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α-Synuclein, Aβ and Alzheimer's disease
Progress in Neuro-Psychopharmacology & Biological Psychiatry 27 (2003) 103 – 108 www.elsevier.com/locate/pnpbp
Review article
A-Synuclein, AB and Alzheimer’s disease Oliver Wirths*, Thomas A. Bayer Department of Psychiatry, Section Neurobiology and European Graduate School of Neuroscience (EURON), University of Saarland Medical Center, Building 90, D-66421 Homburg/Saar, Germany Accepted 18 November 2002
Abstract A-Synuclein is a presynaptic protein that is implicated in the pathogenesis of various neurodegenerative diseases. Missense mutations in the A-synuclein gene are linked to familial cases of Parkinson’s disease (PD), and it has further been shown that A-synuclein is a major constituent of the Lewy bodies in sporadic PD and dementia with Lewy body (DLB). The contribution of A-synuclein to the pathological changes in Alzheimer’s disease (AD) has been currently a matter of scientific debate. Some reports hypothesized that A-synuclein may play a role in amyloid B/A4 protein (AB) aggregation in senile plaques, whereas recent reports challenged this finding by showing a lack of Asynuclein-immunoreactivity in AB plaques. In this review, we report on recent findings on the physiological and pathological role of Asynuclein and try to elucidate its possible contribution to AD pathology. D 2002 Elsevier Science Inc. All rights reserved. Keywords: A-Synuclein; Alzheimer’s disease; Amyloid; Lewy body; Mutations; NAC
1. A-Synuclein protein structure a-Synuclein belongs to a family of small proteins (113 – 143 amino acids) that are highly expressed in nervous tissues with an enrichment in presynaptic terminals (Jakes et al., 1994; Irizarry et al., 1996). The initial study identified a-synuclein in the electric organ of the Pacific electric ray Torpedo californica. The synaptic, but also nuclear localization of the protein led to the nomenclature ‘‘synuclein’’ (Maroteaux et al., 1988). Up to now, three different members, referred to as a-, b- and g-synuclein have been identified. A 35 amino acid peptide was found in the SDS-insoluble fraction from Alzheimer’s disease (AD) patients brain tissue, which was named the non-amyloid b/ A4 protein (Ab) component of Alzheimer’s disease amyloid (NAC). Further analysis revealed that these 35 amino acids are highly hydrophobic and correspond to residues 61 –95
Abbreviations: Ab, amyloid b/A4 protein; AD, Alzheimer’s disease; APP, amyloid precursor protein; DLB, dementia with Lewy bodies; NAC, non-Ab component of AD; NACP, NAC precursor; PD, Parkinson’s disease. * Corresponding author. Department of Psychiatry, Section Neurobiology, University of Saarland Medical Center, Building 90, D-66421 Homburg/Saar, Germany. E-mail address: [email protected] (O. Wirths).
of the larger NAC precursor (NACP), now referred to as asynuclein. In the N-terminus, several imperfect repeats of the motif KTKEGV are located; however, the physiological importance remains so far unclear (Ueda et al., 1993). The C-terminal region is rich in glutamate residues and thus acidic. Weinreb et al. (1996) applied different spectroscopic methods and reported that a-synuclein in solution occurs as a mixture of rapidly equilibrating monomers and is ‘‘natively unfolded,’’ which means that it contains no secondary structure. 2. Genetic association between AD and A-synuclein The human a-synuclein gene (SNCA) was assigned to chromosome 4q21.3 –q22 (Chen et al., 1995; Spillantini et al., 1995). The mRNA is abundantly expressed in the brain, but has also been detected in lungs, placenta and kidneys (Ueda et al., 1993). There are conflicting reports on the genetic association between AD and the a-synuclein gene. Initially, screening of AD families failed to establish any linkage between SNCA and AD; however, there was evidence that one of the SNCA polymorphisms (NACP allele 2) was involved in the development of AD pathology linked to ApoE e4 polymorphisms as a confounding risk factor. The
0278-5846/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. doi:10.1016/S0278-5846(02)00339-1
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NACP allele 2 was four-fold higher represented in agematched healthy controls harboring the ApoE e4 allele than in AD patients with ApoE e4. This suggests a protective effect of the NACP polymorphism against ApoE e4-associated risk for AD (Xia et al., 1996). Hellman et al. (1998) repeated genotyping of the same polymorphism in a comparable number of AD patients and healthy controls but failed to detect any protective effect for the NACP allele 2, or any other NACP alleles. A similar complex dinucleotid repeat than that used in the previous studies was found in the mouse. It is located upstream of the translational start of a-synuclein and seems to account for the control of asynuclein expression (Touchman et al., 2001). Recently, Matsubara et al. (2001) reported that women carrying a common polymorphism in exon 3, had a 2.2-fold increased risk of developing sporadic AD than noncarrier women, independent of ApoE status. Mutational analysis of the a-synuclein gene revealed two missense