In Parkinson’s disease (PD), midbrain dopaminergic neuronal death is linked to the accumulation of aggregated α-synuclein. The familial PD mutant form of α-synuclein, E46K, has not been thoroughly evaluated yet in an organismal model system. Here, we report that E46K resembled wild-type (WT) α-synuclein in Saccharomyces cerevisiae in that it predominantly localized to the plasma membrane, and it did not induce significant toxicity or accumulation. In contrast, in Schizosaccharomyces pombe, E46K did not associate with the plasma membrane. Instead, in one strain, it extensively aggregated in the cytoplasm and was as toxic as WT. Remarkably, in another strain, E46K extensively associated with the endomembrane system and was more toxic than WT. Our studies recapitulate and extend aggregation and phospholipid membrane association properties of E46K previously observed in vitro and cell culture. Furthermore, it supports the notion that E46K generates toxicity partly due to increased association with endomembrane systems within cells. 1. Introduction Parkinson’s disease (PD) is a fatal and incurable neurodegenerative disorder characterized by resting tremors, postural instability, and bradykinesia [1]. The death of midbrain substantia nigra neurons characterizes the pathology of both familial and sporadic forms of PD. Two types of molecules accumulate within these dying cells: misfolded and aggregated α-synuclein in cytoplasmic inclusions called Lewy bodies [2] and oxidative free radicals [1, 3]. Therefore, a major hypothesis is that α-synuclein misfolding and aggregation is linked to neuronal toxicity in PD [4–6]. While the molecular basis of sporadic PD is still unclear, mutations in six genes cause familial PD [7], including three point mutations (A53T, A30P, and E46K) within the α-synuclein gene [8–10]. In addition to self-aggregation [11–13], several other α-synuclein properties that may be relevant to PD include maintenance of neurotransmitter vesicular pools [14, 15], synaptic plasticity [16, 17], phospholipid binding and lipid metabolism [18–21], microtubule binding [22], chaperone-like ability [23–25], and ER-Golgi trafficking [26, 27]. While the A30P and A53T familial mutants are widely studied in how they alter the above properties to cause cytotoxicity and contribute to PD pathogenesis [28], less is known about the third and most recently discovered familial mutant, E46K. Thus far, E46K is known to aggregate into fibrils more rapidly than wild-type α-synuclein in vitro [29, 30] and in cell culture [31]. Additionally, recent biochemical studies
References
[1]
C. W. Olanow and W. G. Tatton, “Etiology and pathogenesis of Parkinson's disease,” Annual Review of Neuroscience, vol. 22, pp. 123–144, 1999.
[2]
M. G. Spillantini, R. A. Crowther, R. Jakes, M. Hasegawa, and M. Goedert, “α-synuclein in filamentous inclusions of Lewy bodies from Parkinson's disease and dementia with Lewy bodies,” Proceedings of the National Academy of Sciences of the United States of America, vol. 95, no. 11, pp. 6469–6473, 1998.
[3]
T. M. Dawson and V. L. Dawson, “Molecular pathways of neurodegeneration in Parkinson's disease,” Science, vol. 302, no. 5646, pp. 819–822, 2003.
[4]
M. S. Goldberg and P. T. Lansbury Jr., “Is there a cause-and-effect relationship between α-synuclein fibrillization and Parkinson's disease?” Nature Cell Biology, vol. 2, no. 7, pp. E115–E119, 2000.
[5]
K. Ono, M. Hirohata, and M. Yamada, “α-synuclein assembly as a therapeutic target of Parkinson's disease and related disorders,” Current Pharmaceutical Design, vol. 14, no. 30, pp. 3247–3266, 2008.
[6]
K. Vekrellis, M. Xilouri, E. Emmanouilidou, and L. Stefanis, “Inducible over-expression of wild type α-synuclein in human neuronal cells leads to caspase-dependent non-apoptotic death,” Journal of Neurochemistry, vol. 109, no. 5, pp. 1348–1362, 2009.
[7]
C. Schiesling, N. Kieper, K. Seidel, and R. Krüger, “Review: familial Parkinson's disease—genetics, clinical phenotype and neuropathology in relation to the common sporadic form of the disease,” Neuropathology and Applied Neurobiology, vol. 34, no. 3, pp. 255–271, 2008.
