Brain Pathology 9: 707-720(1999)

SYMPOSIUM: Tau and in Neuropathology

Alpha-synuclein in Lewy Body Disease and Alzheimer’s Disease

Makoto Hashimoto1 and Eliezer Masliah1,2 pointed to -synuclein, also known as the precursor of the non-A component of plaques (NACP), as a major Departments of 1Neurosciences and 2Pathology, University of California, San Diego, School of Medicine, La Jolla, California factor in the pathogenesis of AD and LBD (76). Alpha- 92093-0624 synuclein was first isolated by Maroteaux and co-work- ers by expression screening of a torpedo cDNA library Alzheimer’s disease (AD) and Lewy body disease using anti-serum against purified synaptic vesicles (72). (LBD) are the most common causes of dementia in the elderly population. Previous studies have shown The term synuclein was proposed because immunoreac- that cognitive alterations in these disorders are tivity was observed in presynaptic termini and nuclei, associated with synaptic loss. Injury and loss of although nuclear immunoreactivity has not been noted synapses might be associated with altered function in subsequent studies. Nakajo and co-workers purified a of synaptic . Among them, recent studies presynaptic phosphoprotein (PNP14) from bovine brain have shown that abnormal aggregation and accumu- that was very similar to synuclein (86, 122), which is lation of synaptic proteins, such as -synuclein, now known as -synuclein. might be associated with plaque formation in AD and In 1993 Saitoh and coworkers identified -synuclein Lewy body formation in LBD. Further reinforcing the as NACP, which was a non-amyloid that co-puri- hypothesis that -synuclein plays a major role in the pathogenesis of these disorders, recent work has fied with amyloid from AD brains (125). NACP is the shown that mutations that alter the conformation of human homologue to murine -synuclein (17). Jakes this molecule are associated with familial forms of and Goedert purified both -synuclein and -synuclein Parkinson’s disease. The mechanisms by which from human brain using monoclonal antibodies raised altered function or aggregation of -synuclein might against tau protein (48). At the same time, Clayton and lead to neurodegeneration are not completely clear; coworkers identified synelfin, the avian homologue of however, new evidence points to a potential role for -synuclein, whose expression was dramatically down- this molecule in synaptic damage and neurotoxicity regulated during a critical period for song learning in the via amyloid-like fibril formation and mitochondrial canary (28), suggesting that synelfin might play a criti- dysfunction. In this manuscript we review the data linking -synuclein to the pathogenesis of AD and cal role in neural plasticity. LBD. More recently, -synuclein was isolated as a synucle- in-like molecule expressed predominantly in peripheral Introduction sympathetic neurons (2). Gamma-synuclein was cloned Alzheimer’s disease (AD) and Lewy body disease from an EST library and identified as the breast cancer (LBD) are the most common causes of dementia in the specific (BCSG1) whose expression was previous- elderly population. Previous studies have shown that ly observed in invasive types of breast cancer (52). In cognitive alterations in these disorders correlate with 1997 Polymeropoulos and colleagues identified a mis- synaptic loss (20, 121). It is postulated that synaptic loss sense mutation (A53T) in -synuclein in Italian and and injury might be associated with altered function of Greek families with autosomal dominant Parkinson’s synaptic proteins (75). Among them, recent studies have disease (PD) (99). Shortly thereafter, Kruger and col- leagues identified another missense mutation (A30P) in -synuclein in a German family (59). Following these Corresponding author: Dr. E. Masliah, Department of Neurosciences, University of seminal observations, neuropathologic and biochemical California, San Diego, La Jolla, CA 92093-0624; Tel.: (858) 534- studies established that abnormal aggregates of -synu- 8992; Fax (858) 534-6232; E-mail: emasliah @ucsd.edu Alpha-synuclein belongs to a family of proteins that includes -synuclein and -synuclein (14) (Figure 1). Beta-synuclein is encoded by a gene in 5 and -synuclein by a gene on chromosome 10 (Table 1). Both -synuclein and -synuclein are abundantly expressed in the central nervous system (CNS) (48), whereas -synuclein is expressed mainly in the periph- eral nervous system (2). Although N-terminal regions of the are highly similar, C-terminal regions are divergent except for the last 10 amino acids, which are conserved between -synuclein and -synuclein (48). Alpha-synuclein shows more homology with -synucle- in than with -synuclein. Although the Ser118 residue of Figure 1. Synuclein proteins: Structure and relationship to neu- -synuclein was previously characterized as a potential rodegenerative disorders. phosphorylation site of calmodulin kinase (87), this ser- ine residue is not conserved in -synuclein and -synu- clein. No studies have been published on the phospho- rylation state of a-synuclein. Interestingly, -synuclein has a deletion of part of the NAC domain, which makes it non-amyloidogenic. The structural relationship between -synuclein and -synuclein is similar to that between amyloid precursor protein (APP) and its relat- ed protein, APLP2 (130). APP is one of several related proteins that belong to the APP superfamily (109). Among them, APLP2 is highly homologous with APP Figure 2. The synuclein family of proteins. except for absence of the amyloidogenic A domain. The biological significance for the evolution of related

Synuclein Chromosome MW (kDa) Disease molecules with amyloidogenic and non-amyloidogenic (approx) domains remains a matter for future study.

alpha $q21.3-q22 19 Lewy body disease Several mRNAs are known to arise from alternative splicing of the -synuclein gene. In rats, two alternative beta 5q35 14 - transcripts have been reported (73). These include gamma 10q23 12 Breast cancer SYN2, which has a distinct C-terminal region, and SYN3, which is truncated at the N-terminal region Table 1. Gene localization of alpha, beta and gamma synucle- in. (Figure 2). In humans, -synuclein 126 and -synucle- in 112 have been identified (12, 126), which result in clein were major components of Lewy bodies (LBs) and deletions from codon 41 to 54 and from 103 to 130, dystrophic neurites in LBD (119). respectively. These alternatively spliced products are far less abundant than -synuclein and their biological sig- The synuclein family of proteins nificance is unknown (Figure 2). Alpha-synuclein is a 140 amino acid protein with lit- tle or no secondary structure (131) encoded by a gene Alpha-synuclein physiology located on chromosome 4. Sequence analysis suggests Although -synuclein is regarded as a presynaptic that it has three major domains: 1) an N terminal region protein with a potential role in neuronal plasticity (28), composed of incompletely an repeated KTKEGV little is known of its actual normal function. motifs, 2) a NAC domain that is extremely hydrophobic, Immunohistochemical analyses using laser scanning and 3) a C-terminal region with a negative charge (14) confocal microscope showed that -synuclein (Figure 1). Interestingly, both the A53T and A30P muta- immunoreactivity was predominantly located in presy- tions lie in the N-terminal region, suggesting that the N- naptic regions (Figure 3). Immunoelectron microscopic terminal domain may be critical for LB formation studies demonstrated loose association with synaptic (Figure 1). vesicles, suggesting that -synuclein might play a role

708 M. Hashimoto and E. Masliah: -synuclein in LBD and AD Figure 3. Alpha-synuclein immunoreactivity in AD and LBD. A) -synuclein colocalization to synapses in normal control; B,C) -synu- clein colocalization with ubiquitin and in LBD; D,E) NAC immunoreactivity in a plaque in AD; F,G) congo red and ultra- structure of -synuclein aggregates in vitro; H) C-term -synuclein immunoreactive LB; I,J) N-term -synuclein immunoreactive LB; K,L) C-term -synuclein immunoreactive neurites; M) C-term -synuclein immunoreactive LB in substantia nigra; O) double labeling for -synuclein and cyt-C; P) LB-like inclusions in tg mice. in function of synaptic vesicles (45). Similarly, ultra- possibility that another function of the synucleins may structural analysis of -synuclein in platelets showed its be in specific signal transduction pathways. In this con- association with secretory vesicles and plasma mem- text, it is noteworthy that expression of -synuclein con- brane (39). More recently, -synuclein has been shown ferred increased invasive properties to breast cancer to interact with a cellular vesicular system (50) (Dale cells (53) and also selectively degraded neurofilaments Bredeson, personal communication) indicating that it in primary cultured neurons (10), suggesting that synu- might play a critical role in the release or uptake of neu- cleins may influence the integrity of cytoskeleton net- rotransmitters. works. Finally, synphilin-1 has recently been identified In developmental studies of the murine brain, expres- using a yeast two-hybrid method as a cytosolic binding sion of -synuclein was first detectable at embryonic protein for -synuclein. Further characterization of this days 12 to 15, followed by a dramatic increase at later molecule may provide clues as to additional physiolog- time points (41). Its expression was detected earlier than ical functions of -synuclein (24). another major synaptic vesicle protein, synaptophysin. These findings support the contention that -synuclein Alpha-synuclein in Lewy body disease might play a role in synaptogenesis. Both -synuclein and -synuclein have been shown to specifically inhibit Alpha-synuclein as a genetic risk factor for phospholipase D2 in a cell free systems (49), raising a Parkinson’s disease. The great majority of PD cases are

