New Approaches to the Pathology and Genetics of Neurodegeneration
2010; Elsevier BV; Volume: 176; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2010.091077
ISSN1525-2191
Autores Tópico(s)Alzheimer's disease research and treatments
ResumoThe pathologies of major neurodegenerative diseases including Parkinson disease and Alzheimer disease have been well known for decades. More recently, advances in molecular genetics have suggested important mechanistic links between the pathology of these disorders and pathogenesis of neuronal dysfunction and death. Numerous animal models have been produced based on the new information emerging from human genetic studies. As a complement to traditional mouse models, a number of investigators have modeled neurodegenerative diseases in simple model organisms ranging from yeast to Drosophila. These simple genetic models often display remarkable pathological similarities to their cognate human disorders, and genetic and biochemical studies have yielded important insights into the pathogenesis of the human disorders. Use of these tractable simple models may become even more important as large amounts of genetic data emerge from genome-wide association studies in Alzheimer disease, Parkinson disease, and other neurodegenerative disorders. The pathologies of major neurodegenerative diseases including Parkinson disease and Alzheimer disease have been well known for decades. More recently, advances in molecular genetics have suggested important mechanistic links between the pathology of these disorders and pathogenesis of neuronal dysfunction and death. Numerous animal models have been produced based on the new information emerging from human genetic studies. As a complement to traditional mouse models, a number of investigators have modeled neurodegenerative diseases in simple model organisms ranging from yeast to Drosophila. These simple genetic models often display remarkable pathological similarities to their cognate human disorders, and genetic and biochemical studies have yielded important insights into the pathogenesis of the human disorders. Use of these tractable simple models may become even more important as large amounts of genetic data emerge from genome-wide association studies in Alzheimer disease, Parkinson disease, and other neurodegenerative disorders. Degenerative diseases of the nervous system are common, devastating to patients and families, and essentially untreatable. Alzheimer disease is the most prevalent neurodegenerative disorder and affects about 10% of people over the age of 70.1Plassman BL Langa KM Fisher GG Heeringa SG Weir DR Ofstedal MB Burke JR Hurd MD Potter GG Rodgers WL Steffens DC Willis RJ Wallace RB Prevalence of dementia in the United States: the aging, demographics, and memory study.Neuroepidemiology. 2007; 29: 125-132Crossref PubMed Scopus (1368) Google Scholar Parkinson disease is next in frequency, with a prevalence of approximately 2% of individuals over the age of 70.2Moghal S Rajput AH Meleth R D'Arcy C Rajput R Prevalence of movement disorders in institutionalized elderly.Neuroepidemiology. 1994; 14: 297-300Crossref Scopus (31) Google Scholar, 3Harris MK Shneyder N Borazanci A Korniychuk E Kelley RE Minagar A Movement disorders.Med Clin North Am. 2009; 93: 371-388Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar Advancing age is the most important risk factor for Alzheimer disease, Parkinson disease, and related neurodegenerative disorders. Because the population of the United States is aging, neurodegenerative diseases will represent an increasing burden on individuals, families, and the health care system in the coming years. Fortunately, the advent of molecular genetics has facilitated important insights into the pathogenesis of neurodegenerative diseases, with critical progress first emerging in rare neurodegenerative disorders. Localization of the gene for Huntington disease to the short arm of chromosome 4 by James Gusella and coworkers using anonymous DNA markers was an early landmark application of molecular genetic technology to the study of neurodegenerative disease.4Gusella JF Wexler NS Conneally PM Naylor SL Anderson MA Tanzi RE Watkins PC Ottina K Wallace MR Sakaguchi AY Young AB Shoulson I Bonilla E Martin JB A polymorphic DNA marker genetically linked to Huntington disease.Nature. 