g\ network may lead to preferential cleavage of AP1.40 (Tischer and Cordell, 1996). 9 R E G U L A T I O N OF S E C R E T I O N N E U R O T R A N S M I T T E R R E G U L A T I O N O F A P P P R O C E S S I N G Regulation of APPS Secretion by Neurotransmitter Receptors in Cell Lines The first study indicating that APP processing can be regulated by neurotransmitters involved FJEK 293 cells overexpressing the human muscarinic acetylcholine receptor (Nitsch et al., 1992). Later studies extended these initial findings in a variety of cell lines overexpressing the muscarinic receptor: cholinergic agonists coincidently increased APPa release and decreased AP production (Hung et al., 1993; Buxbaum et al., 1994; Slack et al., 1995). Similarily, cholinergic regulation of APPS secretion has been shown to occur in cell lines expressing their normal complement of muscarinic receptors (Buxbaum et al., 1992; Wolf et al., 1995). Regulation of APPS release has been subsequently shown to occur for other neurotransmitters acting at G-protein-coupled receptors. These include the metabotropic glutamate receptor (Lee et al., 1997; Lee et al., 1995; Nitsch et al., 1997) and serotonin receptor (Nitsch et al., 1996a). G-protein-coupled receptor stimulation of APPS release has been shown to be selective for receptor subtypes that are coupled to phosphatidylinositol (PI) hydrolysis. For example, stimulation of HEK 293 cells overexpressing the Mi and M 3 receptor subtypes with the muscarinic agonist carbachol significantly increased APPS release while stimulation of the M 2 and M 4 receptor subtypes did not. Therefore, it was implied that stimulation of muscarinic receptors coupled to the phospholipase C (PLC) pathway increased APPS release while 10 muscarinic receptors linked to adenylyl cyclase did not. Similarily, application of serotonin to 3T3 cells stably overexpressing the receptors 5-HT2a or 5-HT2c stimulated PI turnover and APPS release in a dose-dependent manner (Nitsch et al., 1996a). Finally, glutamate increased both APPS release and PI hydrolysis in HEK 293 cells transiently expressing the metabotropic glutamate receptor la subtype (Lee et al., 1995), an effect that was antagonized by the metabotropic glutamate receptor antagonist MCPG (Nitsch et al., 1997). Regulation of APPS Secretion by Neurotransmitter Receptors in Central Neurons Neurotransmitter regulation of APP processing in central neurons has focused predominantly on cholinergic and glutamatergic innervation. Electrical depolarization of hippocampal slices induced a rapid increase in the release of both acetylcholine and APPS, effects that were inhibited by blocking voltage-sensitive sodium channels with tetrodotoxin (Nitsch et al., 1993). Similarily, the muscarinic receptor agonist bethanechol and cholinesterase inhibitors enhanced APPS release from cortical slices of the rat (Mori et al., 1995). Also, exposure of hippocampal slices to the mixed cholinergic agonist carbachol stimulated APPS release in the presence of the M 2 antagonist gallamine (Farber et al., 1995). Moreover, a selective M i agonist, WAL 2014 (Ensinger et al., 1993), enhanced APPS release from cortical slices at low concentrations. In contrast, in vivo studies have argued against positive regulation of APPS by nonselective muscarinic receptor stimulation as APPS release was elevated following lesion of the basal forebrain cholinergic neurons (Wallace et al., 1995). Some of these apparent discrepancies can be explained by the pharmacological complexity of G-protein-coupled receptor regulation 11 of APPS release. Specifically, M 2 receptor activation may be negatively coupled to APPS release thereby explaining both the biphasic nature of the WAL 2014 response (Ensinger et al., 1993) and the neccessity of gallamine in order to observe cholinergic stimulation of APPS release in hippocampal slices (Farber et al., 1995). However, this hypothesis is not consistent with the observation that bethanechol, a full agonist at muscarinic M 2 receptors enhanced APPS release from rat cortical slices (Mori et al., 1995). Like cholinergic receptor regulation of APPS release, glutamatergic regulation has also been shown to be receptor subtype specific, involving metabotropic glutamate receptors coupled to PI hydrolysis and Ca 2 + mobilization (Lee et al., 1995; Ulus and Wurtman, 1997; Kirazov et al., 1997). In hippocampal rat neurons, L-glutamate, QA and ACPD stimulated APPS release, an effect that was antagonized by both the PKC inhibitor GF 109203X and the metabotropic glutamate receptor antagonist L-AP3 (Lee et al., 1995). Likewise, in rat brain cortical or hippocampal slices, L-glutamate or a selective metabotropic agonist increased APPS release (Kirazov et al., 1997; Ulus and Wurtman, 1997) and this effect was blocked by the metabotropic glutamate receptor antagonist MCPG and the PKC inhibitor GF 109203X (Ulus and Wurtman, 1997). Furthermore, ionotropic glutamate agonists had either modest or no effect on APPS release in all of these preparations. However, these experiments were performed in the presence of Mg 2 + an voltage-dependent inhibitor of NMDA receptor function. Nevertheless, taken together, these studies raise questions about the relevance of natural regulation of APP catabolism by neurotransmitters in brain. These same studies demonstrate that selective activation of subsets of neurotransmitter receptors represents a plausible avenue of regulation of Af} production. 12 M U T U A L E X C L U S I V I T Y OF APP„ A N D A B P R O D U C T I O N Traditionally, it has been thought that APPS and A P production were mutually exclusive events (Nitsch and Growdon, 1994; Checler, 1995). However, several studies suggest that regulation of these two pathways does not always occur in a reciprocal manner. For example, in the human neuroblastoma cell line SY5Y, PKC activation stimulated APPS and p3 production while A P release remained unchanged (Dyrks et al., 1994). In addition, carbachol and phorbol 12-myristate 13-acetate (PMA) increased A P production from cells expressing the C-terminal 100 residues of APP (carrying the A P sequence beginning at the N-terminus) implying that the a- and y-secretase were regulated in the same way (Dyrks et al., 1994). A dissociation of phosphorylation events leading to the production of APP fragments was found in a pulse-chase study using a human glioma cell line and primary human astrocytes: although PMA decreased A P release, APPS levels were unchanged (Gabuzda et al., 1993). Also, short-term exposure to the proinflammatory cytokine interleukin-1 increased APPS release while having no effect on A p secretion (Vasilakos et al., 1994; Buxbaum et al., 1992; Buxbaum et al., 1994). Furthermore, in HEK 293 cells overexpressing APP 7 5i caffeine-stimulated Ca 2 + mobilization and the calcium ionophore A 23187 enhanced APP processing via both amyloidogenic and nonamyloidogenic pathways (Querfurth et al., 1997; Querfurth et al., 1994). Finally, in serum deprived human primary neuronal cultures, a four fold increase in A p release was observed while p3 production was unchanged (LeBlanc et al., 1995). Although these studies indicate that there are exceptions to the rule of mutual exclusivity, it is important to remember that they actually represent only a small sampling of the abundant research which 13 imply that, for the most part, these two pathways do appear to compete w i th each other (N i tsch and G rowdon , 1994; Checler, 1995). Nonetheless, it is important to be aware o f these exceptions as measurement o f one o f these pathways may not necessarily be indicative o f the other. 14 SECOND MESSENGER AND EFFECTOR REGULATION OF APP CATABOLISM Protein kinase C Protein kinase C (PKC) comprises a large family of serine/threonine i. kinases, which are activated, at least partially, by phospholipids. These are classified into three groups: conventional, new and atypical. Of these, diacylglycerol or its analog phorbol ester activates all conventional PKCs and new PKC isoforms. Conventional PKCs are the only ones which require Ca2 + as a cofactor. A strong case has been made for the role of PKC activation in the regulation of APP catabolism (for review see Nitsch and Growdon, 1994). Regulation of APP processing has been shown to be influenced by direct activation of PKC by phorbol esters in several continuous cell lines including PC12 (Buxbaum et al., 1990; Caporaso et al., 1992), CHO (Buxbaum et al., 1993), COS (Gabuzda et al., 1993), neuroblastoma (Dyrks et al., 1994), Swiss 3T3 (Slack et al., 1993) and HEK 293 cells (Jacobsen et al., 1994; Nitsch et al., 1992). Short-term activation of PKC by phorbol esters has been shown to coincidently increase APPS and decrease Ap release (Buxbaum et al., 1993; Gabuzda et al., 1993; Hung et al., 1993; Jacobsen et al., 1994) (Table 1). PKC-mediated stimulation of APPS has been shown to be specific insofar as downregulation of PKC blocked both phorbol ester stimulation of APPS release (Buxbaum et al., 1994) and phorbol ester inhibition of Ap release (Buxbaum et al., 1994; Hung et al., 1993). Moreover, these effects were blocked by the protein kinase C inhibitors H-7 (Gabuzda et al., 1993; Slack et al., 1993), staurosporine (Hung et al., 1993) and GF109203X (Slack et al., 1995). Furthermore, regulation of APP processing was not seen for the inactive phorbol ester analogues 4 a-phorbol 12,13-didecanoate (PDBu) 15 (Gabuzda et al., 1993; Caporaso et al., 1992). Finally, an analogue of diacylglycerol, the physiological activator of PKC, mimicked the effects of phorbol esters on APP processing (Gabuzda et al., 1993). Indirect evidence supporting a role for PKC phosphorylation in APP catabolism has come from studies using okadaic acid, an inhibitor of the serine/threonine protein phosphatases PP1 and PP2A (Cohen, 1990), known to inhibit the dephosphorylation and inactivation of PKC substrates. Okadaic acid augmented both phorbol ester stimulation of APPS release (Caporaso et al., 1992) and inhibition of Ap release by phorbol 12,13-dibutyrate (Hung et al., 1993; Buxbaum et al., 1993). The role of various PKC isoenzymes in regulating APP processing has not been addressed extensively. However, one study indicated that the conventional PKC isoenzyme PKC-a regulated APPS release in Swiss 3T3 fibroblast cells: although the total amount of APPS released was unchanged, the EC50 for PMA regulation of APPS release was lower in cell lines overexpressing PKC-a (Slack et al., 1993). Similarily, Kinouchi et al., (1995) found that fibroblast 3Y1 overexpressing the PKC isoenzymes a and s demonstrated increased basal APPS release while the PKC 5 overexpressing cells did not differ from control. Ca2+ The effects of Ca 2 + on APP processing are complex and appear somewhat contradictory. For example, Buxbaum et al. (1994) reported that thapsigargin, an irreversible inhibitor of sarcoplasmic-endoplasmic reticulum Ca 2 + ATPases (Thastrup et al., 1990) that prevents Ca 2 + reuptake in endoplasmic reticular stores, increased formation of APPS from CHO cells and human neuroglioma cells. Likewise, in HEK 293 cells overexpressing APP751, Querfurth et al. (1997) reported that the calcium reuptake inhibitors thapsigargin and cyclopiazonic acid potentiated caffeine-stimulated p3 release, a fragment stoichometrically coupled to APPS and therefore thought to be a product of the nonamyloidogenic secretory pathway. In contrast to these findings, Nitsch et al. (1996) reported that thapsigargin failed to change APP, release in 3T3 cells using the same concentrations of drug. Finally, in an earlier report using the Ca 2 + ionophore A23187 Nitsch et al. (1992) demonstrated that Ca 2 + did not increase APPS release from HEK 293 cells. The effects of Ca 2 + on A(3 release are equally complex. For example, Buxbaum et al. (1994) demonstrated a concentration-dependent effect of thapsigargin on A(3 release, increasing relative A P release at 10 nM while decreasing release at 20 nM. However, direct application of inositol trisphosphate, the second messenger thought to be responsible for releasing cytoplasmic calcium from intracellular stores, had no effect on A P release in permeabilized HEK 293 cells (Querfurth and Selkoe, 1994). Furthermore, a rise in intracellular Ca 2 + generated by A23187 or caffeine was shown to increase A P release from HEK 293 cells stably expressing A P P 7 5 1 (Querferth and Selkoe, 1994). Finally, thapsigargin and cyclopiazonic acid were both shown to potentiate caffeine-stimulated release of A p , presumably by inhibiting reuptake of Ca 2 + (Querfurth et al., 1997). Clearly, these contradictions cannot easily be explained by cell specific differences. However, some of these discrepancies may be explained by effects of these drugs on Ca 2 + within the acidic lumenal environment of the secretory pathway rather than overall intracellular Ca 2 + levels (Querfurth et al., 1997). For example, the weak base NH4C1 decreases A p and results in luminal Ca 2 + depletion of the trans-Go\g\ network and/or secretory vesicles. Likewise, an 17 increase in A3 secretion observed with calcium ionophores and caffeine may be a consequence of increased Ca 2 + sequestration within the acidic post-Golgi vesicles. The fact that N H 4 C I attenuates caffeine or A23187-induced stimulation of A 3 secretion supports this hypothesis (Querfurth et al., 1997). Phospholipase C Stimulation of heterotrimeric G-protein-coupled receptors activates PLCs. Three PLC isoforms designated 3 Y and 8 are activated by at least two different mechanisms. PLC-3 activation through serpentine receptors is mediated via heterotrimeric G proteins whereas PLC-y binds to tyrosine kinases or receptor-associated tyrosine kinases and is activated by phosphorylation. Given that PKC-mediated effects on APP processing were well established it followed that activation of the upstream effector PLC would also modulate APPS release. Indeed, mastoparan and mastoparan X, activators of PLC increased formation of APPS while decreasing production of A 3 (Buxbaum et al., 1993). Phospholipase A2 Stimulation of heterotrimeric G-protein-coupled receptors activates phospholipase A 2 (PLA2) (Farooqui et al., 1997a; Farooqui et al., 1997b). Receptor-mediated activation of PLA 2 generates free fatty acids (i.e. arachidonic acid) and lysophosphatidylcholine from membrane phospholipids (Farooqui et al., 1997a; Farooqui et al., 1997b). An initial study suggested that PLA 2 can partially mediate muscarinic receptor stimulation of APPS formation (Emmerling et al., 1993). Subsequent studies have extended these results to include both serotonergic and glutamatergic regulation of APP processing (Nitsch et al., 1996a; Nitsch et al., 1997). The PLA 2 inhibitors manoalide, dimethyleicosadienoic acid or oleyloxyethyl phosphorylcholine inhibited serotonin stimulation 18 of APPS secretion in a fibroblast cell line overexpressing the serotonergic 5-HT2a receptor (Nitsch et al., 1996a). Similarily, these same inhibitors antagonized glutamate receptor stimulation of APPS release in HEK 293 cells expressing the metabotropic glutamate receptor subtype la (Nitsch et al., 1997). Furthermore, melittin, a peptide which stimulates PLA 2 has been shown to augment APPS release in a variety of cell lines (Nitsch et al., 1997; Nitsch et al., 1996a). Likewise, inhibition of cyclooxygenase an enzyme which metabolizes arachidonic acid increases APPS release in human glioblastoma cells (Kinouchi et al., 1995). Indeed, coupling of the serotonergic 5-HT2c receptor to APPS secretion is thought to require both PLC and PLA 2 activities (Nitsch et al., 1996a). This may be expected as enhanced diacylglyerol-induced activation of some PKC isoforms have been observed by free fatty acids demonstrating a cooperativity between PLA and PLC in PKC activation (Spiegel et al., 1996). Cyclic AMP-Dependent Protein Kinase Recent evidence suggests that like PKC, cyclic AMP-dependent protein kinase (PKA) exerts effects on both constitutive as well as regulated APP processing. PKA mediated effects on constitutive secretory processing of APP vary between studies. Forskolin, an activator of adenylate cyclase, inhibited constitutive stimulation of APPS in a glioma cell line while 1,9-dideoxyforskolin, an inactive analogue, had no effect (Efthimiopoulos et al., 1996). Conversely, two independent studies found that either forskolin or 8-bromo cyclic AMP increased constitutive APPS release from PC 12 and HEK 293 cells (Xu et al., 1996; Marumbaud et al., 1996). Finally, Querfurth and Selkoe (1994) demonstrated that the cyclic AMP analogue, dibutyryl cyclic AMP, had no effect on constitutive Af3 release. Resolution of these apparent discrepancies is still a matter of investigation. In contrast to the variability seen for the effects of PKA on constitutive APPS 19 release, PKA has been shown to inhibit regulation of APP processing in all studies to date. For example, in a glioma cell line, a rise in intracellular cyclic AMP levels induced by stimulation of P-adrenergic receptors was shown to inhibit PKC stimulation of APPS release (Efthimiopoulos et al., 1996). A dose-dependent inhibition of PKC regulation of APP processing was also observed for dibutyryl cyclic AMP and forskolin (Efthimiopoulos et al., 1996). Similarily, phorbol ester and metabotropic glutamate receptor stimulation of APPS release was inhibited by either forskolin or dibutyryl cyclic AMP in cortical astrocyte cultures (Lee and Wurtman, 1997). REGULATION OF APP CATABOLISM IN RESPONSE TO STRESS Recent studies indicate that altered APP catabolism may arise as a result of stressful stimuli associated with either oxidant stress, metabolic compromise or programmed cell death. For example, in COS cells overexpressing APP695, inhibition of oxidative energy metabolism by sodium azide or the mitochondrial uncoupler carbonyl cyanide w-chlorophenylhydrazone increased the activity of (3-secretase resulting in an 80 fold increase in the production of a 11.5 kDa C-terminal derivative (Gabuzda et al., 1994). Radiosequence analysis confirmed that this C-terminal fragment of APP resulted from P-secretase cleavage and therefore was a potential processing intermediate in the generation of AP (Gabuzda et al., 1994). Similarily, both glucose deprivation and sodium azide decreased release of APPS from COS cells within a two hour exposure period while having no effect on APP expression levels (Gasparini et al., 1997). Treatment of COS cells with the antioxidant glutathione completely antagonized azide inhibition of APPS release (Gasparini et al., 1997). 