M. Hashimoto and E. Masliah: -synuclein in LBD and AD 709 Gene Chromosome Inheritance Other or linkages for familial PD have been

alpha-synuclein (PARK1) 4 Autosomal dominant identified, including parkin (PARK2) (55), UCHL-1

parkin (PARK2) 6 Autosomal recessive (65) and a yet-to-be-identified gene linked to 4p (25)

PARK3 2 Autosomal dominant (Table 2). There is also linkage of autosomal dominant PD to chromosome 2p13 (PARK3) (27) (Table 2). It will 4p haplotype 4 Incomplete penetrance be important to determine whether or not these other UCH-L1 4 Autosomal dominant genes are involved in -synuclein physiology. One of the proposed genes for PD is a UCHL-1, which is a thiol Table 2. Genetic risk factors for Parkinson’s disease. protease involved in ubiquitin metabolism. It is hypoth- esized that the mutation may produce disturbance in enzyme activity leading to altered proteolysis, aggrega- tion and ubiquitination of -synuclein. This remains to be determined, but it is notable that LBs contain not only ubiquitinated proteins, but also UCHL-1 immunoreactivity (69). Genetic risk factors that may be relevant to PD include of polymorphisms in CYP2D6 gene (104) and APOE gene (108); however, controversy still exists as to the significance of these findings.

Figure 4. Central role of -synuclein in the mechanisms of neu- Alpha-synuclein in Lewy body disease (LBD). rodegeneration in LBD. Disorders with LBs comprise a diverse group of dis- eases including Lewy body variant of AD (LBV) (34), sporadic, and environmental factors that act by inducing diffuse Lewy body disease (DLBD) (21) and PD (26). oxidative stress are felt to play an important role in its Other terms used for LBV include combined AD+PD pathogenesis (71). Nevertheless, a small percentage of and senile dementia of the LB type (22, 94). A recent PD cases are familial, with either autosomal-dominant consensus conference has proposed clinical and patho- or autosomal-recessive inheritance (128). Recent logic criteria and proposed a terminology, dementia with progress in genetic research in PD has provided new Lewy bodies, in an effort to reconcile differences in cur- insights into the involvement of specific genes in famil- rent terminology (83). In this discussion we will refer to ial PD (Table 2). As mentioned previously, -synuclein these disorders collectively as LBD. The common fac- has been shown to be mutated in some families with tors in LBD are degeneration of select neuronal popula- early onset PD (98, 99, 59). Given an autosomal-domi- tions in the brain, most notably dopaminergic neurons in nant inheritance, it was predicted that these missense the substantia nigra and neurons in the mesial temporal mutations in -synuclein would results in a gain of toxic lobe (23, 62), LB formation (11, 21, 30, 34, 57, 58, 139) function, but this remains to be proven (Table 2). and extensive neuritic degeneration (43, 54). Spillantini Although -synuclein mutations have not been found in and coworkers first reported that LBs and Lewy neurites subsequent cohorts of familial and sporadic PD (13, 25, (116) were immunoreactive for -synuclein. More 33, 127), -synuclein has nevertheless been regarded as recent studies have shown that both cortical and subcor- being critical to the pathogenesis of LBD (Figure 4). tical LBs were strongly stained, and this immunoreac- This may be analogous to the central role of APP in the tivity was observed with several antibodies recognizing pathogenesis of AD. Despite the fact that only a small different regions of -synuclein, including N-terminal percentage of familial AD cases exhibit APP mutations, and C-terminal domains (115, 118, 119) (Figure 3). The A-protein is key factor in the amyloidogenesis of AD. evidence suggests that full-length or partially truncated Thus, other mutations may be found in familial PD that -synuclein accumulates in LBs. Semi-quantification also influence -synuclein biology. In this regard, with immunohistochemistry has revealed that -synu- Kruger and coworkers have recently found a difference clein staining is more sensitive than ubiquitin for LBs between sporadic PD patients and control individuals and Lewy neurites (115). Prior to the discovery of synu- with respect to apolipoprotein E (APOE4) and allele 1 clein the most sensitive markers for LBs were antibod- of the -SYN promoter polymorphism, suggesting a ies to ubiquitin (61) and, to a lesser extent, neurofila- role for interactions or combined actions of these pro- ment (107). Neither -synuclein nor -synuclein teins in pathogenesis of PD (60). immunoreactivity has been found in LBs (115). These

710 M. Hashimoto and E. Masliah: -synuclein in LBD and AD results have been confirmed by a number of laborato- ries. Takeda and co-workers showed that a-synuclein immunoreactivity was detected in all variants of LBD, but not in neurofibrillary tangles, neuropil threads, Pick bodies, ballooned neurons and tau-positive glial lesions (119). Moreover, -synuclein immunoreactivity has also been detected in multiple systemic atrophy (4, 129) and amyotrophic lateral sclerosis (84), suggesting a new category of disease, the synucleinopathies (35). Figure 5. Alpha-synuclein aggregation in vitro. Iwatsubo and colleagues prepared monoclonal anti- bodies using highly purified LBs from diffuse LBD by varying the time, temperature and pH (Figure 5) (36). brains and biochemically corroborated that -synuclein Both electron microscopic and histochemical studies accumulated in LBs (6, 47). Furthermore, their results demonstrated that a-synuclein aggregates formed were suggested that a partially truncated -synuclein may be filamentous and thioflavin-S- and Congo-red-positive. present in LBs (6), although the mechanism of fragmen- Ultrastructurally, they resembled amyloid fibrils (Figure tation was not explained. An altered conformation of a- 3). It is of interest that LBs have also been described to synuclein may be essential for aggregation and accumu- display thioflavin-S fluorescence (36) and that purified lation of -synuclein in LBs and Lewy neurites. fibrils from LBs have amyloid-like fibrils (115). These Supporting this contention, immunoelectron microscop- results support the hypothesis that -synuclein might ic studies have shown that the pattern of immunogold aggregate and form fibrils within neurons. Interestingly, labeling obtained by the C-terminal antibody is distinct preliminary observations suggest that transgenic mice from that with an N-terminal antibody (115). overexpressing -synuclein develop inclusion bodies Furthermore, -synuclein immunoreactivity was signif- and dopaminergic deficits (Figure 3), supporting the icantly enhanced by formic acid and proteinase K treat- contention that abnormal aggregation of -synuclein ment, suggesting that the structure of a-synuclein may might play a central role in LBD. be changed or that the epitope may be masked or altered More recently, using cell-free experimental systems, in the lesions (118). In fact, utilizing a similar approach several authors have reported that -synuclein rapidly to reveal hidden epitopes, we have found that an anti- forms fibrils and that mutated -synuclein (especially body to C-terminus of -synuclein immunostained a A53T type) has a greater propensity to fibril formation few neurofibrillary tangles in AD, progressive supranu- than wild type -synuclein (15, 29, 88), indicating that clear palsy and corticobasal degeneration, as well as single amino-acid mutations confer structural changes. Pick bodies and glial inclusions in progressive supranu- Such subtle differences in predisposition to aggregation clear palsy and corticobasal degeneration (117). In LBD may become significant not only during the nucleation astrocytes and granular neurons were immunostained step of fibril formation in vitro (134), but also during after formic acid pretreatment with our anti-NAC anti- aging over decades in vivo. body. The N-terminal region antibody recognized only LBs, but not of other lesions (Figure 3). These results Modulators of -synuclein aggregation. An unex- support the view that fragments of -synuclein might be plained feature of LBD is that regional expression of a- incorporated into diverse neuronal and glial lesions in synuclein does not correlate with the regional location several neurodegenerative disorders. The significance of of LBs and Lewy neurites (45). Furthermore, the protein these observations awaits biochemical confirmation. level of -synuclein is not increased selectively in the substantia nigra where dopaminergic neurons are most Studies of -synuclein aggregation in vitro. The vulnerable to degeneration. Thus, it is possible that other sequential events leading to formation of LBs is unclear. modulators, which directly or indirectly affect the Since -synuclein immunoreactivity in LBs is detected aggregation of -synuclein, vary regionally in the brain. with antibodies to N- and C-terminal regions, it is like- Analogously, A-protein aggregation is stimulated by ly that full-length -synuclein is involved in LB forma- several factors, including apo-E, a1-antichymotrypsin tion (Figure 3). To investigate the molecular mechanism and proteoglycans (70, 114, 133). These molecules co- underlying the aggregation of -synuclein, an in vitro localize with A-protein in senile plaques, leading to the aggregation system was developed. Human -synuclein concept that pathological chaperons play a role in amy- was induced to aggregate under experimental conditions