mutations (A30P, A53T), which cause inherited forms of Parkinson’s disease (PD) (Polymeropoulos et al., 1997; Kruger et al., 1998). Further studies assessed the question whether the A53T mutation also transmits susceptibility to sporadic PD, dementia with Lewy bodies (DLB) and AD, but no involvement could be detected in these disorders (Higuchi et al., 1998). Screening for further mutational alterations in early onset AD patients revealed no mutations (Campion et al., 1995), leading to the conclusion that asynuclein mutations do not contribute to AD pathology. The expression levels of the synuclein family vary in AD and Lewy body disease. Whereas a-synuclein mRNA levels in the superior temporal cortex of AD patients do not differ from levels in control subjects, b-synuclein levels are decreased and g-synuclein levels seem to be increased in AD patients in this affected brain region (Rockenstein et al., 2001). This finding raises the question whether subtle alterations in the balance of synucleins contribute to the pathological events that lead to various neurodegenerative diseases. 3. Influence of A-synuclein mutations, transport and membrane interaction a-Synuclein is located to presynaptic terminals, which suggests that it is intimately involved in synaptic function (Iwai et al., 1995). Under normal conditions, a-synuclein is axonally transported after synthesis in the cell body, which leads to synaptic protein assembly. All three different types of axonal transport take part in these processes with the slow component b as the main type of transport (Jensen et al., 1999). The normal localization is displaced in some neurodegenerative disorders like PD or DLB. In these pathological situations, a relocalization occurs and a-synuclein accumulates in the cell bodies and neurites of degenerating neurons. The normal function of a-synuclein remains so far unclear, but recent reports suggest that it is involved in the trafficking
of synaptic terminals (Narayanan and Scarlata, 2001) and that its function includes its proper interaction with lipids (Jo et al., 2000). Perrin et al. (2000) have shown that the lipid binding domains are distributed across the N-terminal region of the protein (which corresponds to redidues 1 –102). aSynuclein binding to synthetic phospholipid vesicles is accompanied by a strong increase in a-helicity, an observation consistent with a role in vesicle function at synaptic terminals (Davidson et al., 1998). Sharon et al. identified protein motifs at the N- and C-terminus that were homologous to fatty acid binding proteins. They suggested that asynuclein represents a novel member of this protein family, which might play a role in fatty acid transport between aqueous and phospholipid compartments in neuronal cells (Sharon et al., 2001). Suppression of a-synuclein expression in cultured, primary neurons using antisense oligonucleotides, results in a significant reduction in the distal pool of synaptic vesicles (Murphy et al., 2000), suggesting that regulation of the size of synaptic vesicle pools may be a function of a-synuclein. The ability to bind brain vesicles is influenced by a-synuclein mutations. The familial PD-linked A30P mutant is defective in binding to phospholipid vesicles, whereas the A53T mutation displayed normal membrane-binding activity, comparable to wild-type a-synuclein (Jo et al., 2002). The same issue was reported by Jensen et al., who investigated recombinant A30P and A53T mutants in a vesicle-binding assay. Whereas the A30P mutant was devoid of any vesicle-binding, the A53T mutant bound as well to vesicles as wild-type asynuclein. They suggested that the missing vesicle-binding capability of A30P mutants may disturb axonal transport, resulting in a-synuclein transported only by the slow component b but not by the fast component of axonal transport. (Jensen et al., 1998). This may, in a time-dependent manner, lead to an accumulation of A30P a-synuclein with disruption of the normal localization of the protein. This gives rise to the assembly of Lewy body filaments, consisting of accumulated and aggregated a-synuclein after reaching a critical concentration. Fluorescence resonance energy transfer (FRET) studies showed that the A30P mutation alters the threedimensional conformation of a-synuclein (McLean et al., 2000). a-Synuclein mutations further seem to influence the structural properties of the protein, resulting in modified aggregational behavior. Aggregation of a-synuclein is tightly linked to pathological alterations in many neurodegenerative diseases, like PD or the Lewy body variant (LBV) of AD. The PD-linked mutations A30P and A53T self-aggregate and form more b-sheeted, amyloid-like filaments that the wildtype protein (El-Agnaf et al., 1998). The formation of aggregates is further accelerated by the point mutations (Narhi et al., 1999), which seems to be due to a faster selfassociation of the mutants than the wild-type (Li et al., 2001). a-Synuclein is unstable in its monomeric form and it seems that self-assembly stabilizes partially folded a-synuclein, leading to oligomers, which might evolve into the nucleus of fibrils (Uversky et al., 2001).
