Apoptosis in Neurodegenerative Diseases

Apoptosis in Neurodegenerative Diseases

lkuo Nishimoto" Takashi Okamotot Ugo Giambarella" Takeshi IwatsuboS *Department of Pharmacology and Neurosciences Keio University School of Medicine T...

2MB Sizes 55 Downloads 213 Views

lkuo Nishimoto" Takashi Okamotot Ugo Giambarella" Takeshi IwatsuboS *Department of Pharmacology and Neurosciences Keio University School of Medicine Tokyo 160, Japan tDepartment of Medicine Harvard Medical School Cardiovascular Research Center Massachusetts General Hospital Charlestown, Massachusetts 02 I29 $Department of Neuropathology and Neuroscience Faculty of Pharmaceutical Sciences University of Tokyo Tokyo 113, Japan

Apoptosis in Neurodegenerative Diseases

1. Introduction Apoptosis is primarily a morphological concept, representing a specific type of cell death that is accompanied by striking alterations in nuclear and cytoplasmic shapes [l].It was originally defined as shrinkage necrosis [2], cell death with morphological changes in the nucleus (such as chromatin condensation, nuclear fragmentation and compaction, disappearance of nucleoli, and micronucleation) associated with cytoplasmic changes (such as shrinkage and blebbing followed by micronuclei extrusion) [3-51. Today the term apoptosis usually implies naturally or developmentally occurring cell death (programmed cell death) or cell death triggered by signal transduction mechanisms (signal-based cell death or active cell death). These modes of cell death have considerable overlap. For example, ced-3, the cell death gene of Caenorhabditis elegans involved in programmed cell death, is a homolog of [email protected] enzyme (ICE) [6], which causes rnamAduunces in I'harmacology, V d u m e 4 1

Copyright 0 1997 by Acddcrnic Press. All right5 of reproduction in any tomi reserved 1054-.3589/97 $25.00



lkuo Nishirnoto et a/.

malian apoptosis [7].The fact that Fas stimulation leads to apoptotic death of T lymphocytes indicates that some signal-based cell death is apoptotic. However, developmentally occurring cell death and signal-based cell death represent different concepts. In any given system, the role of apoptosis in these modes of cell death must be examined. The benefit of the study of apoptosis is the unexpected knowledge that cells from microorganisms to humans are furnished with the intracellular machinery to kill themselves by a genetic mechanism (see the chapter by Desnoyers and Hengartner). In multicellular organisms, this self-destroying machinery is turned on in certain cells during embryogenesis [8]; in others, it is never used throughout life. The biological significance of apoptosis in the immune system (reviewed in the chapters by Eischen and Leibson and Ucker) is well established in processes such as clonal deletion [9], irradiation or glucocorticoid-induced immunotoxicity [ 101, and even autoimmune diseases [11,12]. It is an attractive idea that aberrant use of the “suicide” machinery may also cause mammals to suffer from degenerative diseases after birth. In the central nervous system, apoptosis is also important for organogenesis and synapse formation that occur during development [13]. However, the role of apoptosis in neurodegenerative diseases has remained unclear. Several factors contribute to this uncertainty. First, apoptosis usually ends in secondary necrosis 141, making it difficult to ascertain whether cells died initially by apoptosis or necrosis. Second, the slow growth of neurons makes it unclear whether apoptotic changes can occur at significantly increased frequencies in the diseased brain compared to a healthy control [14]. This comparison is rendered more difficult by the fact that apoptotic cells are removed by phagocytosis [ 151, therefore minimizing the opportunity to detect them in clinical specimens. Incomplete understanding of the molecular basis for apoptosis creates other obstacles. Finally, uncertainty regarding the suitability of various methods for detecting apoptosis creates additional problems. Endonuclease-mediated DNA digestion at internucleosomal sites is a characteristic feature of apoptosis, producing small, double-stranded fragments of DNA that migrate in a 180-bp ladder pattern after electrophoresis in agarose gels [16].However, cell death with classic apoptotic morphologies can occur without the ladder-like degradation of DNA [17,18], or even without a nucleus [19]. Moreover, several examples of apoptosis occur with cleavage of DNA into large fragments in the absence of internucleosomal fragmentation [20]. Recent introduction of the TdT-mediated dUDP-associated nick end labeling (TUNEL) method [21] has allowed visualization of DNA strand breaks in situ in the diseased brain and has substantiated the idea that a number of neurodegenerative diseases are associated with significant DNA fragmentation. Although the TUNEL method is sufficiently sensitive to detect a limited number of DNA strand breaks in apoptotic cells, even in the

Apoptosis in Neurodegenerative Diseases


early phases of cell death [21], random DNA cleavages occurring as a late event in necrosis are also occasionally detected by TUNEL [22]. In contrast, the random smear of DNA in necrosis is preferentially detected by the in situ nick translation method, which labels single-strand breaks. Gold et al. [23] combined these two in situ labeling techniques and reported that necrosis and apoptosis could be distinguished. Nonetheless, the idea that cell death can be categorized in only two types, apoptosis and necrosis, has been questioned [13,24,25]. The cellular heterogeneity of the central nervous system also complicates the study of apoptosis in neurodegenerative diseases. Apoptosis has been primarily defined in individual cell types: T cell apoptosis, developmental apoptosis, and apoptosis of cultured cells are all relatively homogeneous. Human diseases afflict various tissues and cells that interact with other types of tissues or cells of a different nature. In a human brain, for example, many adjacent cells may modify the fate of apoptotic neurons or other types of cells. It is also possible that apoptotic cells can release toxic substances and cause surrounding cells to undergo nonapoptotic death. These considerations raise the possibility that cell death with pure apoptotic features may not occur in human brains afflicted by neurodegenerative diseases. Despite the fact that neuronal death is fundamental to neuronal loss in any neurodegenerative disease, little is known about its nature or mechanism. In the following sections, we will summarize the current understanding of apoptosis in neurodegenerative diseases and especially the mechanistic aspects of Alzheimer’s disease.

II. Pathological Features of Neurodegenerative Diseases and Disorders In this chapter, pathological features that characterize human neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, motor neuron diseases, cerebral ischemia, and others, are briefly summarized and discussed in relation to their molecular background and the relevance to apoptotic death in neurons. A. Alzheimer’s Disease

Alzheimer’s disease (AD) afflicts more than 4 million people in the United States and is a major cause of late-life dementia. This disease is characterized pathologically by progressive degeneration and loss of neurons in the brain, particularly in the olfactory bulb, the cerebral neocortex, and the hippocampus. These changes result in memory disturbance and loss of higher cortical functions, eventually leading to dementia. Since its discovery


lkuo Nishimoto et 01.

in 1902, AD has been associated with two types of abnormal microscopic structures: senile plaques and neurofibrillary tangles. Senile plaques are composed of extracellular amyloid deposits intermixed with dystrophic neurites and reactive glial cells (Fig. 1).Senile plaque amyloid is composed of amyloid P protein (AP), a 40- to 43-amino acid hydrophobic polypeptide, that is proteolytically produced by as yet unidentified proteases called P- and y-secretases from a set of transmembrane precursor proteins collectively referred to as P-amyloid protein precursor (APP) [26]. y-Secretase, which cleaves the C terminus of the AP region, produces two major AP species with different C termini, Apl-40 and Apl-42 [27]. Although Apl-40 is the predominant species of physiologically secreted AP, the longer Apl-42 (or 43), which comprises -10% of total secreted A p [27], is more aggregative in vitro [28] and is preferentially deposited in the AD brain [29-311. Thus, it is highly likely that this longer version of AP, not Apl-40, is the major cause for the formation of senile plaques. However, the role of AD deposition or even formation of senile plaques in the pathogenesis of AD remains unknown. This issue will be discussed further in a later section. Another type of abnormal deposit in the AD brain is the neurofibrillary tangle, which accumulates inside the neuronal cytoplasm. The tangles are composed of bundles of abnormal fibrils designated as paired helical fila-

FIGURE I Neuritic dystrophy in Alzheimer’s disease brains. Some dystrophic neurites, darkly impregnated by silver grains, are associated with amyloid plaques (arrows), whereas others are distributed irrespective of amyloid deposits (arrowheads). Modified Bielschowsky’s silver staining. Magnification: X5.50.

Apoptosis in Neurodegenerative Diseases

34 I

ments (PHFs). PHFs are composed of hyperphosphorylated forms of microtubule-associated protein 7 [32]. It is reasonable to assume that excessive phosphorylation of T is due either to hyperactive protein kinases or to defective phosphatases in afflicted neurons. Protein kinases that can phosphorylate T into PHF forms, including MAP kinases [33], GSK3P [34], and cdk.5 [35], have been purified. TDephosphorylation is also attenuated in the AD brain [36]. The relationship of enhanced phosphorylation or impaired dephosphorylation to the pathophysiology of AD is yet to be determined. In contrast to senile plaques, the occurrence of neurofibrillary tangles better correlates with the degree of neuronal loss and clinical dementia [37], suggesting a causative relationship between neurofibrillary tangle formation and neuronal death. However, there is as yet no evidence that PHF is directly toxic to neurons, nor that every dying neuron in AD develops neurofibrillary tangles. Benzing et al. (381 argued against the role of PHF in the pathogenesis of AD by showing that dystrophic neurites in the AD brain initially lack evidence of PHF. Recent advances in the molecular genetic studies of familial AD (FAD) have clearly shown that the polymorphism of apolipoprotein E (apoE), one of the major apolipoproteins in plasma, is one of the determinants for the susceptibility to AD. Among the three common genotypes of apoE (E2, E3, and E4), which result in allelelic variation of two amino acid residues of the apoE polypeptide, the apoE4 genotype increases the risk of developing late-onset AD [39]. Although apoE is a component of senile plaque amyloid and interacts with [email protected] to promote fibril formation in uitro [39], it is still unknown whether apoE4 increases the risk for AD by accelerating senile plaque formation or by other entirely different mechanisms. Recently, genes responsible for early onset FAD located on chromosomes 14 and 1 have been cloned and a number of missense mutations were identified. These genes encode entirely novel, multiple membrane-spanning proteins designated as presenilin 1 (PS-1) [40] and presenilin 2 (PS-2) [41,42]. Although physiological functions of PS-1 and PS-2 as well as their pathological phenotypes in FAD are yet to be elucidated, a relationship of PS-1 and PS-2 to the [email protected] intracellular membranous compartments [43] and a role of PS-2 in apoptosis [44] have been proposed. The mode of neuronal death in AD is a matter of debate. Attempts have been made to determine whether it conforms to one of the classical prototypes of cell death, i.e., apoptosis or necrosis. Several investigators have performed histochemical TUNEL studies of sporadic AD for the detection of DNA strand breaks. Lassmann et al. 1451 found an -30-fold increase in the number of cells showing DNA fragmentation in AD brains compared to age-matched controls. Although 28% of the cells with DNA fragmentation were identified as neurons, the rest were mostly oligodendrocytes and microglial cells. Forty-one percent of neurons with DNA fragmentation contained neurofibrillary tangles, which was 2.5 times higher than in neurons


lkuo Nishirnoto et al.

without DNA fragmentation. Conversely, 21 % of neurofibrillary tanglebearing neurons showed DNA fragmentation, which was 3 times higher than in neurons without neurofibrillary tangles. Thus, there was a significant correlation between neurofibrillary tangle formation and DNA strand breaks, although they do not necessarily occur in the same neuron. The majority of neuronal nuclei with DNA fragmentation did not show the classical morphological characteristics of apoptosis, such as chromatin condensation and nuclear fragmentation. Other recent studies have also provided histochemical evidence for the occurrence of DNA fragmentation in cortical neurons as well as in astrocytes [46-481in AD brains, although an earlier study failed to observe DNA strand breaks [49].Here, we must take into account the stages of disease. In view of the fact that occurrence of neuronal apoptosis drastically differs in various stages of Huntington’s disease (see below), it is plausible that different mechanisms of cell death might underlie the different stages of AD as well. Further confirmatory studies under well-controlled conditions, along with biochemical and other experimental investigations, are needed to elucidate the mechanism underlying neuronal death in AD brains.