M. Hashimoto and E. Masliah: -synuclein in LBD and AD 711 loidogenesis (133). In this context, it is reasonable to trast, activity of SOD is increased (103), indicating that hypothesize that certain molecules may act as patholog- antioxidant protective system may be altered in PD. ical chaperons for aggregation of -synuclein and that Furthermore, it has been shown that free radical forma- candidate molecules might co-localize with -synuclein tion derived from the auto-oxidation of dopamine into in LBs. In this regard, it has been shown that aggrega- neuromelanin may be related to the selective degenera- tion of -synuclein in vitro is modulated by A-protein tion of dopaminergic neurons in PD (1). (51, 91, 138), NAC peptide (91), and lipids (18). It is Iron may also play an essential role in the oxidative noteworthy that Down’s syndrome and many familial stress in PD (140) since the substantia nigra contains AD cases exhibit LB formation, suggesting that A-pro- high iron levels that can act as a free radical catalyst via tein might be involved in the intracellular aggregation of the Fenton reaction. In line with this data, we have -synuclein in AD (67) (Figure 4). Although APP, but recently shown that -synuclein preferentially aggregat- not A-protein, has been detected in LBs (3), additional ed in the presence of ferric ion (37) (Figure 3). Although biochemical and immunocytochemical analysis are war- ferrous ion was not effective by itself, -synuclein was ranted to clarify this issue. aggregated by ferrous ion in the presence of hydrogen Evidence from Iwatsubo and coworkers suggests that peroxide. These results suggest that ferric ion might act -synuclein may be proteolytically cleaved in LBs (6), as contributing factor for the aggregation of -synucle- and it is possible that such proteolysis may yield short in in PD. Indeed, it has been suspected that shift from fragments containing the NAC region, which has been ferrous to ferric ion may be a risk factor for PD. shown to promote aggregation of full length -synucle- Riederer et al (101) showed that the ratio of ferric to fer- in. Fragments of -synuclein might function as seeds or rous ion in the substantia nigra of the patients with nucleation sites that induce aggregation of full-length - advanced PD was remarkably increased compared to synuclein. In this regard, it is noteworthy that mutants controls. Furthermore, it was shown that neuromelanin, with truncation of C-terminus aggregated more readily which acts as an endogenous chelator of ferrous and fer- than wild type -synuclein (16). Lipids may also play a ric ions, differentially regulated the rate of hydroxyl rad- role in -synuclein aggregation. Immunoelectron micro- ical production depending on the redox state of iron scopic studies for -synuclein have shown gold particles (96). Therefore, if iron-catalyzed oxidative reactions decorating synaptic vesicles (45) and secretory vesicles play a significant role in -synuclein aggregation in and plasma membrane in platelets (39). Consistent with vivo, either antioxidant or iron chelation could theoreti- this, synelfin, the avian homologue of -synuclein (28), cally be used as therapeutic agents in PD. Other metal has been shown to associate with membrane fractions. ions may also contribute to the aggregation of -synu- Furthermore, -synuclein binding to phospholipids is clein in neurodegeneration, for example, aluminum ion associated with a conformational change (18). At pres- potently stimulates the aggregation of -synuclein (90). ent, it is yet to be determined whether any of these mol- Furthermore, a recent report described copper ion- ecules are essential for aggregation of -synuclein LBD. induced aggregation of -synuclein in the presence of a Since dozens of molecules have already been identified specific crosslinker (89). as constitutes of LBs (97), some of them might turn out A great deal of evidence has established that dysfunc- to be candidates for chaperone-like molecules that may tion of mitochondria may play a role in oxidative stress promote aggregation of -synuclein. in PD (110). In this regard, it has previously been report- ed that over expression of APP results in reduced func- Oxidative stress and aggregation of -synuclein. It tion and distorted morphological changes of mitochon- has become increasingly clear that oxidative stress plays dria in cultured muscle cells and neuroectodermal cells an important role in the pathogenesis of PD and other (5, 31). Similarly, we have observed striking morpho- neurodegenerative disorders (1). The generation of reac- logical changes in mitochondria, such as giant, flat and tive oxygen species occurs during normal cellular irregularly distorted ones, in addition to formation of metabolism and a number of mechanisms for scaveng- inclusion-like structures in the mitochondrial matrix, of ing free radicals have evolved. These include superoxide -synuclein-expressing GT1-7 neuronal cells, but not in dismutase, which converts superoxide ions to hydrogen vector-transfected cells (L. Hsu, personal communica- peroxide, and glutathione peroxide, which reduces tion). These results suggest that both APP and -synu- hydrogen peroxide to water. In PD brains a correlation clein may cause mitochondrial damage via similar has been found between disease severity and decreases mechanisms. in glutathione in the substantia nigra (101, 111). In con-

712 M. Hashimoto and E. Masliah: -synuclein in LBD and AD Finally, our recent study suggests that mitochondrial cytochrome C may participate in the aggregation of - synuclein (38). In vitro, recombinant -synuclein was induced to aggregate in the presence of cytochrome C and hydrogen peroxide. Aggregation was inhibited by N-acetyl-L-cysteine. Interestingly, cytochrome C immunoreactivity has been detected in LBs in the sub- stantia nigra, but not in cortical LBs (Figure 3). Cytochrome C is normally a component of mitochondr- ial membranes. It translocates to cytoplasm by stimuli that induce apoptosis, where it acts to trigger the apop- Figure 6. Role of aggregation of synaptic proteins with amy- totic cascade (32). Taken together, these results suggest loidogenic domain in neurotoxicity. that aggregation of -synuclein might be closely related to mitochondrial dysfunction and neuronal apoptosis.

Amyloidogenesis and neurotoxicity in LBD. Understanding the mechanisms by which amyloidogen- esis is related to other neuropathological alterations, such as synaptic loss, astrogliosis and neuronal death is a central issue in the study of neurodegeneration. Synaptic injury and loss may play critical roles in early stages of disease. Supporting this, a number of recent reports have shown that in early AD and in experimen- Figure 7. Possible mechanisms of -synuclein amyloidogene- tal animal models synaptic loss precedes neuronal cell sis. loss and amyloid deposition (80, 81). One possible mechanism triggering synaptic damage might be related Emerging evidence suggests that formation of inclu- to gain of a toxic property of amyloidogenic proteins. In sion bodies containing amyloid-like fibrils may be this context, -synuclein is abundantly localized in regarded as a cellular protective strategy rather than a presynaptic termini (45). Therefore, specific mutations toxic event. In polyglutamine disease, such as spin- or other alterations in -synuclein might disrupt normal ocerebellar ataxia type 1 and Huntington disease, for- function synapses in dopaminergic neurons in PD. It is mation of nuclear inclusion bodies are one of patholog- also noteworthy that -synuclein is a major constituent ical hallmarks (19). Klement and coworkers (56) creat- of glial inclusion in multiple systemic atrophy (4, 129) ed transgenic mice carrying either the wild type-ataxin- and in LBD (117) (Figures 1, 3), suggesting that - 1 transgene or mutant ataxin-1, with deletion of the synuclein might have significant effects on oligoden- oligomerization domain. Although aggregates formed droglia, leading to demyelination in this disorder. only in the mice overexpressing wild type ataxin-1, both The molecular mechanism by which fibril formation mice showed neurodegeneration. Saudou and coworkers of -synuclein potentially exerts toxicity on synapses is transfected a huntingtin mutant cDNA with an extended unknown, but one might think of intracellular fibril for- polyglutamine into primary cultures of striatal neurons mation analogous to intracellular amyloid deposits. The and nuclear inclusions were detected (105). conventional idea for the role of amyloidogenesis in Interestingly, transfection of antisense ubiquitin cDNA neurodegeneration is that amyloid fibril formation is in this cellular system suppressed the formation of essential for toxicity. Such a notion is based on experi- inclusion bodies, but was associated with increased neu- ments in which cultured cells were challenged with syn- rotoxicity. Taken together, these results indicate that the thetic A peptides and aggregated peptides with fibrils inclusion body per se is not required for neurotoxicity, were required to cause necrosis or apoptosis (8, 68, 95). but may actually play a protective role in polyglutamine A problem with these systems is that high concentra- disease. A similar concept might be considered for LBs tions (more than 10 M) of A are required to exert tox- in PD. It has previously been reported that the majority icity, which is far beyond physiological concentrations of dopaminergic neurons in the substantia nigra under- in the CNS. going apoptotic cell death do not contain LBs (123, 124). Thus, LB formation in surviving neurons could be