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4. Does A-synuclein interact with AB? The interaction of A-synuclein and AB and the contribution of A-synuclein accumulation in AD plaques has been currently a matter of scientific debates. The B-amyloid peptide is the major constituent of both neuritic plaques and vascular deposits in the walls of cerebral and meningeal arterioles and arteries. These amyloid deposits are mainly composed of aggregates of the AB peptide with 40 –42 residues, which is proteolytically derived from the larger Bamyloid precursor protein (APP). A-Synuclein was initially purified from the SDS-insoluble fraction of the frontal cortex of AD patients. Immunohistochemical analysis of AD brain sections with antibodies against the identified peptide revealed immunostaining of amyloid in diffuse, primitive and mature plaques (Ueda et al., 1993). This observation was corroborated by other studies, which reported NAC immunoreactivity in amyloid plaques, showing more abundant NAC immunoreactivity in the amyloid core than in the periphery (Masliah et al., 1996). Recent studies, however, using different antibodies covering the major portions of the protein, addressed the question whether there is an association between A-synuclein and amyloid plaques. We and others confirmed only the synaptic and Lewy body staining and concluded that A-synuclein is not associated with mature amyloid plaque cores in AD (Bayer et al., 1999; Culvenor et al., 1999). Furthermore, we observed that AD cases with mixed pathology of the LBV revealed accumulation of A-synuclein in Lewy bodies and
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dystrophic neurites decorating AB plaques (Fig. 1) (Wirths et al., 2000). This indicates that A-synuclein accumulation is restricted to dystrophic neurites, and therefore to intracellular compartments. The extracellular amyloid plaque cores are free of A-synuclein accumulation. One possible explanation for these conflicting results could be that the NAC antibody used by Ueda et al. showed cross-reactivity with the AB sequence. Almost the same phenotype seen in AD patients is found in APP transgenic mice, which represent a model system for the investigation of AD-related pathological alterations. Heterozygous Tg2576 mice, which express human APP with the ’’Swedish’’ double mutation (Hsiao et al., 1996), show frequent ubiquitin- and Asynuclein-positive neurites, resembling some aspects of the neuropathological events in cases with the LBV of AD (Yang et al., 2000). In these animals, staining with Asynuclein antibodies reveals a punctate protein distribution in the neuropil, but was clearly absent in the amyloid core. Dystrophic neurites in and around the plaque cores were labeled, but no Lewy or other inclusion bodies were detected in the transgenic mouse brains (Tomidokoro et al., 2001). In the last few years, a couple of human Asynuclein transgenic mice have also been developed, showing various degrees of pathology (Masliah et al., 2000; van der Putten et al., 2000; Giasson et al., 2002; Lee et al., 2002). In a very recent report, Lee et al. generated multiple lines of transgenic mice expressing wild-type A-synuclein, as well as the A53T and the A30P mutants. Interestingly, only the mice with the A53T mutant developed adult-onset
Fig. 1. (A) Ab immunostaining of a typical Alzheimer’s disease case. (B) Parallel section of the same patient stained with an antibody against a-synuclein. (C) a-Synuclein immunostaining of a case with the Lewy body variant of AD. Arrows point to a-synuclein aggregations in dystrophic neurites in the vicinity of an amyloid plaque. (D) a-Synuclein immunoreactive dystrophic neurites stained by an a-synuclein antibody in the cortex of an APP/PS-1 double transgenic mouse. (Scale bars: A: 100 mm; C, D: 20 mm.)
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neurodegenerative defects with progressive motoric dysfunction (Lee et al., 2002). Masliah et al. (2000) showed that wild-type A-synuclein transgenic mice under the control of the PDGF-B promoter harbored prominent A-synuclein-immunoreactive intraneuronal inclusions by 2 months of age, besides loss of dopaminergic neurons and motor impairments. Crossing these mice with human APP transgenic mice revealed mice with enhanced pathology. They show severe learning, memory and motor deficits, as well as prominent age-dependent degeneration of cholinergic neurons and presynaptic terminals. A-Synuclein-immunoreactive inclusions were further more numerous than in Asynuclein single transgenic mice (Masliah et al., 2001). Several studies addressed the question whether AB and synuclein interact in vitro. It was shown that the NAC precursor is able to bind to AB1-38 and AB25-35 immobilized on nitrocellulose membranes (Yoshimoto et al., 1995). Jensen et al. (1997) identified a further AB-binding segment on the A-synuclein protein (57 –97) and showed that B-synuclein is also able to bind to and promote aggregation of AB.
examination revealed significant A-synuclein aggregation in dystrophic neurites decorating AB plaques, as well as marked intraneuronal aggregation in Lewy neurites and Lewy bodies in patients with mixed AD and Lewy body pathology (Wirths et al., 2000). Szpak et al. (2001) also reported recently coexistence of brain stem and cortical Lewy bodies with pathological features of AD in a significant part of dementia cases. Besides Lewy bodies other dense A-synuclein-positive inclusion bodies in pyramidal cells of the medial temporal lobe were reported in AD and the LBV of AD. Lewy bodies were characterized by strong ubiquitin immunoreactivity, a larger size and homogenous texture, whereas the A-synuclein inclusions are smaller and not ubiquitin-immunoreactive (Mukaetova-Ladinska et al., 2000). These inclusions may represent initial aggregates of A-synuclein. A-Synuclein-positive inclusions that differed from Lewy bodies were also reported at high rates in the hippocampus of AD patients (Arai et al., 2001). Staining with a C-teminal Asynuclein antibody reveals immunoreactivity of neurofibrillary tangles in AD and Lewy body disease cases after pretreatment with formic acid (Takeda et al., 2000).