[8]
M. H. Polymeropoulos, C. Lavedan, E. Leroy et al., “Mutation in the α-synuclein gene identified in families with Parkinson's disease,” Science, vol. 276, no. 5321, pp. 2045–2047, 1997.
[9]
R. Krüger, W. Kuhn, T. Müller et al., “Ala30Pro mutation in the gene encoding α-synuclein in Parkinson's disease,” Nature Genetics, vol. 18, no. 2, pp. 106–108, 1998.
[10]
J. J. Zarranz, J. Alegre, J. C. Gómez-Esteban et al., “The new mutation, E46K, of α-synuclein causes Parkinson and Lewy body dementia,” Annals of Neurology, vol. 55, no. 2, pp. 164–173, 2004.
[11]
K. A. Conway, J. D. Harper, and P. T. Lansbury, “Accelerated in vitro fibril formation by a mutant α-synuclein linked to early-onset Parkinson disease,” Nature Medicine, vol. 4, no. 11, pp. 1318–1320, 1998.
[12]
B. I. Giasson, K. Uryu, J. Q. Trojanowski, and V. M. Y. Lee, “Mutant and wild type human α-synucleins assemble into elongated filaments with distinct morphologies in vitro,” Journal of Biological Chemistry, vol. 274, no. 12, pp. 7619–7622, 1999.
[13]
K. A. Conway, J. D. Harper, and P. T. Lansbury Jr., “Fibrils formed in vitro from α-synuclein and two mutant forms linked to Parkinson's disease are typical amyloid,” Biochemistry, vol. 39, no. 10, pp. 2552–2563, 2000.
[14]
P. H. Jensen, M. S. Nielsen, R. Jakes, C. G. Dotti, and M. Goedert, “Binding of α-synuclein to brain vesicles is abolished by familial Parkinson's disease mutation,” Journal of Biological Chemistry, vol. 273, no. 41, pp. 26292–26294, 1998.
[15]
D. D. Murphy, S. M. Rueter, J. Q. Trojanowski, and V. M. Y. Lee, “Synucleins are developmentally expressed, and α-synuclein regulates the size of the presynaptic vesicular pool in primary hippocampal neurons,” Journal of Neuroscience, vol. 20, no. 9, pp. 3214–3220, 2000.
[16]
J. M. George, H. Jin, W. S. Woods, and D. F. Clayton, “Characterization of a novel protein regulated during the critical period for song learning in the zebra finch,” Neuron, vol. 15, no. 2, pp. 361–372, 1995.
[17]
A. Abeliovich, Y. Schmitz, I. Fari？as et al., “Mice lacking α-synuclein display functional deficits in the nigrostriatal dopamine system,” Neuron, vol. 25, no. 1, pp. 239–252, 2000.
[18]
W. S. Davidson, A. Jonas, D. F. Clayton, and J. M. George, “Stabilization of α-synuclein secondary structure upon binding to synthetic membranes,” Journal of Biological Chemistry, vol. 273, no. 16, pp. 9443–9449, 1998.
[19]
R. Sharon, M. S. Goldberg, I. Bar-Josef, R. A. Betensky, J. Shen, and D. J. Selkoe, “α-synuclein occurs in lipid-rich high molecular weight complexes, binds fatty acids, and shows homology to the fatty acid-binding proteins,” Proceedings of the National Academy of Sciences of the United States of America, vol. 98, no. 16, pp. 9110–9115, 2001.
[20]
D. Eliezer, E. Kutluay, R. Bussell, and G. Browne, “Conformational properties of α-synuclein in its free and lipid-associated states,” Journal of Molecular Biology, vol. 307, no. 4, pp. 1061–1073, 2001.
[21]
T. F. Outeiro and S. Lindquist, “Yeast cells provide insight into alpha-synuclein biology and pathobiology,” Science, vol. 302, no. 5651, pp. 1772–1775, 2003.
[22]
M. A. Alim, Q.-L. Ma, K. Takeda et al., “Demonstration of a role for α-synuclein as a functional microtubule-associated protein,” Journal of Alzheimer's Disease, vol. 6, no. 4, pp. 435–442, 2004.