M. Hashimoto and E. Masliah: -synuclein in LBD and AD 713 regarded as a protective strategy against putative toxic with certainty (Figure 3). Secondary structure analysis protofibrils of -synuclein. Recent models of amyloido- has shown that NAC has a strongly tendency to form - genesis have placed increasing emphasis on protofibrils pleated structures (45). Consistent with this, biochemi- rather than fibrils themselves in neurotoxicity (63, 137), cal studies have shown that NAC peptide is extremely and the pathogenic molecules may acquire toxic proper- hydrophobic and easily forms amyloid-like fibrils under ties as a consequence of gene mutation or loss of nega- physiological conditions (45). Immunohistochemical tive regulators (Figure 6). As such the it may not be the studies have revealed that the central portion of amyloid -synuclein fibrils in LBs that are pathogenic, but rather in senile plaques is more strongly stained by anti-NAC protofibrils. In this context, at least two therapeutic antibodies than in the peripheral portion (Figure 3), strategies may be proposed for control of the protofibrils whereas anti-A-protein antibodies stained amyloid (Figures 6 and 7). One strategy is to inhibit formation of plaques evenly (125). protofibrils by suppressing the initial aggregation steps. Genetic linkage studies performed using a polymor- The alternative is to restore the ability of neurons to phic dinucleotide repeat sequence in the -synuclein incorporate pathologic molecules into inclusion bodies. gene showed that a polymorphism (-synuclein allele 2) Further characterization of basic mechanisms of amy- was significantly associated with healthy elderly indi- loidogenesis may provide insight into this issue. viduals who carried APOE 4, which may indicate a possible protective function of the -synuclein allele Alpha-synuclein in Alzheimer’s disease (135). Nevertheless, screening of AD families failed to establish a linkage between -synuclein and AD (9, 12). Identification of NAC in senile plaques of AD. AD Based on these findings, it was suggested that NAC was is characterized by progressive and irreversible deaf- not a principle player in amyloidogenesis, but that it ferentation of the limbic system, association neocortex might be involved in the process of amyloid formation and basal forebrain (40, 42, 79, 92, 93, 132), accompa- or condensation (125) (Figure 6). nied by amyloid plaques, neurofibrillary tangles and neuropil threads [for review see (120)]. How is NAC produced from -synuclein? The above Neurodegeneration is accompanied by reactive scenario is reminiscent of the role of A42 in amy- astrogliosis (7) and microglial activation and prolifera- loidogenesis. A42 secreted into extracellular biologi- tion (78, 102). The precise mechanisms leading to neu- cal fluids is estimated to be approximately 10% of A40 rodegeneration in AD are not completely clear, but the (109). Biochemical studies have shown that A42 is prevailing view is that neuropathology results from more prone to aggregation than A40 (68). toxic gain of function of A or loss of trophic activity Furthermore, histochemical studies have suggested that related to abnormal processing of APP (74, 112). It has A42 precedes A40 deposition in the progression of been suggested that a shift in the processing of APP amyloid formation in senile plaques (46). A prevailing toward the -secretase pathway results in the generation notion is that A42 may seed deposition of other amy- of toxic amyloid fragments (82, 100, 109) as well as in loid peptides. Supporting this contention is the fact that generation of secreted forms of APP that are less effec- familial cases of AD with presenilin or APP mutations tive in promoting removal or clearance of excessive glu- are associated with enhanced secretion of A42 (106). It tamate from the synaptic clefts (66). Aggregation of A- remains to be determined if NAC can serve as a seed for protein is known to be modulated by several amyloid- A deposition. associated proteins in senile plaques, such as 1- The essential issue that needs to be clarified if NAC antichymotrypsin, apo-E, nd heparan sulfate proteogly- plays a role in amyloidogenesis is how NAC is generat- cans (70, 114, 133). Saitoh and colleagues identified ed from its precursor, -synuclein. While NAC is detect- another amyloid associated molecule in 1993, which ed in senile plaque with immunohistochemistry, full- they called NAC (Figure 3). NAC co-purified with A- length or partially truncated -synuclein is not. Alpha- protein in SDS-insoluble fractions. Sequencing indicat- synuclein, in contrast, is located in synapses and dys- ed that NAC was distinct from other amyloid associated trophic neurites around senile plaques (77, 125). Since components (125). The concentration of NAC in senile -synuclein is a cytosolic protein without a signal pep- plaques was estimated to be less than 10% of A-protein tide sequence (14) and since no evidences has been (125). As mentioned previously, NAC is located in the reported on -synuclein in extracellular fluids (e.g., middle of -synuclein, from amino acids 61 to 95, cerebrospinal fluid), it is unlikely that -synuclein is although the amino acid terminus was not determined normally secreted. Furthermore, unlike APP and prese-

714 M. Hashimoto and E. Masliah: -synuclein in LBD and AD nilin there is no direct evidence that -synuclein under- pressing clearance of A-protein in the extracellular goes proteolytic processing in cells. Taken together, it is spaces. This may stimulate aggregation of A-protein natural to conclude that -synuclein may be released and lead to enhanced formation of amyloid plaques. into the extracellular space passively from neurons that Alternatively, but not mutually exclusively, -synuclein are damaged during neurodegeneration or that -synu- might interact intracellularly with A-protein. Recently clein is converted intracellularly into an amyloidogenic several authors have detected A-protein in both deter- protein that has structural alterations that allow it to gent-soluble and insoluble fractions, with being more cross the lipid bilayer of the cell membrane. It may then abundant in the latter than in the former, in both cell cul- be possible for -synuclein to undergo proteolytic tures and the human brain (64, 85, 113, 136). This rais- degradation extracellularly to generate NAC. es the possibility that A-protein in the insoluble frac- tions might be an important source for amyloid. It would Alpha-synuclein expression in early AD. The accu- be intriguing to determine whether -synuclein exists in mulated evidence suggests that NAC accumulation in the same fractions as A-protein and whether -synu- senile plaques may be a downstream event. On the other clein could interact with A-protein from detergent hand, it remains a possibility that changes in -synucle- insoluble fractions. in may be involved in earlier pathogenetic stages. It was shown that -synuclein accumulates in dystrophic neu- Molecular dissection of the interaction of -synu- rites in senile plaques (77) possibly due to synaptic clein and NAC with A-protein. To date, only a few pathology that accompanies AD. studies have attempted to model molecular mechanisms Furthermore, previous studies in our laboratory have for the interaction between -synuclein and A-protein demonstrated that -synuclein expression has novel in cell free systems. Yoshimoto and coworkers described kinetics during AD progression. Semi-quantification of specific binding of -synuclein to A1-38 under physi- -synuclein immunoreactivity using immunohisto- ological conditions (138). The formation of SDS-resist- chemistry and dot blot analyses revealed significantly ant complexes composed of -synuclein and A-protein increased -synuclein expression only during early AD. were detected at 72 hours after incubation, indicating In contrast, previous studies have shown that other that a time lag was required for complex formation. synaptic proteins (including synaptophysin) decreased They further mapped the specific A-protein binding steadily through out the entire course of the disease, site to the carboxyl half of NAC domain (amino acid suggesting that loss of nerve terminals is an early and residues81 to 95) (138). This site was, however, chal- fundamental event in AD (44, 77). Given the different lenged by Jensen and co-workers who analyzed the kinetics of synaptic protein expression, it is worth not- interaction of -synuclein with A-protein in the pres- ing that while -synuclein degradation products (e.g., ence of a chemical crosslinker and suggested that wider NAC) become incorporated in senile plaques, other domains of -synuclein, including its N-terminal synaptic proteins do not. Although the mechanism of the domain, interacted with A in addition to the NAC transient increase in the expression level of -synuclein domain (51). Our preliminary results support the later is unclear, it is predicted that the relative increase of - results. Amino acid residues 81 to 95 of a-synuclein are synuclein compared with other synaptic proteins may be located in the carboxyl portion of the NAC domain, a prerequisite for aggregation of -synuclein. which is conserved with -synuclein (48). Therefore, if synucleins bind to A-protein through this domain, both Site of interaction of alpha-synuclein with A-pro- -synuclein and -synucleins, but not mutant proteins tein. Since in vitro studies showed that -synuclein with this domain deleted, should bind to A-protein. binds to A-protein, it was speculated that altered Our results showed that both wild type and mutant synu- metabolism of a-synuclein may affect the metabolism of cleins formed SDS-resistant complexes with A-protein A-protein or APP, leading to the enhanced accumula- after a time lag and under physiological conditions, indi- tion and aggregation of A. If this were the case, it cating that binding sites other than amino acid residues might be possible to identify specific cellular sites in 81 to 95 may be involved in A-protein interaction. The which -synuclein binds to A-protein or identify - differences in these results may be due to differences in synuclein associated extracellularly with A-protein. experimental systems, and further study is required to Since A-protein is normally secreted from cells, - determine the precise binding sites of -synuclein to synuclein aberrantly released from damaged presynaptic A-protein. The results may be useful for the designing termini may interact with A-protein, possibly sup- rational therapies aimed at suppressing amyloidogenesis