B. Other Neurological Disorders Parkinson’s disease (PD)is another common neurodegenerative disorder afflicting individuals in mid to late life. Extrapyramidal symptoms, including rigidity, tremor, and akinesia, are the characteristic clinical features of PD. These symptoms are caused by a reduction of dopamine in the neostriatum as a result of the loss of neurons in the substantia nigra. Other brain stem neurons, including locus ceruleus and dorsal vagal nucleus, are also affected. Some of the degenerating neurons develop characteristic intracytoplasmic inclusions called Lewy bodies. The precise mechanism of neuronal degeneration in PD is still elusive; however, the clinical and pathological similarities to parkinsonism induced by a neurotoxin l-methyl-4-phenyl-l,2,3,6tetrahydropyridine (MPTP) have attracted the attention of investigators [SO]. Although a recent report showed that MPP+, a toxic derivative of MPTP, can induce apoptosis in primary cultured ventral mesencephalic neurons [51], suggesting a role of apoptosis in MPTP-induced degeneration of catecholaminergic neurons, nigral neurons failed to show DNA fragmentation in a TUNEL histochemical study of the PD brain [Sla]. Huntington’s disease (HD), a hereditary neurodegenerative disease with autosomal-dominant inheritance, presents with choreic involuntary movements and dementia. The site of neurodegeneration in HD is the neostriatum (caudate nucleus and putamen) and cerebral cortex. In the neostriatum, medium-sized spiny neurons that emit striatofugal projections degenerate and decrease in number. This pathology is reminiscent of the patterns of excitotoxic neuronal injury caused by glutamate receptor agonists, including

Apoptosis in Neurodegenerative Diseases


kainate and quinolinic acid [52]. A causative gene for HD has been cloned [ 5 3 ] .It is located on the IT15 locus of chromosome 4p and encodes a protein named huntingtin. The abnormal expansion of the CAG trinucleotide repeat in the N terminus of huntingtin has been identified as a cause of this disease. The normal function of huntingtin is not known nor has any pathogenic effect of the abnormal huntingtin with the elongated glutamine repeat been found. Some of the neostriatal neurons, as well as oligodendrocytes and astrocytes in the H D brain, show evidence for DNA strand breaks [22,47,54]; however, clear DNA ladders have not yet been demonstrated on gel electrophoresis. One of the most important lessons from cell death studies in HD is the realization that the occurrence of DNA fragmentation depends on pathological stages [22]. In grade 0 HD brains, the authors found very few TUNELpositive cells; in grades 1 and 2, numerous neurons in striatum demonstrated DNA fragmentation; and in grades 3 and 4, the TUNEL-positive cells were still prominent, but there were fewer TUNEL-positive neurons in the grade 4 tissue compared to grade 3 . Because neurodegenerative diseases progress in distinct pathological stages, comprehensive studies that characterize the quantitative alterations in DNA fragmentation by stage, as done in HD, are necessary for each neurodegenerative disease. Motor neuron diseases are a group of neurodegenerative disorders affecting lower and/or upper motor neurons, leading to atrophy of the skeletal muscles. Motor neuron diseases include amyotrophic lateral sclerosis (ALS), an adult-onset progressive fatal disorder; and hereditary spinal muscular atrophy, which affects children. Although little has been known as to the mode of neuronal death in ALS, some investigators have shown the presence of TUNEL-positive neurons in the spinal anterior horn of ALS patients [55]. Approximately 20% of patients with familial ALS, which is an autosomal dominant disease, have point mutations in the gene that encodes CulZn superoxide dismutase (SOD1) [56]. Both clinical disease and severe pathological changes with striking similarities to human ALS were produced in transgenic mice overexpressing mutant SOD1 [57]; however, no typical features of apoptosis have been noted. This suggests that neuronal loss and atrophy of the anterior horns in ALS may not be due to apoptosis. In contrast, cloning revealed that a candidate gene for hereditary spinal muscular atrophy encodes a neuronal apoptosis inhibitor protein NAIP, which may act as a negative regulator of motor neuron apoptosis [58]. These observations highlight the potential relevance of apoptosis to the pathophysiology of motor neuron diseases. In a similar vein, Masu et af. [ 5 9 ] reported that targeted disruption of the CNTF gene results in a progressive atrophy and loss of motor neurons in adult mice, suggesting that motor neuron degeneration (by apoptosis) is a process that neurotropic factors antagonize.


lkuo Nishimoto et 01.

Ischemic brain damage is one of the most common causes of neuronal death. Cerebral infarction is the most common form of brain ischemia and is caused by the occlusion of arteries that perfuse a given area of the brain, which usually results in a death process typical of necrosis, i.e., characterized by cellular swelling and lysis at an acute phase. However, a subset of neurons, including those in the CA1 portion of the hippocampus, are susceptible to a particular form of neuronal death, designated delayed neuronal death, when exposed to transient or incomplete ischemia [60]. These neurons show no pathological changes immediately after the ischemic insult, but instead undergo cell death after 2 or 3 days. Recent experimental studies on cerebral ischemia have accumulated evidence that degenerating neurons in delayed neuronal death exhibit apoptosis (i.e., DNA ladders on gel electrophoresis and TUNEL-positive nuclear changes) [61-681. Thus, cerebral ischemia may induce both types of cell death, apoptosis and necrosis, depending on the mode and intensity of the ischemic insult. Neuronal apoptosis has also been implicated in other categories of neurological disorders including traumatic brain injury [69], experimental allergic encephalomyelitis [70], and HIV encephalitis [71].

111. Mechanisms for Cytotoxicity in Alzheimer’s Disease A. A P Hypothesis AD deposition, which is the feature common to sporadic and familial AD and the AD-like disease observed in Down’s syndrome, may directly contribute to neurodegeneration in AD. In further support of this idea, deposition of AD as diffuse plaques is the earliest pathological change so far known in AD and Down’s syndrome brains [30] and precedes the manifestation of dementia. Other support for this hypothesis is offered by the pathological finding that AD deposits specifically associate with dystrophic neurites [72] (Fig. 1) and areas of neuronal loss [73] in brains afflicted with AD. The genetic finding that mutations in APP found in the Swedish type of FAD lead to remarkable overproduction of AD [74,75] also supports a central role of AD deposition in the pathogenesis of AD. Extensive studies have shown that AD peptides induce neurotoxicity in culture [76-871. In vivo, neurotoxicity following intracerebral administration of AD was observed in rats and monkeys [88-921. Learning impairment became manifest in rats after infusion of AD into their cerebral ventricles for 14 days by a perfusion pump [93]. However, others have reported that neurotoxicity of AD is not observed in vitro or in vivo [92,94-961 or is observed only under certain conditions [97]. The inconsistency was attributed, at least in part, to the lot-to-lot variability or “aging” (days after preparation of AD)-dependent difference in the fibril-forming states of AP

Apoptosis in Neurodegenerative Diseases


preparations [98,99]. It was also attributed to differences in the minor components of senile plaques, which may increase the toxicity of AP [loo]. However, it remains unclear whether the neurotoxic effects of AP can account for the neurodegeneration in AD or whether AP-independent mechanisms also exist. Allen et al. [85] reported that neurotoxicity was induced in vitro by non-AP amyloidogenic peptides as well as AP; and Emre et al. [ l o l l observed that neurotoxicity was also evoked in vivo by reverse AP peptide AP40-1 or even by vehicle, casting doubt on the specificity and significance of such AP actions. It is also disputed whether A/3 aggregation accounts for the neurotoxicity elicited by this peptide in vitro [99,102-1051. The mode of AD-induced neuronal death is also confusing. Loo et al. (791reported that AP induces apoptosis in primary cultures of mouse cortical and rat hippocampal neurons. Nitric oxide and N-methybaspartic acid gated channel activation was implicated in AP-induced apoptosis in a neuronal hybrid of substantia nigra and neuroblastoma [106], although Busciglio et al. [lo71 argued against the involvement of excitotoxins. On the other hand, Schubert and colleagues reported that AP causes necrosis in rat pheochromocytoma PC12 cells and rat cortical neurons [84]. This cell death was not prevented by bcl-2 overexpression (821. These contradictory findings may be attributable, at least in part, to the different systems employed, because the ability of AP to induce cytotoxicity depends on the type of cells [ 1081. However, the difference in the systems used by these investigators seems too minor (mouse versus rat) to explain the striking differences in their results. The neurotoxic effects of AP might be mediated by [email protected] cell surface proteins, such as the serpin-enzyme complex (SEC) receptor [109]. Consistent with this idea, physiological levels of [email protected] tyrosine phosphorylation, probably through activation of focal adhesion kinase, and cytosolic calcium levels in neuronal cells [110,111], suggesting a receptormediated specific signaling mechanism for the action of AP. Khalil et al. 11121 showed that AD competes with the action of the ligand for the SEC receptor. However, Schubert et al. [ 1131 argued against the role of this effect in the pathogenesis of AD based on the result that various proteins of other human amyloidoses were as cytotoxic to cultured cells as AP. Accordingly, Arispe et ul. [114] and Mirzabekov et al. (1151 suggested that AD directly affects intracellular Ca2+ homeostasis by acting as a calcium ionophore. Recently, RAGE, the -50-kDa receptor for advanced glycation endproducts, has been shown to specifically bind AP and mediate the generation of thiobarbituric acid-reactive substances and the reduction of MTT [116]. A link between expression of RAGE and the pathophysiology of AD remains to be established. The toxicity of AP has been postulated to be mediated by loss of calcium homeostasis [117-1231, c-Jun induction (1241, impairment of ion-motive ATPase [125], increased outward conductance of choline [126], and oxida-


lkuo Nishimoto et 01.

tive stress [83,99,113,124,327-1311. It has been reported that [email protected] cytotoxicity is inhibited by sulfonated aye [132], Congo red [80,133], Congo rubin [133], calcium channel blockers [134] (although another group [135] reported that calcium channel blockers were ineffective in the same system), expression of a calcium-binding protein [ 1361, heparin sulfate and chondroitin sulfate glycosaminoglycans [137], tumor necrosis factor [ 1381, ceramide [139],growthfactors (basic FGF [140], insulin [141], andTGF-P [142,143]), heat shock [ 1441, and antioxidantdfree radical scavengers (vitamin E [Sl], rifampicin [145], and a synthetic free radical scavenger EUK-8 [146]).Interestingly, nerve growth factor does not protect against AP neurotoxicity 1141 1, but rather potentiates it (761. Antagonism of AP neuropathic effects by substance P [76,76a,89,112] and by thrombin [148] has been reported but remains controversial [103,123,141]. A broad spectrum of evidence suggests that oxidative stress is one of the most likely signals for A0 neurotoxicity. Oxidative stress stimulates production of AD in mammalian lenses [ 1491, allowing a self-perpetuating cycle of [email protected] production and oxidative stress. However, in transgenic mice overexpressing a familial ALS mutant of SOD1 that increases oxidative stress, neuronal death is characterized by vacuolar degeneration of neuronal nuclei [57],a process histochemically distinct from that in AD, diminishing the possibility that oxidative stress plays a central role in the AD type of neurodegeneration. The relevance of the reported [email protected] to the pathogenesis of AD is limited by the fact that most studies were performed with Apl-40 or 25-35 derivatives thereof rather than with Apl-42, the central amyloid protein in the AD brain. Apl-42 is more amyloidogenic than Apl-40 [28] and has been implicated in senile plaque formation [29-311. Secretion of Apl-42 increases in cells expressing V642 type of FAD-APP compared with those expressing normal APP [27,96], whereas secretion of Apl-40 decreases. Therefore, in a genetic form of AD, Apl-42 appears to play a more important role than Apl-40. Because transgenic mice overexpressing V642F APP exhibited massive amyloid plaques consisting mainly of Ap142 [150], neuropathology of this FAD type is at least partly due to the overproduction of Apl-42. However, despite its neurotoxicity in vitro, mice producing excess extracellular Apl-42 showed virtually no neuronal loss [lSl]. In addition, neuronal loss was not evident in transgenic mice overexpressing V642F type of mutants of APP695and [150] and APP7SI [ 150,152] despite significant AP formation. Conversely, no amyloid plaque formation was noted in transgenic mice overexpressing wild-type APPdg5, whereas these mice exhibited multiple phenotypes indicative of hippocampal dysfunction [ 1531. Other transgenic strains carrying extra copies of the gene encoding APP7sI,but not transgenic APPhrsmice, showed age-related learning deficits [154] and early AD-like histopathology related to 7 [155]. However, Perry et al. 11561 reported that marked impairments in the Morris water maze task and deficits in coordinated foot placement and swimming movements

Apoptosis in Neurodegenerative Diseases


observed in the APP7SImice may be secondary consequences of motor deficits and not related to overexpression of the transgene or abnormal deposition of the amyloid protein. In transgenic mice overexpressing APP mutated in the a-secretase cleavage sites, neurodegeneration and disturbed behavior occur without significant deposition of AD [ 1571. These observations indicate that Apl-42 deposits are not sufficient and are not required to cause the neurodegeneration characteristic of AD, suggesting that APP can contribute to neurodegeneration via mechanisms independent of AP-induced neurotoxicity. Yamatsuji et ul. [96] showed that expression of FAD-associated mutants of APP kills neuronal cells via a mechanism independent of AD. In neuronal F11 cells, transient expression of V642 mutants of APP resulted in death with DNA fragmentation. However, DNA strand breaks were scarcely induced by incubating F11 cells with 50 p M synthetic Apl-40 or Apl-42, with the conditioned medium (CM-V642I) obtained from V6421 APP-transfected cells that were undergoing DNA fragmentation, or with a mixture of CMV642I and 50 FM Apl-42. The concentrations of Apl-42 in CM-V642I/ F/G were approximately 10 pM as determined by an enzyme-linked immunosorbent assay. Expression of the V6421 APP mutant lacking the 41st and 42nd residues of the Ap region, which encodes no Apl-42, induced DNA fragmentation to an extent comparable to that induced by V642I APP. In contrast, expression of the V642I APP mutant lacking cytoplasmic amino acids H657-K676 induced little apoptosis, whereas this mutant secreted AP peptides into cultured media at amounts similar to those secreted by V642I APP. Pertussis toxin abolished V642I APP-induced DNA fragmentation with only minor quantitative alterations in secreted AP peptides. These FAD-associated mutants of APP therefore cause neuronal death in a manner independent of A0 secretion.

