Rescuing Defective Vesicular TraffickingProtects against �-Synuclein Toxicity in Cellular andAnimal Models of Parkinson’s DiseaseHilal A. Lashuel†,* and Harald Hirling‡

†Laboratory of Molecular Neurobiology and Neuroproteomics, and ‡Laboratory of Cellular Neurobiology, Brain Mind Institute, EcolePolytechnique Fédérale de Lausanne, Station 15, CH-1015 Lausanne, Switzerland

ABSTRACT Studies in yeast are providingcritical insights into the mechanisms of neurode-generation in Parkinson’s disease (PD). A recentstudy shows that disruption of vesicular traf-ficking between the endoplasmic reticulum (ER)and the Golgi, caused by the overexpressionand/or aggregation of �-synuclein, is linked todegeneration of dopamine neurons. Overexpres-sion of proteins that are known to enhance ER-to-Golgi transport rescue defective trafficking inyeast, worm, fly, and cellular models of PD.

T he failure of proteins to fold correctlyor to remain folded is the primarycause of several systemic and neuro-

degenerative diseases that affect a signifi-cant portion of the world’s population (1). Toprotect against aberrant folding, livingorganisms have evolved efficient proteinsynthesis and quality-control machinery.This system relies on close cooperationbetween the chaperone and the protein-degradation machinery to ensure controlover proper folding, targeting, and degrad-ing of proteins.

Protein folding occurs in the cytosol orwithin the lumen of the endoplasmic reticu-lum (ER). Proteins that are destined to gothrough the secretory pathway must passthrough a series of quality-control check-points to ensure their correct processingand targeting to the extracellular space, theplasma membrane, or their final destinationwithin the cell (Figure 1). The first check-point along this pathway resides in the ER,where soluble proteins are translated intothe ER lumen and transmembrane proteinsare translated and integrated into the ERmembrane. Concomitant to translation, aspecialized set of chaperones and thequality-control machinery of the ER ensurecorrect folding. In addition, post-transla-tional modifications such as disulfidebonds or N-linked glycosylations are intro-duced. Once a protein is properly folded andmodified and has passed the quality-control

process, it is directed to a specialized ER exitsite, where it is integrated into transportvesicles that bud from the ER membrane.These vesicles are anterogradely trans-ported to the Golgi, where they dock andfuse with the membrane of the cis-Golgi(see ref 2 for a detailed description of traf-ficking at the ER and Golgi apparatus). Themajor function of the Golgi is to fine-tune theadded sugar residues and to sort the differ-ent cargo proteins into particular vesicles tobe transported along distinguished traffick-ing pathways. These include routes to theplasma membrane for secretion and inser-tion of surface proteins or to other intracellu-lar compartments such as the endosomal/lysosomal system. Each transport step alsoengages in a retrograde transport activity.This is particularly important for transportbetween the ER and the Golgi in order torecapture components of the vesicle-trafficking machinery and ER residentproteins.

However, this process is not perfect, andmany newly synthesized proteins misfoldand rapidly degrade. Proteins that fail to foldproperly are retrotranslocated at the level ofthe ER to the cytosol for degradation by theubiquitin proteasome system (UPS). If theER folding machinery and the UPS cannotkeep up with protein misfolding, the accu-mulation and/or aggregation of misfoldedproteins induces cellular stress by multiplemechanisms. The result is cellular dysfunc-

*Corresponding author,hilal.lashuel@epfl.ch.

Published online August 18, 2006

10.1021/cb600331e CCC: $33.50

© 2006 by American Chemical Society

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tion, the initiation of ER-induced apoptosis,and ultimately disease manifestation.

Several misfolding diseases are causedby mutations that result in the loss ofprotein function due to improper folding,trafficking, and/or enhanced intracellulardegradation by the UPS (e.g., cystic fibrosis,sickle-cell anemia, �-1-antitrypsin defi-ciency, familial hypercholesterolemia, andsome forms of cancer) (1). If the rate ofprotein misfolding is faster than that of deg-radation because of mutations and/orimpaired quality-control machinery withinthe cell, then the misfolded proteins accu-mulate and self-associate to form highlyordered �-sheet-rich toxic aggregates. Thepresence of aggregates of misfolded proteinin the form of intracellular inclusions orextracellular deposits in the vicinity of dyingneurons is a defining hallmark of severalneurodegenerative diseases (NDDs), includ-ing Alzheimer’s disease (AD), Parkinson’sdisease (PD), amyotrophic lateral sclerosis,and polyglutamine and prion diseases (3).