M. Hashimoto and E. Masliah: -synuclein in LBD and AD 715 of A-protein that may be stimulated by -synuclein 10. Buchman VL, Hunter H, Pinon L, Thompson J, Privalova (Figure 7). EM, Ninkina NN, Davies AM (1998) Persyn, a member of the synuclein family, has a distinct pattern of expression in the developing nervous system. J Neurosci 18: 9335-9341 Concluding remarks 11. Burkhardt CR, Filley CM, Kleinschmidt-DeMasters BK, de During the past 5 years, a wealth of information has la Monte S, Norenberg MD, Schneck SA (1988) Diffuse indicated that -synuclein plays a critical role in the Lewy body disease and progressive dementia. Neurology pathogenesis of LBD and AD. Further progress in the 38: 1520-1528 understanding of normal and abnormal physiology of - 12. Campion D, Martin C, Heilig R, Charbonnier F, Moreau V, synuclein will certainly add valuable insights into the Flaman JM, Petit JL, Hannequin D, Brice A, Frebourg T (1995) The NACP/synuclein gene: chromosomal assign- molecular mechanisms of neurodegeneration, as well as ment and screening for alterations in Alzheimer disease. novel strategies to suppress amyloidogenesis and neuro- Genomics 26: 254-257 toxicity in these neurodegenerative disorders. 13. Chan P, Tanner CM, Jiang X, Langston JW (1998) Failure to find the alpha-synuclein gene missense mutation Acknowledgments (G209A) in 100 patients with younger onset Parkinson’s This work was supported by NIH Grants AG5131 (to disease. Neurology 50: 513-514 EM and MH) and AG10869 (to EM) and by a fellowship 14. Clayton DF, George JM (1998) The synucleins: a family of proteins involved in synaptic function, plasticity, neurode- from the Spencer family (to MH). The authors would generation and disease. TINS 21: 249-254 also like to thank Margaret Mallory for her technical 15. Conway KA, Harper JD, Lansbury PT (1998) Accelerated assistance and Drs. L. Thal and C. Shultz for their sup- in vitro fibril formation by a mutant alpha-synuclein linked port and encouragement. to early-onset Parkinson disease. Nature Med 4: 1318- 1320 References 16. Crowther RA, Jakes R, Spillantini MG, Goedert M (1998) Synthetic filaments assembled from C-terminally truncat- 1. Adams J, Odunze IN (1991) Oxygen free radicals and ed alpha-synuclein. FEBS Lett 436: 309-312 Parkinson’s disease. Free Radic Biol Med 10: 161-169 17. Cuello AC, Garofalo L, Kenisberg RL, Maysinger D (1989) 2. Akopian AN, Wood JN (1995) Peripheral nervous system- Gangliosides potentiate in vivo and in vitro effects of specific genes identified by subtractive cDNA cloning. J on central cholinergic neurons. Proc Biol Chem 270: 21264-21270 Natl Acad Sci USA 86: 2056-2060 3. Arai H, Lee V-Y, Hill WD, Greenberg BD, Trojanowski JO 18. Davidson WS, Jonas A, Clayton DF, George JM (1998) (1992) Lewy bodies contain beta-amyloid precursor pro- Stabilization of alpha-synuclein secondary structure upon teins of Alzheimer’s disease. Brain Res 585: 386-390 binding to synthetic membranes. J Biol Chem 273: 9443- 4. Arima K, Ueda K, Sunohara N, Arakawa K, Hirai S, 9449 Nakamura M, Tonozuka-Euhara H, Kawai M (1998) 19. Davies SW, Turmaine M, Cozens BA, DiFiglia M, Sharp NACP/alpha-synuclein immunoreactivity in fibrillary com- AH, Ross CA, Scherzinger E, Wanker EE, Mangiarini L, ponents of neuronal and oligodendroglial cytoplasmic Bates GP (1997) Formation of neuronal intranuclear inclusions in the pontine nuclei in multiple system atrophy. inclusions underlies the neurological dysfunction in mice Acta Neuropathol 96: 439-444 transgenic for the HD mutation. Cell 90: 537-548 5. Askanas V, McFerrin J, Baque S, Alvarez RB, Sarkozi E, 20. DeKosky ST, Scheff SW (1990) Synapse loss in frontal Engel WK (1996) Transfer of -amyloid precursor protein cortex biopsies in Alzheimer’s disease: correlation with gene using adenovirus vector causes mitochondrial cognitive severity. Ann Neurol 27: 457-464 abnormalities in cultures of normal human muscle. Proc Natl Acad Sci USA 93: 1314-1319 21. Dickson DW, Crystal H, Mattiace LA, Kress Y, Schwagerl A, Ksiezak-Reding H, Davies P, Yen S-HC (1989) Diffuse 6. Baba M, Nakajo S, Tu P-H, Tomita T, Nakaya K, Lee VM- Lewy body disease: light and electron microscopic Y, Trojanowski JQ, Iwatsubo T (1998) Aggregation of - immunocytochemistry of senile plaques. Acta synuclein in Lewy bodies of sporadic Parkinson’s disease Neuropathol 78: 572-584 and dementia with Lewy bodies. Am J Pathol 152: 879- 884 22. Dickson DW, Davies P, Mayeux R, Crystal H, Horoupian DS, Thompson A, Goldman JE (1987) Diffuse Lewy body 7. Beach TG, Walker R, McGeer EG (1989) Patterns of glio- disease. Neuropathological and biochemical studies of six sis in Alzheimer’s disease and aging cerebrum. GLIA 2: patients. Acta Neuropathol 75: 8-15 420-436 23. Eggertson DE, Sima A (1986) Dementia with cerebral 8. Behl C, Davis J, Lesley R, Schubert D (1994) Hydrogen Lewy bodies. A mesocortical dopaminergic effect? Arch peroxide mediates amyloid protein toxicity. Cell 77: 817- Neurol 43: 524-527 827 24. Engelender S, Kaminsky Z, Guo X, Sharp AH, Amaravi 9. Brookes AJ, St.Clair D (1994) Synuclein proteins and RK, Kleiderlein JJ, Margolis RL, Troncoso JC, Lanahan Alzheimer’s disease. Trends Neurosci 17: 404-405 AA, Worley PF, Dawson VL, Dawson TM, Ross CA (1999) Synphilin-1 associates with alpha-synuclein and pro- motes the formation of cytosolic inclusions. Nature Gen 22: 110-114