[23]
J. M. Jenco, A. Rawlingson, B. Daniels, and A. J. Morris, “Regulation of phospholipase D2: selective inhibition of mammalian phospholipase D isoenzymes by α- and β-synucleins,” Biochemistry, vol. 37, no. 14, pp. 4901–4909, 1998.
[24]
N. Ostrerova, L. Petrucelli, M. Farrer et al., “α-synuclein shares physical and functional homology with 14-3-3 proteins,” Journal of Neuroscience, vol. 19, no. 14, pp. 5782–5791, 1999.
[25]
S. Engelender, Z. Kaminsky, G. Xin et al., “Synphilin-1 associates with α-synuclein and promotes the formation of cytosolic inclusions,” Nature Genetics, vol. 22, no. 1, pp. 110–114, 1999.
[26]
A. A. Cooper, A. D. Gitler, A. Cashikar et al., “α-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson's models,” Science, vol. 313, no. 5785, pp. 324–328, 2006.
[27]
A. D. Gitler, B. J. Bevis, J. Shorter et al., “The Parkinson's disease protein α-synuclein disrupts cellular Rab homeostasis,” Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 1, pp. 145–150, 2008.
[28]
B. Caughey and P. T. Lansbury, “Protofibrils, pores, fibrils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders,” Annual Review of Neuroscience, vol. 26, pp. 267–298, 2003.
[29]
W. Choi, S. Zibaee, R. Jakes et al., “Mutation E46K increases phospholipid binding and assembly into filaments of human α-synuclein,” FEBS Letters, vol. 576, no. 3, pp. 363–368, 2004.
[30]
E. A. Greenbaum, C. L. Graves, A. J. Mishizen-Eberz et al., “The E46K mutation in α-synuclein increases amyloid fibril formation,” Journal of Biological Chemistry, vol. 280, no. 9, pp. 7800–7807, 2005.
[31]
N. Pandey, R. E. Schmidt, and J. E. Galvin, “The alpha-synuclein mutation E46K promotes aggregation in cultured cells,” Experimental Neurology, vol. 197, no. 2, pp. 515–520, 2006.
[32]
C. C. Rospigliosi, S. McClendon, A. W. Schmid et al., “E46K Parkinson's-linked mutation enhances C-terminal-to-N-terminal contacts in α-synuclein,” Journal of Molecular Biology, vol. 388, no. 5, pp. 1022–1032, 2009.
[33]
P. T. Lansbury, “Evolution of amyloid: what normal protein folding may tell us about fibrillogenesis and disease,” Proceedings of the National Academy of Sciences of the United States of America, vol. 96, no. 7, pp. 3342–3344, 1999.
[34]
R. A. Fredenburg, C. Rospigliosi, R. K. Meray et al., “The impact of the E46K mutation on the properties of α-synuclein in its monomelic and oligomeric states,” Biochemistry, vol. 46, no. 24, pp. 7107–7118, 2007.
[35]
E. Di Pasquale, J. Fantini, H. Chahinian, M. Maresca, N. Ta？eb, and N. Yahi, “Altered Ion channel formation by the Parkinson's-disease-linked E46K mutant of α-synuclein is corrected by GM3 but not by GM1 gangliosides,” Journal of Molecular Biology, vol. 397, no. 1, pp. 202–218, 2010.
[36]
M. St？ckl, P. Fischer, E. Wanker, and A. Herrmann, “α-synuclein selectively binds to anionic phospholipids embedded in liquid-disordered domains,” Journal of Molecular Biology, vol. 375, no. 5, pp. 1394–1404, 2008.
[37]
C. R. Bodner, A. S. Maltsev, C. M. Dobson, and A. Bax, “Differential phospholipid binding of α-synuclein variants implicated in Parkinson's disease revealed by solution NMR spectroscopy,” Biochemistry, vol. 49, no. 5, pp. 862–871, 2010.
[38]
A. Klegeris, S. Pelech, B. I. Giasson et al., “α-synuclein activates stress signaling protein kinases in THP-1 cells and microglia,” Neurobiology of Aging, vol. 29, no. 5, pp. 739–752, 2008.
