Alpha-synuclein and presynaptic function

  • Published on

  • View

  • Download

Embed Size (px)


<ul><li><p>NeuroMolecular Medicine 115 Volume 2, 2002</p><p>Alpha-Synuclein and Presynaptic Function</p><p>Implications for Parkinsons Disease</p><p>Simon Lykkebo and Poul Henning Jensen*</p><p>Department of Medical Biochemistry, Aarhus University, DK-8000 Aarhus-C, Denmark</p><p>Received April 17, 2002; Accepted May 16, 2002</p><p>Abstract</p><p>This article focuses on -synucleins role in normal and pathological axonal and presynap-tic functions and its relationship to Parkinsons disease. It is not possible to mention all the con-tributions to aspects of this area. Readers interested in -synucleins relation to aggregation,Lewy lesions, and pathological modifications are referred to the many reviews (see Goldbergand Lansbury 2000; Galvin 2001a; Goedert 2001).</p><p>Index Entries: synuclein; synapse; axon; vesicles; Parkinsons disease; Lewy body.</p><p>NeuroMolecular MedicineCopyright 2002 Humana Press Inc.All rights of any nature whatsoever reserved.ISSN1535-1084/02/02:115129/$20.00</p><p>*Author to whom all correspondence and reprint requests should be addressed. E-mail:</p><p>-Synuclein in Parkinsons Disease</p><p>The small presynaptic protein -synuclein hasreceived great attention since missense mutations inits gene were associated with heritable autosomaldominant early-onset Parkinsons disease in 1997-98(Polymeropoulos, 1997; Krger, 1998). It is a memberof the synuclein gene family, wherein the -, -, and-synucleins, encoded by genes on chromosomes4q21, 5q35 and 10q23, (Campion, 1995; Spillantini,1995; Lavedan, 1998), are predominant in humans(Fig. 1). Synucleins were first identified based on theirpresynaptic nature by expression cloning using asynaptic-vesicle-specific antibody on a vector libraryof mRNA from the electromotor nucleus of the rayTorpedo California (Maroteaux, 1988). Homologous</p><p>mRNAs and proteins have been identified in birdsand mammals, thus demonstrating the highly con-served structure between fish and mammals(Maroteaux, 1988) and birds (George, 1995) (Fig. 1).The initial report coined the term synuclein becauseof its localization to the presynaptic terminus and thenuclear envelope. The latter localization was latershown not to be normal for synucleins.</p><p>Parkinsons disease is the second most commonneurodegenerative diseasethe first is Alzheimersdisease (AD). It is primarily a movement disorder,and is characterized by rigidity, tremor, and bradykinesia, but a wider effect on the nervous system isfrequently noticed, as evidenced by autonomic andcognitive dysfunctions. The definitive diagnosisrelies on the neuropathological demonstration of</p></li><li><p>116 Lykkebo and Jensen</p><p>NeuroMolecular Medicine Volume 2, 2002</p><p>nerve-cell loss in the pars compacta of the substan-tia nigra in the brainstem, accompanied by the pres-ence of proteinaceous inclusions, Lewy bodies, andLewy neurites. Similar neurodegenerative changesare often found in other parts of the braine.g., thenucleus basalis of Meynert, hypothalamus, cingu-lated gyrus, entorhinal cortex, sympathetic ganglia,and peripheral parasympathetic neurons, wherethey may explain the autonomic and cognitivesymptomatology. For a recent review on clinical andpathophysiological aspects of Parkinsons disease,see (Lang, 1998). The identification of two missense</p><p>mutations in the -synuclein gene on chromo-some 4q21 causing Parkinsons disease representedthe first demonstration of a single gene as causativefor the disease. Both the mutant Ala53Thr in thelarge Contoursi kindred (Polymeropoulos, 1997)and the Ala30Pro mutations in a German family(Krger, 1998) cause an early-onset Parkinsons dis-ease with a dominant heritability of high penetrancethat is indicative of a gain-of-toxic-function by themutant peptide. Mutations in the -synuclein genehave subsequently turned out to be exceedingly rare. The identification of the mutations in the </p><p>Fig. 1. Sequence comparison among synucleins. (A) Alignment of human -synuclein, -synuclein and -synuclein protein sequences. Gray bars indicate the six KTKEGV consensus repeats in the -synuclein sequence.Note the high degree of sequence identity within the N-terminal 62 residues of the gene products as compared totheir divergent C-termini. (B) Alignment of -synuclein protein sequences from human, rat, bird, and fish synu-clein. Note the high degree of conservation of the -synuclein protein in bird and mammals, and even with theunrelated fish synuclein. Dots indicate amino acids identical with the human -synuclein sequence. Gaps areindicated with a wavy line (~). Alignments were performed using the MegAlign software from DNAstar Inc. Acces-sion numbers used for the alignments are: human - (P37840), human - (Q16143), human - (AAC27738), rat - (P37377), bird - (AAA93538), and fish synuclein (A60887).