716 M. Hashimoto and E. Masliah: -synuclein in LBD and AD 25. Farrer M, Wavrant-De Vrieze F, Crook R, Boles L, Perez- 41. Hsu LJ, Mallory M, Xia Y, Veinbergs I, Hashimoto M, Tur J, Hardy J, Johnson WG, Steele J, Maraganore D, Yoshimoto M, Thal LJ, Saitoh T, Masliah E (1998) Gwinn K (1998) Low frequency of alpha-synuclein muta- Expression pattern of the non-A component of tions in familial Parkinson’s disease. Ann Neurol 43: 394- Alzheimer’s disease amyloid precursor protein (NACP/- 397 synuclein) during brain development. J Neurochem 71: 26. Gaspar P, Graf F (1984) Dementia in idiopathic 338-344 Parkinson’s disease. Acta Neuropathol 64: 43-52 42. Hyman BT, VanHoesen GW, Damasio AR, Barnes CL 27. Gasser T, Muller-Myhsok B, Wszolek ZK, Oehlmann R, (1984) Alzheimer’s disease: Cell-specific pathology iso- Calne DB, Bonifati V, Bereznai B, Fabrizio E, Vieregge P, lates the hippocampal formation. Science 225: 1168-1170 Horstmann RD (1998) A susceptibility locus for 43. Ip NY, Li Y, Yancopoulos GD, Lindsay RM (1993) Cultured Parkinson’s disease maps to chromosome 2p13. Nature hippocampal neurons show responses to BDNF, NT-3, Gen 18: 262-265 and NT-4, but not NGF. J Neurosci 13: 3394-3405 28. George JM, Jin H, Woods WS, Clayton DF (1995) 44. Iwai A, Masliah E, Sundsmo MP, DeTeresa R, Mallory M, Characterization of a novel protein regulated during the Salmon DP, Saitoh T (1996) The synaptic protein NACP is critical period for song learning in the zebra finch. Neuron abnormally expressed during the progression of 15: 361-372 Alzheimer’s disease. Brain Res 720: 230-234 29. Giasson BI, Uryu K, Trojanowski JQ, Lee V-M (1999) 45. Iwai A, Yoshimoto M, Masliah E, Saitoh T (1995) Non-A Mutant and wild type human alpha-synucleins assemble component of Alzheimer’s disease amyloid (NAC) is amy- into elongated filaments with distinct morphologies in loidogenic. Biochemistry 34: 10139-10145 vitro. J Biol Chem 274: 7619-7622 46. Iwatsubo T, Odaka A, Suzuki N, Mizusawa H, Nukina N, 30. Gibb W, Lees AJ (1988) The relevance of the Lewy body Ihara I (1994) Visualization of A beta 42(43) and A beta 40 to the pathogenesis of idiopathic Parkinson’s disease. J in senile plaques with end-specific A beta monoclonals: Neurol Neurosurg Psychiatry 51: 745-752 evidence that an initially deposited species in A beta 31. Grant SM, Shankar SL, Chalmers-Redman R, Tatton WG, 42(43). Neuron 13: 45-53 Szyf M, Cuello AC (1999) Mitochondrial abnormalities in 47. Iwatsubo T, Yamaguchi H, Fujimuro M, Yokosawa H, Ihara neuroectodermal cells stably expressing human amyloid Y, Trojanowski JQ, Lee V-M (1996) Purification and char- precursor protein (hAPP751). NeuroReport 10: 41-46 acterization of Lewy bodies from brains of patients with 32. Green DR, Reed JC (1998) Mitochondria and apoptosis. diffuse Lewy body disease. Am J Pathol 148: 1517-1529 Science 281: 1309-1312 48. Jakes R, Spillantini MG, Goedert M (1994) Identification 33. Group TFPsDS (1998) Alpha-synuclein gene and of two distinct synucleins from human brain. FEBS Lett Parkinson’s disease. Science 279: 1116-1117 345: 27-32 34. Hansen L, Salmon D, Galasko D, Masliah E, Katzman R, 49. Jenco JM, Rawlingson A, Daniels B, Morris AJ (1998) DeTeresa R, Thal L, Pay MM, Hofstetter R, Klauber M Regulation of phospholipase D2: Selective inhibition of (1990) The Lewy body variant of Alzheimer’s disease: a mammalian phospholipase D isoenzymes by - and - clinical and pathologic entity. Neurology 40: 1-7 synucleins. Biochemistry 37: 4901-4909 35. Hardy J, Gwinn-Hardy K (1998) Genetic classification of 50. Jensen PH, Nielsen MS, Jakes R, Dotti CG, Goedert M primary neurodegenerative disease. Science 282: 1075- (1998) Binding of alpha-synuclein to brain vesicles is 1079 abolished by familial Parkinson’s disease mutation. J Biol Chem 273: 26292-26294 36. Hashimoto M, Hsu LJ, Sisk A, Xia Y, Takeda A, Sundsmo M, Masliah E (1998) Human recombinant NACP/-synu- 51. Jensen PH, Hojrup P, Hager H, Nielsen MS, Jacobsen L, clein is aggregated and fibrillated in vitro: Relevance for Olesen OF, Gliemann J, Jakes R (1997) Binding of A to Lewy body disease. Brain Res 799: 301-306 - and -synucleins: identification of segments in -synu- clein/ NAC precursor that bind A and NAC. Biochem J 37. Hashimoto M, Hsu LJ, Xia Y, Takeda A, Sundsmo M, 323: 539-546 Masliah E (1999) Oxidative stress induces amyloid-like aggregate formation of NACP/-synuclein in vitro. 52. Ji H, Liu YE, Wang M, Liu J, Xiao G, Joseph BK, Rosen NeuroReport 10: 717-721 C, Shi YE (1997) Identification of a breast cancer-specific gene, BCSG1, by direct differential cDNA sequencing. 38. Hashimoto M, Takeda A, Hsu LJ, Takenouchi T, Masliah E Cancer Res 57: 759-764 (1999) Role of cytochrome c as a stimulator of -synucle- in aggregation in Lewy body disease. J Biol Chem (in 53. Jia T, Liu YE, Liu J, Shi YE (1999) Stimulation of breast press) cancer invasion and metastasis by synuclein gamma. Cancer Res 59: 742-747 39. Hashimoto M, Yoshimoto M, Sisk A, Hsu LJ, Sundsmo M, Kittel A, Saitoh T, Miller A, Masliah E (1997) NACP, a 54. Kim H, Gearing M, Mirra SS (1995) Ubiquitin-positive synaptic protein involved in Alzheimer’s disease, is differ- CA2/3 neurites in hippocampus coexist with cortical Lewy entially regulated during megakaryocyte differentiation. bodies. Neurology 45: 1768-1770 Biochem Biophys Res Comm 237: 611-616 55. Kitada T, Asakawa S, Hattori N, Matsumine H, Yamamura 40. Hof PR, Cox K, Morrison JH (1990) Quantitative analysis Y, Minoshima S, Yokochi M, Mizuno Y, Shimizu N (1998) of a vulnerable subset of pyramidal neurons in Mutations in the parkin gene cause autosomal recessive Alzheimer’s disease: I. Superior frontal and inferior tem- juvenile parkinsonism. Nature 392: 605-608 poral cortex. J Comp Neurol 301: 44-54

M. Hashimoto and E. Masliah: -synuclein in LBD and AD 717 56. Klement IA, Skinner PJ, Kaytor MD, Yi H, Hersch SM, 71. Markesbery WR, Carney JM (1999) Oxidative alterations Clark HB, Zoghbi HY, Orr HT (1998) Ataxin-1 nuclear in Alzheimer’s disease. Brain Pathol 9: 133-146 localization and aggregation: Role in polyglutamine- 72. Maroteaux L, Campanelli JT, Scheller RH (1988) induced disease in SCA1 transgenic mice. Cell 95: 41-53 Synuclein: a neuron-specific protein localized to the 57. Kosaka K (1978) Lewy bodies in cerebral cortex. Report nucleus and presynaptic nerve terminal. J Neurosci 8: of three cases. Acta Neuropathol 42: 127-134 2804-2815 58. Kosaka K, Yoshimura M, Ikeda K, Budka H (1984) Diffuse 73. Maroteaux L, Scheller RH (1991) The rat brain synucle- type of Lewy body disease. Progressive dementia with ins; family of proteins transiently associated with neuronal abundant cortical Lewy bodies and senile changes of membrane. Mol Brain Res 11: 335-343 varying degree - A new disease? i 3: 183-192 74. Masliah E (1997) Role of amyloid precursor protein in the 59. Kruger R, Kuhn W, Muller T, Woitalla D, Graeber M, Kosel mechanisms of neurodegeneration in Alzheimer’s dis- S, Przuntek H, Epplen JT, Schols L, Reiss O (1998) ease. Lab Invest 77: 197-209 Ala30Pro mutation in the gene encoding -synuclein in 75. Masliah E (1998) Mechanisms of synaptic pathology in Parkinson’s disease. Nature Gen 18: 106-108 Alzheimer’s disease. J Neural Transm 53 (Suppl): 147-158 60. Kruger R, Vieira-Saecker AM, Kuhn W, Berg D, Muller T, 76. Masliah E (1998) The role of synaptic proteins in neu- Kuhnl N, Fuchs GA, Storch A, Hungs M, Woitalla D, rodegenerative disorders. Neurosci News 1: 14-20 Przuntek H, Epplen JT, Schols L, Riess O (1999) 77. Masliah E, Iwai A, Mallory M, Ueda K, Saitoh T (1996) Increased susceptibility to sporadic Parkinson’s disease Altered presynaptic protein NACP is associated with by a certain combined alpha-synuclein/apolipoprotein E plaque formation and neurodegeneration in Alzheimer’s genotype. Ann Neurol 45: 611-617 disease. Am J Pathol 148: 201-210 61. Kuzuhara S, Mori H, Izumiyama N, Yoshimura M, Ihara Y 78. Masliah E, Mallory M, Hansen L, Alford M, Albright T, (1988) Lewy bodies are ubiquitinated. A light and electron Terry R, Shapiro P, Sundsmo M, Saitoh T (1991) microscopic immunocytochemical study. Acta Immunoreactivity of CD45, a protein phosphotyrosine Neuropathol 75: 345-353 phosphatase, in Alzheimer disease. Acta Neuropathol 83: 62. Langlais PJ, Thal L, Hansen L, Galasko D, Alford M, 12-20 Masliah E (1993) Neurotransmitters in basal ganglia and 79. Masliah E, Mallory M, Hansen L, Alford M, DeTeresa R, cortex of Alzheimer’s disease with and without Lewy bod- Terry R (1993) An antibody against phosphorylated neu- ies. Neurology 43: 1927-1934 rofilaments identifies a subset of damaged association 63. Lansbury PT Jr (1999) Evolution of amyloid: What normal axons in Alzheimer’s disease. Am J Pathol 142: 871-882 protein folding may tell us about fibrillogenesis and dis- 80. Masliah E, Mallory M, Hansen L, DeTeresa R, Alford M, ease. Proc Natl Acad Sci USA 96: 3342-3344 Terry R (1994) Synaptic and neuritic alterations during the 64. Lee SJ, Liyanage U, Bickel PE, Xia W, Lansbury PTJ, progression of Alzheimer’s disease. Neurosci Lett 174: Kosik KS (1998) A detergent-insoluble membrane com- 67-72 partment contains A beta in vivo. Nat Med 4: 730-734 81. Masliah E, Sisk A, Mallory M, Mucke L, Schenk D, Games 65. Leroy E, Boyer R, Auburger G, Leube B, Ulm G, Mezey E, D (1996) Comparison of neurodegenerative pathology in Harta G, Brownstein MJ, Jonnalagada S, Chernova T, transgenic mice overexpressing V717F -amyloid precur- Dehejia A, Lavedan C, Gasser T, Steinbach PJ, Wilkinson sor protein and Alzheimer’s disease. J Neurosci 16: 5795- KD, Polymeropoulos MH (1998) The ubiquitin pathway in 5811 Parkinson’s disease. Nature 395: 451-452 82. Mattson MP, Barger SW, Cheng B, Lieberburg I, Smith- 66. Li S, Mallory M, Alford M, Tanaka S, Masliah E (1997) Swintosky VL, Rydel RE (1993) -amyloid precursor pro- Glutamate transporter alterations in Alzheimer’s disease tein metabolites and loss of neuronal Ca2+ homeostasis in are possibly associated with abnormal APP expression. J Alzheimer’s disease. TINS 16: 409-414 Neuropathol Exp Neurol 56: 901-911 83. McKeith IG, Galasko D, Kosaka K, Perry EK, Dickson DW, 67. Lippa CF, Fujiwara H, Mann DM, Giasson B, Baba M, Hansen LA, Salmon DP, Lowe J, Mirra SS, Byrne EJ, Schmidt ML, Nee LE, O’Connell B, Pollen DA, St. George- Quinn NP, Edwardson JA, Ince PG, Bergeron C, Burns A, Hyslop P (1998) Lewy bodies contain altered alpha-synu- Miller BL, Lovestone S, Collerton D, Jansen E, de Vos R, clein in brains of many familial Alzheimer’s disease Wilcock GK, Jellinger KA, Perry RH (1996) Clinical and patients with mutations in presenilin and amyloid precur- pathological diagnosis of dementia with Lewy bodies sor protein genes. Am J Pathol 153: 1365-1370 (DLB): Report of the CDLB International Workshop. 68. Lorenzo A, Yankner BA (1994) -amyloid neurotoxicity Neurology 47: 1113-1124 requires fibril formation and is inhibited by Congo red. 84. Mezey E, Dehejia A, Harta G, Papp M, Polymeropoulos Proc Natl Acad Sci USA 91: 12243-12247 M, Brownstein M (1998) Alpha synuclein in neurodegen- 69. Lowe J, McDermott H, Landon M, Mayer RJ, Wilkinson erative disorders: murderer or accomplice? Nature Med 4: KD (1990) Ubiquitin carboxyl-terminal hydrolase (PGP 755-757 9.5) is selectively present in ubiquitinated inclusion bodies 85. Morishima-Kawashima M, Ihara Y (1998) The presence of characteristic of human neurodegenerative diseases. J amyloid b-protein in the detergent-soluble membrane Pathol 161: 153-160 compartment of human neuroblastoma cells. 70. Ma J, Yee A, Brewer HBJ, Das S, Potter H (1994) Amyloid- Biochemistry 37: 15247-15253 associated proteins a1-antichymotrypsin and apolipopro- tein E promote assembly of Alzheimer -protein into fila- ments. Nature 372: 92-94