20 Agent Spectrum of Cell System Effect on References Action Processing PMA, PDBu and OAG Activator of PKC CHO HEK 293 Human Astrocytes Human Glioma TAPPS l A p Buxbaum et al., 1993; Gabuzda et al., 1993; Hung et al., 1993; Jacobsen et al., 1994 Thapsigargin T[Ca2 + 1, CHO f or <H> APPS Human Neuroglioma Variable on Ap 3T3 Buxbaum et al., 1994 Nitsch etal., 1996a IP3 T[Ca 21, HEK 293 <-> Ap Querfurth and Selkoe, 1994 A23187 Ca + ionophore HEK 293 <-> APPS Nitsch et al., 1992; TAP Querfurth and Selkoe, 1994 Mastoparan Activator of PLC CHO TAPPS lAp Buxbaum et al., 1993 Melittin Activator ofPLA 2 3T3 HEK 293 TAPPS Nitsch et al., 1996a; Nitsch et al., 1997 Indomethacin Inhibitor of cyclooxygenase Human Glioblastoma TAPR Kinouchi et al., 1995 Forskolin Activator of Adenylate Rat Glioma, PC 12, t or I APPS Cyclase HEK 293 Efthimiopoulos et al. 1996; Xu et al., 1996; Marumbaud et al, 1996 dibutyryl cyclic AMP cyclic AMP analogue HEK 293 <-> Ap Querfurth and Selkoe, 1994 Table 1. A Summary o f Second Messengers and Ef fectors K n o w n to Regulate A P P Catabo l ism 21 Altered APP catabolism resulting from stressful stimuli in neuronal cell lines or central neurons has also been observed. For example, oxidative stress in neuroblastoma cells increased secretion of A P although APP expression levels were also increased (Yan et al., 1995). Similarly, serum free media induced apoptosis in human primary neuronal cultures resulting in a three fold increase in A P release and a corresponding, though somewhat more modest, decrease in APPS production (LeBlanc, 1995). Also, PC 12 cells maintained in serum free media with or without additional injurious agents released a 60 kDa C-terminal fragment containing the intact A P sequence (Baskin et al., 1991). Although extremely interesting, stress induced regulation of APP catabolism by these various stimuli remain nothing more than a phenomenology at present. Both the signaling cascades and the receptors involved in these regulatory pathways are presently unknown. However, a potential role for PKC has been suggested for altered APP catabolism induced by apoptosis (LeBlanc, 1995). TYROSINE KINASE-DEPENDENT REGULATION OF APP PROCESSING Stimulation of a wide range of receptors having intrinsic or associated tyrosine kinase activity have been shown to regulate APP processing. These include receptors for growth factors, (Schubert et al., 1989a; Refolo et al., 1989; Fukuyama et al., 1993; Clarris et al., 1994; Ringheim et al., 1997) cytokines, (Buxbaum et al., 1992; Buxbaum et al., 1994; Vasilakos et al., 1994), thrombin, (Davis-Salinas et al., 1994) and neurotransmitters (Slack et al., 1995). Studies on tyrosine kinase receptor stimulation of APP processing were among the first 22 indicating that APPS release was a regulated event. For example, stimulation of various tyrosine kinase receptors with either nerve growth factor, fibroblast growth factor or epidermal growth factor have all been shown to increase APPS (Schubert et al., 1989a; Refolo et al., 1989; Fukuyama et al., 1993; Clarris et al., 1994; Ringheim et al., 1997). These studies focused on the effects of long-term exposure to growth factors, at which time alterations of APP expression levels have been reported (Mobley et al., 1988; Gray and Patel, 1993; Lahiri and Nail, 1995; Ringheim et al., 1997). However, recent studies provide evidence that transient stimulation of growth factor receptors increase APPS release (Slack et al., 1997). Specifically, exposure of a human epidermoid carcinoma cell line to epidermal growth factor stimulates both APPS and PI turnover within 30 minutes (Slack et al., 1997). An increase in protein tyrosine phosphorylation has also been associated with stimulation of G-protein-coupled neurotransmitter receptors. However, tyrosine kinase activity has only recently been suggested to underly neurotransmitter regulation of APP processing (Slack et al., 1995) (see below). P K C - M E D I A T E D E F F E C T S OF N E U R O T R A N S M I T T E R S U P O N A P P P R O C E S S I N G Regulation of APP processing by G-protein-coupled neurotransmitter receptors has been shown to be mediated by PKC in a variety of cell types. For example, the PKC inhibitor staurosporine antagonized cholinergic receptor stimulation of APPS release in HEK 293 cells overexpressing the M i or M 3 receptor subtype (Nitsch et al., 1992; Slack et al., 1995). The PKC inhibitor staurosporine as well as the more specific inhibitor chelerythrine chloride inhibited glutamate stimulation of APPS release in HEK 293 cells overexpressing the metabotropic glutamate receptor subtype la (Nitsch et al., 1997). Down regulation of PKC by prolonged pretreatment with PMA also blocked glutamatergic stimulation of APPS release in these cells (Nitsch et al., 1997). A selective metabotropic glutamate receptor agonist stimulated APPS release from cortical astrocyte cultures. This effect was suppressed by the PKC inhibitor GF109203X (Lee and Wurtman, 1997). These PKC-mediated effects have been extended to include central neurons as the PKC inhibitor GF109203X inhibited APP8 release evoked by metabotropic glutamate receptor stimulation in rat hippocampal cultures (Lee et al., 1995). PROTEIN KINASE C-INDEPENDENT MEDIATED EFFECTS OF NEUROTRANSMITTERS UPON APP CATABQLISM Ca2*-Dependent Although PKC activation can regulate APP processing, neurotransmitter regulation of APP catabolism has been shown to occur in cells lacking functional PKC. Specifically, bethanochol was shown to regulate APPS and A(3 release in HEK 293 cells following downregulation of PKC (Buxbaum et al., 1994). Therefore, it was suggested that G-protein-coupled receptors regulated APP catabolism via either the PLC/PKC cascade or PLC/Ca 2 + cascade. Indeed, thapsigargin, a compound which raises cytoplasmic Ca 2 + by irreversibly inhibiting uptake of Ca 2 + into the endoplasmic reticulum, mimicked the effect of cholinergic agonists on APPS regulation in cells lacking functional PKC (Buxbaum et al., 1994). Tyrosine Kinase-Dependent Stimulation of growth factor receptors phosphorylates PLC-y, thereby increasing its enzymatic activity (Meisenhelder et al., 1989; Ronnstrand et al., 1992; Middlemas et al., 1994). Therefore, it has been suggested that growth factor stimulation of APPS secretion is mediated by the PLC/PKC signaling pathway. However, recent evidence suggests that tyrosine kinase receptor stimulation of APP processing may be largely PKC-independent. Epidermal growth factor receptor regulation of APPS was found to be predominantly PKC-independent as GF109203 X decreased the effect of epidermal growth factor by 35% at a concentration which completely inhibited the release of APPS by PKC (Slack etal., 1997). PKC-independent regulation of APP processing via G-protein-coupled receptors may also involve tyrosine kinases (Slack et al., 1995). In HEK 293 cells overexpressing the muscarinic acetylcholine receptors M i or M 3 , cholinergic receptor stimulation of APPS was only partially reduced by the PKC inhibitor GF109203X, while the response to PMA was abolished. Moreover, the increase in APPS elicited by carbachol was accompanied by increased tyrosine phosphorylation of several unidentified proteins and was reduced by the tyrosine kinase inhibitors genestein and tyrphostin A25. Finally, inhibition of tyrosine phosphatases by vanadyl hydroperoxide enhanced APPS release indicating that PKC-independent regulation of APPS release may involve tyrosine kinases (Slack et al., 1995). Phospholipase A^Dependent PLA 2 is also a potential candidate as the effector mediating PKC-independent regulation of APPS release via G-protein-coupled receptors. For example, in 3T3 cells overexpressing the 5-HT2a receptor, serotonin regulated APPS secretion was not inhibited by either pharmacological inhibition or down-regulation of PKC (Nitsch et al., 1996a). The hypothesis that PLA 2 may be the critical effector in the signaling cascade that couples 5-HT2a receptor to APPS was supported by the finding that several PLA 2 inhibitors blocked 5-HT2a receptor-mediated increase in APPS secretion. In contrast, both PLA 2 and PKC appear to be critical effectors in mediating regulation by other G-protein-coupled receptors (Nitsch et al., 1997). Whether these differences reflect cell-specific effects or receptor-specific effects is currently unknown. STEROL REGULATED APP PROCESSING A unique mechanism of regulation of APP cleavage was recently reported which correlates with cell membrane cholesterol content (Racchi et al., 1997; Bodovitz and Klein, 1996). Cellular cholesterol content is controlled either through intracellular synthesis or by uptake of cholesterol through the low-density lipoprotein (LDL) receptor pathway (Brown and Goldstein, 1986). A dose-dependent inhibition of APPS release was observed when COS cells were incubated with increasing concentrations of cholesterol (Racchi et al., 1997). Similarily, cholesterol, solubilized by methyl-(3-cyclodextrin or ethanol, reduced APPS release, while having no effect on cellular expression levels in HEK 293 cells (Bodovitz and Klein, 1996). This inhibition was specific as cholesterol increased secretion of several other cellular proteins. Both synthesis and uptake of cholesterol is tightly regulated by sterol regulating element binding proteins (SREBPs), membrane-bound transcription factors which are proteolytically 26 cleaved and then translocate to the nucleus where they regulate transcription of genes involved in cholesterol biosynthesis and uptake (Brown and Goldstein, 1997). Interestingly, several parallels have been drawn between APP and SREBPs. Like APP, membrane-associated proteolytic cleavage of the SREBPs is also highly dependent on sterol membrane content as cholesterol inhibits proteolytic processing of SREBP-1 and SREBP-2 in cultured cells (Brown and Goldstein, 1997). Also, SREBPs and APP are the only proteins known to be cleaved within a membrane-spanning segment suggesting that proteases involved in SREBPs and APP processing may be members of the same family (Brown and Goldstein, 1997). That both processes are regulated by membrane cholesterol content suggests that these proteins exhibit multiple functional similarities. The recent cloning of a putative metalloprotease required for cleavage of SREBPs may shed light on a similar intramembrane cleavage within the APP (Rawson et al., 1997). In neurons, it has been suggested that apolipoprotein E may regulate phospholipid and cholesterol content (Igbavboa et al., 1997): apolipoprotein E-lipoprotein complexes enter neurons by binding to the LDL receptor and LDL receptor-related protein thereby increasing cellular cholesterol content. Therefore, cholesterol effects on APP and SREBPs may be mediated by apolipoprotein E and its receptor. This idea is supported by experiments in transgenic mice where circulating cholesterol and apoE levels were inversely related to amounts of secreted APPS and A(3 in the brain (R. Scott: personal communication). Differential cholesterol and lipid uptake by apolipoprotein e3 and s4 (Poirier, 1994; Poirier et al., 1995) may also underly effects of these proteins on APPS release. In PC 12 cells, nanomolar levels of apolipoprotein s3 induced a rapid decrease in the secretion of APPS while 27 apolipoprotein s4 increased secretion of APPS (Wolozin et al., 1996). If, as in transgenic mice, APPS and Ap levels are regulated in the same manner, apolipoprotein s4 would be expected to increase secretion of A P as well. The actual mechanims whereby cholesterol alters cleavage of membrane bound proteins is not known. However, it has been suggested that increased cellular cholesterol, resulting in increased cholesterol membrane content, increases membrane rigidity (Yeagle, 1991) thereby decreasing the interaction of the various secretases with their substrate (Racchi et al., 1997). Altered activities of intrinsic membrane enzymes by changes in membrane lipid and cholesterol content sets a precedent for this mechanism of control (Mitchell et al., 1990; Criado et al., 1982). T H E M A P K P A T H W A Y : A POINT OF C O N V E R G E N C E FOR M U L T I P L E SIGNALS The data cited above predict the existence of an effector system that can be regulated in either a PKC-dependent or -independent fashion and may involve activation of tyrosine kinases. The mitogen-activated protein kinase (MAPK) signaling pathway meets all of these criteria. MAPKs also known as extracellular signal-regulated protein kinases (ERKs) are the terminal enzymes in a three level kinase cascade involving the sequential activation of Raf, MEK1/2 and ERK1/2. The MAPK pathway is activated by the same first and second messengers capable of regulating APP processing (Fig. 2). Moreover, both processes occur in a PKC-dependent and -independent manner. In the remaining chapters we investigate the signaling pathways which regulate APP catabolism in the CNS giving special consideration of the roles 28 played by PKC and MAPK. Identifying critical effectors in the signaling pathways controlling APP processing will undoubtedly increase our current understanding of the factors which contribute to the amyloid 'cascade' seen in AD. N M D A Figure 2. MAPK and APP catabolism. Many of the same first messengers regulate both APP catabolism and MAPK activation. This can occur in a both PKC-dependent and -independent manner. GENERAL HYPOTHESES Loss of basal forebrain cholinergic neurons projecting to the cortex and hippocampus is an invariant feature of AD pathology and is strongly correlated with the clinical phenotype of AD. Studies by Nitsch et al. (1992) showed that muscarinic receptor stimulation increased nonamyloidogenic processing of APP. These studies suggested for the first time that loss of 29 cholinergic input may be causally linked to the increased amyloid burden seen in Alzheimer's disease. However, these studies were limited by the fact that that these observations were made in cell lines overexpressing muscarinic receptors. Therefore, in our first study we repeated these experiments using central cortical neurons. Our general hypothesis is: loss of cholinergic innervation to the cortex and hippocampus increase AB deposition in AD. The findings from our first study and those of others (Wallace et al., 1995) cautioned against acceptance of this hypothesis. Moreover, it became apparent in the literature that the downstream effects of various first messengers known to regulate APP processing were more complex than had first been thought. We postulated that regulation of APP processing is dependent on activation of intracellular 'targets' or effectors rather than stimulation of specific receptors. At the time these experiments were initiated PKC was a well characterized effector, however, its activation was not an absolute requirement for regulation of APP catabolism. An alternative candidate which appeared to meet all of the criteria for an effector mediating regulation of APP catabolism was MAPK. Our general hypothesis is: altered signal transduction and decreased MAPK activation increase brain amyloid burden in AD. Finally, in addition to amyloid, a second hallmark of AD is the presence of neurofibrillary tangles (NFT) composed primarily of another insoluble protein. Tau, a microtubule associated protein is hyperphosphorylated in AD leading to the formation of these NFTs. Kinases within the MAPK family associated with stress activated pathways phosphorylate tau and may increase this activity in the presence of the neurotoxic peptide A(3. Therefore, our final hypothesis is: A B stimulates SAPK activation in neurons resulting in tau hyperphosphorylation. 