5. Lewy body pathology in AD Different groups reported A-synuclein-immunoreactive Lewy bodies in AD patients. Lippa et al. investigated whether A-synuclein pathology plays a significant role in familial AD cases using A-synuclein immunohistochemistry. The authors detected A-synuclein-immunoreactive Lewy bodies in 22% of the investigated AD brains, most numerous in the amygdala, where they in part co-localized with T-positive neurofibrillary tangles. Over 60% of cases with amygdala samples available had A-synuclein-immunoreactive Lewy bodies (Lippa et al., 1998). A recent report addresses the same question in a large cohort of sporadic AD patients and shows that more than 60% of the cases harbored A-synuclein positive Lewy bodies. Moreover, in all of these cases, the amygdala was involved with numerous Lewy bodies in this particular area, suggesting that the amygdala is the most commonly affected brain region (Hamilton, 2000). Analysis of Down’s syndrome patient samples revealed that more than 50% of the investigated cases displayed A-synuclein-positive Lewy bodies and dystrophic neurites in the amygdala, adding further evidence to the fact that this brain region is highly susceptible for these pathological alterations (Lippa et al., 1999). It is now well established that the mixed pathology, consisting of Lewy bodies and AB plaques, represents a distinct subtype of AD, which is referred to as LBV of AD (Hansen et al., 1990; Forstl et al., 1993; Petersen, 1998). In a study that focused on the differences in the pattern of progression between AD and AD with Lewy bodies, Lopez et al. reported a faster progression of extrapyramidal signs in patients with AD and Lewy bodies. The rate of cognitive decline however was not different between pure AD patients and patients with the LBV (Lopez et al., 2000). Histological
6. Conclusion In cases with the LBV of AD, overlapping pathological alterations regarding A-synuclein and AB are obvious. Trafficking of synaptic terminals and lipid interaction seems to be important functions of A-synuclein. In pure AD cases, impaired trafficking and resulting aberrant processing of APP are the key events that may lead to the pathological deposition of AB peptides. Therefore, changes in the lipid composition of membranes and altered membrane fluidity during aging might have a significant impact on transport of both proteins. Several recent studies suggested brain cholesterol alterations as an important event in the progression of AD (Frears et al., 1999; Runz et al., 2002). Furthermore, interactions of AB with anionic phospholipids were reported (Chauhan et al., 2000). APP, as well as A-synuclein, becomes axonally transported (Jensen et al., 1999; Kamal et al., 2000). A direct interaction of A-synuclein and APP, however, seems unlikely and there is no evidence that APP and A-synuclein become transported by the same vesicles. However, it has been shown that A-synuclein and AB can interact in vitro (Jensen et al., 1997). Further studies on membrane interactions and the impact of changes in membrane composition during aging will help to elucidate the connection between both proteins in neurodegeneration.
Acknowledgements With kind support from the Fritz Thyssen Foundation, the Alzheimer Forschung Initiative (to TAB) and the European Community (Quality of Life and Management
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of Living Resources, QLK6-CT-2000-60042, QLK6-GH00-60042-02 to OW).
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Szpak, G.M., Lewandowska, E., Lechowicz, W., Bertrand, E., WierzbaBobrowicz, T., Gwiazda, E., Pasennik, E., Kosno-Kruszewska, E., Lipczynska-Lojkowska, W., Bochynska, A., Fiszer, U., 2001. Lewy body variant of Alzheimer’s disease and Alzheimer’s disease: a comparative immunohistochemical study. Folia Neuropathol. 39, 63 – 71. Takeda, A., Hashimoto, M., Mallory, M., Sundsumo, M., Hansen, L., Masliah, E., 2000. C-terminal alpha-synuclein immunoreactivity in structures other than Lewy bodies in neurodegenerative disorders. Acta Neuropathol. (Berl.) 99, 296 – 304. Tomidokoro, Y., Harigaya, Y., Matsubara, E., Ikeda, M., Kawarabayashi, T., Shirao, T., Ishiguro, K., Okamoto, K., Younkin, S.G., Shoji, M., 2001. Brain Abeta amyloidosis in APPsw mice induces accumulation of presenilin-1 and tau. J. Pathol. 194, 499 – 505. Touchman, J.W., Dehejia, A., Chiba-Falek, O., Cabin, D.E., Schwartz, J.R., Orrison, B.M., Polymeropoulos, M.H., Nussbaum, R.L., 2001. Human and mouse alpha-synuclein genes: comparative genomic sequence analysis and identification of a novel gene regulatory element. Genome Res. 11, 78 – 86. Ueda, K., Fukushima, H., Masliah, E., Xia, Y., Iwai, A., Yoshimoto, M., Otero, D.A., Kondo, J., Ihara, Y., Saitoh, T., 1993. Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease. Proc. Natl. Acad. Sci. USA 90, 11282 – 11286. Uversky, V.N., Lee, H.J., Li, J., Fink, A.L., Lee, S.J., 2001. Stabilization of partially folded conformation during alpha-synuclein oligomerization in both purified and cytosolic preparations. J. Biol. Chem. 276, 43495 – 43498. van der Putten, H., Wiederhold, K.H., Probst, A., Barbieri, S., Mistl, C., Danner, S., Kauffmann, S., Hofele, K., Spooren, W.P., Ruegg, M.A., Lin, S., Caroni, P., Sommer, B., Tolnay, M., Bilbe, G., 2000. Neuropathology in mice expressing human alpha-synuclein. J. Neurosci. 20, 6021 – 6029. Weinreb, P.H., Zhen, W., Poon, A.W., Conway, K.A., Lansbury Jr., P.T., 1996. NACP, a protein implicated in Alzheimer’s disease and learning, is natively unfolded. Biochemistry 35, 13709 – 13715. Wirths, O., Weickert, S., Majtenyi, K., Havas, L., Kahle, P.J., Okochi, M., Haass, C., Multhaup, G., Beyreuther, K., Bayer, T.A., 2000. Lewy body variant of Alzheimer’s disease: alpha-synuclein in dystrophic neurites of Abeta plaques. NeuroReport 11, 3737 – 3741. Xia, Y., Rohan de Silva, H.A., Rosi, B.L., Yamaoka, L.H., Rimmler, J.B., Pericak-Vance, M.A., Roses, A.D., Chen, X., Masliah, E., DeTeresa, R., Iwai, A., Sundsmo, M., Thomas, R.G., Hofstetter, C.R., Gregory, E., Hansen, L.A., Katzman, R., Thal, L.J., Saitoh, T., 1996. Genetic studies in Alzheimer’s disease with an NACP/alpha-synuclein polymorphism. Ann. Neurol. 40, 207 – 215. Yang, F., Ueda, K., Chen, P., Ashe, K.H., Cole, G.M., 2000. Plaque-associated alpha-synuclein (NACP) pathology in aged transgenic mice expressing amyloid precursor protein. Brain Res., Jan. 24. 853, 381 – 383. Yoshimoto, M., Iwai, A., Kang, D., Otero, D.A., Xia, Y., Saitoh, T., 1995. NACP, the precursor protein of the non-amyloid beta/A4 protein (Abeta) component of Alzheimer disease amyloid, binds Abeta and stimulates Abeta aggregation. Proc. Natl. Acad. Sci. USA 92, 9141 – 9145.
Review article
A-Synuclein, AB and Alzheimer’s disease Oliver Wirths*, Thomas A. Bayer Department of Psychiatry, Section Neurobiology and European Graduate School of Neuroscience (EURON), University of Saarland Medical Center, Building 90, D-66421 Homburg/Saar, Germany Accepted 18 November 2002
Abstract A-Synuclein is a presynaptic protein that is implicated in the pathogenesis of various neurodegenerative diseases. Missense mutations in the A-synuclein gene are linked to familial cases of Parkinson’s disease (PD), and it has further been shown that A-synuclein is a major constituent of the Lewy bodies in sporadic PD and dementia with Lewy body (DLB). The contribution of A-synuclein to the pathological changes in Alzheimer’s disease (AD) has been currently a matter of scientific debate. Some reports hypothesized that A-synuclein may play a role in amyloid B/A4 protein (AB) aggregation in senile plaques, whereas recent reports challenged this finding by showing a lack of Asynuclein-immunoreactivity in AB plaques. In this review, we report on recent findings on the physiological and pathological role of Asynuclein and try to elucidate its possible contribution to AD pathology. D 2002 Elsevier Science Inc. All rights reserved. Keywords: A-Synuclein; Alzheimer’s disease; Amyloid; Lewy body; Mutations; NAC
1. A-Synuclein protein structure a-Synuclein belongs to a family of small proteins (113 – 143 amino acids) that are highly expressed in nervous tissues with an enrichment in presynaptic terminals (Jakes et al., 1994; Irizarry et al., 1996). The initial study identified a-synuclein in the electric organ of the Pacific electric ray Torpedo californica. The synaptic, but also nuclear localization of the protein led to the nomenclature ‘‘synuclein’’ (Maroteaux et al., 1988). Up to now, three different members, referred to as a-, b- and g-synuclein have been identified. A 35 amino acid peptide was found in the SDS-insoluble fraction from Alzheimer’s disease (AD) patients brain tissue, which was named the non-amyloid b/ A4 protein (Ab) component of Alzheimer’s disease amyloid (NAC). Further analysis revealed that these 35 amino acids are highly hydrophobic and correspond to residues 61 –95
Abbreviations: Ab, amyloid b/A4 protein; AD, Alzheimer’s disease; APP, amyloid precursor protein; DLB, dementia with Lewy bodies; NAC, non-Ab component of AD; NACP, NAC precursor; PD, Parkinson’s disease. * Corresponding author. Department of Psychiatry, Section Neurobiology, University of Saarland Medical Center, Building 90, D-66421 Homburg/Saar, Germany. E-mail address: [email protected] (O. Wirths).