[39]
C. B. Kunst, E. Mezey, M. J. Brownstein, and D. Patterson, “Mutations in SOD1 associated with amyotrophic lateral sclerosis cause novel protein interactions,” Nature Genetics, vol. 15, no. 1, pp. 91–94, 1997.
[40]
J. Ma and S. Lindquist, “De novo generation of a PrP-like conformation in living cells,” Nature Cell Biology, vol. 1, no. 6, pp. 358–361, 1999.
[41]
S. Krobitsch and S. Lindquist, “Aggregation of huntingtin in yeast varies with the length of the polyglutamine expansion and the expression of chaperone proteins,” Proceedings of the National Academy of Sciences of the United States of America, vol. 97, no. 4, pp. 1589–1594, 2000.
[42]
P. J. Muchowski, K. Ning, C. D'Souza-Schorey, and S. Fields, “Requirement of an intact microtubule cytoskeleton for aggregation and inclusion body formation by a mutant huntingtin fragment,” Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 2, pp. 727–732, 2002.
[43]
P. Zabrocki, K. Pellens, T. Vanhelmont et al., “Characterization of α-synuclein aggregation and synergistic toxicity with protein tau in yeast,” FEBS Journal, vol. 272, no. 6, pp. 1386–1400, 2005.
[44]
C. Dixon, N. Mathias, R. M. Zweig, D. A. Davis, and D. S. Gross, “α-synuclein targets the plasma membrane via the secretory pathway and induces toxicity in yeast,” Genetics, vol. 170, no. 1, pp. 47–59, 2005.
[45]
N. Sharma, K. A. Brandis, S. K. Herrera et al., “α-synuclein budding yeast model: toxicity enhanced by impaired proteasome and oxidative stress,” Journal of Molecular Neuroscience, vol. 28, no. 2, pp. 161–178, 2006.
[46]
K. A. Brandis, I. F. Holmes, S. J. England, N. Sharma, L. Kukreja, and S. K. DebBurman, “α-synuclein fission yeast model: concentration-dependent aggregation without plasma membrane localization or toxicity,” Journal of Molecular Neuroscience, vol. 28, no. 2, pp. 179–192, 2006.
[47]
S. Willingham, T. F. Outeiro, M. J. DeVit, S. L. Lindquist, and P. J. Muchowski, “Yeast genes that enhance the toxicity of a mutant huntingtin fragment or α-synuclein,” Science, vol. 302, no. 5651, pp. 1769–1772, 2003.
[48]
M. Okochi, J. Walter, A. Koyama et al., “Constitutive phosphorylation of the Parkinson's disease associated α-synuclein,” Journal of Biological Chemistry, vol. 275, no. 1, pp. 390–397, 2000.
[49]
H. Fujiwara, M. Hasegawa, N. Dohmae et al., “α-synuclein is phosphorylated in synucleinopathy lesions,” Nature Cell Biology, vol. 4, no. 2, pp. 160–164, 2002.
[50]
K. E. Paleologou, A. Oueslati, G. Shakked et al., “Phosphorylation at S87 is enhanced in synucleinopathies, inhibits α-synuclein oligomerization, and influences synuclein-membrane interactions,” Journal of Neuroscience, vol. 30, no. 9, pp. 3184–3198, 2010.
[51]
O. M. A. El-Agnaf, R. Jakes, M. D. Curran et al., “Aggregates from mutant and wild-type α-synuclein proteins and NAC peptide induce apoptotic cell death in human neuroblastoma cells by formation of β-sheet and amyloid-like filaments,” FEBS Letters, vol. 440, no. 1-2, pp. 71–75, 1998.
[52]
R. Bussell and D. Eliezer, “Effects of Parkinson's disease-linked mutations on the structure of lipid-associated α-synuclein,” Biochemistry, vol. 43, no. 16, pp. 4810–4818, 2004.
[53]
T. S. Ulmer and A. Bax, “Comparison of structure and dynamics of micelle-bound human α-synuclein and Parkinson disease variants,” Journal of Biological Chemistry, vol. 280, no. 52, pp. 43179–43187, 2005.
[54]
R. Jakes, M. G. Spillantini, and M. Goedert, “Identification of two distinct synucleins from human brain,” FEBS Letters, vol. 345, no. 1, pp. 27–32, 1994.