B. Normal Function of APP Does APP possess any normal function?APP consists of 695-770 amino acids encoded by differentially spliced mRNAs transcribed from a single gene located on human chromosome 21 [26]. The 695-amino acid form of APP, referred to as is expressed abundantly and rather specifically in the brain, whereas all other identified forms of APP bear a Kunitz protease inhibitor domain (which is absent in APP,,,) in their extracellular portions and are expressed widely [158-1611. APP has a structure and transmembrane orientation resembling a polypeptide hormone receptor [ 158,1621. It consists of an extracellular, a transmembrane, and a cytoplasmic domain. Only the extracellular region is glycosylated. The cytoplasmic region, which is not glycosylated, is well conserved and almost identical across species [163]. These are common features that


lkuo Nishimoto et ol.

a number of cell surface receptors share, suggesting that APP may function as a cell surface receptor. A number of observations also suggest that APP might function as a receptor. Neve and colleagues [164] provided the first evidence that the cytoplasmic domain of APP generates intracellular signals relevant to cell death. Recently, they have found that the C-terminal portion of APP binds intracellular enzymes, including APP-BP1, which is homologous to ubiquitin-activating enzymes [165]. Fiore et al. [166] found, using a yeast two-hybrid system, that the cytoplasmic domain of APP binds to Fe65, which has a phosphotyrosine binding (PTB) domain homologous to that of Shc, an oncogene product. The PTB domain of Shc mediates the transduction of signals from upstream receptors to downstream effectors. At residues 3 16-337 in its extracellular domain, APT',,, has a high-affinity binding site for heparin; and the affinity for heparin is increased two- to fourfold in the presence of micromolar zinc (11),another ligand for APP, raising the possibility that APP might be an allosteric protein 11671. Collectively, these observations suggest that APP might be a functional receptor. Consistent with the role of APP as a receptor, we previously demonstrated that APP can bind Go [168], a finding subsequently confirmed by other investigators [169,170]. A clue was the presence in APP of a heterotrimeric G protein activator sequence motif, which specifies the G proteincoupled regions in receptors [171], including the adrenergic, muscarinic, and dopaminergic receptors [ 172-1741. Searches for G protein-linked sequences in receptors conducted by other investigators 1175-1781 verified the validity of the motif theory. Based on this motif, Nishimoto et al. 11681 found the G protein activator sequence in APP,,, to be H657-K676. This domain activated G, (we use this term for its trimeric form) selectively among G,,, G,?,G,,, G,, Ras, and Rab 3A. Immunoprecipitates obtained from brain membranes using anti-APP antibody contained Gaoimmunoreactivity. Recombinant coprecipitated purified Go, whereas the APP,95 lacking H6.57-K676 lost its G,, association. Treatment with GTPyS resulted in dissociation of G,, from APPhri, consistent with typical features of receptor-G protein interactions. Okamoto et al. [ 1791 demonstrated, using reconstituted phospholipid vesicles, that APP,,, behaves like a normal receptor that couples to G,, in a manner dependent on ligand binding. Because the natural ligand of APP remains unknown, the authors examined the effect of cross-linking APP using a monoclonal antibody. Antibodies for the extracellular domains of receptors may act as their agonists, as OKT3 does for T cell receptor [ 1801. In reconstituted APP6,,/G,, vesicles, 22C11, a monoclonal antibody against K66-E81 of APP,,, [181], activated G,, in an antibody-specific, dose-dependent manner. Liganded promoted the GDP/GTP exchange of G, but not its intrinsic GTP hydrolysis activity, consistent with G protein activation by typical G-coupled receptors. The stimulatory effect of 2 2 C l l on G,,

Apoptosis in Neurodegenerative Diseases


was attenuated by a synthetic peptide corresponding to APP K66-E81. A monoclonal antibody against APP H657-K676 inhibited the 22C1 l-induced G, activation, whereas control antibodies against other domains did not. These findings provide direct evidence that APP functions as a G,,-linked signaling receptor. Ferreira et al. [182] and Culvenor e t a / . [183] demonstrated the presence of APP in surface membranes and clathrin-coated vesicles of neurons and transfected HeLa cells, respectively, lending additional credence to the notion that APP is a functional membrane receptor. Colocalization of APP with G,, as well as GAP-43 in growth cones and presynapses of neurons [182,184,185] is another strong argument that G, acts as a transducer of signals from cell surface APP. In further support, it has been shown that APP [Sla,186-190] and Go [193-1961 are involved in identical functions of neurons, such as neurite outgrowth, synaptic contact, and cell-cell adhesion. Because GAP-43 serves as a Go-coupled receptor-specific potentiator [ 1971, colocalization of GAP-43 with APP and G, further strengthens this theory. In animal models [192,198,199], APP has been shown to be engaged in spatial learning and exploratory behavior. In C. elegans, Ga, also plays an important role in behavior [200,201], at least partly consistent with the notion that G, is a neuronal transducer of APP. However, we do not exclude the possibility that the G, and G12subclasses of G proteins or other types of transducers may also be linked to APP. Murayama et al. [202] provided evidence that APP acts as a cell surface receptor that upregulates MAP kinases in living cells. When COS-NK1 cells, a neuron-like transformant of COS-7 cells [203], were treated with 22Cl1, slight but significant stimulation of MAP kinases was observed over a rapid time frame. Murayama et al. [202] also showed that 2 2 C l l stimulates the p42 MAP kinase ERK2 but not the p44 MAP kinase ERKl in glioma cells overexpressing Pertussis toxin blocked ERK2 activation induced by 22C11. The suggestion that APP6g5selectively activates ERK2 by a pertussis toxin-sensitive G-linked pathway is consistent with the report that thrombin, a stimulant of a typical G protein-coupled (G-coupled) receptor, selectively activates ERK2 in platelets [204]. Crespo et al. [205] found that the GPy subunit of heterotrimeric G proteins stimulates the Ras and MAP kinase pathway, suggesting that APP induces the release of Goy from G, and thereby activates the MAP kinase cascade. In addition, Ga,, is also capable of activating MAP kinases by a protein kinase C-dependent mechanism [206]. Although Greenberg et al. [207] reported that MAP kinases are activated when cells are treated extracellularly with soluble APP, it seems unlikely that 22C11 activates MAP kinases through the secretion of soluble APP because this antibody blocks the action of soluble APP 11871. Instead, it is tempting to hypothesize that soluble APP activates intracellular MAP kinases through interaction with the cell surface APP because soluble APP may aggregate with membrane-bound APP and promote its conversion to


lkuo Nishimoto et ol.

an active conformation. Indeed, activation of transmembrane APP is likely mediated by its aggregation [ 1791, Aggregation is also required for activation of signaling by pl,4-galactosyltransferase, another G,-coupled receptor with a single transmembrane domain [178], and rhodopsin, a representative Gcoupled receptor with a heptahelical structure 12.081. Alternatively, soluble APP may act on cell surface APP-binding proteins, such as LDL, receptorrelated protein [209]. C. V642 Type of FAD Mutants of APP In early onset FAD, missense mutations V6421, V642F, and V642G have been identified within the transmembrane domain in (V642 corresponds to V717 in APP,,, [210-2141. These mutations cosegregate with the AD phenotype 12151 and account for most, if not all, of the evidence showing a linkage of AD to chromosome 21 [216]. However, little has been known about how these mutations cause AD or what abnormality they induce in APP. As mentioned previously, these mutants of APP are associated with increased Apl-42 production and decreased Apl-40 production [27]. In addition, each mutant APP (FAD-APP) behaves as a constitutively active receptor in vitro and in vivo. In reconstituted vesicles, the three recombinant FAD-APP proteins activate purified G, without ligands; for this activation, the H657-K676 domain is required [2171. Expression of FAD-APP stimulates the effects of Go-MAP kinase activation and cAMP response element suppression-in living cells, confirming the constitutive action of FAD-APP on G,, in vivo 1202,2181. Ikezu et al. [218] investigated the coupling of V642F APP to chimeric Ga, proteins. They constructed various Ga, chimeras consisting of Gas (1-389), which lacks the original five residues of Ga, at the C terminus, and the C-terminal five residues of known a subunits of the G; family (Gas/ a x ) Because , the C-terminal five-residue region is the receptor contact site and Gasactivates all isoforms of adenylyl cyclase, Gaslaxis recognized and activated by Ga,-coupled receptors, leading to the stimulation of adenylyl cyclase. Among Gasla;,Ga,/a,, GaJa,, and Gas,expression of V642F APP selectively activated Gasla,,and increased cAMP levels. In contrast, Gasla,, was not activated by the expression of normal APP. These findings suggest that V642F APP is coupled selectively to Ga,, among similar G proteins in living cells. Collectively, the results suggest that FAD-APPs are gain-offunction mutants that constitutively trigger G,-linked pathways. Although the concept that the signaling function of APP is a target of the FAD mutations is a new one, it is not without precedent. Several point mutations are known to create constitutive signaling by other G-coupled receptors in human diseases [219-2221: a mutant thyrotropin receptor in thyroid adenoma, a mutant luteinizing hormone receptor in familial male precocious puberty, a mutant parathyroid hormone receptor in metaphyseal

Apoptosis in Neurodegenerative Diseases

35 I

chondrodysplasia, and mutant rhodopsins in retinitis pigmentosa. The V642 type of FAD is another example in which constitutively active G-coupled receptor mutants are implicated. G, is engaged in neural network formation [193,194,196] and synaptic long-term potentiation of hippocampus [223,224]; Therefore, abnormal activation of Go by FAD-APP would disturb pivotal functions of neurons, potentially leading to pathogenic processes of FAD. Gocan activate multiple effectors phospholipase C [225,226], MAP kinases and protein kinase C [206], and inhibit calcium channels [227]. Multiple observations suggest that activity of G, may be upregulated in patients with sporadic AD. First, intracellular adenylyl cyclase is suppressed in AD tissues [22,228-23 11. Because Go is a physiological inhibitor of adenylyl cyclases in neuronal cells [232-2361, sustained activation of G, is likely to suppress adenylyl cyclases in the AD brain. It has also been reported that high-affinity forms of muscarinic receptors disappear in the AD brain [237,238]. This seems to be a typical feature of activation of G proteins that are coupled to muscarinic receptors, probably G, and G,. It has also been observed that Ca2+influx is suppressed [239] and polyphosphoinositide turnover is enhanced in AD tissues [240]. These findings are again consistent with activation of Go in AD tissues, because Go is the only known G protein that can both inhibit calcium channels and potentiate polyphosphoinositide turnover [241], Finally, activation of Gohas been implicated in apoptosis of neurons [242], and constitutively activated mutations of a typical G-coupled receptor rhodopsin turn on apoptosis pathways in retinal neurons, leading to retinitis pigmentosa [243]. Based on these previous studies linking Go activation and apoptosis, the effect of FAD-APP on apoptosis was examined in cultured cells transfected with each FAD-APP cDNA [203]. These studies were performed using COSNK1 cells, which, in contrast with usual COS cells, express Ga, endogenously. Four different approaches (enzyme-linked immunosorbent assay using antibodies against histones and DNAs, DNA ladder formation, alteration of nuclear morphology, and TUNEL staining) consistently revealed that all three FAD-APPs, but not normal APP,ys, cause NK1 cells to undergo apoptosis. The notion that FAD-APP cytotoxicity is apoptotic was further confirmed by the finding that Bcl-2 overexpression prevented DNA fragmentation. The intermediary role of AP peptides was excluded by the methods used in the study of F11 neuronal cells (see AP hypothesis). Instead, an intermediary role of Go was suggested by the observation that FAD-APPs were unable to induce apoptosis in usual COS cells lacking Go. Furthermore, apoptosis by V642I APP was blocked by pertussis toxin (a blocker of GI/ Go), by cotransfection of dominantly interfering Ga, mutant, and by the deletion of the H657-K676 domain whose only known target is Go, confirming the intermediary role of G, in FAD-APP-mediated cell death. Given the fact that FAD-APPs are receptor-like molecules that constitutively activate