1983; 306: 234-238Crossref PubMed Scopus (1654) Google Scholar Ten years later the same group reported the molecular cloning of the locus, revealing that huntingtin is a very large (>350-kDa) novel protein of unknown function.5The Huntington Disease Collaborative Research GroupA novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington disease chromosomes.Cell. 1993; 72: 971-983Abstract Full Text PDF PubMed Scopus (7050) Google Scholar Cloning of the Huntington disease gene provided significant insight into the pathogenesis of the disorder: CAG trinucleotide expansion occurs within the gene. When an allele has expanded sufficiently (>34 units), neurodegeneration ensues. Understanding the fundamental genetic basis of Huntington disease provided the knowledge and tools needed to create models of the disorder. Two approaches were taken initially. First, the mouse homolog of the Huntington disease gene was inactivated. Mice completely lacking gene function or having reduced levels of huntingtin die in early embryogeneis.6Nasir J Floresco JB O'Kusky JR Diewert VM Richman JM Zeisler J Borowski A Marth JD Phillips AG Hayden MR Targeted disruption of the Huntington disease gene results in embryonic lethality and behavioral and morphological changes in heterozygotes.Cell. 1995; 81: 811-823Abstract Full Text PDF PubMed Scopus (674) Google Scholar, 7Duyao MP Auerbach AB Ryan A Persichetti F Barnes GT McNeil SM Ge P Vonsattel JP Gusella JF Joyner AL Macdonald ME Homozygous inactivation of the mouse Hdh gene does not produce a Huntington disease-like phenotype.Science. 1995; 269: 407-410Crossref PubMed Scopus (571) Google Scholar Although these findings leave open the possibility that loss of Huntington disease gene function contributes to some aspect of the disorder,8Leavitt BR Guttman JA Hodgson JG Kimel GH Singaraja R Vogl AW Hayden MR Wild-type huntingtin reduces the cellular toxicity of mutant huntingtin in vivo.Am J Hum Genet. 2001; 68: 313-324Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 9Zhang S Feany MB Saraswati S Littleton JT Perrimon N Inactivation of Drosophila Huntingtin affects long-term adult functioning and the pathogenesis of a Huntington disease model.Dis Model Mech. 2009; 2: 247-266Crossref PubMed Scopus (62) Google Scholar simply reducing gene function does not provide a good model for the disorder. In contrast, mice that overexpress mutant polyglutamine-expanded (the nucleotide triplet CAG encodes glutamine) forms of huntingtin have progressive neurological phenotypes, and in some models, neuronal cell loss and early death.10Heng MY Detloff PJ Albin RL Rodent genetic models of Huntington disease.Neurobiol Dis. 2008; 32: 1-9Crossref PubMed Scopus (129) Google Scholar Murine models of Huntington disease based on expression of mutant forms of human or mouse huntingtin have been used extensively to test hypotheses regarding the pathogenesis of the disorder and to assess candidate therapeutics. Despite the undoubted utility of vertebrate animal models like Huntington disease transgenic mice, certain experimental approaches, particularly genetic ones, are limited by the time and expense of breeding and maintaining mice. In contrast, simple invertebrate models can be used for genome-wide forward genetic analyses, drug screens, and other higher throughput approaches. Thus, once the principles of polyglutamine pathogenesis had been established using human molecular genetics and mouse modeling, invertebrate models were created using a similar approach. Transgenic flies, worms, and yeast were created that expressed mutant polyglutamine-expanded versions of huntingtin.11Jackson GR Salecker I Dong X Yao X Arnheim N Faber PW MacDonald ME Zipursky SL Polyglutamine-expanded human huntingtin transgenes induce degeneration of Drosophila photoreceptor neurons.Neuron. 