of the larger NAC precursor (NACP), now referred to as asynuclein. In the N-terminus, several imperfect repeats of the motif KTKEGV are located; however, the physiological importance remains so far unclear (Ueda et al., 1993). The C-terminal region is rich in glutamate residues and thus acidic. Weinreb et al. (1996) applied different spectroscopic methods and reported that a-synuclein in solution occurs as a mixture of rapidly equilibrating monomers and is ‘‘natively unfolded,’’ which means that it contains no secondary structure. 2. Genetic association between AD and A-synuclein The human a-synuclein gene (SNCA) was assigned to chromosome 4q21.3 –q22 (Chen et al., 1995; Spillantini et al., 1995). The mRNA is abundantly expressed in the brain, but has also been detected in lungs, placenta and kidneys (Ueda et al., 1993). There are conflicting reports on the genetic association between AD and the a-synuclein gene. Initially, screening of AD families failed to establish any linkage between SNCA and AD; however, there was evidence that one of the SNCA polymorphisms (NACP allele 2) was involved in the development of AD pathology linked to ApoE e4 polymorphisms as a confounding risk factor. The
0278-5846/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. doi:10.1016/S0278-5846(02)00339-1
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NACP allele 2 was four-fold higher represented in agematched healthy controls harboring the ApoE e4 allele than in AD patients with ApoE e4. This suggests a protective effect of the NACP polymorphism against ApoE e4-associated risk for AD (Xia et al., 1996). Hellman et al. (1998) repeated genotyping of the same polymorphism in a comparable number of AD patients and healthy controls but failed to detect any protective effect for the NACP allele 2, or any other NACP alleles. A similar complex dinucleotid repeat than that used in the previous studies was found in the mouse. It is located upstream of the translational start of a-synuclein and seems to account for the control of asynuclein expression (Touchman et al., 2001). Recently, Matsubara et al. (2001) reported that women carrying a common polymorphism in exon 3, had a 2.2-fold increased risk of developing sporadic AD than noncarrier women, independent of ApoE status. Mutational analysis of the a-synuclein gene revealed two missense mutations (A30P, A53T), which cause inherited forms of Parkinson’s disease (PD) (Polymeropoulos et al., 1997; Kruger et al., 1998). Further studies assessed the question whether the A53T mutation also transmits susceptibility to sporadic PD, dementia with Lewy bodies (DLB) and AD, but no involvement could be detected in these disorders (Higuchi et al., 1998). Screening for further mutational alterations in early onset AD patients revealed no mutations (Campion et al., 1995), leading to the conclusion that asynuclein mutations do not contribute to AD pathology. The expression levels of the synuclein family vary in AD and Lewy body disease. Whereas a-synuclein mRNA levels in the superior temporal cortex of AD patients do not differ from levels in control subjects, b-synuclein levels are decreased and g-synuclein levels seem to be increased in AD patients in this affected brain region (Rockenstein et al., 2001). This finding raises the question whether subtle alterations in the balance of synucleins contribute to the pathological events that lead to various neurodegenerative diseases. 3. Influence of A-synuclein mutations, transport and membrane interaction a-Synuclein is located to presynaptic terminals, which suggests that it is intimately involved in synaptic function (Iwai et al., 1995). Under normal conditions, a-synuclein is axonally transported after synthesis in the cell body, which leads to synaptic protein assembly. All three different types of axonal transport take part in these processes with the slow component b as the main type of transport (Jensen et al., 1999). The normal localization is displaced in some neurodegenerative disorders like PD or DLB. In these pathological situations, a relocalization occurs and a-synuclein accumulates in the cell bodies and neurites of degenerating neurons. The normal function of a-synuclein remains so far unclear, but recent reports suggest that it is involved in the trafficking
of synaptic terminals (Narayanan and Scarlata, 2001) and that its function includes its proper interaction with lipids (Jo et al., 2000). Perrin et al. (2000) have shown that the lipid binding domains are distributed across the N-terminal region of the protein (which corresponds to redidues 1 –102). aSynuclein binding to synthetic phospholipid vesicles is accompanied by a strong increase in a-helicity, an observation consistent with a role in vesicle function at synaptic terminals (Davidson et al., 1998). Sharon et al. identified protein motifs at the N- and C-terminus that were homologous to fatty acid binding proteins. They suggested that asynuclein represents a novel member of this protein family, which might play a role in fatty acid transport between aqueous and phospholipid compartments in neuronal cells (Sharon et al., 2001). Suppression of a-synuclein expression in cultured, primary neurons using antisense oligonucleotides, results in a significant reduction in the distal pool of synaptic vesicles (Murphy et al., 2000), suggesting that regulation of the size of synaptic vesicle pools may be a function of a-synuclein. The ability to bind brain vesicles is influenced by a-synuclein mutations. The familial PD-linked A30P mutant is defective in binding to phospholipid vesicles, whereas the A53T mutation displayed normal membrane-binding activity, comparable to wild-type a-synuclein (Jo et al., 2002). The same issue was reported by Jensen et al., who investigated recombinant A30P and A53T mutants in a vesicle-binding assay. Whereas the A30P mutant was devoid of any vesicle-binding, the A53T mutant bound as well to vesicles as wild-type asynuclein. They suggested that the missing vesicle-binding capability of A30P mutants may disturb axonal transport, resulting in a-synuclein transported only by the slow component b but not by the fast component of axonal transport. (Jensen et al., 1998). This may, in a time-dependent manner, lead to an accumulation of A30P a-synuclein with disruption of the normal localization of the protein. This gives rise to the assembly of Lewy body filaments, consisting of accumulated and aggregated a-synuclein after reaching a critical concentration. Fluorescence resonance energy transfer (FRET) studies showed that the A30P mutation alters the threedimensional conformation of a-synuclein (McLean et al., 2000). a-Synuclein mutations further seem to influence the structural properties of the protein, resulting in modified aggregational behavior. Aggregation of a-synuclein is tightly linked to pathological alterations in many neurodegenerative diseases, like PD or the Lewy body variant (LBV) of AD. The PD-linked mutations A30P and A53T self-aggregate and form more b-sheeted, amyloid-like filaments that the wildtype protein (El-Agnaf et al., 1998). The formation of aggregates is further accelerated by the point mutations (Narhi et al., 1999), which seems to be due to a faster selfassociation of the mutants than the wild-type (Li et al., 2001). a-Synuclein is unstable in its monomeric form and it seems that self-assembly stabilizes partially folded a-synuclein, leading to oligomers, which might evolve into the nucleus of fibrils (Uversky et al., 2001).