30 H. CHOLINERGIC AGONISTS DO NOT REGULATE THE RELEASE OF APP S IN CULTURED RAT CORTICAL NEURONS I N T R O D U C T I O N AB, the principle constituent of senile plaques found in the AD brain (Hardy, 1997; Selkoe, 1997), is derived by proteolysis of an integral membrane protein known as the APP via at least two pathways. One involves activation of an unidentified enzyme known as oc-secretase, cleaving APP within the Ap sequence (Sisodia et al., 1990; Anderson et al., 1991; Wang et al., 1991), precluding Ap generation and releasing a soluble APPS into the extracellular space. The alternative route involves two unidentified enzymes termed P- and y-secretase, which cleave APP on the amino- and carboxy-termini of AP, respectively. The resultant AP is then released into the extracellular space (Haass et al., 1992; Shoji et al., 1992). Some early onset autosomal dominant forms of AD are strongly linked to mutations in the APP gene (see Hardy, 1992 for review). These mutations are associated with increased production of AP (Cai et al., 1993; Citron et al., 1992; Suzuki et al., 1994) or an increased formation of more amyloidogenic forms of AP in vitro. Transgenic mice expressing one of these mutations in the APP gene exhibit many of the pathological signs of AD, including neuritic plaques with thioflavin-S positive deposits of AP (Games et al., 1995). Decreased release of APPS has been documented in the cerebrospinal fluid of AD patients (Farlow et al., 1992, Van Nostrand et al., 1992). These and other data strongly suggest that mismetabolism of APP or Ap is central to the disease process (Selkoe, 1993). Accelerated metabolism of APP via the nonamyloidogenic a-secretory pathway as a means of decreasing Ap production has been proposed as a potential area of therapeutic intervention (Buxbaum et al., 1993). Studies on the cellular mechanisms which control APP metabolism have emphasized the role of PKC and neurotransmitter receptors linked to the PLC signal transduction pathways (Nitsch and Growdon, 1994). Activation of PKC in continuous cell lines has been demonstrated to accelerate the processing and release of APPS while coincidently inhibiting soluble AP production (Hung et al., 1993; Gabuzda et al., 1993; Buxbaum et al., 1993). Similarily, cholinergic agonists have caused an increase in the release of APPS in cell lines over-expressing muscarinic receptors (Buxbaum et al., 1992; Nitsch et al., 1992) and concomitant decrease in AP release (Hung et al., 1993). Although the PKC signal transduction pathway has been implicated in the regulation of APPS release, few studies have tested this hypothesis in central neurons (Farber et al., 1995; Mori et al., 1995). In the present investigation, we examined whether direct or indirect stimulation of PKC increases the release of APPS in primary cultures of rat cortical neurons. We show that stimulation of PKC by the addition of phorbol esters does indeed increase the release of APPS but activation of a well known PKC-coupled cell surface receptor did not significantly increase the release of APPS. 32 M A T E R I A L A N D M E T H O D S Cell Culture Timed pregnant Sprague-Dawley rats were anesthetized with halothane at 15-17 days gestation and the cerebral cortex was removed from the rat embryos (Soderback et al., 1989). Tissue was collected and stored in Hanks buffer (4°C). Following the dissection, Hanks buffer was aspirated and the tissue pieces were dissociated by mild trituration in Dulbecco's Modified Minimum Essential Media containing NI supplements (Bottenstein et al., 1980) + 10% fetal calf serum. A single cell suspension was plated on 12 well tissue culture plates precoated with poly-D-lysine (0.1 mg/ml) at 1. lxlO6 cells/well. After 7 days in vitro, cells were exposed to cytosine arabinoside (10 5M) in media overnight to halt nonneuronal cell growth and enrich for cortical neurons: approximately 50% of the cells stained positively for neuron specific enolase at the time cultures were exposed to various pharmacological agents. Media replacement was carried out on a biweekly schedule and cells were maintained at 37°C in 5% C0 2 . After 15-16 days in vitro cultures were washed with Hepes-buffered saline and immediately exposed to the pharmacological agents phorbol esters, oxotremorine-M, atropine or KC1 in Minimum Essential Media containing 25 mM Hepes or carbachol in Dulbecco's Modified Minimum Essential Media containing NI supplements without fetal calf serum for 1 hour. Phorbol esters were diluted from concentrated (20mM) stocks made up in dimethyl sulfoxide. The response of cells to media containing the drug vehicle was not different from media alone as determined by two-tailed t test (p > 0.05; n=6). Measurement of APP and Amyloid Precursor-Like Protein Immediately following the 1 hour exposure period, media was removed and phenylmethylsulfonyl fluoride (PMSF) was added (2 mM final concentration). The media was subsequently centrifuged at 10 000 xg for 15 min. to remove cellular debris. One ml of the media was then concentrated to 0.2 ml by low speed centrifugation (4200 x g) at 4°C using Millipore filters having a molecular weight cut off of 30, 000. One ml of double distilled H 2 0 (4°C) containing 2 mM PMSF was added and the sample was concentrated as before. This step was repeated 6 times to ensure that concentrated sample was completely desalted. Following concentration and desalting, the sample was frozen and lyophilized overnight. Cells were lysed in an extraction buffer containing 1% Nonidet P-40 and 1% sodium deoxycholate and centrifuged for 10 min. at 16 000 x g to remove detergent-insoluble material. Laemmli SDS sample buffer was added to concentrated secreted proteins or lysates and the samples were boiled for 2 min. Secreted and cellular protein was measured with the bicinchoninic acid assay (Pierce). Unless stated otherwise, reconstituted culture media proteins corresponding to 4 p.g of total secreted protein and cellular protein corresponding to 4 u.g of total cellular protein were separated by SDS-polyacrylamide gel electrophoresis on 10% gels. Following gel electrophoresis proteins were transferred to Hybond™ ECL™ nitrocellulose membrane at low voltage (30 °V) overnight. The membranes were blocked in Tris Buffered Saline containing 0.05% Tween-20 (TBS-T) and 5% milk. Membranes were rinsed 3 times with TBS and incubated in TBS-T containing 1% Bovine Serum Albumin and 1° antibody overnight. The 1° antibodies used and their dilutions are as follows: 22C11 (1: 200; anti-PreA4 monoclonal antibody, clone 22C11, Weidemann et al., 1989, purchased from Boehringer Mannheim) that recognizes a region between amino acids 66 and 81 of APP695 34 (Hilbich et al., 1993) or the affinity purified anti-GID polyclonal antibody (1:500; Cole et al., 1991) while the amyloid precursor-like protein (APLP; 1: 2000) was detected using polyclonal antibody D2-I raised against mouse APLP2 (Thinkakaran and Sisodia, 1994) (Fig. 3). The secondary horseradish peroxidase-linked antibodies (1:5000) were visualized by enhanced chemiluminescence (Amersham) using hyperfilm. Immunoreactive bands were compared densitometrically with the use of a Molecular Dynamics image quantifier. Densitometric measurements were performed in the linear range as determined by standard dilution curves of secreted cellular proteins. For each trial, densitometric analysis of control APPS secretion was arbitrarily defined as 1; measurement of pharmacological effects upon APPS secretion from the same western blot was then scaled to the control measurement. Each trial represents cells harvested from a different animal and plated separately. All comparisons within a single trial were made on matched sister cultures derived from a single plating. Analysis of variance followed by Fisher's post-hoc analysis was used to determine the significance of observed differences. Measurement of Phosphoinositide Turnover Accumulation of [3H]CDP-Diacylglycerol formation ([3H]CDP-DAG) (Godfrey, 1989; Hwang et al., 1990) was measured using cultures kept in vitro for 14-16 days according to the method of Murphy et al. (1992). In brief, cultures were prelabelled with 1.5 uCi/ml of [TTJcytidirie (25 u. Ci/mmol; no.32, 206-7; Sigma Chemical Co.) in a Hanks' balanced salt solution for 40 minutes. 2.5 mM LiCl was added to all cultures in the presence of 1.5 pCi/ml of [3H]cytidine 20 minutes before drug treatment. Cells were incubated with cholinergic drugs in the presence of 2.5 mM LiCl and 1.5 uCi/ml of [3H]cytidine for 1 hour. Incubations 35 were terminated with the addition of 1 ml ice-cold chloroform/methanol (1:2 vol/vol). Phase separation was accomplished by adding 0.8 ml of chloroform and 0.8 ml of water and centrifuging at low speed. The organic phase was collected, washed once with 2.0 ml of 1M HCl/methanol, evaporated to dryness and radioactivity counted by liquid scintillation spectrophotometry. Measurement of [3H] NMS Binding to Intact Cortical Neurons Measurement of N-[3H] methylscopolamine ([3H] NMS) binding to intact cortical neurons was performed using a modified assay of Eva et al. (1990). In brief, cortical cultures were washed with phosphate-buffered saline with Ca 2 + and Mg 2 + (PBS+) and incubated in saturating concentrations (1 nM) of [3H] NMS in PBS+ for 1 hour (37°C). Nonspecific binding was determined by binding in the presence of 1 uM atropine. Following this incubation period, cultures were washed with PBS+ and phosphate buffered saline lacking Ca 2 + and Mg 2 + (PBS") containing 1% SDS and 1% Triton-X was added to lyse the cells. Radioactivity of the cell lysate was counted by liquid scintillation spectrophotometry. Cellular protein was measured in sister wells with the bicinchoninic acid assay (Pierce). 36 R E S U L T S Detection of APPS in the Culture Medium of Rat Cortical Neurons. Soluble proteins in the medium of rat cortical neurons were subjected to Western blot analysis using the monoclonal antibody 22C11 raised against a full-length APP fusion protein (Fig. 3). The immunoreactive proteins with apparent molecular masses ranging from 100-130 kDa are similar to those of secreted amino-terminal APPS derivatives previously described in cell culture media and human cerebrospinal fluid (Nitsch and Growdon, 1994). 22C11 A n t i - G I D N B a A I WO-2 o— o— o— 0— 0— 0— 0— o— —0 —0 —0 -0 -0 —0 —0 —0 —0 Figure 3. Schematic of APP with antibodies specific for different epitopes of APP or Ap. The N-terminal antibodies 22C11 and Anti-GID recognize both APPS generated by a-secretase as well as the truncated APPS generated by P-secretase while the Ap antibody WO-2 recognizes only the APPS generated by a-secretase. Phorbol Esters Increase APPS Release PKC stimulation by phorbol esters has been shown to dramatically increase the release of amino-terminal APPS derivatives in a wide variety of cell lines (Buxbaum et al., 1993; 37 Gabuzda et al., 1993; Hung et al., 1993; Buxbaum et al., 1992; Caporaso et al., 1992). To determine if phorbol esters can regulate APPS production in central neurons we examined primary cultures of rat cortical neurons. A one hour time course was chosen as this representated a time interval long enough for APPS detection and short enough to avoid a PKC-mediated increase in cellular expression levels of APP (Trejo et al., 1994; Lahiri and Nail 1995). Cultures were exposed to phorbol esters or drug vehicle for 1 hour and media was collected immediately following this exposure period. The levels of APPS increased significantly when the cells were incubated with either the phorbol ester PDBu or PMA. PDBu markedly stimulated APPS in a dose-dependent manner. Neuronal cultures incubated with 1 uM PDBu and 10 uM PDBu increased APPS release by 210.3 % ± 27.4 and 277.2 % ± 31.4, respectively (n=3 p < 0.05; Figs. 4a and 4b). Cortical cultures incubated with 1 uM PMA increased APPS generation by 199.3 % ± 34.0 (n=3 p < 0.05; Figs. 4a and 4b). Cholinergic Receptor Stimulation Does Not Increase APPS Release APP processing can be regulated in cell lines by the stimulation of cell-surface receptors linked to activation of phospholipase C and PKC (Buxbaum et al., 1992; Nitsch et al., 1992; Buxbaum et al., 1993; Hung et al., 1993). In particular, an increase in APPS production has been demonstrated with drugs that stimulate the muscarinic acetylcholine receptors Ml or M 3 (Nitsch et al., 1992; Hung et al., 1993). To determine whether or not muscarinic receptor stimulation can augment APPS production in neuronal cultures we examined the effect of the muscarinic receptor agonist oxotremorine-M on basal levels of APPS. We observed that APPS production in culture wells incubated for 1 hour with 10 uM oxotremorine-M was not 38 B 4.0 0.0 o O O Q-Q °-1 - o A P P C kD 9 7 . 4 -Figure 4. PKC stimulation increases APPS release. A. Densitometric analysis of the effect of PDBu and PMA on basal APPS. Cultures were exposed to phorbol esters or drug vehicle for 1 hour and media was collected immediately following this exposure period. Results are the means ± S.E.M. of 3 experiments performed in duplicate.*, Different from control (p < 0.05); , Different from all other treatment groups (p < 0.05). B. Representative western blot of APPs fragments released by rat cortical neurons alone or in the presence of 1 and 10 pM PDBu or 1 uM PMA, as indicated above. Cortical APPS fragments were detected with the monoclonal antibody anti-Pre A4. 39 statistically different from control (w=5 p > 0.05; Fig 5a and 5b). This did not appear to be caused by saturation of the receptor by endogenously released neurotransmitter, as APPS release in culture wells incubated with the muscarinic receptor antagonist atropine was not different from control wells. Similar results were found for the cholinergic agonist carbachol (Fig. 6). Cultured Cortical Neurons Express Functional Muscarinic Receptors In order to test for expression of muscarinic receptors in our rat cortical cultures, we carried out binding studies using [3H] NMS. In 3 experiments, [3H] NMS binding was 71.8 fmol/mg protein +11.1 (S.E.M.). This level of expression is comparable to that of other rat cortical cultures (Eva et al., 1990). However, it is worth noting that the percentage of cultured cortical neurons expressing muscarinic receptors as well as the specific subtypes expressed is unknown. In order to determine whether the muscarinic receptors in our cortical cultures were functionally coupled to the PLC pathway, we measured PI turnover following exposure to muscarinic receptor agonists by assaying for stimulation of CDP-DAG accumulation. CDP-DAG accumulation was measured in the presence of lithium which decreases intracellular inositol by inhibiting inositol monophosphatase thus preventing further metabolism of CDP-DAG to PI. Although diacylglycerol formation can be produced by phospholipase D, the presence of lithium selects for formation of diacylglycerol due to PI turnover. Moreover, CDP-DAG formation has been shown be a particularly accurate measure of inositol phosphate A B Figure 5. Oxotremorine-M does not increase the formation of APPS. A. Densitometric analysis of the effect of oxotremorine-M on basal APPS. Results are the means ± S.E.M of 5 experiments performed in duplicate. None of the treatments were significantly different from control (p > 0.05). B. Representative western blot of APP fragments secreted in 1 hour by cultured rat cortical neurons alone or in the presence of 10 uM oxotremorine-M (OX-M) with or without 1 pM atropine (ATR) as indicated above. Cortical APPS fragments were detected with the monoclonal antibody anti-Pre A4. 41 A Figure 6. Carbachol does not increase the release of APPS. A. Densitometric analysis of the effect of carbachol on basal APPS corresponding to 500 pg of cellular protein. Results are the means ± S.E.M of 3 experiments measured on separate neuronal culture preparations. Sister cultures exposed to carbachol were not different from control (p > 0.05 by paired t-test). B. Representative western blot of APPS fragments secreted by cultured rat cortical neurons alone or in the presence of 1 mM carbachol (Carb). Cortical APPS fragments were detected with the monoclonal anti-Pre A4. 