1998; 21: 633-642Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar, 12Faber PW Alter JR MacDonald ME Hart AC Polyglutamine-mediated dysfunction and apoptotic death of a Caenorhabditis elegans sensory neuron.Proc Natl Acad Sci USA. 1999; 96: 179-184Crossref PubMed Scopus (255) Google Scholar, 13Muchowski PJ Ning K D'Souza-Schorey C Fields S Requirement of an intact microtubule cytoskeleton for aggregation and inclusion body formation by a mutant huntingtin fragment.Proc Natl Acad Sci USA. 2002; 99: 727-732Crossref PubMed Scopus (116) Google Scholar These models all replicated substantial toxicity of mutant huntingtin. In addition, invertebrate models proved an excellent system in which to study another feature of Huntington disease: abnormal protein aggregation. Although the presence of intranuclear inclusions had been described in Huntington disease by electron microscopy previously,14Roizin L Stellar S Willson N Whittier J Liu JC Electron microscope and enzyme studies in cerebral biopsies of Huntington chorea.Trans Am Neurol Assoc. 1974; 99: 240-243PubMed Google Scholar identification of huntingtin and the subsequent development of antibodies recognizing the protein led to the description of significant intranuclear and neuritic aggregation of mutant huntingtin in the brains of affected patients (Figure 1A).15DiFiglia M Sapp E Chase KO Davies SW Bates GP Vonsattel JP Aronin N Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain.Science. 1997; 277: 1990-1993Crossref PubMed Scopus (2312) Google Scholar Animal models of the disorder, including simple genetic models, recapitulate the abnormal aggregation of mutant huntingtin (Figure 1, B and C). We now know that Huntington disease is only one example of a group of disorders caused by expansion of a CAG repeat that encodes a polyglutamine stretch within the host protein. Other polyglutamine disorders include spinal bulbar muscular atrophy (SBMA), dentatorubropallidoluysian atrophy (DRPLA), and six types of spinocerebellar ataxia (SCA1, 2, 3, 6, 7, and 17). These are all Mendelian genetic disorders with dominant inheritance, and are all thought to be caused by a dominant gain of function mechanism reflecting toxicity, perhaps through abnormal aggregation, of the expanded polyglutamine protein sequences.16Bauer PO Nukina N The pathogenic mechanisms of polyglutamine diseases and current therapeutic strategies.J Neurochem. 2009; 110: 1737-1765Crossref PubMed Scopus (146) Google Scholar A number of comprehensive genetic and chemical screens have been carried in simple genetic models of Huntington disease and related polyglutamine expansion disorders. Many of these screens have provided strong evidence for a causative role for protein aggregation in the disorders,17Fernandez-Funez P Nino-Rosales ML de Gouyon B She WC Luchak JM Martinez P Turiegano E Benito J Capovilla M Skinner PJ McCall A Canal I Orr HT Zoghbi HY Botas J Identification of genes that modify ataxin-1-induced neurodegeneration.Nature. 2000; 408: 101-106Crossref PubMed Scopus (547) Google Scholar, 18Zhang X Smith DL Meriin AB Engemann S Russel DE Roark M Washington SL Maxwell MM Marsh JL Thompson LM Wanker EE Young AB Housman DE Bates GP Sherman MY Kazantsev AG A potent small molecule inhibits polyglutamine aggregation in Huntington disease neurons and suppresses neurodegeneration in vivo.Proc Natl Acad Sci USA. 2005; 102: 892-897Crossref PubMed Scopus (232) Google Scholar, 19Bilen J Bonini NM Genome-wide screen for modifiers of ataxin-3 neurodegeneration in Drosophila.PLoS Genet. 2007; 3: 1950-1964Crossref PubMed Scopus (165) Google Scholar although the precise species of toxic aggregate remains undefined. In addition, novel therapeutic pathways have emerged from genetic studies in model organisms. Pioneering work from Leslie Thompson's group demonstrated a role for mutant huntingtin in control of histone acetylation and further showed that inhibiting histone deacetylases provided therapeutic benefit to flies expressing mutant huntingtin.20Steffan JS Bodai L Pallos J Poelman M McCampbell A Apostol BL Kazantsev A Schmidt E Zhu YZ Greenwald M Kurokawa R Housman DE Jackson GR Marsh JL Thompson LM Histone deacetylase inhibitors arrest polyglutamine-dependent neurodegeneration in Drosophila.Nature. 2001; 413: 739-743Crossref PubMed Scopus (1045) Google Scholar These findings have been replicated in vertebrate models of polyglutamine toxicity, and histone deacetylase inhibitors are promising therapeutic compounds in the human diseases.21Chuang DM Leng Y Marinova Z Kim HJ Chiu CT Multiple roles of HDAC inhibition in neurodegenerative conditions.Trends Neurosci. 