718 M. Hashimoto and E. Masliah: -synuclein in LBD and AD 86. Nakajo S (1990) Purification and characterization of a 101. Riederer P, Sofic E, Rausch W-D, Schmidt B, Reynolds novel brain-specific 14-kDa protein. J Neurochem 55: GP, Youdim MB (1989) Transition metals, ferritin, glu- 2031-2038 tathione, and ascorbic acid in Parkinsonian brains. J 87. Nakajo S, Tsukada K, Omata K, Nakamura Y, Nakaya K Neurochem 52: 515-520 (1993) A new brain-specific 14-kDa protein is a phospho- 102. Rogers J, Luber-Narod J, Styren SD, Civin WH (1988) protein. It’s complete amino acid sequence and evidence Expression of immune system-associated antigens by for phosphorylation. Eur J Biochem 217: 1057-1063 cells of the human central nervous system: relationship to 88. Narhi L, Wood SJ, Steavenson S, Jiang Y, Wu GM, Anafi the pathology of Alzheimer’s disease. Neurobiol Aging 9: D, Kaufman SA, Martin F, Sitney K, Denis P, Louis JC, 339-349 Wypych J, Biere AL, Citron M (1999) Both familial 103. Saggu H, Cooksey J, Dexter D, Wells FR, Lees A, Jenner Parkinson’s disease mutations accelerate alpha-synucle- P, Marsden CD (1989) A selective increase in particulate in aggregation. J Biol Chem 274: 9843-9846 superoxide dismutase activity in parkinsonian substantia 89. Paik SR, Shin HJ, Lee JH, Chang CS, Kim J (1999) nigra. J Neurochem 53: 692-697 Copper(II)-induced self-oligomerization of alpha-synucle- 104. Saitoh T, Xia Y, Chen X, Masliah E, Galasko D, Shults C, in. Biochem J 340: 821-828 Thal LJ, Hansen LA, Katzman R (1995) The CYP2D6B 90. Paik SR, Lee JH, Kim DH, Chang CS, Kim J (1997) mutant allele is overrepresented in the Lewy body variant Aluminum-induced structural alterations of the precursor of Alzheimer’s disease. Ann Neurol 37: 110-112 of the non-A beta component of Alzheimer’s disease amy- 105. Saudou F, Finkbeiner S, Devys D, Greenberg ME (1998) loid. Arch Biochem Biophys 344: 325-334 Huntingtin acts in the nucleus to induce apoptosis but 91. Paik SR, Lee JH, Kim DH, Chang CS, Kim YS (1998) Self- death does not correlate with the formation of intranuclear oligomerization of NACP, the precursor protein of the non- inclusions. Cell 95: 55-66 amyloid beta/A4 protein (A beta) component of 106. Scheuner D, Eckman C, Jensen M, Song X, Citron M, Alzheimer’s disease amyloid, observed in the presence of Suzuki N, Bird TD, Hardy J, Hutton M, Kukull W, Larson E, a C-terminal A beta fragment (residues 25-35). FEBS Lett Levy-Lahad E, Viitanen M, Peskind E, Poorkaj P, 421: 73-76 Schellenberg G, Tanzi R, Wasco W, Lannfelt L, Selkoe D, 92. Palmer AM, Gershon S (1990) Is the neuronal basis of Younkin S (1996) Secreted amyloid beta-protein similar to Alzheimer’s disease cholinergic or glutamatergic? FASEB that in the senile plaques of Alzheimer’s disease is J 2745: 2752 increased in vivo by the presenilin 1 and 2 and APP muta- tions linked to familial Alzheimer’s disease. Nature Med 2: 93. Perry EK, Perry RH, Blessed G, Tomlinson BE (1977) 864-870 Neurotransmitter enzyme abnormalities in senile demen- tia: CAT and GAD activities in necropsy tissue. J Neurol 107. Schmidt ML, Murray J, Lee VM-Y, Hill WD, Wertkin A, Sci 34: 247-265 Trojanowski JQ (1991) Epitope map of neurofilament pro- tein domains in cortical and peripheral nervous system 94. Perry RH, Irving D, Blessed G, Fairbairn A, Perry EK Lewy bodies. Am J Pathol 139: 53-65 (1990) Senile dementia of Lewy body type. A clinically and neuropathologically distinct form of Lewy body 108. Seeman P, Tedesco JL, Lee T, Chau-Wong M, Muller P, dementia in the elderly. J Neurol Sci 95: 119-139 Bowles J, Whitaker PM, McManus C, Tittler M, Weinreich P, Friend WC, Brown GM (1978) Dopamine receptors in 95. Pike CJ, Walencewicz AJ, Glabe CG, Cottman CW (1991) the central nervous system. Fed Proc 37: 131-136 In vitro aging of -amyloid protein causes peptide aggre- gation and neurotoxicity. Brain Res 563: 311-314 109. Selkoe D (1994) Cell biology of the amyloid -protein pre- cursor and the mechanisms of Alzheimer’s disease. Ann 96. Pilas B, Sarna T, Kalyanaraman B, Schwartz HM (1988) Rev Cell Biol 10: 373-403 The effect of melanin on iron associated decomposition of hydrogen peroxide. Free Rad Biol Med 4: 285-293 110. Shigenaga MK, Hagen TM, Ames BN (1994) Oxidative damage and mitochondrial decay in aging. Proc Natl Acad 97. Pollanen MS, Dickson DW, Bergeron C (1993) Pathology Sci USA 91: 10771-10778 and biology of the Lewy body. J Neuropathol Exp Neurol 52: 183-191 111. Sian J, Dexter DT, Lees AJ, Daniel S, Agid Y, Javoy-Agid F, Jenner P, Mardsen CD (1994) Alterations in glutathione 98. Polymeropoulos MH, Higgins JJ, Golbe LI, Johnson WG, levels in Parkinson’s disease and other neurodegenera- Ide SE, Di Iorio G, Sangers G, Stenroos ES, Pho LT, tive disorders affecting basal ganglia. Ann Neurol 36: 348- Schaffer AA, Lazzarini AM, Nussbaum RL, Duvoisin RC 355 (1996) Mapping of a gene for Parkinson’s disease to chromosome 4q21-q23. Science 274: 1197-1199 112. Sisodia SS, Price DL (1995) Role of the beta-amyloid pro- tein in Alzheimer’s disease. FASEB J. 9: 366-370 99. Polymeropoulos MH, Lavedant C, Leroy E, Ide SE, Dehejia A, Dutra A, Pike B, Root H, Rubenstein J, Boyer 113. Skovronsky DM, Doms RW, Lee VMY (1998) Detection of R, Stenroos ES, Chandrasekharappa S, Athanassiadou a novel intraneuronal pool of insoluble amyloid -protein A, Papapetropulos T, Johnson WG, Lazzarini AM, that accumulates with time in culture. J Cell Biol 141: Duvoisin RC, Di Iorio G, Golbe LI, Nussbaum RL (1997) 1031-1039 Mutation in the -synuclein gene identified in families with 114. Snow AD, Seikiguchi R, Nochlin D, Fraser P, Kimata K, Parkinson’s disease. Science 276: 2045-2047 Mizutani A, Arai M, Schreier WA, Morgan DG (1994) An 100. Price DL, Sisodia SS, Gandy SE (1995) Amyloid beta important role of heparan sulfate proteoglycan (Perlecan) amyloidosis in Alzheimer’s disease. Curr Opin Neurol 8: in a model system for the deposition and persistence of 268-274 fibrillar A beta-amyloid in rat brain. Neuron 12: 219-234