42 formation for muscarinic receptor activation in the cortex and hippocampus (Heacock et al., 1993). Application of 10 uM oxotremorine-M to cortical neurons resulted in a 10-fold increase in PI turnover, and this effect was readily blocked by the muscarinic receptor antagonist atropine; 1 mM carbachol resulted in an even larger increase in PI turnover (Table 1). In order to determine whether these increases in PI turnover were reflective of PKC activity, we carried out preliminary experiments (n=2) on neuronal cultures exposed to the various pharmacological agents for 10 min. Histone HI phosphorylation in the cytosolic fraction expressed as percent control for 10 u,M PDBu, 10 oxotremorine-M and 1 mM carbachol was 79%, 115% and 121%, respectively while in the membrane fraction it was 282%, 108% and 123%. Therefore, the phorbol ester PDBu appeared to increase translocation of PKC and robustly stimulate particulate PKC activity while the cholinergic agonists produced comparatively little PKC translocation and only a modest increase in cytosolic PKC activity. These data suggest that muscarinic receptor stimulation of the PKC pathway is not sufficient to alter APPS release in cortical neuronal cultures. The Effect of Membrane Depolarization on APPS Regulation of APP processing by electrical depolarization has been demonstrated using rat hippocampal slices (Nitsch et al., 1993). To determine whether neuronal depolarization can also increase APPS release in primary cultures, cortical neurons were exposed to 35 mM KC1 for 1 hour. KCl-induced depolarization did not significantly alter APP processing (w=3 p > 0.05; Fig 7a and 7b). Because cortical cultures are spontaneously active, we considered the 43 Treatment % of control [ 3 H]CDP-DAG Oxotremorine-M (10 uM) 1189 ±282 Oxotremorine-M (lOuM) + Atropine (luM) 162 ± 7 2 Carbachol (ImM) 3156 =t 677 Table 2. PI turnover associated with cholinergic receptor stimulation. Results have been normalized to control (cultures that were treated only with 2.5 mM LiCl). The cpm for the control samples were 2169 ± 717 S.E.M. 10 uM oxotremorine-M and 1 mM carbachol produced significant elevations in [3H] CDP-DAG content as determined by two-tailed t test (p < 0.05). Data are expressed as mean ± S.E.M. (n=3). 44 A Figure 7. KCl-induced depolarization does not increase the formation of APPS. A. Densitometric analysis of the effect of KC1 on basal APPS release. Results are the means ± S.E.M. of 3 experiments performed in duplicate. None of the treatments were significanly different from control (p > 0.05). B. Representative western blot of APPS release from cortical neuronal cultures under basal conditions, or in the presence of 35 mM KC1 with or without 1 uM tetrodotoxin (TTX). Cortical APPS release was detected using the monoclonal antibody anti-Pre A4. 45 possibility that spontaneously released transmitter might be sufficient to activate phosphoinositol turnover (Murphy et al., 1992) and thereby tonically increase APPS release. However, application of 1 uM TTX, a treatment which blocks voltage-sensitive sodium channels necessary for the generation of action potentials, did not alter basal APPS release (Fig 7). Ideally, a pre-equilibration period in media containing TTX would ensure that basal levels of tonic activity were lowered. However, a long exposure in serum free conditions would not be ideal due to the potential onset of apoptosis (LeBlanc 1995). The Effect of Cholinergic Receptor Stimulation and PKC Stimulation on APPS and APLP Release. During the course of these experiments it was discovered that the anti-Pre A4 used in this study cross-reacts with members of the APLP family (Wasco et al., 1992; Wasco et al., 1993; Slunt et al., 1994). As further confirmation that the pattern of protein release was representative of APPS additional trials were performed and secreted APPS was quantitated using the affinity purified anti-GID polyclonal antibody raised against peptide 175-186 of APP (Cole et al., 1991). The sequence of APLP shows poor homology in the APP176-186 region and therefore, the anti-GID antibody should not cross-react with APLP. As with APPS release analyzed using the anti-PreA4 antibody, APPS production in culture wells incubated with 1 uM PDBu was well above basal levels, while those incubated with 10 uM oxotremorine-M were not different from control (n=3; Fig 8). The effect of these same drug treatments on APLP2 release was also studied. APLP2 is a protein within the APP family of membrane glycoproteins and is recognized by the anti-pre A4 antibody (Thinkakaran and Sisodia, 1994). Parallel western blots were probed with the polyclonal antibody D2-I raised against mouse 46 APLP2 (Thinkakaran and Sisodia, 1994). D2-I is specific for APLP2 and does not cross-react with APP or APLP1 (Thinkakaran and Sisodia, 1994). Like APPS, APLP2 release in cortical cultures increased following exposure to 1 u.M PDBu while those exposed to 10 u.M oxotremorine-M were similar to control (n=3; Fig 8). 47 A B A P P kD C e l l C M C e l l C M 1 2 3 4 5 6 97- ' m * mm mm * 4 mm 68-46-31-20-A P L P C e l l C M C e l l C M 1 2 3 4 5 6 kD • 97-68-46-31-20-mm 9m mj ** W^^' ^^^^ Figure 8. PKC stimulation but not cholinergic receptor stimulation increases release of APPS and APLP. Parallel western blots of cellular (cell) and secreted (CM, conditioned media) proteins from cultured rat cortical neurons alone (lanes 1, 2, 4, 5) or in the presence of either 1 pM PDBu (lane 3) or 10 uM oxotremorine-M (lane 6). A. APP was detected using the polyclonal antibody anti-GID. B. APLP was detected using the polyclonal antibody D2-I. Immunoblots are representative of 3 separate experiments performed in parallel. 48 DISCUSSION In the present study, we examined whether or not first and second messengers of cholinergic neurotransmission increase APPS release. We found that while APPS release can indeed be regulated by direct activation of PKC with phorbol esters, muscarinic receptor stimulation did not statistically increase APPS release above basal levels. However, a potential confound in the interpretation of the present data is the fact that the N-terminal antibodies which we employed recognized APPS generated by both a-secretase and P-secretase cleavage (Fig. 3). Although aware of this complication, antibodies which recognized APPS generated by the nonamyloidogenic processing route were not available at the time these studies were performed. Although the data show an overall increase in APPS by PKC activation, additional experiments using the more selective antibody WO-2 (Fig. 3) would need to be performed in order to firmly establish that this increase in APPS production correlates with a decrease in A P production (Fig. 13). Nevertheless, a large number of studies have shown that PKC regulates APPS and A P release in a mutually excusive manner (Mills et al., 1997; Gabuzda et al., 1993; Buxbaum et al., 1994; Buxbaum et al., 1993; Hung et al., 1993). Regulation of APP processing has been shown to be influenced by direct activation of PKC by phorbol esters in a number of cell lines including PC 12 pheochromocytoma (Buxbaum et al., 1990, Caporaso et al., 1992), CHO (Buxbaum et al., 1993), human glioma (Hs 683) (Buxbaum et al., 1993), COS (Gabuzda et al., 1993), and human embryonic kidney 293 cells (Gillespie et al., 1992). Our data indicate that this phenomenon can be extended to include cortical neurons in culture and is in agreement with recent reports showing that phorbol esters increase APPS release in primary hippocampal neuronal cultures (Lee et al., 1995). Therefore 4 9 the relevance of PKC-mediated regulation of APPS both in cell lines and central neurons is firmly established. Cell lines over-expressing muscarinic acetylcholine receptors subtypes M i or M 3 show increased release of APP, upon addition of cholinergic agonists (Buxbaum et al., 1992, Nitsch et al., 1992, Buxbaum et al., 1994, Slack et al., 1995), an effect that is antagonized by a variety of protein kinase inhibitors (Nitsch et al., 1992, Slack et al., 1995). Similarily, cholinergic regulation of APPS secretion has been shown to occur in cell lines expressing their normal complement of muscarinic receptors (Buxbaum et al., 1992, Wolf et al., 1995). In contrast, we were unable to demonstrate any statistically significant change in APP, release from cortical neurons when their natural complement of muscarinic receptors were stimulated. These receptors were fully functional, as evidenced by increases in PI turnover which were comparable to those seen in cortical tissue taken from the adult rat brain (Godfrey, 1989). However, the observation that only modest PKC stimulation was evoked by muscarinic receptor activation may suggest that only those conditions which produce robust increases in PKC activity enhance APP, secretion in central neurons. The evidence that cholinergic agonists regulate APP, release in central neurons is somewhat contradictory at present. In agreement with our findings, recent in vivo studies argue against positive regulation of APP, by acetylcholine, as APP, release was elevated following lesion of the basal forebrain cholinergic neurons (Wallace et al., 1995). Similarily, Farber et al., (1995) found that the nonspecific cholinergic agonist carbachol had no effect on APP, release from rat hippocampal or cortical slices. 50 In contrast to these studies, other data indicate that cholinergic neurotransmission can alter APPS release in the mammalian brain. Exposure of hippocampal slices to carbachol in the presence of the selective M 2 antagonist gallamine significantly increases APPS production (Farber et al., 1995). Moreover, a more slective Mi agonist WAL 2014 (Ensinger et al., 1993) enhanced APPS release from cortical slices at low concentrations while lacking this effect at higher concentrations. The biphasic nature of the WAL 2014 response and the effect of gallamine suggest that M 2 receptor activation inhibits APPS production. Both cholinesterase inhibitors and muscarinic receptor stimulation with bethanechol enhanced APPS release from cortical slices of the rat (Mori et al., 1995). If, as suggested above, the M2 receptor is negatively coupled to APPS production, this finding is surprising given that bethanechol is a full agonist at the M 2 receptor and only a partial agonist at muscarinic receptors positively coupled to phospholipase C (Richard, 1990). Finally, in vivo data from patients receiving lithium or antidepressants, drugs possessing relatively non-specific anticholinergic properties have lower CSF APPS levels than controls (Clarke et al., 1993). Taken together, these findings suggest that cholinergic receptor stimulation of APPS production in central neurons is pharmacologically complex. Critical assessment of APPS regulation by the various cholinergic receptor subtypes and their corresponding second messenger pathways may explain these apparent discrepancies. Electrical depolarization of hippocampal slices has been shown to increase release of APPS with increasing stimulation frequencies from 0 to 30 Hz. This effect was presumably due to the release of endogenous transmitters (Nitsch et al., 1993). However, when we exposed 51 cortical neuronal cultures to 35 mM KC1, a treatment which should result in marked depolarization accompanied by both calcium entry and massive neurotransmitter release, APPS release was unchanged. This finding is puzzling as in both cortical cultures and hippocampal slices, the main neurotransmitter released by such treatment would be glutamate, which would be expected to activate protein kinase C either via metabotropic glutamate receptor stimulation, or by calcium entry through NMDA receptor-gated channels . Indeed both depolarization-induced increases in intracellular Ca 2 + (Querfurth and Selkoe, 1994; Buxbaum et al., 1994) and metabotropic glutamate receptor stimulation (Lee et al., 1995) would be expected to stimulate an increase in APPS production. Nevertheless discrepancies in the two preparations are worthy of mention. In the hippocampal slice preparation, the terminals of hippocampal afferents would be intact, and depolarization could result in release of a considerable array of neurotransmitter agents that would undoubtedly differ from those released in cortical cultures (for review see Nicoll et al., 1990). These include, acetylcholine, norepinephrine and dopamine. In addition, in the hippocampal slice preparation, depolarization was induced electrically while in cortical neuronal cultures depolarization was induced chemically. Finally, PKC isoenzyme expression and compartmentalization may differ between intact and dissociated neuronal cell preparations (Shimohama et al., 1991). One or more of these discrepancies may enable electrical stimulation to activate PKC in hippocamal slices, sufficiently, so as to result in the regulation of APPS release. From these observations we conclude that regulated APPS release can occur in cortical neurons, as stimulation of PKC with phorbol esters reliably increased APPS release. However, stimulation of the natural complement of muscarinic receptors (as expressed in cultured 52 cortical neurons) did not result in a statistically significant change in APPS. Several possible explanations for this apparent discrepancy exist. PKC activation by phorbol esters is more robust and more likely to result in PKC translocation than that evoked by muscarinic receptor stimulation (Nishizuka 1992). In this regard, a particular limitation of the present study is the fact that the PKC concentrations used are clearly supraphysiological. The effect of nanamolar concentrations would better mimic the magnitude of PKC activation expected by neurotransmitter receptor stimulation (Miyamae et al., 1995; Zoukhri et al., 1997) and allow for a more accurate comparison. Preliminary experiments using a mutant herpes simplex virus to overexpress the human M i receptor were initiated as a follow-up to these studies in order to augment muscarinic receptor stimulation of PKC. However, technical difficulties prevented the completion of these studies (Appendix I). PKC activation by phorbol esters may also be more prolonged and result in differential PKC translocation (Zoukhri et al., 1997) than that evoked by muscarinic receptor stimulation (Nishizuka 1992; Newton, 1995). Phorbol esters may also stimulate signaling pathways distinct from those stimulated by cholinergic agonists. Recent observations indicate that partially divergent signal transduction pathways exist for regulating APPS release (Buxbaum et al., 1994; Slack et al., 1995). Convergence of these pathways may occur downstream of PKC activation at a tyrosine phosphorylation-dependent step (Slack et al., 1995). These findings are important when considering both the etiology of AD and therapeutic strategies for ameliorating its pathology (Haass and Selkoe, 1993). Because the increased release of APPS is accompanied by a decrease in AS release (Buxbaum et al., 1993; Hung et 53 al., 1993), it is generally assumed that activation of the a-secretase pathway is beneficial. Moreover, it has been implied that the well-documented loss of cholinergic innervation of the cerebral cortex in AD (Coyle et al.,1983) might reduce APPS release, thereby contributing to the deposition of amyloid (Buxbaum et al, 1992; Lahiri et al.,1992; Nitsch and Growdon, 1994). Our data, and those of others (Wallace et al., 1995) would tend to caution against hasty acceptance of the hypothesis that the loss of cholinergic neurons is a primary event altering APPS in AD. With respect to treatment strategies, cholinomimetic replacement therapy has met with little success (Reiner and Fibiger, 1995). Assuming that the deposition of amyloid is the primary pathological feature of AD (Hardy and Allsop, 1991), our data would predict that cholinomimetic replacement therapy would not mitigate amyloid deposition in the cerebral cortex. On the other hand, it is clear that APPS release can be regulated in cortical neurons. Unraveling the molecular pathways responsible for such regulation will be an important challenge for future studies. 54 m. REGULATION OF APP CATABOLISM INVOLVES THE MAPK SIGNAL TRANSDUCTION PATHWAY INTRODUCTION Within the last several years APP catabolism has been shown to be regulated by a diverse array of first and second messengers including calcium (Nitsch et al., 1992; Buxbaum et al., 1994), cAMP (Hu et al., 1996), growth factors (Schubert et al., 1989a; Fukuyama et al.,1993), cytokines (Buxbaum et al., 1992; Buxbaum et al., 1994), and estrogen (Jaffe et al., 1994). Although the effector PKC can alter APP catabolism it is not required for this regulation as a PKC-independent pathway has recently been discovered (Buxbaum et al., 1994). Moreover, PKC-independent regulation of APPS may involve activation of protein-tyrosine kinases (Slack et al., 1995; Nitsch et al., 1996b). These observations predict the existence of a pathway activated by multiple first and second messengers, capable of regulating APP catabolism in both a PKC-dependent and -independent fashion. These criteria are met by the MAPK signal transduction pathway (Pelech and Charest, 1996; Graves et al., 1995; Malarkey et al., 1995; Cobb and Goldsmith, 1995). MAPKs, also known as extracellular signal-regulated protein kinases (ERKs), are the terminal enzymes in a three level kinase cascade involving the sequential activation of Raf, MEK and ERK (Pelech and Charest, 1996). As MEKs are the only known physiological activators of ERKs (Bardwell and Thorner, 1996), MEKs provide a useful target for manipulating ERK activity. We used both PD 98059, a selective inhibitor of MEK1 (Alessi et al., 1995; Lazar et al., 1995; Dudley et al., 1995), and over-expresssion of a kinase-dead MEK1 mutant (Seger et 55 al., 1994) to test the hypothesis that E R K activation was necessary for regulation o f A P P processing. 