2009; 32: 591-601Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar Although the ability to model the toxicity and abnormal aggregation of polyglutamine disorders successfully in simple genetic model organisms suggested some utility for the approach, it was not initially clear that similar success would obtain for more common neurodegenerative diseases. Most cases of Alzheimer disease and Parkinson disease do not have an obvious genetic basis. However, like the polyglutamine disorders, both Alzheimer disease and Parkinson disease are characterized by the presence of abnormal protein aggregates in affected brain tissue: amyloid plaques, neurofibrillary tangles, and Hirano bodies in Alzheimer disease, and Lewy bodies and Lewy neurites in Parkinson disease. Significant encouragement was given to the idea of creating simple genetic models of Parkinson disease in 1997 when genetic cloning of the first Parkinson disease gene was reported.22Polymeropoulos MH Lavedan C Leroy E Ide SE Dehejia A Dutra A Pike B Root H Rubenstein J Boyer R Stenroos ES Chandrasekharappa S Athanassiadou A Papapetropoulos T Johnson WH Lazzarini AM Duvoisin RC Di Iorio G Golbe LI Nussbaum RL Mutation in the α-synuclein gene identified in families with Parkinson disease.Science. 1997; 276: 2045-2047Crossref PubMed Scopus (6600) Google Scholar, 23Krüger R Kuhn W Müller T Woitalla D Graeber M Kösel S Przuntek H Epplen JT Schöls L Riess O Ala30Pro mutation in the gene encoding α-synuclein in Parkinson disease.Nat Genet. 1998; 18: 106-108Crossref PubMed Scopus (3297) Google Scholar The autosomal dominant PARK1 locus encodes α-synuclein, an abundant neuronal protein of unknown function. So far, three missense mutations linked to familial Parkinson disease have been identified in α-synuclein: A53T, A30P, and E46K.24Zarranz JJ Alegre J Gomez-Esteban JC Lezcano E Ros R Ampuero I Vidal L Hoenicka J Rodriguez O Atares B Llorens V Gomez Tortosa E del Ser T Munoz DG de Yebenes JG The new mutation. E46K, of alpha-synuclein causes Parkinson and Lewy body dementia.Ann Neurol. 2004; 55: 164-173Crossref PubMed Scopus (2145) Google Scholar Missense mutations in α-synuclein are a very rare cause of Parkinson disease; however, the identification of α-synuclein mutations has been remarkably informative. Description of mutations in the α-synuclein gene quickly led to the discovery that α-synuclein is a major protein component of Lewy bodies and Lewy neurites (Figure 1D).25Spillantini MG Schmidt ML Lee VM Trojanowski JQ Jakes R Goedert M Alpha-synuclein in Lewy bodies.Nature. 1997; 388: 839-840Crossref PubMed Scopus (6110) Google Scholar, 26Baba M Nakajo S Tu PH Tomita T Nakaya K Lee VM Trojanowski JQ Iwatsubo T Aggregation of alpha-synuclein in Lewy bodies of sporadic Parkinson disease and dementia with Lewy bodies.Am J Pathol. 1998; 152: 879-884PubMed Google Scholar Because these inclusion bodies are present not only in the rare patient carrying mutations in the α-synuclein gene, but in sporadic Parkinson disease as well, substantial support was given to the hypothesis that α-synuclein may play a critical role in both genetic and sporadic forms of the disorder. Taking a cue from the work on Huntington disease and related polyglutamine disorders, a number of investigators overexpressed normal and mutant versions of human α-synuclein in experimental animals to model Parkinson disease. In addition to the Drosophila model developed in our laboratory and discussed in detail below, expression of human α-synuclein in yeast,27Outeiro TF Lindquist S Yeast cells provide insight into alpha-synuclein biology and pathobiology.Science. 2003; 302: 1772-1775Crossref PubMed Scopus (615) Google Scholar, 28Willingham S Outeiro TF DeVit MJ Lindquist SL Muchowski PJ Yeast genes that enhance the toxicity of a mutant huntingtin fragment or alpha-synuclein.Science. 2003; 302: 1769-1772Crossref PubMed Scopus (346) Google ScholarC. elegans,29Lakso M Vartiainen S Moilanen AM Sirviö J Thomas JH Nass R Blakely RD Wong G Dopaminergic neuronal loss and motor deficits in Caenorhabditis elegans overexpressing human alpha-synuclein.J Neurochem. 2003; 86: 165-172Crossref PubMed Scopus (300) Google Scholar mice,30Masliah E Rockenstein E Veinbergs I Mallory M Hashimoto M Takeda A Sagara Y Sisk A Mucke L Dopaminergic loss and inclusion body formation in alpha-synuclein mice: implications for neurodegenerative disorders.Science. 