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4. Does A-synuclein interact with AB? The interaction of A-synuclein and AB and the contribution of A-synuclein accumulation in AD plaques has been currently a matter of scientific debates. The B-amyloid peptide is the major constituent of both neuritic plaques and vascular deposits in the walls of cerebral and meningeal arterioles and arteries. These amyloid deposits are mainly composed of aggregates of the AB peptide with 40 –42 residues, which is proteolytically derived from the larger Bamyloid precursor protein (APP). A-Synuclein was initially purified from the SDS-insoluble fraction of the frontal cortex of AD patients. Immunohistochemical analysis of AD brain sections with antibodies against the identified peptide revealed immunostaining of amyloid in diffuse, primitive and mature plaques (Ueda et al., 1993). This observation was corroborated by other studies, which reported NAC immunoreactivity in amyloid plaques, showing more abundant NAC immunoreactivity in the amyloid core than in the periphery (Masliah et al., 1996). Recent studies, however, using different antibodies covering the major portions of the protein, addressed the question whether there is an association between A-synuclein and amyloid plaques. We and others confirmed only the synaptic and Lewy body staining and concluded that A-synuclein is not associated with mature amyloid plaque cores in AD (Bayer et al., 1999; Culvenor et al., 1999). Furthermore, we observed that AD cases with mixed pathology of the LBV revealed accumulation of A-synuclein in Lewy bodies and
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dystrophic neurites decorating AB plaques (Fig. 1) (Wirths et al., 2000). This indicates that A-synuclein accumulation is restricted to dystrophic neurites, and therefore to intracellular compartments. The extracellular amyloid plaque cores are free of A-synuclein accumulation. One possible explanation for these conflicting results could be that the NAC antibody used by Ueda et al. showed cross-reactivity with the AB sequence. Almost the same phenotype seen in AD patients is found in APP transgenic mice, which represent a model system for the investigation of AD-related pathological alterations. Heterozygous Tg2576 mice, which express human APP with the ’’Swedish’’ double mutation (Hsiao et al., 1996), show frequent ubiquitin- and Asynuclein-positive neurites, resembling some aspects of the neuropathological events in cases with the LBV of AD (Yang et al., 2000). In these animals, staining with Asynuclein antibodies reveals a punctate protein distribution in the neuropil, but was clearly absent in the amyloid core. Dystrophic neurites in and around the plaque cores were labeled, but no Lewy or other inclusion bodies were detected in the transgenic mouse brains (Tomidokoro et al., 2001). In the last few years, a couple of human Asynuclein transgenic mice have also been developed, showing various degrees of pathology (Masliah et al., 2000; van der Putten et al., 2000; Giasson et al., 2002; Lee et al., 2002). In a very recent report, Lee et al. generated multiple lines of transgenic mice expressing wild-type A-synuclein, as well as the A53T and the A30P mutants. Interestingly, only the mice with the A53T mutant developed adult-onset
Fig. 1. (A) Ab immunostaining of a typical Alzheimer’s disease case. (B) Parallel section of the same patient stained with an antibody against a-synuclein. (C) a-Synuclein immunostaining of a case with the Lewy body variant of AD. Arrows point to a-synuclein aggregations in dystrophic neurites in the vicinity of an amyloid plaque. (D) a-Synuclein immunoreactive dystrophic neurites stained by an a-synuclein antibody in the cortex of an APP/PS-1 double transgenic mouse. (Scale bars: A: 100 mm; C, D: 20 mm.)