</p></li><li><p>Alpha-Synuclein and Presynaptic Function 117</p><p>NeuroMolecular Medicine Volume 2, 2002</p><p>-synuclein gene prompted the immunohistochem-ical examination of brain tissue affected by Parkin-sons disease, and this showed that -synuclein inthe disease is accumulated in the Lewy bodies(Spillantini, 1997). More neurodegenerative diseasesare associated with the development of Lewy bodiesor similar inclusions in which -synuclein is a prominent component, dementia with Lewy bodies,Lewy body variant of AD, motor neuron disease,Hallervorden-Spatz disease (for recent reviews seeDickson, 2001; Goedert, 2001) and multiple systematrophy, where the inclusions occur in the astrocytes(Gai, 1998). Accordingly, these diseases are collec-tively designated as -synucleinopathies. Lewybodies were discovered by Frederic Lewy (Lewy,1912), and were for decades identified histochemi-cally by their affinity for the dye eosin. The sensi-tivity of their detection increased significantly withthe development of immunohistochemistry, forwhich ubiquitin antibodies have represented thestandard for their detection until recently. However,anti--synuclein antibodies have increased the sensitivity even further by demonstrating the exis-tence of subsets of ubiquitin-negative Lewy bodies(McKeith, 1999). Lewy bodies and Lewy neuritescontain large amounts of protein filaments (Forno,1987), and thus resemble the neuropathological</p><p>lesions in other late-onset neurodegenerative dis-eases, such as neuritic tangles in Alzheimers dis-ease and intranuclear inclusions in Huntingtonsdisease. The filaments in Lewy bodies and glialcytoplasmic inclusions contain highly insoluble -synuclein, which is partly modified by proteoly-sis, phosphorylation, and oxidations (Baba, 1998; Gai,1999; Giasson, 2000; Jensen, 2000; Fujiwara, 2002),and they resemble the filaments that can be formedin vitro from recombinant human -synuclein basedon immuno-electronmicroscopical criteria (Conway,1998; Crowther, 1998; Giasson, 1999). This suggeststhat-synuclein is the building block for the core fil-aments that may possess an additional coat of asso-ciated auxiliary cytoplasmic proteins. Structuralanalysis of in vitro-formed filaments have demon-strated that they possess a large content of -foldedstructure and exhibit characteristics of classical amyloid filaments such as the amyloid peptide(A)-filaments in Alzheimers disease (Hashimoto,1998; Conway, 2000a). The accumulation of -synuclein in the Lewy bodies and neurites repre-sents a dramatic relocalization of the protein from itsnormal presynaptic localization (Fig. 2). A less pro-nounced abnormal accumulation of -synuclein and-synuclein in non-Lewy-body structures has alsobeen demonstrated in Parkinsons disease, but no fil-</p><p>Fig. 2. Topological and structural changes in neuronal -synuclein in Parkinsons disease. -synuclein is nor-mally a presynaptic protein. It is synthesized in the cell body and targeted to the nerve terminal by axonal trans-port. A dramatic relocalization from the nerve terminal to the cell body and neurites occurs in the degeneratingneurons during the course of Parkinsons disease. A structural change takes place concomittant with this relocal-ization as -folded amyloid-type -synuclein filaments accumulates in the Lewy bodies and in the neurites. Colorimage available for viewing at</p></li><li><p>118 Lykkebo and Jensen</p><p>NeuroMolecular Medicine Volume 2, 2002</p><p>aments have been detected for these synucleins(Galvin, 1999). Concomitant with the -synucleinrelocalization occurs the above-mentioned structuraltransition of some monomeric unfolded -synucleininto -folded filaments. A biochemical correlate tothe filamentous transition is a decreased solubilityof-synuclein in isolated filaments and in brain tissuecontaining numerous -synuclein-positive inclu-sions (Dickson 1999; Gai 1999; Jensen 2000). A sim-ilar decreased solubility has been demonstrated intransgenic mice expressing human -synuclein anddisplaying pathological symptoms (Kahle, 2001).</p><p>The aggregation of recombinant human -synuclein in vitro represents a nucleation-dependent aggregation process in which filamentformation is preceded by a lag phase where fila-mentogene oligomers build up (Conway, 1998;Wood, 1999). The oligomers represents an ill-definedstructural entity whose existence has been demon-strated biochemically and by atomic forcemicroscopy (Conway, 2000a, 2000b; Rochet, 2000;Volles, 2001). However, the oligomers ability to seedfilament growth emphasizes the importance of fac-tors that increase their concentration. Such factorsmay be subject to dynamic regulation in vivo andcomprise disease-causing mutations, C-terminaltruncation (Crowther, 1998, Jensen, 2000), oxidativemodifications (Hashimoto, 1999; Paik, 2000; Souza,2000), phosphorylations (Fujiwara, 2002), calciumbinding (Nielsen, 2001), and possibly certain -synuclein binding proteins such as synphilin(Engelender, 1999).</p><p>The direct link between -synuclein alterationsand the development of Parkinsons diseaseestablished by the pathogenic mutations in the -synuclein gene, and the demonstration of anabnormal -synuclein aggregation in all cases withLewy bodies, including Parkinsons diseasehavehad a tremendous impact by allowing directed molecular investigations of disease mechanisms.