lkuo Nishimoto et al

G,, 12171, the G-protein-linked pathway most likely triggers apoptosis. A novel mechanism is therefore proposed: Expression of FAD-APP causes cell death by turning on the apoptosis pathway via an intracellular receptor mechanism linked to a G-protein signaling cascade. To substantiate the relationship between the pathogenesis of FAD and induction of apoptosis by FAD-APP, the apoptosis-causing ability of all 19 possible V642 APP mutants was examined in NK1 cells, which originated in the kidney [203]. Despite similar expression and cellular distribution of APP695,three FAD-APPs, and all the other 16 mutants in NK1 cells, the three FAD-APPs yielded the highest incidence of apoptosis among all the mutants; normal APP,, yielded the lowest incidence of apoptosis. Therefore, induction of apoptosis by APP mutants was phenotypically linked to the FAD trait in the NK1 system, suggesting that induction of apoptosis by FAD-APPs reflects a key pathological process of FAD. Yamatsuji et al. [96] have also utilized TUNEL to demonstrate that DNA fragmentation is induced by the expression of the three FAD-APPs in neuronal FI1 cells, a hybrid of primary neurons and neuroblastoma cells. Again, little fragmentation is induced by suggesting that a point mutation in the transmembrane segment of APP turns on a pathway that leads to neurotoxicity. The same study also excluded the intermediary role of Apl-42 (see Section III,A) and defined the involvement of G,, in this neurotoxicity. The demonstration that Bcl-2 overexpression can antagonize the FAD-APP-induced fragmentation of DNA in F11 cells (I. Nishimoto, unpublished observation) suggests that this neurotoxicity is mediated by an apoptotic mechanism. This notion is consistent with the fact that DNA fragmentation by FAD-APP-induced Goactivation in NK1 cells reflects apoptosis [203] and that Go or pertussis toxin-sensitive G proteins are involved in apoptosis in cultured neurons [242] and the immune system [244-2461. The elucidation of a mechanism by which FAD-APP can cause neuronal cell death has important implications for therapy. The observation that the cytoplasmic amino acids H657-K676 of V642I APP play a central role for neurotoxicity raises the possibility that manipulating the function encoded by this domain would allow for the regulation of FAD-APP neurotoxicity. Likewise, the presence of a Go-mediated cascade raises the possibility that interference with downstream components of this pathway might modulate FAD-APP neurtotoxicity. Elucidation of the role of Go-linked pathways in the pathogenesis of FAD also has important implications for the pathogenesis of nonfamilial AD. Go is a major protein in the brain (1or 2 % of the total proteins in brain membranes) [247] and is concentrated in the brain areas that correspond well to those severely afflicted by AD. The intermediary role of G, in the action of FAD-APP suggests a novel idea that various insults causing activation of Go (or the G proteins that share the same downstream pathways with Go)

Apoptosis in Neurodegenerative Diseases


may be able to induce biological abnormalities similar to those induced by FAD-associated mutants of APP (Fig. 2). Also, insults may stimulate this pathway independent of G, by activating points lower in the cascade. Thus, the outcome, DNA fragmentation in selected neurons, may be the same for all insults, including FAD-APP, which activates this pathway. This cascade theory allows for the possibility that various causes induce similar or the same AD pathophysiology and may well explain the conclusion of genetic analysis that AD is heterogeneous. The products of PS genes responsible for other types of FAD encode membrane integral proteins with seven or eight transmembrane domains [40-421. Because such proteins may be G-coupled receptors, this discovery might provide support for the concept that abnormalities in G-protein-linked pathways are common promoters of AD, although the real function of PS remains unknown. Recently, PS-2 has been implicated in apoptosis [44]. Employing a “death trap” system to select genes involved in apoptosis of a mouse T cell hybridoma, six apoptosislinked genes (ALG-1 to -6) were isolated. ALG-3, which encodes the 103 carboxyl-terminal amino acids of PS-2, protected the T cells from T cell receptor- and Fas-induced apoptosis. Although it will be necessary to define the function of PS-2 using full-length PS-2 and neuronal cells, PS-2 may regulate apoptotic pathways, either positively or negatively. The concept that a point mutation in the transmembrane domain of a cell surface molecule can contribute to a disease is not without precedent. c-ErbB2 is a normal receptor tyrosine kinase and is converted to the oncogene Neu by the transmembrane mutation V664E (Fig. 2). Neu has constitutive kinase activity and causes transformation [248], whereas c-ErbB2 has no transforming activity. Interestingly, a similar mechanism appears to be at work in the FAD-APP-induced cell death system (Fig. 2). In this system, activation of the downstream machinery by the FAD mutations probably causes cell death with DNA fragmentation. In both systems, point mutations in the transmembrane domains of cell surface proteins, probably by facilitating aggregation, induce constitutive activity in their effector systems. The resulting cellular malfunctions (transformation and apoptosis) are probably essential processes in the mechanisms of the respective diseases of cancer and AD. It is also similar that only a small fraction of each disease is caused by these mutations. Because studies of tyrosine kinases have provided a growing body of information in cancer research, further studies of FAD-APPinduced apoptosis are expected to contribute to analyzing and developing therapies for AD. If the apoptotic effect of FAD-APP is so rapid, why does it take many years for patients with FAD to present clinically? One possibility is that expression of the cell death apparatus, which is necessary for this effect of FAD-APP, is age dependent. Alternatively, the cell death effect of FAD-APP may be antagonized by defense mechanisms that deteriorate with aging. A

FIGURE 2 Transmembraneactivation of FAD-APPand c-ErbB2 systemsby their intramem-

branous mutations. (Bottom) FAD-APP induces cell death with DNA fragmentation through C protein activation. Thus, other C-coupled receptor mutants with constitutive activity could induce biological abnormalities similar to those induced by FAD-APP. This theory allows for the possibility that the insults bypassing the G protein and activating its downstream pathway could cause cellular malfunction similar to FAD. (Top)c-ErbB2 is a normal receptor tyrosine kinase and is converted to oncogenic Neu by the V644E mutation in its transmembrane domain. Neu has constitutive kinase activity and causes transformation, whereas c-ErbB2 has n o transforming activity. In the FAD-APP system, activation of the downstream machinery by FAD mutations causes apoptotic cell death, whereas normal APP has no such activity. In both cases, point mutations of a receptor or a receptor-like protein induce dominant abnormality in their effector systems and cause cellular malfunctions that are probably critical processes of each disease.

Apoptosis in Neurodegenerative Diseases


similar discrepancy has been noted between the in vitro and in vivo effects of FAD-APP. In vitvo (in cultured neuronal cells), overexpression of FADAPP leads to neuronal death. At this time, transgenic mice overexpressing FAD-APP genes have not developed convincing neurodegeneration similar to that observed in patients with FAD. This discrepancy may again suggest the existence of suppressors that ameliorate FAD-APP-mediated neurotoxicity in vivo, as Fukuchi et al. [249] have posed. If this is true, the cellular systems described previously might prove useful for identifying cellular mechanisms that protect against FAD-APP-mediated cytotoxicity.

D. Down’s Syndrome Down’s syndrome (DS), or trisomy 21, is a common genetic cause of mental retardation. Development of the DS brain is associated with decreased number and abnormal differentiation of neurons. Adults with DS subsequently develop AD. No histochemical study with TUNEL has examined the mode of neuronal death occurring in the DS brain. In culture, however, cortical neurons from fetal DS and age-matched control brain differentiate normally, but DS neurons subsequently degenerate and undergo apoptosis, whereas normal neurons remain viable [250],suggesting that apoptosis again underlies the DS neurodegeneration. Degeneration of DS neurons was prevented by treatment with free radical scavengers or catalase, suggesting that oxidative stress is involved in apoptosis of DS neurons. Overexpression of normal nonmutated APP in the nervous system is probably responsible for the pathophysiology of AD observed in DS. Obviously, increased [email protected] production is one of the possible causes. However, transgenic mice overexpressing wild-type developed an age-related central nervous system disorder without AP deposition [153], suggesting that [email protected] are not a prerequisite for degeneration of neurons overexpressing APP. Moechars etal. [156] reported that expression in brain of APP mutated in the a-secretase site causes disturbed behavior, neuronal degeneration, and premature death in transgenic mice. Again, the lack of detectable [email protected] deposition or plaque formation was noted. Because normal degradation of APP is attenuated by such mutations, this mutant APP protein should have a long half-life. Therefore, these mice may potentially mimic APP overexpression in DS. These results suggest that overexpression of normal APP potentially causes neuronal loss without [email protected] The observation that an increased density of G-coupled receptors on the cell surface spontaneously activates G proteins and switches on Gcoupled intracellular pathways [251J raises the possibility that long-term overexpression of APP can activate Go to a significant degree in a ligandindependent fashion. In other words, a locally augmented density of APP


lkuo Nishimoto et 01.

on the cell surface may increase the fraction of aggregated APP, which then activates intracellular transducers [217]. Therefore, overexpression of APP, even if the APP is normal, may activate the G,-linked pathway and cause programmed death in neurons [96,242]. Alternatively, neuronal death by APP overexpression in DS may proceed through the same mechanism as in the sporadic form of AD (see below). In support of this latter model, Fukuchi et al. [252] suggested that overexpression of APP in human neuronal cells leads to the same aberrant cleavage of APP as occurs in sporadic AD.

E. Sporadic AD Except for AP deposition, there are few clues about the mechanism responsible for neuronal loss in sporadic AD, the most common form of this disease. However, significantly increased frequencies of DNA fragmentation in the brain with sporadic AD provide the first clue. Although apoptosis was once thought to be irrelevant to neuronal loss in sporadic AD, a number of laboratories independently observed that DNA fragmentation is a major feature in pathology in brains from sporadic AD donors [45-481. Because AD peptides do not always induce DNA fragmentation of cells, the observed DNA fragmentation in sporadic AD is unlikely to be explained simply by deposited AD. The second clue is that constitutive activation of the receptor function of APP, which is probably mediated by its aggregation [179], was responsible for the induction of neuronal death by FAD-associated mutations [96,217]. This neurotoxicity proved to be independent of AP secretion. Nevertheless, the deposition of AD is a common feature of all sporadic forms of AD, which provides us with the third clue. Clearly, the deposition of AD suggests that augmented N-terminal cleavage from APP has occurred locally, i.e., in the cells nearby. The simplest interpretation of these findings is that the AP cleavage, not secreted AP, is involved in the mechanism that triggers DNA fragmentation of neurons. The conversion of EGF receptor to v-erbB, a truncated EGF receptor that functions as an oncogene product, highlights another mechanism in which a receptor becomes constitutively active: A receptor signal can be turned on by losing an extracellular N-terminal domain. During the process for the cleavage of AP from normal APP, several intermediate fragments are potentially produced. It is conceivable that some of the APP fragments become constitutively active and trigger the death signal encoded by their cytoplasmic domains (Fig. 3 ) . In this regard, the study of Neve and colleagues [164], which was subsequently confirmed by other groups [253,254], seems quite important. They observed that expression of the C-terminal lOO-amino acid tail (C100) of APP causes demise of neuronal cells. This death was apoptotic because


EGF receptor

LPP fragment







PROLIFERATION (the same output as point mutants induce)







FIGURE 3 A putative mechanism, except for the AP hypothesis, whereby C-terminal fragments of APP cause neurotoxicity. (Left) Although the ECF receptor is a normal receptor tyrosine kinase that regulates cell proliferation, v-ErbB, the N-terminally truncated EGF receptor, is a constitutively active receptor and induces transformation. This suggests that deletion of extracellular portions from receptors is another way to switch on signaling through the cytoplasmic domains of the receptor. (Right) During the process of cleavage of Ap from normal APP, several intermediate fragments are potentially produced. The analogy with the oncogenic conversion of the EGF receptor to v-ErbB suggests that some of the APP fragments might become constitutively active and trigger the death signal encoded by the cytoplasmic domain of APP.


lkuo Nishimoto et 01.

it was associated with cellular shrinkage and detachment from plates. Conditioned medium, acquired from cells expressing the C terminus, was not cytotoxic [253].Overexpression of ClOO caused dystrophic neurites in transgenic mice 125.71. ClOO spans the region from the first residue of the A 0 sequence to the extreme cytoplasmic end of APP, and thereby consists of the entire [email protected] region and the whole cytoplasmic domain. Generation of a similar fragment by N-terminal cleavage of APP at the site of AP might be the mechanism underlying pathogenesis of sporadic AD [256-259). Because N-terminal deletions from APP at the site of AP enhance aggregation of the AP region [260], C100, the APP with the N-terminus deletion just before AP, would tend to aggregate, which could constitutively turn on the intracellular death signal through its cytoplasmic domain. In support of this model, Lynn et al. [261] found that overexpression of a C-terminal 97-amino acid fragment of APP in PC12 cells resulted in a continuous intracellular Ca2' wave, indicating that the C-terminal fragment of APP has a capacity to constitutively turn on intracellular signaling pathways. Therefore, the AP sequence may be important for aggregation of APP, which switches on the intracellular machinery after deletion of the large extracellular N terminus of APP. Further research will be necessary to determine whether expression of ClOO generates signals and outputs similar or identical t o V642 mutants of APP and whether G protein-mediated DNA fragmentation is implicated in the C100-induced neuronal death.