[55]
R. J. Perrin, W. S. Woods, D. F. Clayton, and J. M. George, “Interaction of human α-synuclein and Parkinson's disease variants with phospholipids: structural analysis using site-directed mutagenesis,” Journal of Biological Chemistry, vol. 275, no. 44, pp. 34393–34398, 2000.
[56]
M. M. Tompkins, W. P. Gai, S. Douglas, and S. J. Bunn, “α-synuclein expression localizes to the Golgi apparatus in bovine adrenal medullary chromaffin cells,” Brain Research, vol. 984, no. 1-2, pp. 233–236, 2003.
[57]
H. J. Lee, S. Patel, and S. J. Lee, “Intravesicular localization and exocytosis of α-synuclein and its aggregates,” Journal of Neuroscience, vol. 25, no. 25, pp. 6016–6024, 2005.
[58]
L. Maroteaux and R. H. Scheller, “The rat brain synucleins; family of proteins transiently associated with neuronal membrane,” Molecular Brain Research, vol. 11, no. 3-4, pp. 335–343, 1991.
[59]
T. Shibayama-Imazu, I. Okahashi, K. Omata et al., “Cell and tissue distribution and developmental change of neuron specific 14 kDa protein (phosphoneuroprotein 14),” Brain Research, vol. 622, no. 1-2, pp. 17–25, 1993.
[60]
M. C. Irizarry, W. Growdon, T. Gomez-Isla et al., “Nigral and cortical Lewy bodies and dystrophic nigral neurites in Parkinson's disease and cortical Lewy body disease contain α-synuclein immunoreactivity,” Journal of Neuropathology and Experimental Neurology, vol. 57, no. 4, pp. 334–337, 1998.
[61]
P. J. McLean, H. Kawamata, S. Ribich, and B. T. Hyman, “Membrane association and protein conformation of α-synuclein in intact neurons. Effect of Parkinson's disease-linked mutations,” Journal of Biological Chemistry, vol. 275, no. 12, pp. 8812–8816, 2000.
[62]
P. J. Kahle, M. Neumann, L. Ozmen et al., “Subcellular localization of wild-type and Parkinson's disease-associated mutant α-synuclein in human and transgenic mouse brain,” Journal of Neuroscience, vol. 20, no. 17, pp. 6365–6373, 2000.
[63]
R. Borghi, R. Marchese, A. Negro et al., “Full length α-synuclein is present in cerebrospinal fluid from Parkinson's disease and normal subjects,” Neuroscience Letters, vol. 287, no. 1, pp. 65–67, 2000.
[64]
P. H. Lee, G. Lee, H. J. Park, O. Y. Bang, I. S. Joo, and K. Huh, “The plasma alpha-synuclein levels in patients with Parkinson's disease and multiple system atrophy,” Journal of Neural Transmission, vol. 113, no. 10, pp. 1435–1439, 2006.
[65]
T. Tokuda, S. A. Salem, D. Allsop et al., “Decreased α-synuclein in cerebrospinal fluid of aged individuals and subjects with Parkinson's disease,” Biochemical and Biophysical Research Communications, vol. 349, no. 1, pp. 162–166, 2006.
[66]
A. L. Pidoux and J. Armstrong, “The BiP protein and the endoplasmic reticulum of Schizosaccharomyces pombe: fate of the nuclear envelope during cell division,” Journal of Cell Science, vol. 105, no. 4, pp. 1115–1120, 1993.
[67]
A. E. Roux, P. Chartrand, G. Ferbeyre, and L. A. Rokeach, “Fission yeast and other yeasts as emergent models to unravel cellular aging in eukaryotes,” The Journals of Gerontology Series A, vol. 65, no. 1, pp. 1–8, 2010.
[68]
S. Fernandez, M. J. Homann, S. A. Henry, and G. M. Carman, “Metabolism of the phospholipid precursor inositol and its relationship to growth and viability in the natural auxotroph Schizosaccharomyces pombe,” Journal of Bacteriology, vol. 166, no. 3, pp. 779–786, 1986.