2000; 287: 1265-1269Crossref PubMed Scopus (1552) Google Scholar, 31Giasson BI Duda JE Quinn SM Zhang B Trojanowski JQ Lee VM Neuronal alpha-synucleinopathy with severe movement disorder in mice expressing A53T human alpha-synuclein.Neuron. 2002; 34: 521-533Abstract Full Text Full Text PDF PubMed Scopus (923) Google Scholar, 32Lee MK Stirling W Xu Y Xu X Qui D Mandir AS Dawson TM Copeland NG Jenkins NA Price DL Human alpha-synuclein-harboring familial Parkinson disease-linked Ala-53 –> Thr mutation causes neurodegenerative disease with alpha-synuclein aggregation in transgenic mice.Proc Natl Acad Sci U S A. 2002; 99: 8968-8973Crossref PubMed Scopus (639) Google Scholar rats,33Kirik D Rosenblad C Burger C Lundberg C Johansen TE Muzyczka N Mandel RJ Bjorklund A Parkinson-like neurodegeneration induced by targeted overexpression of alpha-synuclein in the nigrostriatal system.J Neurosci. 2002; 22: 2780-2791Crossref PubMed Google Scholar, 34Lo Bianco C Ridet JL Schneider BL Deglon N Aebischer P alpha-Synucleinopathy and selective dopaminergic neuron loss in a rat lentiviral-based model of Parkinson disease.Proc Natl Acad Sci USA. 2002; 99: 10813-10818Crossref PubMed Scopus (427) Google Scholar and monkeys35Kirik D Annett LE Burger C Muzyczka N Mandel RJ Bjorklund A Nigrostriatal alpha-synucleinopathy induced by viral vector-mediated overexpression of human alpha-synuclein: a new primate model of Parkinson disease.Proc Natl Acad Sci USA. 2003; 100: 2884-2889Crossref PubMed Scopus (340) Google Scholar has provided a number of models of Parkinson disease. Many of these models replicate key biochemical and cell biological features of Parkinson disease and show significant promise in furthering the understanding of Parkinson disease pathogenesis and the development of therapies for the disorder. As in Huntington disease and related polyglutamine expansion diseases, knocking out α-synuclein in mice does not provide a faithful model of Parkinson disease.36Abeliovich A Schmitz Y Fariñas I Choi-Lundberg D Ho WH Castillo PE Shinsky N Verdugo JM Armanini M Ryan A Hynes M Phillips H Sulzer D Rosenthal A Mice lacking alpha-synuclein display functional deficits in the nigrostriatal dopamine system.Neuron. 2000; 25: 239-252Abstract Full Text Full Text PDF PubMed Scopus (1388) Google Scholar When we expressed wild-type or Parkinson disease–linked mutant forms of human α-synuclein in Drosophila we observed age-dependent degeneration of dopaminergic neurons and progressive locomotor dysfunction.37Feany MB Bender WW A Drosophila model of Parkinson disease.Nature. 2000; 404: 394-398Crossref PubMed Scopus (1694) Google Scholar Although mutant forms of α-synuclein (A30P, A53T) were somewhat more toxic that wild-type α-synuclein, we observed very similar pathologies when expressing either wild-type or mutant versions of the protein. These findings are consistent with a critical role for α-synuclein in both genetic and sporadic forms of the disorder, and suggest that increased expression of the protein may underlie neurotoxicity. Indeed, the recent description of duplications and triplications of the α-synuclein locus in familial Parkinson disease38Singleton AB Farrer M Johnson J Singleton A Hague S Kachergus J Hulihan M Peuralinna T Dutra A Nussbaum R Lincoln S Crawley A Hanson M Maraganore D Adler C Cookson MR Muenter M Baptista M Miller D Blancato J Hardy J Gwinn-Hardy K alpha-Synuclein locus triplication causes Parkinson disease.Science. 2003; 302: 841Crossref PubMed Scopus (3469) Google Scholar lends strong support to the hypothesis that wild-type α-synuclein can cause neuronal death and dysfunction in patients when levels of the protein are elevated. As for the polyglutamine disorders, modeling in Drosophila also recapitulates abnormal aggregation of the overexpressed protein. Flies expressing wild-type or mutant human α-synuclein develop α-synuclein–rich intraneuronal inclusion bodies with advancing age (Figure 1E). These inclusions form in both neuronal cell bodies and in neuritic processes. At the electron microscopic level the inclusions are filamentous and resemble cortical-type Lewy bodies from Parkinson disease patients.37Feany MB Bender WW A Drosophila model of Parkinson disease.Nature. 