56 M A T E R I A L A N D M E T H O D S Cell Lines and Transfections HEK 293 cells were transiently transfected with pCMV695, an expression vector for APP695 (Selkoe et al., 1988), pCMVfj, an expression vector for bacterial P-galactosidase (Clontech Laboratories) and either pCDNAK97A, an expression vector for kinase-inactive MEK, or the expression vector alone using a high-efficiency calcium phosphate transfection protocol (Chen and Okayama, 1987) as previously described (Raymond et al., 1996). Transfection efficiency was assessed by staining for P-galactosidase and determining the percentage of positively stained cells according to the method of Raymond et al. (1996). HEK 293 cells stably transfected with a construct carrying the Alzheimer's disease-linked double ('Swedish') mutation (K695sw), known to secrete elevated levels of both Ap 4 0 and Ap 4 2 (Citron et al., 1996), were cultured in DMEM supplemented in 10% fetal calf serum and 400 mg/ml geneticin. HEK 293 cells were cultured in MEM supplemented with 10% fetal calf serum as previously described (Raymond et al., 1996). Rat pheochromocytoma (PC 12) cells were cultured in DMEM supplemented with 10% horse serum and 5% fetal calf serum as previously described (Buxbaum et al., 1990). One day prior to stimulation, HEK 293 cells or PC 12 cells were exposed to culture media containing charcoal inactivated calf serum at the same percentage previously used for cell maintenance. All cell lines were exposed to drugs for 15 min. PC 12 cells were exposed to drugs in DMEM according to the method of Buxbaum et al.(1990). HEK 293 cells were exposed to drugs in MEM supplemented with 1 mg/ml glucose while K695sw cells were exposed to drugs in DMEM. 57 Cortical Cell Cultures and Drug Treatment Timed pregnant Sprague-Dawley rats were anesthetized with halothane at 18 days of gestation and the cerebral cortex was removed from rat embryos and dissociated using a method previously described (Murphy and Baraban, 1990), with the exception that the plating medium L-cystine concentration was supplemented to 300 uM. Culture maintenance and drug exposure was carried out using the method of Fiore et al.(1993) with minor modifications. In brief, prior to drug treatment, cells were washed once with 1 ml Hanks' balanced salt solution and pre-exposed to PD 98059 or drug vehicle for 1 hr. Both PD 98059 and phorbol esters were diluted from 10 mM stocks, made up in dimethyl sulfoxide. Quantification of APPS and Ap in Culture Media Following drug exposure, media was centrifuged for 10 min. at 16 000 x g to remove cellular debris. For APPS detection, the media was subsequently desalted and concentrated by centrifugation in the presence of protease inhibitors (17 pg/ml phenylmethanesulfonyl fluoride, 2 ug/ml leupeptin, 10 ug/ml aprotinin and 2 ug/ml pepstatin) according the method of Mills and Reiner (1996). APP was detected by Western blot analysis using an anti-APP N-terminal antibody (anti-PreA4 monoclonal antibody; Boehringer Mannheim, Laval, Quebec) or WO-2, a monoclonal antibody generated against the first sixteen amino acids of the N-terminal region of AB (Ida et al., 1996, anti-1-16) as previously described (Mills and Reiner, 1996). All western blots were probed first with the anti-PreA4 monoclonal antibody (22C11). In some experiments, membranes were subsequently stripped of antibodies and re-probed with the APP-selective antibody WO-2 (1:400) to prevent detection of secreted APLP (Slunt et al., 1994). For AB detection, proteins were precipitated by TCA according to the method of 58 Hames (1981). Ap was separated by Tris/Tricene SDS-PAGE according to the method of Klafki et al. (1996b) and detected by Western blot analysis according to the method of Ida et al. (1996) using the monoclonal antibody WO-2. Following densitometric measurements, ANOVA followed by Fisher's post hoc analysis was used to determine the significance of observed differences. Data are expressed as mean ± SEM and unless otherwise stated, are representative of three separate trials. Western Blots of MAPK, MEK and Cellular APP Cells were lysed in an extraction buffer containing 1% Nonidet P-40, 1% sodium deoxycholate, 4 mM /?-nitrophenylphosphate and 1 mM sodium vanadate and the lysate was centrifuged to remove detergent-insoluble material. Twenty-five pg of cellular protein was separated by SDS-PAGE on 10% 20 cm gels or 12.5% low bis (acrylamide:bis ratio is 118.5:1 instead of 37.5:1) mini gels for Western blots of either ERK or MEK Following gel electrophoresis, proteins were transferred electrophorectically to a nitrocellulose membrane and probed using a rabbit polyclonal antibody specific for ERK (1:25 000; Erkl-CT, UBI, Lake Placid, NY), phosphorylated ERK (1:1000; phospho-MAPK; New England Biolabs, Mississauga, Ontario) or MEK (1:25 000; Mekl-NT, UBI, Lake Placid, NY). Five pg of cellular protein was separated on 10% mini gels for western blots of APP and membranes were subsequently probed with anti-PreA4 monoclonal antibody. Western blots in a given panel are representative of 3 separate trials which may or may not have been taken from the exact same trial. Each trial was performed on a separate primary culture or cell plating. R E S U L T S Pharmacological Inhibition of MEK Antagonizes NGF Receptor Stimulation ofAPP\ Secretion and ERK Activation Activation of a wide variety of growth factor receptors having intrinsic or associated tyrosine kinase activity has been shown to stimulate ERK activation (Pelech and Sanghera 1992; Pelech et al., 1993). Included among these are receptors for NGF, epidermal growth factor and fibroblast growth factor whose stimulation has also been shown to increase APPS release in cell lines (Schubert et al., 1989a; Fukuyama et al., 1993; Refolo et al., 1989). These observations implicate the involvement of ERK in growth factor receptor mediated regulation of APPS release. Activation of ERK by growth factors involves the small GTP-binding protein p21Ras, which binds to Raf when in the GTP-bound state (Marshall, 1996). To determine whether or not regulation of APPS secretion by NGF involves stimulation of a Ras-dependent ERK pathway, we used both wild type PC 12 cells and PC 12 cell lines stably expressing inducible forms of either dominant inhibitory or constitutively active mutants of Ras (GSrasDNl and GSras2, respectively) (Thomas et al., 1992) under control of the dexamethasone-inducible mouse mammary tumor virus promotor. Surprisingly, a 12 hour preexposure of wild type PC 12 cells (the control cell line) to dexamethasone (0.3 uM) altered stimulation of APPS release making these results difficult to interpret (Appendix II). To avoid long-term drug effects and study the neccessity of ERK activation in NGF receptor-dependent stimulation of APPS release more directly, we examined PC 12 cells stimulated with NGF in the presence of the MEK1 inhibitor PD 98059. This pharmacological agent has 60 previously been shown to antagonize tyrosine kinase receptor stimulation of ERK1 (Alessi et al., 1995; Lazar et al., 1995; Dudley et al., 1995; Pang et al., 1995) with an IC 5 0 of approximately 10 uM (Dudley et al., 1995). APPS production increased significantly when cells were incubated with 100 ng/ml of NGF for 15 min. and this increase was antagonized in the presence of 10 uM of PD 98059 (2.0 ± 0.3 and 1.0 + 0.2 respectively; n=3, p < 0.05; Fig. 9A). For ERK to become activated, it must first be phosphorylated by the dual specificity kinase MEK on both a tyrosine and threonine residue in the TEY motif (Pague et al., 1991; Anderson et al., 1990). Phosphorylated ERK can be detected using Western blotting by a gel shift assay where the electrophoretic mobility of phosphorylated ERK is retarded relative to its nonphosphorylated form (Posada and Cooper, 1992) or by using antibodies raised against the phosphorylated TEY consensus sequence. The phosphorylation state of ERK was measured using these methods to ensure 10 pM PD 98059 antagonized NGF receptor stimulation of ERK activation. A 15 min. exposure to NGF activated ERK1 and ERK2, and this activation was inhibited by PD 98059 (Fig. 9B). Pharmacological Inhibition of MEK Antagonizes PKC Stimulation of APPS Release and ERK Activation PKC stimulation by phorbol esters has been shown to dramatically increase the release of APPS in a wide variety of cell lines (Buxbaum et al., 1992; Caporaso et al., 1992; Gabuzda et al., 1993; Hung et al., 1993). To determine if ERKs are necessary for PKC-mediated regulation of APP catabolism, HEK 293 cells were exposed to 0.1 pM phorbol 12-myristate 61 13-acetate (PMA) with or without 10 uM PD 98059. Stimulation of APPS release by PMA was inhibited by PD 98059 during a 15 min. drug exposure as determined using the monoclonal antibody 22C11 (7.7 ± 1.5 and 4.4 ± 1.4, respectively; n=5, p < 0.05; Fig. 10A) or WO-2 (3.9 ± 0.5 and 2.0 ± 0.6, respectively; n=3, p < 0.05; Fig 10A). To ensure that PD 98059 antagonized PMA-stimulated ERK activation in HEK 293 cells, the phosphorylation state and mobility of ERK was measured in Western blots. The experiments revealed that the PMA induced electrophoretic shift was antagonized by PD 98059 (Fig. 10B upper, n = 3), as was the increase in phospho-ERK immunoreactivity induced by PMA (Fig. 10B lower, n=3). PKC-dependent regulation of APPS release has also been observed in primary cultures of hippocampal and cortical neurons (Lee et al., 1995; Mills and Reiner, 1996). To determine whether or not ERK activation is necessary for PKC-mediated regulation of APPS release in neurons, primary cultures of rat cortical neurons were incubated with PDBu (1 uM) with or without PD 98059 (10 uM) for 1 hr. Levels of APPS in the culture media increased significantly in the presence of PDBu and this increase was antagonized in the presence of PD 98059 (6.52 ±1.51 and 3.01 ± 0.90, respectively n =5, p < 0.05; Fig. 11A). Moreover, phorbol ester stimulation of ERK activation was also suppressed in the presence of PD 98059 as seen by western blot analysis of ERK mobility (upper) or phospho-ERK (lower) (Fig. 1 IB) Kinase-Inactive MEK Antagonizes PKC Stimulation of APPS Release and ERK Activation Overexpression of mutant proteins has proven to be a powerful tool for studying the role of signaling pathways in various cellular processes. A kinase-inactive MEK mutant, K97A, was generated by mutating lysine 97 to alanine (Charest and Pelech, unpublished data). This lysine 62 3 . 0 2 . 5 2 . 0 1.5 1 .0 0 . 5 0 . 0 APR. kD 97.4-vehicle PD98059 B p44 p42 +/-NGF r p h o s - M A P K phos -MAPK Vehicle PD98059 - — r — ; n Figure 9. The MEK inhibitor PD 98059 antagonizes N G F receptor stimulation of APPS secretion and ERK activation in PC 12 cells. A: (Upper) Densitometric analysis of the effect of N G F (100 ng/ml) on basal APPS release with or without PD 98059 (10 pM). Data are mean ± SEM of three experiments, p < 0.05, different from all other treatment groups. (Lower) Representative Western blot of APPS fragments released in 15 min. by PC 12 cells alone or in the presence of N G F with or without PD 98059. B: Representative Western blot of phospho-ERK in PC 12 cells following a 15 min. drug exposure. The increase in immunoreactivity of the phospho-ERK specific antibody in the presence of N G F was inhibited by PD 98059. 63 is critical to MEK's activity as it is found in the ATP-binding site (Seger et al., 1994). Previously, the K97A mutant has been shown to act in a 'dominant negative' fashion as its overexpression in N1H 3T3 cells inhibited phorbol ester stimulation of endogenous MEK and its downstream substrate ERK (Seger et al., 1994). Stimulation of APPS by 0.1 uM PMA was measured in HEK 293 cells transiently overexpressing human APP 6 9 5 together with the K97A mutant or vector alone. Densitometric analysis revealed that PMA stimulation of APPS secretion was significantly inhibited in the presence of the kinase-inactive MEK as compared to vector alone as determined using 22C11 (3.0 ± 0.3 and 1.9 + 0.2, respectively n=3; p < 0.05 Fig. 12A ) or W-2 (2.3 ± 0.2 and 1.4 ± 0.4, respectively n=3; p < 0.05 Fig. 12A ). Moreover, Western blots using the gel shift assay indicate that the PMA induced increase in ERK phosphorylation was antagonized by expression of the K97A mutant (Fig. 12B). Incomplete antagonism of ERK activation may be attributed in part to transfection efficiency. P-galactosidase staining indicated that the percentage of transfected cells was 81.4% ±1.6 %, n=3. Overexpression of the K97A mutant was confirmed using a rabbit polyclonal antibody raised against the N-terminus of MEK1 (UBI) (Fig. 12B). Cellular levels of APP695 were not affected by overexpression of the dominant negative MEK (Fig. 12B). Parmacological Inhibition of MEK Antagonizes PKC Regulation of Ap Release Activation of PKC is also known to regulate AP secretion. Specifically, a reduction of Ap secretion has been observed after phorbol ester treatment (Jacobsen et al., 1994; Hung et al., 1993; Buxbaum et al., 1993; Gabuzda et al., 1993; Querfurth et al., 1994), direct activation of phospholipase C (Buxbaum et al., 1993) and first messengers (Hung et al., 1993) known to activate the PLC/PKC pathway. However, the cellular mechanisms underlying this regulation 64 Figure 10. The MEK inhibitor PD 98059 antagonizes phorbol ester stimulation of APPS release and ERK activation in HEK 293 cells. A: (Upper) Densitometric analysis of PMA (0.1 uM) stimulation of APPs secretion with or without PD 98059 (10 uM). Data are mean ± SEM and represent five experiments for 22C11 (solid columns) or three experiments for WO-2 (hatched columns), p < 0.05, different from all other treatment groups. (Lower) Representative Western blot of the effect of PMA on basal APPS release alone or in the presence of PD 98059. B: Representative Western blot of ERK isoforms with ERK1 C-terrninus antibody (upper) or phospho-ERK forms (lower) in HEK 293 cells following a 15 min. drug exposure. The PMA induced 'electrophoretic shift' was inhibited by PD 98059. Similarily, the increase in phospho-ERK immunoreactivity in the presence of PMA was antagonized by PD 98059. 65 CD CO O Q ° - 'I ° - J Vehicle PD98059 APR s kD 97.4-B +/-PDBu r p44 p42' M A P K M A P K p44 p42 p h o s - M A P K p h o s - M A P K Vehicle PD98059 1 Figure 11. The MEK inhibitor PD 98059 antagonizes PKC stimulation of APPS secretion and ERK activation in cortical neurons. A: (Upper) Densitometric analysis of PDBu (1 uM) stimulation of APPS secretion in rat cortical cultures with or without PD 98059 (10 uM). Data are mean ± SEM of five experiments. *p < 0.05, different from all other treatment groups. (Lower) Representative Western blot of the effect of PD 98059 on PDBu stimulation of APPS release in 15 min.. B: Representative Western blot of ERK iso forms with ERK C-terminus antibody (upper) or phospho-ERK forms (lower) in cortical cultures following a 1 hour drug exposure: phorbol ester induced increase in the phosphorylation state of ERK was antagonized by pharmacological inhibition of MEK. 66 are poorly understood. To determine whether or not ERKs are involved in PKC regulation of AB secretion, HEK 293 cells overexpressing human APP695 carrying the Swedish mutation were exposed to 1 uM PMA for 15 min. with or without 10 uM PD 98059. Densitometric analysis revealed that PMA inhibition of A3 secretion was antagonized by PD 98059 (0.48 ± 0.05 and 0.83 ± 0.07, respectively n=7; p < 0.05 Fig 13). 67 B +/-PMA r M A P K Vector K97A + II -p44 M A P K p42 MEK APP 695 Figure 12. The kinase-dead MEK mutant K97A inhibits phorbol ester stimulation of APPS secretion and ERK activation in HEK 293 cells. A: (Upper) Densitometric analysis of PMA (0.1 pM) stimulation of APPS secretion in cells expressing the MEK mutant (K97A) or vector alone. Data are mean ± SEM and represent three experiments for both 22C11 (solid columns) and WO-2 (hatched columns), p < 0.05, different from all other treatment groups. (Lower) Representative Western blot of the effect of PMA on basal APPS release following transient transfection of vector alone or the K97A mutant. B: (Upper) Western blot of ERK isoforms with an ERK C-terminus antibody in HEK 293 cells transfected with the kinase-dead MEK mutant or vector alone. The 'electrophoretic shift' induced by PMA treatment in cells expressing vector alone was inhibited in cells expressing the kinase-dead MEK mutant. (Middle) Western blot of MEK1 using a rabbit polyclonal antibody raised against the N-terminus of MEK1. (Lower) Western blot of cellular APP using a monoclonal antibody generated against the N-terminus of APP. Although Western blots of APPS and cell lysate were not taken from the exact same trial the results are representative. 