M. Hashimoto and E. Masliah: -synuclein in LBD and AD 719 115. Spillantini MG, Crowther RA, Jakes R, Hasegawa M, 129. Wakabayashi K, Yoshimoto M, Tsuji S, Takahashi H Goedert M (1998) alpha-synuclein in filamentous inclu- (1998) -synuclein immunoreactivity in glial cytoplasmic sions of Lewy bodies from Parkinson’s disease and inclusions in multiple system atrophy. Neurosci Lett 249: dementia with Lewy bodies. Proc Natl Acad Sci USA 95: 180-182 6469-6473 130. Wasco W, Gurubhagavatula S, Paradis MD, Romano CM, 116. Spillantini MG, Schmidt ML, Lee V-Y, Trojanowski JQ, Sisodia SS, Hyman BT, Neve RL, Tanzi RE (1993) Jakes R, Goedert M (1997) a-Synuclein in Lewy bodies. Isolation and characterization af APLP2 encoding a Nature 388: 839-840 homologue of the Alzheimer’s associated amyloid beta 117. Takeda A, Hashimoto M, Mallory M, Sundsmo M, Hansen protein precursor. Nature Gen 5: 95-100 L, Masliah E (1999) C-terminal -synuclein immunoreac- 131. Weinreb PH, Zhen W, Poon AW, Conway KA, Lansbury tivity in structures other than Lewy bodies in neurodegen- PT Jr (1996) NACP, a protein implicated in Alzheimer’s erative disorders. Acta Neuropathol (in press) disease and learning, is natively unfolded. Biochemistry 118. Takeda A, Hashimoto M, Mallory M, Sundsmo M, Hansen 35: 13709-13715 L, Sisk A, Masliah E (1998) Human NACP/-synuclein 132. Wilcock GK, Esiri MM, Bowen DM, Hughes AO (1988) distribution in Lewy body disease. Lab Invest 78: 1169- The differential involvement of subcortical nuclei in senile 1177 dementia of Alzheimer’s type. J Neurol Neurosurg 119. Takeda A, Mallory M, Sundsmo M, Honer W, Hansen L, Psychiatry 51: 842-849 Masliah E (1998) Abnormal accumulation of NACP/a- 133. Wisniewski T, Castano EM, Golabek A, Vogel T, Frangione synuclein in neurodegenerative disorders. Am J Pathol B (1994) Acceleration of Alzheimer’s fibril formation by 152: 367-372 apolipoprotein E in vitro. Am J Pathol 145: 1030-1035 120. Terry RD, Hansen L, Masliah E. (1994) Structural alter- 134. Wood SJ, Wypych J, Steavenson S, Louis JC, Citron M, ations in Alzheimer disease. In: Terry RD, Katzman R Biere AL (1999) Alpha-synuclein fibrillogenesis is nucle- (eds) Alzheimer Disease. Raven Press, New York pp. 179- ation dependent. Implications for the pathogenesis of 196 Parkinson’s disease. J Biol Chem 274: 19509-19512 121. Terry RD, Masliah E, Salmon DP, Butters N, DeTeresa R, 135. Xia Y, de Silva H, Rosi BL, Yamaoka LH, Rimmler JB, Hill R, Hansen LA, Katzman R (1991) Physical basis of Pericak-Vance MA, Roses AD, Chen X, Masliah E, cognitive alterations in Alzheimer disease: synapse loss is DeTeresa R, Iwai A, Sundsmo M, Thomas RG, Hofstetter the major correlate of cognitive impairment. Ann Neurol CR, Gregory E, Hansen LA, Katzman R, Thal LJ, Saitoh 30: 572-580 T (1996) Genetic studies in Alzheimer’s disease with an 122. Tobe T, Nakajo S, Tanaka A, Mitoya A, Omata K, Nakaya NACP/alpha-synuclein polymorphism. Ann Neurol 40: K, Tomita M, Nakamura Y (1992) Cloning and characteri- 207-215 zation of the cDNA encoding a novel brain-specific 14 136. Yanagisawa K, Odaka A, Suzuki N, Ihara Y (1995) GM1 kDa protein. J Neurochem 59: 1624-1629 ganglioside-bound amyloid beta-protein (A beta): a possi- 123. Tompkins MM, Basgall EJ, Zamrini E, Hill WD (1997) ble form of preamyloid in Alzheimer’s disease. Nature Apoptotic-like changes in Lewy-body-associated disor- Med 1: 1062-1066 ders and normal aging in substantia nigra neurons. Am J 137. Yang AJ, Chandswangbhuvana D, Shu T, Henschen A, Pathol 150: 119-131 Glabe CG (1999) Intracellular accumulation of insoluble, 124. Tompkins MM, Hill WD (1997) Contribution of somal Lewy newly synthesized Abeta1-42 in amyloid precursor pro- bodies to neuronal death. Brain Res 775: 24-29 tein-transfected cells that have been treated with Abeta1- 42. J Biol Chem 274: 20650-20656 125. Ueda K, Fukushima H, Masliah E, Xia Y, Iwai A, Otero D, Kondo J, Ihara Y, Saitoh T (1993) Molecular cloning of a 138. Yoshimoto M, Iwai A, Kang D, Otero D, Xia Y, Saitoh T novel component of amyloid in Alzheimer’s disease. Proc (1995) NACP, the precursor protein of non-amyloid /A4 Natl Acad Sci USA 90: 11282-11286 protein (A ) component of Alzheimer disease amyloid, binds A and stimulates A aggregation. Proc Natl Acad 126. Ueda K, Saitoh T, Mori H (1994) Tissue-dependent alter- Sci USA 92: 9141-9145 native splicing of mRNA for NACP, the precursor of non-A beta component of Alzheimer’s disease amyloid. Biochem 139. Yoshimura M (1983) Cortical changes in the parkinsonian Biophys Res Comm 205: 1366-1372 brain: a contribution to the delineation of diffuse Lewy body disease. J Neurol 229: 17-32 127. Vaughan J, Durr A, Tassin J, Bereznai B, Gasser T, Bonifati V, De Michele G, Fabrizio E, Volpe G, Bandmann 140. Youdim MB, Ben-Schachar D, Riederer P (1989) Is O, Johnson WG, Golbe LI, Breteler M, Meco G, Agid Y, Parkinson’s disease a progressive siderosis of substantia Brice A, Marsden CD, Wood NW (1998) The alpha-synu- nigra resulting in iron and melanin induced neurodegen- clein Ala53Thr mutation is not a common cause of famil- eration? Acta Neurol Scand 126: 47-54 ial Parkinson’s disease: a study of 230 European cases. Ann Neurol 44: 270-273 128. Veldman BA, Wijn AM, Knoers N, Praamstra P, Horstink MW (1998) Genetic and environmental risk factors in Parkinson’s disease. Clin Neurol Neurosurg 100: 15-26

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