IV. Closing Remarks In this chapter, we have reviewed pathological features of various neurodegenerative diseases, the relevance of apoptosis in their pathogenesis, and underlying genetic abnormalities. We have also described proposed mechanisms for AD neurodegeneration. Although little has been known about the molecular mechanisms for other neurodegenerative diseases, it is possible to define the neurotoxic mechanisms in each disease by applying approaches similar to those used in AD studies. Elucidation of the neurotoxic mechanisms will also highlight the normal functions of the disease-related genes. In this regard, it is extremely important to identify the native ligand that turns on the receptor function of APP, if any, and to clarify the physiological role played by the signaling function of APP. In addition, further technological progress is required to clarify the role of apoptosis in neurodegenerative diseases. Indeed, the TUNEL technique has drastically advanced the study of apoptosis in these diseases. However, the DNA fragmentation detected by this method potentially reflects both apoptosis and necrosis. Therefore, an advanced method of TUNEL that can specifically stain the 180-bp nucleosomal-sized fragmentation of DNA in situ would open a new horizon in this research field.

Apoptosis in Neurodegenerative Diseases


One hundred years ago, researchers explored the mechanisms of diseases by examining samples microscopically. Now, we have alternative approaches to investigate disease mechanisms at a molecular level. For instance, we can overexpress or deplete particular molecules in a cell or even in an individual animal and observe the consequences. We can also identify the gene abnormalities responsible for human familial diseases by gene linkage analysis with chromosome markers. Furthermore, as reviewed elsewhere in this volume (e.g., the chapters by Desnoyers and Hengartner, Thornberry et al., Canman and Kastan, and Reed) the identification of several genes that govern apoptosis, e.g., bcl-2 [262], C. eleguns cell death genes [263], 953 [264], c-myc [265], nur77 [266], and ICE [7], and its family [267], has provided a molecular foothold from which to analyze the key steps in the activation and execution of apoptosis. Therefore, the best strategy to elucidate the role of apoptosis in neurodegenerative diseases would be to characterize the mechanisms responsible for apoptotic and nonapoptotic death of neurons and then to examine whether either or both mechanisms are activated by gene abnormalities responsible for each neurodegenerative disease. Acknowledgments We thank Ken Bloch and T. Bernard Kinane for critical reading of the manuscript and Dovie Wylie, Keisuke Kouyama, and Tomo Yoshida for expert technical assistance.

References 1. 2. 3. 4. 5. 6.

7. 8. 9. 10. 11.

12. 13. 24. 15. 16. 17.

D. Hockenbery, A m . J . Pathol. 146, 16-19 (1995). J. F. R. Kerr,]. Pathol. 105, 13 (1971). J. F. Kerr, A. H. Wyllie, and A. R. Currie, BY. J . Cancer 26, 239-257 (1972). A. Wyllie, J. F. R. Kerr, and A. R. Currie, Int. Rev. Cytol. 68, 251-306 (1980). A. H. Wyllie, R. G. Morris, A. L. Smith, and D. Dunlop, J. Patbol. 142, 67-77 (1984). J. Yuan, S. Shaham, S. Ledoux, H. M. Ellis, and H. R. Horvitz, Cell 75, 641-652 (1993). M. Miura, H. Zhu, R. Rotello, E. A. Hartwieg, and J. Yuan Cell 75, 653-660 (1993). J. R. Tata, Dev. Biol. 13, 77-94 (1966). G. G. Singer and A. K. Abbas, Immunity 1, 365-371 (1994). J. J. Cohen and R. C. DukeJ. Immunol. 132, 38 (1984). J. Wu, T. Zhou, J. Zhang, J. He, W. C. Gause, and J. D. Mountz, Proc. Nutl. Acad. Sci. USA 91,2344-2348 (1994). S. Nagata and T. Suda, Immunol. Today 16, 39-43 (1995). E. M. Johnson and T. L. Deckwerth, Annu. Rev. Neurosci. 16, 31-46 (1993). D. E. Bredesen, in “Neuronal Apoptosis: Genetic and Biochemical Modulation in Apoptosis II” (L. D. Tomei and F. 0. Frederick, Eds.), pp. 397-421. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1994). R. E. Ellis and H. R. Horvitz, Cell 44, 817-829 (1986). A. H. Wyllie, Nature 284, 555-556 (1980). L. D. Tomei, J. P. Shapiro, and F. 0.Cope, Proc. Natl. Acad. Sci. USA 90,853-857 (1993).


lkuo Nishimoto et ol,

18. E. Falcieri, A. M . Martelli, R. Bareggi, A. Cataldi, and L. Cocco, Bzochem. Biophys. Res. Commun. 193, 19-25 (1993). 19. M. D. Jacobson, J. F. Burne, and M. C. Raff, EMBO J. 13, 1899-1910 (1994). 20. F. Oherhammer, J . W. Wilson, C. Dive, I. D. Morris, J. A. Hickman, A. E. Wakeling, P. R. Walker, and M. Sikorska, EMBO J . 12, 3679-3684 (1993). 21. Y . Gavrieli, Y. Sherman, and S. A. Ben-Sasson, J. Cell Biol. 119, 493-501 (1992). 22. L. B. Thomas, D. J. Gates, E. K. Richfield, T. F. O’Brien, J. B. Schweitzer, and D. A. Steindler, Exp. Neurol. 133, 265-272 (1995). 23. R. Gold, M . Schmied, G. Giegerich, H. Breitschopf, H. P. Hartung, K. V. Toyka, and H. Lassman, Lab Invest. 71, 219-225 (1994). 24. A. H. Wyllie, Int. Rev. Cytol. 17, 755-785 (1987). 25. D. L. Vaux, Proc. Natl. Acad. Sci. USA 90, 786-789 (1993). 26. D. J. Selkoe, Annu. Rev. Cell. Biol. 10, 373-403 (1994). 27. N. Suzuki, T. T. Cheung, X.-D. Cai, A. Odaka, L. Otvos, Jr,, C. Eckman, T. E. Golde, and S. G. Younkin, Science 264, 1336-1340 (1994). 28. J. T. Jarrett, E. P. Berger, and P. T. Lansbury, Jr., Biochemistry 32, 4693-4697 (1993). 29. T. Iwatsubo, A. Odaka, N. Suzuki, H . Mizusawa, N . Nukina, and Y. Ihara, Neuron 13, 45-53 (1994). 30. T. Iwatsubo, D. M. A. Mann, A. Odaka, N. Suzuki, and Y. Ihara, Ann. Neurol. 37, 294-299 (1995). 31. A. Tamaoka, T. Kondo, A. Odaka, N. Sahara, N. Sawamura, K. Ozawa, N. Suzuki, S. Shoji, and H. Mori, Biochem. Biophys. Res. Commun. 205, 834-842 (1994). 32. V. M.-Y. Lee, B. J. Balin, L., Jr., Otvos, and J. Q. Trojanowski, Science 251, 675678 (1991). 33. G. Drewes, B. Lichtenberg-Kraag, F. Diiring, E.-M. Mandelkow, J. Biernat, J. Goris, M. DorCe, and E. Mandelkow, EMBO J. 11,2131-2138 (1992). 34. K. Ishiguro, A. Shiratsuchi, S. Sato, A. Omori, M . Arioka, S. Kobayashi, T. Uchida, and K. Imahori, FEBS Lett. 325, 167-172 (1993). 35. S. Kobayashi, K. Ishiguro, A. Omori, M. Takamatsu, M . Arioka, K. Imahori, and T. Uchida, FEBS Lett. 335, 171-17.5 (1993). 36. J. Q. Trojanowski and V. M. Lee, FASEB J. 9, 1570-1576 (1995). .37. P. A. Arriagada, J. H. Growdon, E. T. Hedley-White, and B. T. Hyman, Newofogy 42, 631-639 (1992). 38. W. C. Benzing, D. R. Brady, E. J. Mufson, and D. M. Armstrong, Brain Res. 619,55-68. 39. W. J. Strittmatter, A. M . Saunders, D. Schmechel, M. Pericak-Vance, J. Enghild, G. S . Salvesen, and A. D. Roses, Proc. Natl. Acad. Sci. USA 90, 1977-1981 (1993). 40. R. Sherrington, E. I. Rogaev, Y. Liang, E. A. Rogaeva, G. Levesque, M. Ikeda, H. Chi, C. Lin, G. Li, K. Holman, T. Tsuda, L. Mar, J.-F. Foncin, A. C. Bruni, M. P. Montesi, S. Sorbi, I. Raniero, L. Pinessi, L. Nee, I. Chumakov, D. Pollen, A. Brookes, P. Sanseau, R. J. Polinsky, W. Wasco, H. A. R. Da Silva, J. L. Haines, M. A. Pericak-Vance, R. E. Tanzi, A. D. Roses, P. E. Fraser, J. M. Rommens, and P. H. St. George-Hyslop, Nature 375, 754-760 (1995). 41. E. I . Rogaev, R. Sherrington, E. A. Rogaeva, G. Levesque, M . Ikeda, Y. Liang, H. Chi, C. Lin, K. Holman, T. Tsuda, L. Mar, S. Sorhl, B. Nacmias, S. Piacentini, I,. Amaducci, I. Chumakov, D. Cohen, L. Lannfelt, P. E. Fraser, J. M. Rommens and P. H. St. GeorgeHyslop Nature 376, 775-778 (1995). 42. E. Levy-Lahad, W . Wasco, P. Poorkaj, D. M. Romano, J. Oshima, W. H. Pettingel, C.-E. Yu, P. D. Jondro, S. D. Schmidt, K. Wang, A. C. Crowley, Y.-H. Fu, S. Y. Guenette, D. Galas, E. Nemens, E. M. Wijsman, T. D. Bird, G. D. Schellenberg, and R. E. Tanzi, Science 269, 973-977 (1995). 43. D. Scheuner, C. Eckman, M. Jensen, X. Song, M. Criron,N. Suzuki,T. D. Bird, J. Hardy, M. Mutton, W. Kukll, E. Larson, E. Levy-Lahad, M. Vitanen, E. Peskind, P. Poorkaj,