68 Figure 13. The MEK inhibitor PD 98059 antagonizes phorbol ester inhibition of AB secretion in K695sw cells. (Upper) Densitometric analysis of PMA (1 uM) inhibition of AB secretion in K695sw cells with or without PD 98059 (10 pM). Data are mean ± SEM of seven experiments. *p < 0.05, different from control (vehicle alone). (Lower) Representative Western blot of the effects of the MEK antagonist PD 98059 on PMA inhibition of Ap release. 69 DISCUSSION The major finding of the present study is that activation of the MAPK pathway is necessary for regulation of the secretory processing of APP. Antagonism of MEK inhibits phorbol ester and NGF receptor stimulation of APPS release as well as phorbol ester-mediated inhibition of AP release. Similar results have recently been found in an independent study (Desdouits-Magnen et al., 1998). The strength of the current study derives from the use of two distinct approaches for inhibiting the MAPK cascade, the pharmacological agent PD 98059 and gene transfer with a kinase-dead MEK mutant, both of which provided mutually supportive results. Moreover, the effects we have observed are manifest in several different cell lines including neurons, suggesting that they are likely to be general rather than cell-specific. Different mechanisms of MEK inhibition can be implicated in this process as dominant-negative MEK interferes with raf activity and reduces basal MEK activity whereas PD 98059 does not (Seger et al., 1994; Dudley et al., 1995). Specifically, the dominant-negative nature of kinase-dead MEK is thought to be correlated with both its expression levels and stability of association with raf. Consequently, in addition to inhibiting MEK activation K97A would also inhibit raf activity. Therefore, K97A may be inhibiting regulation of APPS release by antagonizing raf activity. However, this is unlikely, given the fact that the antagonist PD 98059, which does not inhibit rafs activity also inhibited stimulation of APPS release. These results suggest that both PD 98059 and K97A inhibit regulation of APP catabolism by antagonizing MEK activation. 70 These data also have broader implications for the function of ERKs. Correlative evidence has suggested that secretory stimuli activate ERKs in a variety of cells (Frodin et al., 1995; Stratton et al., 1991; Cox et al., 1996), but evidence demonstrating a requirement for ERK activation in secretion has not been obtained. Moreover, it has been shown that activation of the MAPK pathway is not required in some instances (Khoo and Cobb, 1997). The present experiments clearly implicate ERKs in regulation of APP secretory processing, and thereby provide the first direct evidence for the necessity of the MAPK pathway in secretory events. These results are relevant to our understanding of the molecular mechanisms by which APP catabolism is regulated in cells. A strong case has been made for the role of PKC activation in the regulation of APP catabolism (Nitsch and Growdon, 1994). PKC regulation of APP processing has been extensively characterized and shown to occur in a wide variety of cell lines (Buxbaum et al., 1990; Caporaso et al., 1992; Buxbaum et al., 1993; Gabuzda et al., 1993) and in central neurons (Mills and Reiner, 1996; Lee et al., 1995). However, the downstream effectors remain unknown. Our studies using both pharmacological and gene transfer approaches imply that MEK and/or ERK are necessary effectors for PKC-mediated stimulation of APPS release in both cell lines and neurons. Antagonism of PKC-mediated inhibition of Ap secretion with the MEK inhibitor PD 98059 indicates that the MAPK pathway is also downstream of PKC regulation of AP production, and that activation of MEK and/or ERK may reduce AP secretion. 71 The best characterized means of stimulating the MAPK pathway is by activation of receptor tyrosine kinases (Pelech and Sanghera, 1992; Cobb and Goldsmith, 1995). Upon ligand binding, these receptors autophosphorylate, promoting the association of ras with GTP leading to the sequential activation of rafl, MEK and ERK. Autophosphorylation also promotes interaction of the receptor with a number of alternative target proteins including PLC-y (Meisenhelder et al., 1989; Ronnstrand et al., 1992, Middlemas et al., 1994, Eriksson et al., 1995). Because of the abundant evidence that PKC activation regulates APP catabolism (Nitsch and Growdon, 1994), it is tempting to hypothesize that regulation of APP catabolism via receptor tyrosine kinases might be mediated by activation of PLC-y. However, it is equally plausible that the "direct route" of ERK activation by receptor tyrosine kinases may be sufficient for regulation of APP catabolism by growth factor receptors. Regardless of the detailed molecular circuitry involved, our data demonstrate that ERK activation is necessary for growth factor stimulation of APPS secretion The mechanism by which the MAPK pathway regulates APP catabolism is unknown. Because of the time course involved in the present experiments, ERKs are unlikely to increase APPS secretion by increasing overall expression of cellular APP. Rather, it seems likely that ERKs are acting to phosphorylate one or more targets within the cell to modify APP catabolism. Direct phosphorylation of the APP holoprotein is unlikely as activated ERK does not phosphorylate the cytoplasmic domain of APP under conditions in which it is able to hyperphosphorylate x (Aplin et al., 1996). Alternatively, ERKs may regulate APP processing indirectly by phosphorylating proteins involved in intracellular trafficking. For example, like PKC, ERK may increase APPS secretion by phosphorylating a tightly associated trans-Go\gi 72 network protein thereby altering the formation of constitutive secretory vesicles containing mature APP (Xu et al., 1995). Also, presenilin-1, another protein thought to alter APP processing via its effects on protein trafficking (Lemere et al., 1996; Citron et al., 1997; Borchelt et al., 1996; Weidemann et al., 1997) has a consensus sequence for ERK-dependent phosphorylation and has recently been shown to be a substrate for PKC (Walter et al., 1997a; Walter et al., 1997b; Seeger et al., 1997). Of course, the yet-to-be-identified secretases which cleave APP remain potential candidates for phosphorylation by the MAPK cascade, either directly or indirectly. A number of structurally unrelated membrane proteins undergo cleavage and subsequent release of their ectodomains into the extracellular medium, much like APP (Mattson et al., 1997; Echlers and Riordon, 1991); many of these share a common mechanism of regulation (Arribas and Massague, 1995). In addition to APP, PKC regulation of membrane protein processing has been observed for proTGF-a (Pandiella and Massague, 1991) colony-stimulating factor 1 (Stein and Rettenmier, 1991), colony-stimulating factor 1 receptor (Downing et al., 1989) and LAR transmembrane protein tyrosine phosphatase (Mullberg et al., 1992). Our data implicating the MAPK cascade in regulation of APP catabolism suggests that this mechanism of regulation may be relevant to these membrane proteins as well. Juxtamembrane cleavage serves to liberate APPS which may act as a paracrine signaling factor. For example, APPS has been shown to stimulate a cGMP-dependent protein kinase (Furukawa et al., 1996) as well as ERKs (Greenberg et al., 1994; Greenberg et al., 1995) and this function may be altered by phosphorylation of the ectodomain (Walter et al., 1997a; Walter et 73 al., 1997b). ERK activation by APPS is intriguing in light of the present findings, as it suggests that there may be a positive-feedback pathway whereby activation of ERK stimulates APPS release, which in turn activates the MAPK pathway. Cell surface receptors known to regulate APP processing include heterotrimeric G-protein-coupled receptors and tyrosine kinase-coupled receptors (for reviews see Nitsch et al., 1996b; Beyreuther et al., 1996; Buxbaum and Greengard 1996). The effector system responsible can be regulated in either a PKC-dependent or -independent fashion (Buxbaum et al., 1994, Slack et al., 1995, Nitsch et al., 1996b) and may involve activation of tyrosine kinases (Slack et al., 1995). All of these criteria are met by the MAPK signal transduction pathway, Our data for the first time implicate MEK and/or ERK in both PKC and tyrosine kinase receptor regulation of APP catabolism. Indeed, a recent study our laboratory suggests that MEK and/or ERK are critically involved in iV-methyl-D-aspartate receptor stimulation of APPS secretion (see Chapter IV) suggesting that the MAPK pathway may be critical for regulation of APP catabolism by a number of first messengers. It is widely hypothesized that production and deposition of amyloid is an early event in AD, and may be the key pathological event which triggers the disease process (Hardy, 1997; Selkoe, 1997). As such, any manipulation which diminishes the production of A3 is of potential therapeutic utility. The results presented herein suggest that strategies aimed at activating the MAPK cascade may be a viable approach in this regard. 74 IV. REQUIREMENT FOR MAPK SIGNALING IN NMDA RECEPTOR REGULATION OF APP S SECRETION I N T R O D U C T I O N The neurotransmitter glutamate is predominantly responsible for excitatory neurotransmission in the mammalian brain. Glutamate receptors can be broadly categorized into two groups namely, ionotropic, mediating ion influxes and G-protein-coupled receptors, initiating activation of intracellular effectors (Nakanishi, 1992; Seeburg, 1993). Glutamatergic regulation of APPS catabolism has been observed in cell lines, primary neuronal cultures and slice preparations (Lee et al., 1997; Lee et al., 1995; Nitsch et al., 1997; Ulus and Wurtman, 1997; Kirazov et al., 1997). However, to date, glutamatergic regulation of APPS secretion has focused predominantly on G-protein-coupled receptor subtypes linked to the PLC/PKC signaling system. Stimulation of glutamatergic, serotonergic or cholinergic G-protein-coupled-receptors have been shown to increase APPS and concomitantly decrease A(3 production (Hung et al., 1993; Buxbaum et al., 1994; Slack et al., 1995; Buxbaum et al., 1992; Wolf et al., 1995; Lee et al., 1995; Nitsch et al., 1997; Nitsch et al., 1996a). Of the ionotropic glutamate receptors, the NMDA receptor is unique in that it is highly permeable to Ca 2 + (McBain and Mayer, 1994). NMDA receptors control several important physiological processes initiated by changes in intracellular Ca 2 + including long term potentiation (LTP), a cellular model of learning and memory (Regehr and Tank, 1990). In AD the glutamatergic corticocortical connections and major projections of the hippocampus degenerate early in the disease process (Francis et al., 1993). Moreover, in postmortem AD brain, glutamate concentrations are significantly decreased (Hyman et al., 1987). Because these brain regions accumulate amyloid and are intimately involved in learning and memory, it has been suggested that glutamatergic hypoactivity may contribute both to increased brain amyloid burden and memory dysfunction (Lee et al., 1995). Presently, little is known of the role of ligand-gated Ca 2 + channels in regulating APP catabolism. However, Ca 2 + regulation of APP catabolism has been demonstrated previously in a variety of cell lines (Buxbaum et al., 1994; Querfurth et al., 1994; Querfurth et al., 1997). Ca 2 + regulation of APP catabolism is thought to involve a PKC-independent signaling pathway and may involve activation of protein-tyrosine kinases. These criteria are met by the MAPK signal transduction pathway, a signaling system recently implicated in PKC and growth factor regulation of APP processing (Mills et al., 1997). Unlike the serine/threonine kinase PKC, Ca 2 + does not interact with MAPK directly. Ca 2 + activation of MAPK is indirect via activation of the small guanine nucleotide-binding protein p21Ras (Rosen et al., 1994; Farnsworth et al., 1995). Activation of p21Ras in turn leads to serial activation of the kinases Raf, MEK and ERK. Stimulation of the NMD A receptor, a ligand-gated Ca 2 + channel, has been shown to increase MAPK activity in cortical cultures (Xia et al., 1996). Therefore, we hypothesize that (1) NMD A receptors regulate APP processing and (2) ERK is required for this NMDA receptor-mediated regulation. In order to test these hypotheses, we transiently overexpressed HEK 293 cells with the NMDA receptor and a kinase-dead MEK1 mutant (Seger et al., 1994) or vector expressing B-galactosidase. 76 M A T E R I A L A N D M E T H O D S Cell culture and transfections HEK 293 cells were cultured in MEM supplemented with 10% fetal calf serum as described previously (Mills et al., 1997). Fully functional NMDA receptors having a high Ca 2 + conductance are formed by the expression of the two NMDA receptor subunits NR1 and NR2A (Raymond et al., 1996). Therefore, HEK 293 cells were transiently transfected with pCMV695 (an expression vector for APP 6 9 5: Selkoe et al., 1988), pCDNANRl and pCDNANR2A (expression vectors for the NMDA receptor subunits) and either pCDNAK97A (an expression vector for kinase-inactive MEK) or vector expressing bacterial P-galactosidase (Clontech Laboratories) using a high-efficiency calcium phosphate transfection protocol (Chen and Okayama, 1987). Transfection efficiency was assessed by staining for P-galactosidase. At the end of the 12 hour incubation with the DNA containing solution, cells were washed and 2 x 106 cells were replated onto poly-D-lysine precoated 60-mm dishes in complete media containing 1 mM D, L 2-amino-5-phosphonovalerate (APV). Thirty-six hours before stimulation cells were exposed to culture media containing 1 mM APV and 10% charcoal-inactivated calf serum. At the end of this period, HEK 293 cells were exposed to drugs in Ringers solution containing in mM: 140 NaCl, 5.4 KC1, 1.4 CaCl2, 1.2 NaH2P04, 21 glucose and 26 NaHC03. Media for control cultures contained 1 mM APV and 1 mM Mg 2 + to antagonize basal levels of NMDA receptor stimulation while stimulated cultures contained 100 uM NMDA and 50 pM glycine. Quantification of APPS and A/3 in Culture Media Following drug exposure, media was centrifuged for 10 min. at 16 000 x g to remove cellular debris. Media was subsequently desalted and concentrated by centrifugation in the presence of protease inhibitors (17 ug/ml phenylmethanesulfonyl fluoride, 2 ug/ml leupeptin, 10 ug/ml aprotinin and 2 u,g/ml pepstatin) according the method of Mills and Reiner (1996). APP was detected by Western blot analysis using an anti-APP N-terminal antibody (anti-PreA4 monoclonal antibody; Boehringer Mannheim) or WO-2, a monoclonal antibody generated against the first sixteen amino acids of the N-terminal region of Ap (anti-1-16) as previously described (Mills et al., 1997). Following densitometric measurements, ANOVA followed by Fisher's post hoc analysis was used to determine the significance of observed differences. Data are expressed as mean ± SEM and unless otherwise stated, is representative of three separate trials. Western Blots ofMAPKs, MEKJ, NMDA and Cellular APP Cells were lysed in an extraction buffer containing 1% Nonidet P-40, 1% sodium deoxycholate, 4 mM/?-nitrophenylphosphate and 1 mM sodium vanadate and the lysate was briefly tip sonicated on ice. Twenty-five ug of cellular protein was separated by SDS-PAGE on 12.5% low bis (acrylamide:bis ratio is 118.5:1) mini gels for Western blots of ERK, MEK, c-Jun N-terminal kinase stress-activated protein kinases (JNK/SAPK), p38/HOGl, APP or NR1. Following gel electrophoresis, proteins were transferred electrophoretically to a nitrocellulose membrane and probed using an antibody specific for phosphorylated ERK (1:1000; phospho-MAPK; New England Biolabs), MEK (1:25 000; Mekl-NT, UBI), phosphorylated p38/HOGl (1:500; phospho-p38, New England Biolabs), phosphorylated 78 JNK/SAPK (1:1000; phospho-SAPK/JNK, New England Biolabs), APP (1:200; anti-PreA4 monoclonal antibody, Boehringer Mannheim) orNRl (1:500; Anti-Rat NR1, CT, Upstate Biotechnology). Western blots, taken from the same trial, were run in parallel and are representative of 3 separate trials. 