2000; 404: 394-398Crossref PubMed Scopus (1694) Google Scholar Work in simple model organisms has contributed substantially to understanding the relationship of Lewy pathology to the pathogenesis of Parkinson disease. Soon after the development of the α-synuclein transgenic fly model of Parkinson disease, expression of human HSP70 was reported to ameliorate neurotoxicity of the protein,39Auluck PK Chan HY Trojanowski JQ Lee VM Bonini NM Chaperone suppression of alpha-synuclein toxicity in a Drosophila model for Parkinson disease.Science. 2002; 295: 865-868Crossref PubMed Scopus (1053) Google Scholar, 40Auluck PK Bonini NM Pharmacological prevention of Parkinson disease in Drosophila.Nat Med. 2002; 8: 1185-1186Crossref PubMed Scopus (253) Google Scholar implicating abnormal protein folding in pathogenesis. α-synuclein is one of a number of aggregation prone proteins that are natively unstructured but have a propensity to form β sheet secondary structure as part of the aggregation process.41Rochet JC Lansbury Jr, PT Amyloid fibrillogenesis: themes and variations.Curr Opin Struct Biol. 2000; 10: 60-68Crossref PubMed Scopus (996) Google Scholar Subsequent studies have shown that aggregation of α-synuclein is strongly dependent on phosphorylation of the protein. α-synuclein is a natively unstructured molecule of 140 amino acids that can be divided into three distinct domains (Figure 2). The highly conserved N terminus contains imperfect repeats including the sequence motif KTKEGV. The three missense mutations linked to familial Parkinson disease lie within the repeat region. The central nonamyloid component (NAC) region of α-synuclein is relatively hydrophobic, whereas the C-terminal tail is acidic. Many of the documented phosphorylation sites are in the acidic tail. Serine 129 is extensively phosphorylated in brain tissue from patients with Parkinson disease and in α-synuclein transgenic mouse models,42Fujiwara H Hasegawa M Dohmae N Kawashima A Masliah E Goldberg MS Shen J Takio K Iwatsubo T alpha-Synuclein is phosphorylated in synucleinopathy lesions.Nat Cell Biol. 2002; 4: 160-164Crossref PubMed Scopus (159) Google Scholar, 43Neumann M Kahle PJ Giasson BI Ozmen L Borroni E Spooren W Muller V Odoy S Fujiwara H Hasegawa M Iwatsubo T Trojanowski JQ Kretzschmar HA Haass C Misfolded proteinase K-resistant hyperphosphorylated alpha-synuclein in aged transgenic mice with locomotor deterioration and in human alpha-synucleinopathies.J Clin Invest. 2002; 110: 1429-1439Crossref PubMed Scopus (294) Google Scholar, 44Saito Y Kawashima A Ruberu NN Fujiwara H Koyama S Sawabe M Arai T Nagura H Yamanouchi H Hasegawa M Iwatsubo T Murayama S Accumulation of phosphorylated alpha-synuclein in aging human brain.J Neuropathol Exp Neurol. 2003; 62: 644-654Crossref PubMed Scopus (303) Google Scholar suggesting a role for serine 129 phosphorylation in disease pathogenesis. We have tested the role of serine 129 phosphorylation in our Drosophila model of Parkinson disease and found that phosphorylation at serine 129 is a key mediator of dopaminergic toxicity and α-synuclein aggregation.45Chen L Feany MB Alpha-synuclein phosphorylation controls neurotoxicity and inclusion formation in a Drosophila model of Parkinson disease.Nat Neurosci. 2005; 8: 657-663Crossref PubMed Scopus (518) Google Scholar Importantly, we found that the formation of large Lewy body-like inclusion bodies correlated with neuroprotection rather than neurotoxicity. To clarify the role of α-synuclein aggregation in neurotoxicity we then deleted the 11 amino acids that constitute the central NAC domain (Figure 2). Prior studies had demonstrated that the NAC domain was essential for aggregation of α-synuclein in vitro.46Bodles AM Guthrie DJ Greer B Irvine GB Identification of the region of non-Abeta component (NAC) of Alzheimer disease amyloid responsible for its aggregation and toxicity.J Neurochem. 2001; 78: 384-395Crossref PubMed Scopus (126) Google Scholar, 47Giasson BI Murray IV Trojanowski JQ Lee VM A hydrophobic stretch of 12 amino acid residues in the middle of alpha-synuclein is essential for filament assembly.J Biol Chem. 2001; 276: 2380-2386Crossref PubMed Scopus (757) Google Scholar, 48Zibaee S Jakes R Fraser G Serpell LC Crowther RA Goedert M Sequence determinants for amyloid fibrillogenesis of human alpha-synuclein.J Mol Biol. 2007; 374: 454-464Crossref PubMed Scopus (61) Google Scholar We confirmed that the NAC domain is required for aggregation in vivo because flies expressing α-synuclein without NAC did not form large aggregates.49Periquet M Fulga T Myllykangas L Schlossmacher MG Feany MB Aggregated alpha-synuclein mediates dopaminergic neurotoxicity in vivo.J Neurosci. 