[69]
I. McVeigh and E. Bracken, “The nutrition of Schizosaccharomyces pombe,” Mycologia, vol. 47, no. 1, pp. 13–25, 1955.
[70]
V. M. McDonough and T. M. Roth, “Growth temperature affects accumulation of exogenous fatty acids and fatty acid composition in Schizosaccharomyces pombe,” Antonie van Leeuwenhoek, vol. 86, no. 4, pp. 349–354, 2005.
[71]
R. Harada, N. Kobayashi, J. Kim et al., “The effect of amino acid substitution in the imperfect repeat sequences of α-synuclein on fibrillation,” Biochimica et Biophysica Acta, vol. 1792, no. 10, pp. 998–1003, 2009.
[72]
M. J. Volles and P. T. Lansbury, “Relationships between the sequence of α-synuclein and its membrane affinity, fibrillization propensity, and yeast toxicity,” Journal of Molecular Biology, vol. 366, no. 5, pp. 1510–1522, 2007.
[73]
J. Li, V. N. Uversky, and A. L. Fink, “Effect of familial Parkinson's disease point mutations A30P and A53T on the structural properties, aggregation, and fibrillation of human α-synuclein,” Biochemistry, vol. 40, no. 38, pp. 11604–11613, 2001.
[74]
E. Yeger-Lotem, L. Riva, L. J. Su et al., “Bridging high-throughput genetic and transcriptional data reveals cellular responses to alpha-synuclein toxicity,” Nature Genetics, vol. 41, no. 3, pp. 316–323, 2009.
[75]
M. Fiske, S. Valtierra, M. Solvang, et al., “Contribution of alanine-76 and serine phosphorylation in α-synuclein membrane association and aggregation in yeasts,” Parkinson's Disease. In press.
[76]
J. H. Soper, S. Roy, A. Stieber et al., “α-synuclein-induced aggregation of cytoplasmic vesicles in Saccharomyces cerevisiae,” Molecular Biology of the Cell, vol. 19, no. 3, pp. 1093–1103, 2008.
[77]
T. R. Flower, L. S. Chesnokova, C. A. Froelich, C. Dixon, and S. N. Witt, “Heat shock prevents alpha-synuclein-induced apoptosis in a yeast model of Parkinson's disease,” Journal of Molecular Biology, vol. 351, no. 5, pp. 1081–1100, 2005.
[78]
N. Thayanidhi, J. R. Helm, D. C. Nycz, M. Bentley, Y. Liang, and J. C. Hay, “α-synuclein delays endoplasmic reticulum (ER)-to-Golgi transport in mammalian cells by antagonizing ER/Golgi SNAREs,” Molecular Biology of the Cell, vol. 21, no. 11, pp. 1850–1863, 2010.
[79]
J. L. Webb, B. Ravikumar, J. Atkins, J. N. Skepper, and D. C. Rubinsztein, “α-synuclein is degraded by both autophagy and the proteasome,” Journal of Biological Chemistry, vol. 278, no. 27, pp. 25009–25013, 2003.
[80]
A. M. Cuervo, L. Stafanis, R. Fredenburg, P. T. Lansbury, and D. Sulzer, “Impaired degradation of mutant α-synuclein by chaperone-mediated autophagy,” Science, vol. 305, no. 5688, pp. 1292–1295, 2004.
[81]
H. J. Lee, F. Khoshaghideh, S. Patel, and S. J. Lee, “Clearance of α-synuclein oligomeric intermediates via the lysosomal degradation pathway,” Journal of Neuroscience, vol. 24, no. 8, pp. 1888–1896, 2004.
[82]
J. A. Lee and F. B. Gao, “Roles of ESCRT in autophagy-associated neurodegeneration,” Autophagy, vol. 4, no. 2, pp. 230–232, 2008.
[83]
T. Kuwahara, A. Koyama, S. Koyama et al., “A systematic RNAi screen reveals involvement of endocytic pathway in neuronal dysfunction in α-synuclein transgenic C . elegans,” Human Molecular Genetics, vol. 17, no. 19, pp. 2997–3009, 2008.
[84]
D. Burke, D. Dawson, and T. Stearns, Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2000.
[85]
C. Alfa, Experiments with Fission Yeast, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1993.