79 R E S U L T S Kinase-inactive MEK antagonizes NMDA receptor stimulation of APPS release Stimulation of APPS was measured in HEK 293 cells transiently expressing the NMDA receptor, APP 6 9 5 together with the K97A mutant or vector expressing P-galactosidase. Densitometric analysis revealed that levels of APPS in the culture media increased significantly in the presence of 1 OOuM NMDA (1.00 ± 0.09 and 2.16 ± 0.28, respectively; n=4, p < 0.05) (Fig. 14A). This increase was significantly inhibited in the presence of the kinase dead MEK1 mutant K97A as compared with vector alone (2.16 + 0.28 and 1.42 ± 0.22, respectively; n=4, p < 0.05) (Fig. 14A). Constitutive release of APPS from HEK 293 cells transfected with cDNA expression constructs encoding P-galactosidase or K97A were not significantly different from one another. Preliminary results using the MEK1 antagonist PD 98059 complement these findings. Specifically, NMDA receptor stimulation of APPS release was completely antagonized by PD 98059 while having no effect on constitutive APPS release (J.M., P.B.R., unpublished observations). Kinase-dead MEK antagonizes NMDA receptor stimulation of ERK NMDA receptor stimulation of ERK activity has been previously shown to be inconsistent (Fiore et al., 1993; Bading and Greenberg, 1991; Xia et al., 1996, Wang and Durkin, 1995). To ensure that NMDA receptor stimulation increased ERK phosphorylation and therefore its activation (Canagarajah et al., 1997), Western blots of HEK 293 cells were probed using a phospho-specific MAPK antibody. Immunoreactivity of phospho-ERK 1 and phospho-ERK2 were both increased by NMDA receptor stimulation and this increase was inhibited by K97A 80 overexpression (Fig. 14B). (3-galactosidase staining indicated that the percentage of transfected cells was 56 ± 4 (n=3). Basal levels of phospho-ERK activity also appeared to be increased by expression of the NMDA receptor. Although this activity might be caused by release of endogenous glutamate synthesized and secreted by the cells, it is somewhat unlikely given that the antagonists APV and Mg 2 + are present in the control media. The mammalian MAPK family members have recently expanded to include the JNK/SAPK (Derijard et al., 1994; Kyriakis et al., 1994) and p38/HOGl (Han et al., 1994). Unlike ERKs these family members have been implicated in the transduction of stress signals. However, activation of SAPK/JNK or p38/HOGl has been shown to occur following receptor-mediated rises in intracellular Ca2+(Schwarzschild et al., 1997; Mitchell et al., 1995; Kramer et al., 1995; Watanabe et al., 1997; Mitchell et al., 1995; Kawasaki et al., 1997). Activation of SAPK/JNK or p38/HOGl did not appear to be increased by NMDA receptor stimulation even when measured in the same lysates that contained elevated ERK activity (Fig. 14B). Overexpression of the K97A mutant was confirmed using a rabbit polyclonal antibody raised against the N terminal of MEK1 (Upstate Biotechnology) (Fig. 14B). Cellular expression levels of either APP695 or the NMDA receptor were not affected by overexpression of the dominant negative MEK1 mutant (Fig. 14B). 81 Figure 12. The kinase-dead MEK mutant K97A inhibits NMDA receptor stimulation of APPS secretion and ERK activation in HEK 293 cells. A: (Top) Densitometric analysis of APPS released during a 15 min. exposure to NMDA in cells expressing the MEK mutant (K97A) or vector alone (Vector). Data are mean ± SEM and represent four experiments using an anti-APP N-terminal antibody 22C11. (Bottom) Representative Western blots of the effect of NMDA on basal APPS release. Western blots, run in parallel were probed with either 22C11 or WO-2. B: Twenty-five pgs of cell lysates were run in parallel and Western blots were probed using phospho-specific antibodies for ERK, SAPK/JNK or p38/HOG: ERK was the only MAPK whose phosphorylation state was increased by a 15 min. exposure to NMDA (100 u.M). 82 B 1 Mock "" +/-NMDA - + phos-MAPK p44 . „ phos-MAPK p42 MEK p54 p46 phos-SAPK phos-SAPK p38 phos-MAPK APP 695 Vector 1 1 K97A 1 + - + NR1 t i in* i .«y.i.lint mu g DISCUSSION We provide evidence that NMDA receptor stimulation promotes nonamyloidogenic APP processing and that the MAPK pathway is involved in this regulation. Specifically, overexpression of a kinase-dead MEK1 mutant blocked NMDA receptor stimulation of both APPS release and ERK activity. These results extend an earlier study from our laboratory showing that MAPK is necessary for PKC and growth factor regulation of APP catabolism (Mills et al., 1997). Our findings indicate that the MAPK pathway also underlies neurotransmitter regulation of APP processing. Glutamatergic regulation of APP processing has been observed in cell lines, primary cultures and rat brain slices (Ulus and Wurtman, 1997; Kirazov et al., 1997; Lee et al., 1995; Lee et al., 1997; Nitsch et al., 1997). These studies have emphasized the role of the metabotropic glutamate receptor, a G-protein-coupled receptor. To date, NMDA receptor stimulation of APPS has not been observed (Lee et al., 1995; Lee et al., 1997; Ulus and Wurtman, 1997). However, NMDA receptor coupling to the effector MAPK may not have occurred in these preparations as drug exposures were performed in the presence of 0.2 mM M g 2 + , which would presumably contribute to a voltage-dependent block. NMDA receptor stimulation of primary rat cortical cultures did not increase MAPK activity in the presence of this concentration of Mg 2 + (Fiore et al., 1993). The intracellular messenger Ca 2 + has been traditionally associated with NMDA receptor signaling. Ca2+-dependent regulation of APP catabolism has been previously shown in several 84 different cell lines (Buxbaum et al., 1994; Nitsch et al., 1992; Querfurth et al., 1995; Querfurth et al., 1997). However, effects of simply increasing intracellular Ca 2 + levels on APP processing appear complex and somewhat contradictory. For example, thapsigargin, a drug which prevents Ca 2 + reuptake in the endoplasmic stores, had varied effects on APPS release (Buxbaum et al., 1994; Nitsch et al., 1996a). Furthermore, a rise in intracellular Ca 2 + by IP3, A23187 or caffiene were shown to alter Ap release differentially. Some of these discrepancies may be explained in part by effects of these drugs on Ca 2 + within the luminal enviroment of the secretory pathway (Querfurth et al., 1997). Neurotransmitter receptor regulation of APPS release that is Ca2+-dependent has also been suggested (Buxbaum et al., 1994). Specifically, incomplete suppression of APPS release by muscarinic receptor agonists after PKC inhibition or down regulation (Buxbaum et al., 1994; Slack et al., 1995) led to the conclusion that neurotransmitter regulation of APP catabolism can occur in either a PLC/PKC or PLC/Ca 2 + dependent manner (Buxbaum and Greengard, 1996). However, direct evidence for Ca 2 + as a PKC-independent means of neurotransmitter regulation of APP catabolism does not presently exist. MAPKs are central transducers of a wide variety of first messengers, including neurotransmitters and Ca 2 + (Malarkey et al., 1995; Graves et al., 1995; Cobb and Goldsmith, 1995). Several different MAPK pathways are now known to exist including the ERKs, SAPK/JNK (Derijard et al., 1995; Kyriakis et al., 1994) and p38/HOGl (Han et al., 1994). Traditionally, these kinases have been categorized as those activated during differentiation (ERKs) or those activated by stress (SAPK/JNK and p38/HOGl) (Davis, 1994; Waskiewicz and Cooper, 1995). However, it has recently been discovered that many extracellular signals 85 do not regulate MAPKs selectively. The selectivity of the NMDA receptor for altering ERK activation over the stress activated MAPKs argues for a discrete mechanism of NMDA receptor regulation of ERK in these cells. Presently, the mechanism of NMDA receptor activation of ERK remains unknown. NMDA receptor regulation of ERK may be mediated by the first messenger Ca 2 +. Rises in intracellular Ca 2 + have been shown to activate the small guanine nucleotide-binding protein p21Ras which in turn lead to the activation of the MAPK cascade (Rosen et al., 1994; Farnsworth et al., 1995). Alternatively, the NMDA receptor may directly couple to signaling proteins upstream of ERK activation (Niethammer et al., 1996; Gurd and Bissoon, 1997). However, direct evidence for this does not exist at present. NMDA receptor regulation of APPS release may provide a negative feedback mechanism for decreasing NMDA receptor-mediated Ca 2 + influx. APPS has been shown to induce a selective concentration-dependent decrease in NMDA receptor currents (Furukawa and Mattson, 1998). This effect is thought to occur via activation of receptors linked to membrane-associated guanylate cyclase (Furukawa and Mattson, 1998). Specifically, it has been suggested that APPS affects intracellular Ca 2 + by altering receptors linked to cGMP production and dephosphorylation of NMDA receptor channels (Mattson, 1997). Therefore, NMDA receptor regulation of APPS release may provide a mechanism of negative control. The idea that altered signal transduction underlies neurodegenerative disorders such as AD has been previously suggested. Specifically, it is thought that altered signal transduction may lead to increased brain amyloid burden (Nitsch et al., 1992; Selkoe, 1993; Buxbaum and Greengard, 1996). These studies have largely emphasized the role of G-protein-coupled 86 receptors in regulating APP catabolism. The results presented in this study indicate neurotransmitter regulation of APP catabolism includes ligand-gated Ca 2 + channels, thereby extending our understanding of signal transduction mechanisms underlying APP processing events. 87 V. AP-INDUCED TAU HYPERPHOSPHORYLATION: A ROLE FOR C-JUN N-TERMINAL KINASE STRESS-ACTIVATED PROTEIN KINASES In addition to extracellular filamentous deposits described as plaques, the AD brain contain neuronal filamentous (neurofibrillary) tangles. Neurofibrillary tangles (NFT) occur within neuronal cell bodies and are composed of pathological fibrils known as paired helical filaments (PHF). The primary constituent of these filaments is the microtubule associated protein tau which is hyperphosphorylated. Anatomical evidence suggests that NFT may occur in regions associated with amyloid plaque formation in the AD brain (Selkoe, 1991). Although a potential relationship between these structural alterations have been implied, the underlying biochemical mechanisms are only now being explored (Yankner, 1996). A link between A(3 deposition and PHF's have been suggested as AP fibrils induce tau hyperphosphorylation leading to a loss of microtubule binding capacity and cell degeneration. In neuronal cell lines, AP neurotoxicity was accompanied by an increase in immunoreactivity of phosphorylation-dependent anti-tau antibodies (Lambert et al., 1994; Le et al., 1997). Moreover, in human cortical cultures, AP fibrils induced hyperphosphorylation of tau resulting in altered microtuble binding (Busciglio et al., 1995). Ap-induced tau hyperphosphorylation may be a result of either increased kinase or decreased phosphatase activity. In particular, 9 sites that are hyperphosphorylated in PHF-tau are Ser/Thr-Pro sequences (Lee et al., 1991; Biernat et al., 1992) suggesting that disregulation of proline-directed kinases or their corresponding phosphatases occur in AD (Billingsley and 88 Kincaid, 1997; Trojanowski and Lee, 1995; Pelech, 1995). Although the proline directed kinases ERKs were once thought to be candidates for this disregulation (Drewes et al., 1992; Lu et al., 1993) experiments using tau-expressing cells have shown that ERK1 and ERK2 did not produce PFfF-like tau (Lovestone et al., 1994; Latimer et al., 1995). Likewise, the MEK1 inhibitor PD 98059 did not block okadaic acid induced tau hyperphosphorylation in cultured rat cortical neurons despite a block of ERK activation by this inhibitor (Ho et al., 1997). Though the involvement of ERKs in inducing PHF-tau seems unlikely, other stress-activated kinases within the MAPK family appear to be potential candidates (Reynolds et al., 1997a; Reynolds et al., 1997b). In particular, treatment of cultured neurons with A B was shown to induce c-jun immunoreactivity (Anderson et al., 1995) and produce tyrosine phosphorylation of proteins having an electrophoretic mobility similar to SAPK/JNK (Luo et al., 1995). Therefore, preliminary experiments were undertaken to determine whether or not A P induces activation of SAPK in neurons. Specifically, mixed rat cortical cultures prepared according the methods of Mills et al. (1997) were preincubated in serum free media containing N 2 supplements for 30 minutes and A P (20 uM) was added to the culture wells for 90 minutes. Twenty-five ug of cellular protein was separated by SDS-PAGE, transferred to a nitrocellulose membrane and probed with a phosphorylated JNK/SAPK (phospho-SAPK/JNK, New England Biolabs) antibody as previously described (Mills et al., 1997). SAPK phosphorylation was increased by both A P i . 4 0 and its longer more amyloidogenic counterpart A P i . 4 2 (Appendix III). Subsequent studies indicate that this effect was observed using physiological concentrations and was maximal at 200 nM A P i . 4 2 (B. Connup: personal communication). 89 VI. GENERAL DISCUSSION C L E A V A G E O F M E M B R A N E PROTEINS W I T H S O L U B L E C O U N T E R P A R T S : T H E R O L E OF P K C A N D M A P K I N R E G U L A T I N G R E L E A S E A variety of structurally unrelated membrane proteins undergo cleavage and subsequent release of their ectodomain (Echlers and Riordon, 1991). In spite of their varied structure these transmembrane proteins have the following in common; (1) they are released in a slow constitutive manner (2) agonists such as PKC and calcium inonophores regulate rapid release of their ectodomains and (3) the enzymes responsible, attack peptide bonds at a certain distance from the membrane, being somewhat independent of sequence (Sisodia, 1992; Arribas and Massague, 1995). The role of PKC and MAPK in these various cleavage events is currently unknown. The idea that these effectors may be altering protein secretion by increasing overall cellular metabolism is unlikely given the fact that their stimulation causes APPS release to be increased while A(3 release is decreased (Mills et al., 1997; Desdouits-Magnen et al., 1998; Gabuzda et al., 1993; Buxbaum et al., 1994; Buxbaum et al., 1993; Hung et al., 1993). Moreover, pulse-chase metabolic labeling indicated that cellular APP decreased proportionally to increased soluble APPS under constitutive and regulated conditions (Gabuda et al., 1993; Buxbaum et al., 1992). These findings suggested that the increase in APPS release was due to cleavage of its precursor. Alternatively, the MAPK pathway may represent a common pathway responding to multiple activators and initiating regulation of cleavage of a wide variety of membrane proteins. Indeed it would be interesting to determine whether or not regulation of other membrane anchored proteins such as transforming growth factor a 90 also involves ERK. Unraveling ERKs targets would help to predict the selectivity of their involvement in these various cleavage events (see below). R E G U L A T I O N OF A P P C A T A B O L I S M : I N V O L V E M E N T OF M A P K A N D D O W N S T R E A M E F F E C T O R S The discovery that a novel effector ERK was involved in PKC, growth factor and neurotransmitter regulation of APP catabolism is especially significant for two reason. Firstly, it revealed for the first time a signaling system capable of being activated by the diverse range of first messengers known also to regulate APP processing. Secondly, several important downstream targets have been uncovered as a direct consequence of this finding. Although ERK has been shown to be involved in regulation of APP processing it does not appear that ERK activation is necessary. The partial antagonism of both PKC and NMDA receptor stimulation of APPS release speak to the existence of an ERK-independent means of regulating APPS release. One such signaling system may involve members of the MAPK pathway other than the MEK1 substrates ERK1 and ERK2. Although MEK1 is the only known activator of these ERK isoforms, MEK has been shown to phosphorylate other downstream effectors. For example, a Golgi-associated MAPK has been identified as a phosphorylation target for MEK1 (Acharya et al., 1998) raising the possibility that MEK may phosphorylate other ERK isoforms. Phosphorylation of this unidentified ERK isoform could potentially regulate APP catabolism by altering its movement through the secretory pathway. 