2007; 27: 3338-3346Crossref PubMed Scopus (240) Google Scholar Importantly, we were also able to identify soluble oligomeric species biochemically. These oligomeric species also required NAC sequences. Animals unable to form either oligomeric assemblies or large Lewy-body like aggregates showed no toxicity of α-synuclein to dopaminergic neurons. These findings strongly implicate abnormal aggregation of α-synuclein in mediating neurotoxicity, but do not clearly define the species of aggregate responsible for the deleterious effects of the protein. To address this issue we extended our prior analysis of α-synuclein phosphoryation. In addition to serine phosphorylation, α-synuclein can also be phosphorylated at three C-terminal tyrosine residues: tyrosine 125, 133, and 136.50Ellis CE Schwartzberg PL Grider TL Fink DW Nussbaum RL alpha-synuclein is phosphorylated by members of the Src family of protein-tyrosine kinases.J Biol Chem. 2001; 276: 3879-3884Crossref PubMed Scopus (127) Google Scholar, 51Chen L Periquet M Wang X Negro A McLean PJ Hyman BT Feany MB Tyrosine and serine phosphorylation of alpha-synuclein have opposing effects on neurotoxicity and soluble oligomer formation.J Clin Inves. 2009; 119: 3257-3265Crossref PubMed Scopus (51) Google Scholar In contrast to the neurotoxic effect of phosphorylation at serine 129, we see a neuroprotective effect for tyrosine phosphorylation. Most importantly, we found that the levels of soluble oligomers correlated with neurotoxicity in flies with increases or decreases in serine or tyrosine phosphorylation of α-synuclein (Figure 3).51Chen L Periquet M Wang X Negro A McLean PJ Hyman BT Feany MB Tyrosine and serine phosphorylation of alpha-synuclein have opposing effects on neurotoxicity and soluble oligomer formation.J Clin Inves. 2009; 119: 3257-3265Crossref PubMed Scopus (51) Google Scholar These findings correlate well with recent work from Karpinar et al52Karpinar DP Balija MB Kügler S Opazo F Rezaei-Ghaleh N Wender N Kim HY Taschenberger G Falkenburger BH Heise H Kumar A Riedel D Fichtner L Voigt A Braus GH Giller K Becker S Herzig A Baldus M Jäckle H Eimer S Schulz JB Griesinger C Zweckstetter M Pre-fibrillar alpha-synuclein variants with impaired beta-structure increase neurotoxicity in Parkinson disease models.EMBO J. 2009; 28: 3256-3268Crossref PubMed Scopus (354) Google Scholar in which a series of α-synuclein point mutations were assayed for their ability to form oligomers and fibrils in vitro and neurotoxicity in vivo using Drosophila and C. elegans models of α-synuclein dopaminergic neurotoxicity. Similar to work in our laboratory, formation of oligomeric species correlated with toxicity, whereas aggregation into higher order fibrillar species was neuroprotective. These data all together are consistent with a model in which prefibrillar species of α-synuclein are toxic. Levels of these toxic species can be elevated by increasing the total amount of α-synuclein, or by specific posttranslational modifications. Other posttranslation modifications may promote formation of fibrillar species that then aggregate into large Lewy body inclusions. Fibrils and the subsequent accumulation of fibrils into Lewy bodies and Lewy neurites may constitute a protective cellular response (Figure 3). The importance of posttranslational modifications in determining neurotoxicity of aggregation-prone proteins has also become apparent in experimental models of Alzheimer disease and related tauopathies. Neurofibrillary tangles, one of the major pathological hallmarks of Alzheimer disease, comprise abnormally hyperphosphorylated and aggregated tau protein.53Grundke-Iqbal I Iqbal K Tung YC Quinlan M Wisniewski HM Binder LI Abnormal phosphorylation of the microtubule-associated protein tau (tau) in Alzheimer cytoskeletal pathology.Proc Natl Acad Sci U S A. 1986; 83: 4913-
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