</p><p>Expression</p><p>The synucleins are abundant proteins that rep-resent about 0.1% of the total protein in the brain(Shibayama-Imazu, 1993). They are consideredneuron-specific proteins because their levels in theperipheral and central nervous system (CNS) arefar higher than in other tissues (Jakes, 1994; Ueda,1994; Lavedan, 1998), although significant expres-</p><p>sion does exist in other tissues. The synucleins are small acidic and cytosolic proteins of about125140 amino acids with -synuclein comprisedof 140 residues. The synuclein signature was imme-diately noticed after the first cloning as the pres-ence of repeats of 11 amino acids with the coreconsensus sequence KTKEGV (Maroteaux, 1988;Fig. 1B). These repeats extend over the N-terminalabout two-thirds of the peptide, and this region ishighly conserved between the different synucleinsand between species. The C-terminal one-third ishighly negatively charged and displays the mostvariable segment between the synucleins and con-trast, therefore, to the positively charged repeatregion (Fig. 1A).</p><p>The developmental expression of -synucleinbegins at an early stage in mammals (Hsu, 1998)and has been detected from gestational wk 15 and16 in the human substantia nigra and cortex (Bayer,1999; Galvin, 2001b). The level slowly rises duringthe gestational period, and increases more rapidlyduring the end of pregnancy and in the early post-partum period (Hsu, 1998; Petersen, 1999). The -synuclein is initially localized in the cell body andproximal neurites, and the mature presynaptic local-ization in the neuropil is first acquired by about ges-tational wk 18 (Galvin, 2001b). Interestingly, theclose homologous -synuclein and -synucleinare first expressed in the substantia nigra at aboutthe time when -synuclein has acquired its synaptic localization (Galvin, 2001b). Moreover,they first reach their mature presynaptic localiza-tion after being present for several weeks in the cell body (Galvin, 2001b). This indicates either anasynchronous maturation of their respective trans-port mechanisms or expression in different neu-ronal populations. Coexpression of -synuclein and-synuclein within the same synaptic terminal hasbeen demonstrated in vivo (Fig. 3) and in culturedneurons (Murphy 2000). The synaptic targeting of-synuclein displays a pattern very similar to synap-tophysin (Hsu, 1998; Galvin, 2001b), indicating aparallel maturation of the axonal transport mecha-nisms for the two proteins in vivo as compared tothe later maturation for synaptotagmin and synap-tobrevin (Galvin, 2001b). A discrepancy existsbetween the previously mentioned in vivo data andin vitro neuronal cell-culture experimentsthelatter demonstrate an earlier expression of andsynaptic translocation of synaptophysin andsynapsin-1 as compared to -synuclein (Withers,</p></li><li><p>Alpha-Synuclein and Presynaptic Function 119</p><p>NeuroMolecular Medicine Volume 2, 2002</p><p>1997; Murphy, 2000). This probably reflects the non-physiological differentiation and polarization of cultured neurons.</p><p>Catabolism</p><p>The-synucleinopathies are characterized by theaccumulation of -synuclein, some of which dis-plays a decreased solubility. This suggests thatabnormal catabolism of -synuclein could play apathogenic role. -synuclein is a stable protein withT-1/2 &gt; 24 h in transfected mitotic cell lines (Ben-nett, 1999; Okochi, 2000). -synuclein accumulatesin cell lines treated with proteasome inhibitors,although no ubiquitination of native -synucleinhas been demonstrated (Bennett, 1999; Ancolio, 2000;Tofaris, 2001). However, unmodified -synucleincan be directly degraded by the 20S proteasome in vitro (Tofaris, 2001), indicating the existence of anunconventional proteasomal catabolism of thesmall-unfolded -synuclein peptide. The signifi-cance of this observation awaits corroborative stud-</p><p>ies in non-mitotic neurons. The subcellular sitesresponsible for the -synuclein degradation in neu-rons are unsolved, but axons and nerve terminalsare likely to represent major sites as the anterogradetransport exceeds the retrograde transport (Jensen,1999b). Proteasomal degradation at these sites hasbeen demonstrated to play an important role inaxonal organization (Campbell, 2001). Interactionsbetween the ubiquitin proteasome system and apparently unmodified -synuclein have beenreported by the coimmunoprecipitation of the Park-insons disease-related ubiquitin ligase parkin and-synuclein (Choi, 2001; Jensen, unpublished obser-vation). A prerequisite for a specific -synucleinmodificationan O-glycosylationin supportingthe binding and subsequent -synuclein ubiquitina-tion, has been reported in a study of human braintissue (Shimura, 2001). This -synuclein modifica-tion has not yet been confirmed by other groups, but O-glycosylated -synuclein was demonstrated in rat nerve terminals (Cole, 2001). The regulation of -synuclein catabolism is not a trivial matter, considering that the accumulation of misfolded </p><p>Fig. 3. -synuclein and -synuclein colocalize in some nerve terminals. Rat hippocampal sections were labeledfor...</p></li></ul>