Apoptosis in Neurodegenerative Diseases

36 I

G. Schellenberg, R. Tanzi, W. Wasco, L. Lannfelt, D. Selkoe, and S. Younkin, Nut. Med. 2, 864-871 (1996). 44. P. Vito, E. Lacanh, and L. DAdamio, Science 271, 521-525 (1996). 45. H. Lassmann, C. Bancher, H. Breitschopf, J. Wegiel, M. Bobinski, K. Jellinger, and H. M. Wisniewski, Acta Neuropathol. 89, 35-41 (1995). 46. J. H. Su, A. J. Anderson, B. J. Cummings, and C. W. Cotman, Neuroreport 5, 25292533 (1994). 47. M. Dragunow, R. L. M. F a d , P. Lawlor, E. J. Beilharz, K. Singleton, E. B. Walker, and E. Mee, Neuroreport 6, 1053-1057 (1995). 48. G. Smale, N. R. Nichols, D. R. Brady, C. E. Finch, and W. E. Horton, Exp. Neurol. 133, 250-230 (1995). 49. A. Migheli, P. Cavalla, S. Marino, and D. Schiffer, J. Neuropathol. Exp. Neurol. 53, 606-616 (1994). 50. J. W. Langston, P. Ballard, J. W. Tetrud, and I. Irwin, Science 219, 979-980 (1983). 52. H. Mochizuki, N. Nakamura, K. Nishi, and Y. Mizuno, Neurosci. Lett. 170, 191194 (1994). 5 l a . D. H. Small, V. Nurcombe, G. Reed, H. Clarris, R. Moir, K. Beyreuther, and C. L. Masters, J. Neurosci. 14, 2117-2127 (1994). 52. M. F. Beal, N. W. Kowall, D. W. Ellison, M. F. Mazurek, K. J. Swartz, and J. B. Martin, Nature 321, 168-171 (1986). 53. The Huntington’s Disease Collaborative Research Group, Cell 72, 971-983 (1993). 54. C. P. Portera-Cailliau, J. C. Hedreen, D. L. Price, and V. E. Koliatsos, J. Neurosci. 15, 3775-3787 (1995). 55. Y. Yoshiyama, T. Yamada, K. Asanuma, and T. Asahi, Acta Neuropathol. 88, 207211 (1994). 56. M. E. Gurney, H. Pu, A. Y. Chiu, M. C. Dal Canto, C. Y. Polchow, D. D. Alexander, J. Caliendo, A. Hentati, Y. W. Kwon, H. X. Deng, et al., Science 264,1772-1775 (1994). 57. M. C. Dal Canto and M. E. Gurney, Brain Res. 676, 25-40 (1995). 58. P. Liston, N. Roy, K. Tamai, C. Lefebvre, S. Baird, G. Cherton-Horvat, R. Farahani, M. McLean, J. Ikeda, A. MacKenzie, and R. G. Kornrluk, Nature 379,349-353 (1996). 59. Y. Masu, E. Wolf, B. Holtmann, M. Sendtner, G. Brem, and H. Thoenen, Nature 365, 27-32 (1993). 60. T. Kirino, Brain Res. 239, 57-69 (1982). 61. M. Okamoto, M. Matsumoto, T. Ohtsuki, A. Taguchi, K. Mikoshiba, T. Yanagihara, and T. Kamada, Biochem. Biophys. Res. Commun. 196, 1356-1362 (1993). 62. A. Heron, H. Pollard, F. Dessi, J. Moreau, F. Lasbennes, Y. Ben-Ari, and C. CharriautMariangue, 1. Neurochem. 61, 1973-1976 (1993). 63. J. P. MacManus, A. M. Buchan, I. E. Hill, I. Rasquinha, and E. Preston, Neurosci. Lett. 164, 89-92 (1993). 64. S. Kihara, T. Shiraishi, S. Nakagawa, K. Toda, and K. Tabuchi, Neurosci. Lett. 175, 133-136 (1994). 65. I. Ferrer, A. Tortosa, A. Macaya, A. Sierra, D. Moreno, F. Munell, R. Blanco, and W. Squier, Brain l’athol. 4, 115-122 (1994). 66. T. Nitatori, N. Sato, S. Waguri, Y. Karasawa, H. Araki, K. Shibanai, E. Kominami, and Y. Uchiyama, J. Neurosci. 15, 1001-1111 (1995). 67. Y. Li, V. G. Sharov, N. Jiang, C. Zaloga, H. N. Sabbah, and M. Chopp, Am. J. Pathol. 146,1045-1051 (1995). 68. T. Iwai, A. Hara, M. Niwa, M. Nozaki, T. Uematsu, N. Sakai, and H. Yamada, Brain Res. 671, 305-308 (1995). 69. A. Rink, K.-M. Fung, J. Q. Trojanowski, V. M.-Y. Lee, E. Neugabauer, and T. K. Mclntosh, Am. J. Pathol. 147, 1575-1583 (1995). 70. M. P. Pender, K. B. Nguyen, P. A. McCombe, and J. F. R. Kerr, J. Neurol. Sci. 104, 81-87 (1991).


lkuo Nishimoto et ol.

71. C. K. Petito and B. Roberts, Am. 1. Pathol. 146, 1121-1130 (1995). 72. G. Perry, P. Cras, S. L. Siedlak, M. Tabaton, and M. Kawai, A m . J. Pathol. 140, 283-290 (1992). 73. W. C. Benzing, E. J. Mufson, and D. M. Armstrong, Brain Res. 606, 10-18 (1993). 74. M. Citron, T. Oltersdorf, C. Haass, L. McConlogue, A. Y. Hung, P. Seubert, C. VigoPelfrey, I. Lieberburg, and D. J. Selkoe, Nature 360, 672-674 (1992). 75. X.-D. Cai, T. E. Golde, and S. G. Younkin, Science 259, 514-516 (1993). 76. B. A. Yankner, A. Caceres, and L. K. Duffy, Proc. Natl. Acad. Sci. USA 87, 90209023 (1990). 7hu. B. A. Yankner, L. K. Duffy, and D. A. Kirschner, Science 250, 279-282 (1990). 77. A. E. Roher, M. J. Ball, S. V. Bhave, and A. R. Wakade, Biochem. Biophys. Res. Commun. 174, 572-579 (1991). 78. C. J. Pike, D. Burdick, A. J. Walencewicz, C. G. Glabe, and C. W. Cotman, J. Neurosci. 13,1676-1687 (1993). 79. D. T. Loo, A. Copani, C. J. Pike, E. R. Whittemore, A. J. Walencewicz, and C. W. Cotman, Proc. Natl. Acad. Sci. USA 90, 7951-7955 (1993). 80. A. Lorenzo and B. A. Yankner, Proc. Natl. Acad. Sci. USA 91, 12243-12247 (1994). 82. C. Behl, J. Davis, G. M. Cole, and D. Schubert, Biochem. Biophys. Res. Commun. 186, 944-950 (1992). 82. C . Behi, L. Hovey, 111, S . Krajewski, D. Schubert, and J. C. Reed, Biochem. Biophys. Res. Commun. 197, 949-956 (1993). 83. C . Behl, J. B. Davis, R. Lesley, and D. Schubert, Cell 77, 817-827 (1994). 84. C. Behl, J. B. Davis, F. G. Klier, and D. Schubert, Brain Res. 645, 253-264 (1994). 85. Y. S . Allen, P. H. Devanathan, and G. P. Owen, Clin. Exp. Pharmacol. Physiol. 22, 370-371 (199s). 86. L. L. Inversen, R. 1 . Mortishire-Smith, S. J. Pollack, and M. S. Shearman, Biochem. J. 311, 1-16 (1995). 87. C. Exley, Biochem. 1. 314, 709-710 (1996). 88. S. A. Frautschy, A. Baird, and G. M. Cole, Proc. Natl. Acad. Sci. USA 88, 83628366 (1991). 89. N. W. Kowall, M. F. Bed, J. Busciglio, I.. K. Duffy, and B. A. Yankner, Proc. Natl. Acad. Sci. USA 88, 7247-7251 (1991). 90. N. W. Kowall, A. C. McKee, B. A. Yankner, and M. F. Beal, Neurobiol. Aging 13, 537-542 (1992). 91. J. Waite, G. M. Cole, S. A. Frautschy, D. J. Connor, and L. J. Thal, Neurobiol. Aging 13, 595-599 (1992). 92. D. K. Rush, S. Aschmies, and M. C. Merriman, Neurobiol. Aging 13, 591-594 (1992). 93. A. Nitta, A. Itoh, T. Hasegawa, and T. Nabeshima, Neurosci. Lett. 170, 63-66 (1994). 94. J. S . Whitson, D. J. Selkoe, and C. W. Cotman, Science 243, 1488-1490 (1989). 95. D. Games, K. M. Khan, F. G. Soriano, P. S. Keim, D. L. Davis, K. Bryant, and I. Leiberburg, Neurobiol. Agirrg 13, 569-576 (1992). 96. T. Yamatsuji, T. Okamoto, S. Takeda, H. Fukumoto, T. Iwatsubo, N . Suzuki, A. AsamiOdaka, S. Ireland, T. B. Kinane, and I. Nishimoto, Science 272, 1349-1352 (1996). 97. M. D. Smyth, J. P. Kesslak, B. J. Cummings, and C. W. Cotman, Neurobiol. Aging 15, 153-159 (1994). 98. P. C. May, B. L). Gitter, D. C. Waters, L. K. Simmons, G. W. Becker, J. S. Small, and P. M. Robinson, Neurobiol. Aging 13, 605-607 (1992). 99. P. S . Puttfarcken, A. M . Manelli, J. Neilly, and D. E. Frail, Exp. Neurol. 138, 73-81 (1996). 100. A. E. Roher, K. C. Palmer, J. Capodilupo, A. R. Wakade, and M. J. Ball, Can. J. Neurol. Sci. 18, 408-410 (1991). 101. M. Emre, C. Geula, B. J. Ransi, and M. M. Mesulam, Neurobiol. Aging 13, 553-559 (1992).

Apoptosis in Neurodegenerative Diseases


102. T. Giordano, J. B. Pan, L. M. Monteggia, T. F. Holzman, S. W. Snyder, G. Krafft, H. Ghanbari, and N. W. Kowall, Exp. Neurol. 125, 175-182 (1994). 103. K. Ueda, Y. Fukui, and H. Kageyama, Bruin Res. 639,240-244 (1994). 104. D. R. Howlett, K. H. Jennings, D. C. Lee, M. S. Clark, F. Brown, R. Wetzel, S. J. Wood, P. Camilleri, and G. W. Roberts, Neurodegenerution 4,23-32 (1995). 105. C. J. Pike, A. J. Walencewicz-Wasserman, J. Kosmoski, D. H. Cribbs, C. G. Glabe, and C. W. Cotman, J . Neurochem. 64, 253-265 (1995). 106. W.-D. Le, L. V. Colom, W-J. Xie, G. Smith, M. Alexianu, and S. H. Appel, Bruin Res. 686, 49-60 (1995). 107. J. Busciglio, J. Yeh, and B. A. Yankner, J. Neurochem. 61, 1565-1568 (1993). 108. M. Gschwind and G . Huber, ]. Neurochem. 65, 292-300 (1995). 109. G. J o s h , J. E. Krause, A. D. Hershey, S. P. Adams, R. J. Fallon, and D. H. Perlmutter, J. Biol. Chem. 266,21897-21902 (1991). 110. C. Zhang, M. P. Lambert, C. Bunch, K. Barber, W. S. Wade, G. A. Krafft, and W. L. Klein, ]. Biol. Chem. 269, 25247-25250 (1994). 111. Y. Q. Luo, N. Hirashima, Y. H. Li, D. L. Alkon, T. Sunderland, R. Etcheberrigaray, and B. Wolozin, Bruin Res. 68, 65-74 (1995). 112. Z. Khalil, K. Sanderson, P. Isberg, M. Bassirat, B. Livett, and R. Helme, Bruin Res. 651, 227-235 (1994). 113. D. Schubert and M . Chevion, Biochem. Biophys, Res. Commun. 216, 702-707 (1995). 114. N. Arispe, E. Rojas, and H. B. Pollard, Proc. Nutl. Acud. Sci. USA 90, 567-571 (1993). 115. T. Mirzabekov, M. C. Lin, W. L. Yuan, P. J. Marshall, M. Carman, K. Tomaselli, I. Lieberburg, and B. L. Kagan, Biochem. Biophys. Res. Commun. 202,1142-1148 (1994). 116. S. D. Yan, X. Chen, J. Fu, M. Chen, H. Zhu, A. Roher, T. Slattery, L. Zhao, M. Nagashima, J. Morser, A. Migheli, P. Nawroth, D. Stern, and A. M. Schmidt, Nature 382, 685-691 (1996). 117. R. Joseph and E. Han, Biochem. Biophys. Res. Commun. 184, 1441-1447 (1992). 118. M. P. Mattson, B. Cheng, D. Davis, K. Bryant, I. Lieberburg, and R. E. Rydel, J . Neurochem. 12, 376-389 (1992). 119. A. Eckert, H. Hartmann, and W. E. Muller, FEBS Lett. 330, 49-52 (1993). 120. R. Fukuyama, K. C. Wadhwani, 2. Galdzicki, S. I. Rapoport, and G. Ehrenstein. Bruin Res. 667; 269-272 (1994). 121. A. R. Korotzer, E. R. Whittemore, and C. W. Cotman, Eur. J. Phurmacol. 288, 125130 (1995). 122. J. R. Brorson, V. P. Bindokas, T. Iwama, C. J. Marcuccilli, J. C. Chisholm, and R. J. Miller, J . NeurobioL. 26, 325-338 (1995). 123. V. L. Smith-Swintosky, S. Zimmer, J. W. Fenton, Jr., and M. P. Mattson,]. Neurochem. 65, 1415-1418 (1995). 124. A. J. Anderson, C. J. Pike, and C. W. Cotman, J. Neurochem. 65, 1487-1498 (1995). 125. R. J. Mark, K. Hensley, D. A. Butterfieid, and M. P. Mattson, J. Neurochem. 15, 6239-6249 (1995). 126. Z. Galdzicki, R. Fukuyama, K. C. Wadhwani, S. I. Rapoport, and G . Ehrenstein, Bruin Res. 646, 332-336 (1994). 127. K. Hensley, J. M. Carney, M. P. Mattson, M. Aksenova, M. Harris, J. F. Wu, R. A. Floyd, and D. A. Butterfield, Proc. Nutl. Acud. Sci. USA 91, 3270-3274 (1994). 128. M. E. Harris, K. Hensley, D. A. Butterfield, R. A. Leedle, and J. M. Carney, Exp. Neurol. 131, 193-202 (1995). 129. Deleted in proof. 130. A. M. Manelli and P. S. Puttfarcken, Bruin Res. Bull. 38, 569-576 (1995). 131. Y. Sagara, R. Dargusch, F. G. Klier, D. Schubert, and C. Behl, J. Neurosci. 16, 497505 (1996). 132. S. J. Pollack, I. 1. Sadler, S. R. Hawtin, V. J. Tailor, and M. S. Shearman, Neurosci. Lett. 197, 211-214 (1995).


lkuo Nishimoto et 01.