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neurodegenerative defects with progressive motoric dysfunction (Lee et al., 2002). Masliah et al. (2000) showed that wild-type A-synuclein transgenic mice under the control of the PDGF-B promoter harbored prominent A-synuclein-immunoreactive intraneuronal inclusions by 2 months of age, besides loss of dopaminergic neurons and motor impairments. Crossing these mice with human APP transgenic mice revealed mice with enhanced pathology. They show severe learning, memory and motor deficits, as well as prominent age-dependent degeneration of cholinergic neurons and presynaptic terminals. A-Synuclein-immunoreactive inclusions were further more numerous than in Asynuclein single transgenic mice (Masliah et al., 2001). Several studies addressed the question whether AB and synuclein interact in vitro. It was shown that the NAC precursor is able to bind to AB1-38 and AB25-35 immobilized on nitrocellulose membranes (Yoshimoto et al., 1995). Jensen et al. (1997) identified a further AB-binding segment on the A-synuclein protein (57 –97) and showed that B-synuclein is also able to bind to and promote aggregation of AB.
examination revealed significant A-synuclein aggregation in dystrophic neurites decorating AB plaques, as well as marked intraneuronal aggregation in Lewy neurites and Lewy bodies in patients with mixed AD and Lewy body pathology (Wirths et al., 2000). Szpak et al. (2001) also reported recently coexistence of brain stem and cortical Lewy bodies with pathological features of AD in a significant part of dementia cases. Besides Lewy bodies other dense A-synuclein-positive inclusion bodies in pyramidal cells of the medial temporal lobe were reported in AD and the LBV of AD. Lewy bodies were characterized by strong ubiquitin immunoreactivity, a larger size and homogenous texture, whereas the A-synuclein inclusions are smaller and not ubiquitin-immunoreactive (Mukaetova-Ladinska et al., 2000). These inclusions may represent initial aggregates of A-synuclein. A-Synuclein-positive inclusions that differed from Lewy bodies were also reported at high rates in the hippocampus of AD patients (Arai et al., 2001). Staining with a C-teminal Asynuclein antibody reveals immunoreactivity of neurofibrillary tangles in AD and Lewy body disease cases after pretreatment with formic acid (Takeda et al., 2000).
5. Lewy body pathology in AD Different groups reported A-synuclein-immunoreactive Lewy bodies in AD patients. Lippa et al. investigated whether A-synuclein pathology plays a significant role in familial AD cases using A-synuclein immunohistochemistry. The authors detected A-synuclein-immunoreactive Lewy bodies in 22% of the investigated AD brains, most numerous in the amygdala, where they in part co-localized with T-positive neurofibrillary tangles. Over 60% of cases with amygdala samples available had A-synuclein-immunoreactive Lewy bodies (Lippa et al., 1998). A recent report addresses the same question in a large cohort of sporadic AD patients and shows that more than 60% of the cases harbored A-synuclein positive Lewy bodies. Moreover, in all of these cases, the amygdala was involved with numerous Lewy bodies in this particular area, suggesting that the amygdala is the most commonly affected brain region (Hamilton, 2000). Analysis of Down’s syndrome patient samples revealed that more than 50% of the investigated cases displayed A-synuclein-positive Lewy bodies and dystrophic neurites in the amygdala, adding further evidence to the fact that this brain region is highly susceptible for these pathological alterations (Lippa et al., 1999). It is now well established that the mixed pathology, consisting of Lewy bodies and AB plaques, represents a distinct subtype of AD, which is referred to as LBV of AD (Hansen et al., 1990; Forstl et al., 1993; Petersen, 1998). In a study that focused on the differences in the pattern of progression between AD and AD with Lewy bodies, Lopez et al. reported a faster progression of extrapyramidal signs in patients with AD and Lewy bodies. The rate of cognitive decline however was not different between pure AD patients and patients with the LBV (Lopez et al., 2000). Histological
6. Conclusion In cases with the LBV of AD, overlapping pathological alterations regarding A-synuclein and AB are obvious. Trafficking of synaptic terminals and lipid interaction seems to be important functions of A-synuclein. In pure AD cases, impaired trafficking and resulting aberrant processing of APP are the key events that may lead to the pathological deposition of AB peptides. Therefore, changes in the lipid composition of membranes and altered membrane fluidity during aging might have a significant impact on transport of both proteins. Several recent studies suggested brain cholesterol alterations as an important event in the progression of AD (Frears et al., 1999; Runz et al., 2002). Furthermore, interactions of AB with anionic phospholipids were reported (Chauhan et al., 2000). APP, as well as A-synuclein, becomes axonally transported (Jensen et al., 1999; Kamal et al., 2000). A direct interaction of A-synuclein and APP, however, seems unlikely and there is no evidence that APP and A-synuclein become transported by the same vesicles. However, it has been shown that A-synuclein and AB can interact in vitro (Jensen et al., 1997). Further studies on membrane interactions and the impact of changes in membrane composition during aging will help to elucidate the connection between both proteins in neurodegeneration.
Acknowledgements With kind support from the Fritz Thyssen Foundation, the Alzheimer Forschung Initiative (to TAB) and the European Community (Quality of Life and Management
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of Living Resources, QLK6-CT-2000-60042, QLK6-GH00-60042-02 to OW).
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