91 A second ERK-independent signaling pathway may involve PLA 2 (Fig. 15), a phospholipase recently implicated in G-protein-coupled receptor regulation of APPS release (Emmerling et al., 1993; Nitsch et al., 1996a; Nitsch et al., 1997). Receptor-mediated activation of PLA 2 occurs either via a Ca2+-dependent translocation of PLA 2 or agonist-induced MAPK phosphorylation (Farooqui et al., 1997a; Farooqui et al., 1997b): both mechanisms appear important for maximal stimulation of PLA2-induced arachidonic acid release (Schievella et al., 1995; Lin et al., 1993). In particular, NMDA receptor stimulation has been shown to produce a marked increase in PLA 2 stimulation , an effect which may be mediated by either of these mechanisms (Schievella et al., 1995; Lin et al., 1993). Therefore, PLA2-mediated regulation of APP catabolism may occur in a MAPK-dependent or -independent manner. Although PKC regulation of APP catabolism likely involves an ERK-dependent pathway (Mills et al., 1997; Desdouits-Magnen et al., 1998) PKC may also affect APP processing independently through trafficking of the holoprotein (see below). The implications for the PLA 2 signaling pathway in mediating APP processing are especially interesting given the recent finding that antagonism of arachidonic acid metabolism by the cyclooxygenase inhibitor indomethicin increases nonamyloidogenic processing of APP (Kinouchi et al., 1995). Precisely how the MAPK pathway intersects with the PLA 2 signaling system will be a matter of great interest, especially given the potential therapeutic utility of non-steroidal antiinflammatory drugs in the prevention of AD (McGeer and McGeer, 1996). 92 NMDA NGF T G N Proteasomes P S Figure 15. Regulation of APP catabolism via a MAPK-dependent or -independent pathway. MAPK may alter APP catabolism by phosphorylating PLA 2, secretases or proteins involved in APP trafficking (see below). Novel signaling pathways implicated by the present studies are indicated by hollow arrows. PS: presenilin; TGN: trans-Golgi network. M E C H A N I S M OF R E G U L A T I O N The mechanism by which various kinases regulate the secretory processing of APP are unknown. Although APP is phosphorylated by PKC (Suzuki et al., 1992) direct regulation by PKC through phosphorylation of the APP holoprotein is unlikely as mutants lacking the phosphate acceptor residues are still cleaved and secreted following PKC activation (DA Cruz 93 E Silva et al., 1993; Hung and Selkoe, 1994; Efthimiopoulos et al., 1994). Alternatively, protein kinases may also have a direct effect on the yet-to-be-identified secretases (Fig. 15) by altering their activity through phosphorylation. Indirect evidence for this has been suggested by studies using an APP construct resistant to proteolysis which was no longer susceptible to PKC-dependent regulation (Hung and Selkoe, 1994). Secretase activity would be reduced, indirectly, if these enzymes and their substrates were compartmentalized and if access of APP to the secretase-containing compartment were somehow altered. Indeed, evidence is now accumulating that a-, P- y- secretases are themselves localized differentially within the cell (Gabuzda et al., 1994; Checler, 1995). Given the short half-life of APP even subtle changes in the rate of APP transport through these various secretase containing compartments may have large effects on the net steady-state levels of APPS and Ap. Although the targets have not yet been identified, several examples of kinases regulating APP processing indirectly by phosphorylating proteins involved in intracellular trafficking now exist (Fig. 13). PKC and PKA have been shown to phosphorylate a tightly associated trans-Golgi network protein thereby altering the formation of constitutive secretory vesicles containing mature APP (Xu et al., 1995; Xu et al., 1996). Similarily, PKC and PKA may exert their effects by phosphorylating substrates in the APP secretory pathway which affect proteasome activity (Marambaud et al., 1996; Marambaud et al., 1997a; Marambaud et al., 1997b). Also, presenilin-1, another protein thought to alter APP processing via its effects on protein trafficking (Lemere et al., 1996; Citron et al., 1997; Borchelt et al., 1996; Weidemann et al., 94 1997; Ancolio et al., 1997), has a consensus sequence for ERK-dependent phosphorylation and has recently been shown to be a substrate for PKC (Walter et al., 1997a; Walter et al., 1997b; Seeger et al., 1997). Interestingly, presenilin-1 has recently been suggested to be localized to the nuclear membrane (Li et al., 1997), the endoplasmic reticulum (Thinkakaran et al., 1996; Kovacs et al., 1996; Cook et al., 1996) and the plasma membrane (Dewji and Singer, 1997). Therefore, presenillin regulation of APP trafficking could potentially occur at many sites within the cell. THERAPEUTIC APPROACHES RELATED TO A B PRODUCTION Therapeutic approaches aimed at reducing Ap production have focused on the yet-to-be identified secretases responsible for generating APP fragments. The observation that APP9 is dramatically stimulated by a number of first messengers and effectors has suggested that this may provide a mechanism for inhibiting AP production. However, what remains to be demonstrated is that there is a concomitant reduction in Ap production by these same stimuli in mature central neurons and that such findings relate to what is occuring in vivo. Interestingly, a recent in vivo study using a transgenic mouse model of AD expressing humanized AP indicated that PKC activation was coupled to a decrease in AP release although APPS was unchanged (Savage et al., 1998). Although such studies are encouraging, unrestrained activation of either PKC or MAPK has other potential drawbacks that could result in cellular dysfunction. In particular, prolonged stimulation of MAPK would likely interfere, adversely, with a variety of processes including signal transduction, transcription, cell cycle events, regulation of cytoskeletal elements and vesicular trafficking (Pelech 1995; 95 Pelech and Sanghera 1992; Pelech and Charest 1996). Although kinases have been a therapeutic target for other diseases such as inflammation and cancer, the strategy for drug development in these diseases has been to develope selective tyrosine kinase inhibitors (Levitzki and Gazit, 1995). Nevertheless, in time, it may be possible to develop agonists for MAPKs expressed preferentially in AD target neurons (Mohit et al., 1995). At present, such an approach to drug development in neurodegenerative disorders is still in its infancy. From a drug discovery perspective a more straightforward way of inhibiting A|3 formation is direct inhibition of either P- or y-secretases. As these enzymes remain unidentified, no specific inhibitors are currently available. Despite this limitation, a number of compounds have been identified which appear to preferentially inhibit y-secretase generating Api_4o. These studies suggested that cleavages at residues 40 and 42 are accomplished by different enzyme activities and that generation of preferential inhibitors of A P i _ 4 2 may also be possible (Klafki et al., 1996a; Citron et al., 1996) . Although offering the most promise, these compounds presumably block processing of APP to Ap through the disruption of vesicular transport and are therefore unlikely to be specific. Recent cloning of the only other known protease involved in intramembrane proteolysis (Rawson et al., 1997) may aid in identifying these y-secretases thereby providing a promising drug target. M A P K A C T I V I T Y A N D A D To date, emphasis has been placed on the upregnlation of kinase activity as implied by tau hyperphosphorylation in the AD brain. A number of proline directed kinases such as glycogen synthase kinase and cyclin dependent kinase are increased in sites of neuronal cell death in the 96 AD brain and have been shown to regulate both acetylcholine synthesis and tau phosphorylation in an inverse manner (Imahori and Uchida 1997; Busser et al., 1998; Lovestone et al., 1994). As MAPK was once thought to also be a contributor in these pathological events, studies have been designed to test increases in MAPK activity. Notably, one such study reported a slight reduction in ERK expression levels (Trojanowski et al., 1993). With the design of phosphospecific antibodies recognizing selective MAPK family members, the discovery of a novel MAPK expressed in a subset of neurons targeted in AD pathology (Mohit et al., 1995), it will be important to measure MAPK activity levels in the cortex and hippocampus of the AD brain to determine if changes in MAPK activity correlate with senile plaques, NFT and neuronal cell loss. In particular, I predict that activity of the novel MAPK p493F12 kinase, found in human pyramidal neurons of the hippocampus and neocortex decrease in relation to glutamatergic hypoactivity (see Chapter IV). A D A N D AB D E P O S I T I O N : U N A N S W E R E D Q U E S T I O N S It is widely hypothesized that the production and deposition of AB are early events in AD and may be a central pathological event in the disease process (Hardy, 1997; Selkoe, 1997). However, amyloid as a therapeutic target remains an elusive enemy. Although not necessarily aimed at decreasing the brain amyloid burden per se, many of the current therapeutic strategies for AD would likely decrease AP levels. These include (1) replacement of neurotransmitters (Morh et al., 1994; Schorderet, 1995; Giacobini, 1996) neurotrophins (Schorderet, 1995; Koliatsos, 1996) and hormones (Wickelgren, 1997) and (2) screening of antioxidants and nonsteroidal antiinflammatory agents (Schorderet, 1995; Munch et al., 1997; 97 O'Banion and Finch, 1996; McGeer and McGeer, 1996). With advances in our understanding of signal transduction mechanisms underlying APP processing events, the consequences of these treatment strategies on the ability of various cell types to process APP can be predicted. However, given the cell specific nature of APP processing, the net effect on the brain amyloid burden remains unknown. The idea that altered signal transduction underlies neurodegenerative disorders such as AD has been previously suggested (Morrison and Hof, 1997). For example, it is thought that altered signal transduction may lead to increased brain amyloid burden (Nitsch et al., 1992; Selkoe, 1993; Buxbaum and Greengard, 1996). Moreover, reductions of synaptic inputs have been shown to strongly correlate with the clinical phenotype of AD (Terry et al., 1991; Dekosky et al., 1990) suggesting that cognitive deficits are caused by a disruption of cortical and hippocampal neural circuits (Morrison and Hof, 1997). Altered signaling is also apparent by the quantitative differences in either kinase (Jin and Saitoh, 1995) or phosphatase activity (Gong et al., 1993) between the normal aging brain and the AD brain. This is especially interesting given the putative role played by kinases in memory processes (Schwartz, 1993; Kornhauser and Greenberg, 1997). If tyrosine kinase activity is, as has been suggested (Ullrich and Schlessinger, 1990), the primary indicator of signal transduction, these effectors may offer targets for sophisticated drug development in degenerative disease processes (Levitzki and Gazit 1995). Perhaps the most intriguing lines of inquiry regarding regulation of APP processing are those addressing the role of signal transduction in regulating trafficking of APP. These studies are 98 particularity interesting given the notion that different pools of APP exist in the cell which are differentially transported and therefore cleaved differently. Trafficking, and therefore cleavage of APP, may be cell specific. For example, although polarized cells such as neuronal cell lines and central neurons, do not express significantly more cellular APP, they appear to produce proportionally more intracellular Ap (Wertkin et al., 1993; De Strooper et al., 1995; Wild-Bode et al., 1997; Turner et al., 1996; Hartmann et al., 1997). As secreted Ap and intracellular AP appear to be generated by different mechanisms (Tienari et al, 1997; Wild-Bode et al., 1997; De Strooper et al., 1995; Hartmann et al., 1997) cell signaling events controlling the production of these two pools of Ap may be distinct. Underlying the effects of trafficking on APP regulation may be subcellular localization of secretase isoenzymes. Some precedent for the existence of differential localization of isoenzymes already exists for the y-secretases. Given that AP1.40 or its more amyloidogenic counterpart AP1.42 are produced in different compartments (Hartmann et al., 1997; Cook et al., 1997) it is conceivable that the y-secretase isoenzymes that produce them are regulated differentially. Finally, different pools of AP may be produced by secretases that cleave either constitutively or in a regulated manner. These secretases may or may not be one and the same. The idea that AP production can be regulated in both a temporal and spatial fashion demands further consideration as a means of reducing the brain amyloid burden through activation of signal transduction cascades. 99 T H E A M Y L O I D C A S C A D E HYPOTHESIS REVISITED The amyloid cascade hypothesis is presently the dominant hypothesis of AD. The strength of the amyloid hypothesis derives from the fact that it can accomodate all known and molecularily defined causes of AD. However, its limitations include the fact that (1) amyloid deposition poorly correlates with the degree of dementia, synapse loss, neuronal loss, upregulation of endosomes and NFT and (2) amyloid deposition does not appear to preceed other neuropathological features or behavioural deficits (Fraser et al., 1997; Neve and Robakis, 1998). The first criticism has been easily dismissed by the argument that it makes the assumption that 'pathology waits to be counted' (Hardy, 1997). However, the second inconsistency in the amyloid hypothesis not easily dismissed by even the most ardent of Paptists. Opponents of the amyloid hypothesis have used this as a platform for the argument that amyloid depoisits are not, in and of themselves neurotoxic. Rather, they suggest that the process underlying peptide fibril formation is responsible for the neurotoxicity of AP instead of the fibril structures themselves (Mattson, 1997). Evidence for this comes from the fact that AP is most toxic when its rate of aggregation is maximal: fully aggregated AP is actually less toxic (Mattson, 1997). If indeed plaque formation is a dynamic and protective process, it follows that Ap deposition would not correlate well with other neuropathological features of AD. Paptist opponents have suggested that mechanisms other than those mediated by Ap deposition could also contribute to the neurodegeneration observed in AD. These include altered release of soluble APP fragments known to have numerous cellular functions at 100 physiological concentrations or the effects of C-terminus fragments generated following 0-secretase cleavage of APP (Fraser et al., 1997; Neve and Robakis, 1998; Mattson, 1997; Mattson et al., 1997; Auld et al., 1998; Suh, 1997). APPS has been shown to be neuroprotective in vitro and in vivo, reducing intracellular Ca 2 + and suppressing neuronal activity by increasing K + channel conductance. Conversely, low concentrations of AP have been shown to be neurotoxic, increasing intracellular Ca 2 + by acting on voltage-sensitive Ca 2 + channels, K + channels and receptors that mediate IP3-induced intracellular Ca 2 +. Moreover, physiological concentrations of Ap have been shown to induce cholinergic hypofunction inhibiting cholinergic enzyme activity and acetylcholine release in vitro and in vivo. 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Infection of rat cortical neurons with a mutant herpes simplex virus A. Cholinergic regulation of APPS secretion in mixed cortical cultures overexpressing the human muscarinic receptor M i . At 10-14 days in vitro cultures were infected with a mutant herpes simplex virus encoding the M i receptor. Following infection, APPS was measured from the culture media after a 1 hour drug exposure. Lanes: 1. Control; 2. Oxotremorine-M; 3. Oxotremorine-M + Atropine; 4. PDBu. ([3H] N-methylscopolarnine binding sites were approximately 10 fold above background). B. A mutant herpes simplex virus containing P-galactosidase infects rat cortical cultures enriched for neurons. Enriched neuronal cultures (< 10% GFAP positive cells) were stained for P-galactosidase following infection with a mutant herpes simplex virus encoding this prokaryotic enzyme (Left Panel: mock infected; Right Panel: virus infected). However, [3H] N-methylscopolamine binding sites of enriched neuronal cultures infected with the same virus encoding the M i receptor did not differ from mock infected or virus alone suggesting that viral infection does not increase expression of M i receptors in neurons. 124 APPENDIX II P C 1 2 D N 1 APR D E X D E X Figure 15. Effect of dexamethasone on basal APPS release. The glucocorticoid receptor agonist dexamethasone (0.3 uM) decreased basal APPS release following a 12 hour preexposure. 125 APPENDIX III Figure 16. SAPK/JNK phosphorylation in mixed rat cortical cultures is increased by Ap. Representative Western blots of 25 pg of cell lysate probed with either a phospho-SAPK/JNK or SAPK/JNK antibody following a 90 min. exposure to 20 pM APMO or ApY 4 2 (n=2). Blots were probed first with a phospho-SAPK/JNK antibody, stripped and then reprobed with a SAPK/JNK antibody. 126 

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