233. M. C. Burgevin, M. Passat, N. Daniel, M. Capet, and A. Doble, Neuroreport 5,24292432 (1994). 134. J. H. Weiss, C. J. Pike, and C. W. Cotman, f. Neurochem. 62, 372-375 (1994). 135. J. S. Whitson and S. H. Appel, Neurobiol. Aging 16, 5-10 (1995). 136. C. J. Pike and C. W. Cotman, Brain Res. 671, 293-298 (1995). 137. A. G. Woods, D. H. Cribbs, E. R. Whittemore, and C. W. Cotman, Bruin Res. 697, 53-67 (1995). 138. S. W. Barger, D. Horster, K. Furukawa, Y. Goodman, J. Krieglstein, and M. P. Mattson, Proc. Nutl. Acud. Sci. USA 92, 9328-9332 (1995). 139. Y. Goodman and M. P. Mattson, J. Neurochem. 66, 869-872 (1996). 140. M. P. Mattson, K. J. Tomaselli, and E. E. Rydel, Bruin Res. 621, 35-49 (1993). 141. T. Takadera, N. Sakura,T. Mohri, andT. Hashimoto, Neurosci. Lett. 161,41-44 (1993). 142. C. C. Chao, S. Hu, F. H. Kravitz, M. Tsang, W. R. Anderson, and P. K. Peterson, Mol. Chem. Neuroputhol. 23, 159-178 (1994). 143. J. H. Prehn, V. P. Bindokas, J. Jordan, M. F. Galindo, G. D. Ghadge, R. P. Roos, L. H. Boise, C. B. Thompson, S. Krajewski, J. C. Reed, and R. J. Miller, Mol. Pharmacol. 49, 319-328 (1996). 144. C. Behl and D. Schubert, Neurosci. Lett. 154, 1-4 (1993). 245. T. Tomiyama, A. Shoji, K. Kataoka, Y. Suwa, S. Asano, H. Kaneko, and N. Endo, J. Biol. Chem. 271, 6839-6844 (1996). 146. A. J. Bruce, B. Malfroy, andM. Baudry, Proc. Nutl. Acud. Sci. USA 93,2312-2316 (1996). 147. Deleted in proof. 248. C. J, Pike, P. J. Vaughan, D. D. Cunningham, and C. W. Cotman, J . Neurochem. 66, 1374-1382 (1996). 249. P. H. Frederikse, D. Garland, J. S. Zigler, and J. Piatigorsky, f. Biol. Chem. 271,1016910174 (1996). 150. D. Games, D. Adams, R. Alessandrini, R. Barbour, P. Berthelette, C. Blackwell, T. Carr, J. Clemens, T. Donaldson, F. Gillespie, T. Guido, S. Hagoplan, K. Johnson-Wood, K. Khan, M. Lee, P. Leibowitz, I. Lieberburg, S. Little, E. Masliah, L. McConlogue, M. Montoya-Zavala, L. Mucke, L. Paganini, E. Pennirnan, M. Power, D. Schenk, P. Seubert, B. Snyder, F. Sorlano, H. Tan, J. Vitale, S. Wadsworth, B. Wolozin, and J. Zhao, Nature 373, 523-527 (1995). 151. F. M. LaFerla, B. T. Tinkle, C. J. Bieberich, C. C. Haudenschild, and G. Jay, Nature Genet. 9, 21-29 (1995). 252. D. Quon, Y. Wang, R. Catalano, J. M. Scardina, K. Murakami, and B. Cordell, Nature 352,239-241 (1991). 153. K. K. Hsiao, D. R. Borchelt, K. Olson, R. Johannsdotir, C. Kitt, W. Yunis, S. Xu, C. Eckman, S. Younkin, D. Price, C. Ladecola, H. B. Clark, and G. Carlson, Neuron 15, 1203-1218 (1995). 154. P. M. Moran, L. S. Higgins, B. Cordell, and P. C. Moser, Proc. Nutl. Acad. Sci. USA 92, 5341-5345 (1995). 155. L. S. Higgins, J. M. Rodems, R. Catalano, D. Quon, and B. Cordell, Proc. Nutl. Acud. Sci. USA 92, 4402-4406 (1995). 256. T. A. Perry, E. Torres, C. Czech, K. Beyreuther, S.-J. Richards, and S. B. Dunnett, Alzheimer’s Res. 1, 5-14 (1995). 157. D. Moechars, K. Lorent, B. De Strooper, I. Dewachter, and F. van Leuven, EMBO f. 15, 1265-1274 (1996). 158. J. Kang, H.-G. Lemaire, A. Unterback, J. M. Salbaurn, C. L. Masters, K. H. Grezeschik, G. Multhaup, K. Beyreuther, and B. Mitller-Hill, Nature 325, 733-736 (1995). 259. P. Ponte, P. Gonzalez-DeWhitt, J. Schilling, J. Miller, D. Hsu, B. Greenberg, K. Davis, W. Wallace, I. Lieberburg, F. Fuller, and B. Cordell, Nature 311, 525-527 (1988). 260. R. E. Tanzi, A. I. McClatchey, E. D. Lamperti, L. Villa-Kornaroff, J. F. Gusella, and R. L. Neve, Nature 331, 528-530 (1988).

Apoptosis in Neurodegenerative Diseases


161. N. Kitaguchi, Y. Takahashi, Y. Tokushima, S. Shiojiri, and H. Ito, Nature 331, 530532 (1988). 162. T. Dyrks, A. Weidemann, G. Multhaup, J. M . Salbaum, H.-G. Lemaire, J. Kang, B. Muller-Hill, C. L. Masters, and K. Beyreuther, EMBO J. 7, 949-957 (1988). 163. T. Yamada, H. Sasaki, H. Furuya, T. Miyata, I. Goto, and Y. Sakaki, Biochem. Biophys. Res. Commun. 149, 665-671 (1987). 164. B. A. Yankner, L. R. Dawes, S. Fisher, L. Villa-Komaroff, M. L. Oster-Granite, and R. L. Neve, Science 245, 417-420 (1989). 165. N. Chow, J. R. Korenberg, X. N. Chen, and R. L. Neve, J. Biol. Chem. 271, 1133911346 (1996). 166. F. Fiore, N. Zambrano, G. Minopoli, V. Donini, A. Duilio, and T. Russo,J. Biol. Chem. 270, 30853-30856 (1995). 167. G. Multhaup, H. Mechler, and C. L. Masters, J. Mol. Recognition 8, 247-257 (1995). 168. I. Nishimoto, T. Okamoto, Y. Matsuura, T. Okamoto, Y . Murayama, and E. Ogata, Nature 362, 75-79 (1993). 169. S. L. Borowicz and L. A. Dokas, Abstr. SOL. Neurosci. 21(Part 1), 207 (1995). 170. J. Lang, I. Nishimoto, T. Okamoto, R.Regazzi, C. Kiraly, U. Weller, and C. B. Wollheim, EMBO J. 14, 3635-3644 (1995). 171. T. Okamoto, T. Katada, Y. Murayama, M . Ui, E. Ogata, and I. Nishimoto, Cell 62, 709-717 (1990). 172. T. Okamoto, Y. Murayama, Y. Hayashi, M. Inagaki, E. Ogata, and I. Nishimoto, Cell 67, 723-730 (1991). 173. T. Okamoto and I. Nishimoto, J . Biol. Chem. 267, 8342-8346 (1992). 174. T. Ikezu, T. Okamoto, E. Ogata, and I. Nishimoto, FEBS Lett. 311, 29-32 (1992). 175. A. H. Dittman, J. P. Weber, T. J. Hinds, E. J. Choi, J. C. Migeon, N. M. Nathanson, and D. R. Storm, Biochemistry 33, 943-951 (1994). 176. M. G. Eason and S. B. Liggett, J. Biol. Chem. 270, 24753-24760 (1995). 177. H. Sun, J. M. Seyer, and T. B. Patel, Proc. Natl. Acad. Sci. USA 92,2229-2233 (1995). 178. X. Gong, D. H. Dubois, D. J. Miller, and B. D. Shur, Science 269, 1718-1721 (1995). 179. T. Okamoto, S. Takeda, Y. Murayama, E. Ogata, and I. Nishimoto, J . Biol. Chem. 270, 4205-4208 (1995). 180. J. B. Imboden and J. D. Stobo, J. Exp. Med. 161, 446-456 (1985). 181. C. Hilbich, U. Monning, C. Grund, C. L. Masters, and K. Beyreuther, J . Biol. Chem. 268,26571-26577 (1993). 182. A. Ferreira, A. Caceres, and K. S. Kosik, J. Neurosci. 13, 3112-3123 (1993). 183. J. G. Culvenor, A. Friedhuber, S. J. Fuller, K. Beyreuther, and C. L. Masters, Exp. Cell Res. 220, 474-481 (1995). 184. S. M . Strittmatter, D. Valenzuela, T. E. Kennedy, E. J. Neer, and M. C. Fishman, Nature 344, 836-841 (1990). 185. W. Schubert, R. Prior, A. Weidemann, H. Dircksen, G. Multhaup, C. L. Masters and K. Beyreuther, Brain Res. 563, 184-194 (1991). 186. K. C. Breen, M. Bruce, and B. H. Anderton, J. Neurosci. Res. 28, 90-100 (1991). 187. E. A. Milward, R. Papadopoulos, S. J. Fuller, R. D. Moir, D. Small, K. Beyreuther, and C. L. Masters, Neuron 9, 129-137 (1992). 188. A. C. LeBlanc, D. M. Kovacs, H. Y. Chen, F. Villarh, M. Tykocinski, L. Autilio-Gambetti, and P. Gambetti, J . Neurosci. Res. 31, 635-645 (1992). 189. Deleted in proof. 190. L.-W. Jin, H. Ninomiya, J.-M. Roch, D. Schubert, E. Masliah, D. A. Otero, and T. Saitoh, J. Neurosci. 14, 5461-5470 (1994). 191. B. Allinquant, P. Hantraye, P. Mailleux, K. Moya, C. Bouillot, and A. Prochiantz, J . Cell. B i d . 128, 919-927 (1995). 192. H. Zheng, M . Jiang, M. E. Trumbauer, D. J. S. Sirinathsinghji, R. Hopkins, D. W. Smith, R. P. Heavens, G. R. Dawson, S . Boyce, M. W. Conner, K. A. Stevens, H. H. Slunt, S. S. Sisodia, H. Y. Chen, and L. H. T. Van der Ploeg, Cell 81, 525-531 (1995).


lkuo Nishimoto et a/.

19.3. P. Doherty, S. V. Ashton, S. E. Moore, and F. S. Walsh, Cell 67, 21-33 (1991). 194. U. Schuch, M. J. Lohse, and M. Schachner, Neuron 3, 13-20 (1989). 19.5. K. Sebok, D. Woodside, A. Al-Aoukaty, A. D. Ho, S. Gluck, and A. Z. Maghazachi, J. Immunol. 150, 1524-1534 (1993). 196. S. M. Strittrnatter, M. C. Fishman, and X . 2 . Zhu, J. Neurosci. 14, 2327-2338 (1994). 197. S. M. Strittmatter, S. C. Cannon, E. M. Ross, T. Higashijirna, and M. C. Fishman, Proc. Nutl. Acad. Sci. USA 90, 5327-5331 (1993). 198. L. Luo, T. Tully, and K. White, Neuron 9, 595-605 (1992). 199. U. Muller, N. Cristina, Z.-W. Li, D. P. Wolfer, H.-P. Lipp, T. Rulicke, S. Brandner, A. Aguzzi, and C. Weissmann, Cell 79, 755-765 (1994). 200. J. E. Mendel, H. C. Korswagen, K. S. Liu, Y. M. Hajdu-Cronin, M. I. Simon, R. H. A. Plasterk, and P. W. Sternberg, Science 267, 1652-1655 (1995). 201. L. Skgalat, D. A. Elkes, and J. M. Kaplan, Science 267, 1648-1651 (1995). 202. Y. Murayama, S. Takeda, K. Yonezawa, E. Ogata, and I. Nishimoto, Gerontology 42, 2-11 (1996). 20.3. T. Yamatsuji, T. Okamoto, S. Takeda, Y. Murayama, N. Tanaka, and I. Nishimoto, EMBO J. 15,498-509 (1996). 204. J. Papkoff, R.-H. Chen, J. Blenis, and J. Forsman, Mol. Cell. Biol. 14, 463-472 (1994). 205. P. Crespo, N. Xu, W. F. Simonds, and J. S. Gutkind, Nature 369, 418-420 (1994). 206. T. van Biesen, B. E. Hawes, J. R. Raymond, L. M. Luttrell, W. J. Koch, and R. J. Lefkowitz, J . Biol. Chem. 271, 1266-1269 (1996). 207. S. M. Greenberg, E. H. Koo, D. J. Selkoe, W. Q. Qiu, and K. S. Kosik, Proc. Nutl. Acad. Sci. USA 91, 7104-7108 (1994). 208. H. Borochov-Neori, P. A. Fortes, and M. Montal, Biochemistry 22, 206-213 (1983). 209. M. 2. Kounnas, R. D. Moir, G. W. Rebeck, A. I. Bush, W. S. Argraves, R. E. Tanzi, B. T. Hyman, and D. K. Strickland, Cell 82, 331-340 (1995). 210. A. Goate, M.-C. Chartier-Harlin, M. Mullan, J. Brown, F. Crawford, L. Fidani, L. Giuffra, A. Haynes, N. Irving, L. James, R. Mant, P. Newton, K. Rooke, P. Roques, C. Talbot, M. Pericak-Vance, A. Roses, R. Williamson, M. Rossor, M. Owen, and J. Hardy, Nature 349, 704-706 (1991). 21 1 . S. Naruse, S. Igarashi, H. Kobayashi, K. Aoki, T. Inuzuka, K. Kaneko, S. Shimizu, K. Iihara, T. Kojima, T. Miyatake, and S. Tsuji, Lancet 337, 978-979 (1991). 212. J. Murrell, M. Farlow, B. Ghetti, and M. D. Benson, Science 254, 97-99 (1991). 21.3. M.-C. Chartier-Harlin, F. Crawford, H. Houlden, A. Warren, D. Hughes, L. Fidani, A. Goate, M. Rossor, P. Roques, J. Hardy, and M. Mullan, Nature 353, 844-846 (1991). 224. K. Yoshioka, T. Miki, T. Katsuya, T. Ogihara, and Y. Sakaki, Biochem. Biophys. Res. Commun. 178, 1141-1146 (1991). 215. H. Karlinsky, G. Vaula, J. L. Haines, J. Ridgley, C. Bergeron, M. Mortilla, R. G. Tupler, M. E. Percy, Y. Robitaille, N. E. Noldy, T. C. K. Yip, R. E. Tanzi, J. F. Gusella, R. Becker, J. M. Berg, D. R. C. McLachlan, and P. H. St. George-Hyslop, Neurology 42, 1445-1453 (1992). 216. J. Hardy, Nature Genet. 1, 233-234 (1992). 217. T. Okamoto, S. Takeda, U. Giambarella, Y. Murayama, T. Matsui, T. Katada, Y . Matsuura, and I. Nishimoto, EMBO J . 15, 3769-3777 (1996). 218. T. Ikezu, T. Okamoto, K. Komatsuzaki, T. Matsui, J. A. Martyn, and I. Nishimoto, EMBO J . 15, 2468-2475 (1996). 219. A. Shenker, L. Laue, S. Kosugi, J. J .Merendino, Jr., T. Minegishi, and G. B. Cutler, Jr., Nature 365, 652-654 (1993). 220. J. l’arrna, L. Duprez, J. Van Sande, P. Cochauz, C. Gervy, J. Mockel, 1. Dumount, and G. Vassart, Nature 365, 649-651 (1993). 221. V. R. Rao, G. B. Cohen, and D. D. Oprian, Nature 367, 639-642 (1994). 222. E. Schipani, K. Kruse, and H. Juppner, Science 268, 98-100 (1995).

Apoptosis in Neurodegenerative Diseases


223. 1. Ito, D. Okada, and H. Sugiyama, Neurosci. Lett. 90, 181-185 (1988). 224. J. W. Goh and P. S. Pennefather, Science 244, 980-983 (1989). 225. A. Kikuchi, 0. Kozawa, K. Kaihuchi, T. Katada, M. Ui, and Y. Takai, J . Biol. Chem. 261, 11558-11562 (1986). 226. T. M. Moriarty, E. Padrell, D. J. Carty, G. Omri, E. M . Landau, and R. Iyengar, Nature 343, 79-82 (1990). 227. J. Hescheler, W. Rosenthal, W. Trautwein, and G. Schultz, Nature 325,445-447 (1987). 228. R. F. Cowburn, C. O’Neill, R. Ravid, I. Alafuzoff, B. Winhlad, and C. J. Fowler, J. Neurochem. 58, 1409-1419 (1992). 229. R. F. Cowhurn, M. Vestling, C. J. Fowler, R. Ravid, B. Winblad, and C. O’Neill, Neurosci. Lett. 155, 163-166 (1993). 230. H.-M. Huang and G. E. Gibson, J. Biol. Chem. 268, 14616-14621 (1993). 231. A. Schnecko, K. Witte, J. Bohl, T. Ohm, and B. Lemmer, Brain Res. 644,291-296 (1994). 232. N. Charperntier, L. PrCzeau, J. Carrette, R. Bertorelli, G. L. Cam, 0.Manzoni, J. Bockaert, and V. Homhurger, J. Biol. Chem. 268, 8980-8989 (1993). 233. B. D. Carter and F. Medzihradsky, Proc. Natl. Acad. Sci. USA 90, 4062-4066 (1993). 234. R. Taussig, W.-J. Tang, J. R. Hepler, and A. G. Gilman, J . Biol. Chem. 269, 60936100 (1994). 235. J. C. Migeon, S. L. Thomas, and N. M. Nathanson, J . Biol. Chem. 269, 2914629152 (1994). 2.36. E. R. Seaquist, M. 8. Armstrong, T. W. Gettys, and T. F. Walseth, Diabetes 44, 85-89 (1995). 237. C. J. Smith, E. K. Perry, and N. J. M. Birdsall, Biochem. Soc. Trans. 17,202-203 (1989). 238. D. D. Flynn, D. A. Weinstein, and D. C. Mash, Ann. Neurol. 29, 256-262 (1991). 239. E. Ito, K. Oka, R. Etcheberrigaray, T. J. Nelson, D. L. McPhie, B. Tofel-Grehl, G. E. Gibson, and D. L. Alkon, Proc. Natl. Acad. Sci. USA 91, 534-538 (1994). 240. G. E. Gibson and L. Toral-Barza, Mech. Ageing Deu. 63, 1-9 (1992). 241. D. E. Claphain and E. J. Neer, Nature 365, 403-406 (1993). 242. G.-M. Yan, S.-2. Lin, R. P. Irwin, and S. M. Paul,]. Neurochem. 65,2425-2431 (1995). 243. G. Q. Chang, Y. Hao, and F. Wong, Neuron 11,595-605 (1993). 244. R. Ramirez, J. Carracedo, N. Zamzami, M. Castedo, and G. Kroemer, J. E x p . Med. 180, 1147-1152 (1994). 245. J. A. Gonzalo, A. Gonzalez-Garcia, E. Baixeras, N. Zamzami, R. Tarazona, R. Rappuoli, C. Marinez, G. Kroemer, and R. J. Terezone, J . Immunol. 152, 4291-4299 (1994). 246. J. Carracedo, R. Ramirez, P. Marchetti, 0. C. Pintado, E. Baixeras, C. Martinez, and G. Kroemer, Eur. J . Immunol. 25, 3094-3099 (1995). 247. R. M. Huff, J. M. Axton, and E. J. Neer, J . Biol. Chem. 260, 10864-10871 (1985). 248. C. I. Bargmann, M. C. Hung, and R. A. Weinberg, Cell 45, 649-657 (1986). 249. K. Fukuchi, C. E. Ogburn, A. C. Smith, D. D. Kunkel, C. E. Furlong, S. S. Deeh, D. Nochlin, S. M. Sumi, and G. M. Martin, Ann. N Y Acad. Sci. 695, 217-223 (1993). 250. J. Busciglio and B. A. Yankner, Nature 378, 776-779 (1995). 251. R. A. Bond, P. Leff, T. D. Johnson, C. A. Milano, H. A. Rockman, T. R. McMinn, S. Apparsundaram, M . F. Hyek, T. P. Kenakin, L. F. Allen, and R. J. Lefkowitz, Nature 374,272-276 (1995). 252. K. Fukuchi, K. Kamino, S. S. Deeb, C. E. Furlong, J. A. Sundstrom, A. C. Smith, and G. M. Martin, Brain Res. Mol. Brain Res. 16, 37-46 (1992). 253. B. L. Sopher, K. Fukuchi, A. C. Smith, K. A. Leppig, C. E. Furlong, and G. M. Martin, Brain Res. Mol. Brain Res. 26, 207-217 (1994). 254. K. I. Fukuchi, D. D. Kunkel, P. A. Schwartzkroin, K. Kamino, C. E. Oghurn, C. E. Furlong, and G. M. Martin, Exp. Neurol. 127, 253-264 (1994). 255. M. L. Oster-Granite, D. L. McPhie, J. Greenan, and R. L. Neve, J . Neurochem. 16, 6732-6741 (1996).


lkuo Nishimoto et al.

256. B. Tate-Ostroff, R. E. Majocha, and C. A. Marotta, Proc. Natl. Acad. Sci. USA 86, 745-749 (1989). 257. G. M. Cole, E. Masliah, E. R. Shelton, H. W. Chan, R. D. Terry, and T. Saitoh, Neurobiol. Aging 12, 85-91 (1991). 2.78. P. Seubert, T. Oltersdorf, M. G. Lee, R. Barbour, C. Blomquist, D. L. Davis, K. Bryant, L. C. Fritz, D. Glasko, L. J. Thal, et al., Nature 361, 260-263 (1993). 259. K. Maruyama, Y. Kawamura, S. Asada, S. Ishiura, and K. Obata, Biochem. Biophys. Res. Commun. 202, 1517-1523 (1994). 260. C. J. Pike, M. J. Overman, and C. W. Cotman,J. Biol. Chem. 270,23895-23898 (1995). 261. B. D. Lynn, C. A. Marotta, and J. I. Nagy, Neurosci. Lett. 199, 21-24 (1995). 262. D. L. Vaux, S. Cory, and J. M. Adams, Nature 335, 440-442 (1988). 263. R. E. Ellis, J. Y. Yuan, and H. R. Horvitz, Ann. Rev. Cell Biol. 7, 663-698 (1991). 264. P. Shaw, R. Bovey, S. Tardy, R. Sahli, B. Sordat, and J. Costa, Proc. Natl. Acad. Sci. USA 89, 4495-4499 ( 1 992). 265. G. I. Evan, A. H. Wyllie, C. S. Gilbert, T. D. Littlewood, H. Land, M. Brooks, C. M. Waters, L. Z. Penn, and D. C. Hancock, Cell 69, 119-128 (1992). 266. Z. G. Liu, S. W. Smith, K. A. McLaughlin, L. M. Schwartz, and B. A. Osborne, Nature 367, 281-284 (1994). 267. A. J. Darmon, D. W. Nicholson, and R. C. Bleackley, Nature 377, 446-448 (1995).