Increasing evidence from neuropatho-logic, genetic, animal modeling, biochemi-cal, and biophysical sources points towardprotein misfolding and aggregation as theprimary cause of several NDDs. However,the exact mechanisms by which these pro-

cesses cause neurodegeneration and celldeath remain a subject of intense investiga-tion and debate. Studies on cellular andanimal models of protein-aggregation dis-eases suggest that the pathogenesis is com-plicated, and it is likely that neurodegenera-tion occurs by more than one mechanism.Oxidative stress, membrane disruption, ERstress, altered chaperone activity, impair-ment of the UPS, mitochondrial deficit, tran-scriptional dysregulation, axonal transportabnormalities, and Golgi fragmentation areall possible consequences of protein aggre-gation and are thought to play key roles inthe initiation and/or progression of neuro-degeneration.

Yeast Sheds Light on Neurodegenerationin PD. The discovery of disease-associatedmutations in the genes encoding the aggre-gating proteins inspired the development ofgenetic animal and cellular models as toolsfor understanding the relationship betweenprotein aggregation and disease. The exist-ing genetic models of protein aggregationdiseases are all based on the massive over-expression of the gene coding for the wild-type protein or disease-associated mutantsin mouse, rat, Drosophila, and Caenorhab-ditis elegans. These models recapitulatesome features of the disease, but none has

been shown to reproduce the completedisease phenotype observed in humans.

Baker’s yeast (Saccharomyces cerevi-siae), a single-celled organism with �6000genes, was once thought to be too simplefor modeling complex pathologies of thenervous system, but it is now emerging as apowerful tool for modeling NDDs. Despitesignificant differences between yeast cellsand neurons, many of the basic cellular pro-cesses, such as protein folding and thequality-control machinery, are conserved inboth eukaryotic cells. In a recent study pub-lished in Science Express, Antony Cooper(University of Missouri, Kansas City), SusanLindquist’s team (Massachusetts Institute ofTechnology), and colleagues from severalother research groups (4) took advantage ofthese similarities by using a yeast model toelucidate a new molecular mechanismunderlying the pathogenesis of PD. Thisneurodegenerative movement disorder ischaracterized by the loss of dopamine (DA)neurons from the substantia nigra (SN) andthe formation of intraneuronal proteina-ceous inclusions, referred to as Lewybodies (LBs).

�-Synuclein Aggregation and DefectiveTrafficking. Lindquist and colleagues (4, 5)created a yeast model of PD based on

Figure 1. Vesicular trafficking in neurons. a) The extended morphology of neurons showing the cell body, dendrites, axons, and two synapses thatare the contact sites with upstream or downstream cells in the network. b) Like all eukaryotic cells, neurons possess a set of organelles that formthe secretory pathway. Protein synthesis takes place on ribosomes that associate with the ER membrane. Chaperones assist in proper folding ofthese newly translated polypeptide chains. A complex quality-control machinery that recognizes misfolded proteins allows only correctly foldedproteins to reach ER exit sites, where transport vesicles containing the cargo proteins are formed. These vesicles are transported to the cis-side ofthe Golgi, where they dock and fuse with its membrane. This process is dependent on the small GTPase Rab1. After further protein modification,cargo proteins are packaged into specific vesicles that take on diverse transport routes in the cell.

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increased expression of the presynapticprotein �-synuclein, the primary constituentof LBs. Expression of mutant �-synuclein orthe wild-type protein is sufficient to causefamilial PD (6–9). �-Synuclein aggregationand fibrillogenesis are also implicated in thepathogenesis of several NDDs, including AD,multiple-system atrophy, dementia withLBs, Down syndrome, and neurodegenera-tion with brain iron accumulation, collec-tively referred to as synucleinopathies (10).

In the yeast system, the expression of�-synuclein (wild-type or disease-associated mutant A53T) can be tightlyexperimentally regulated and its effectsmonitored in real time; thus, detection ofthe early events involved in �-synuclein tox-icity is possible (4). Within the first 4–8 h,the presence of �-synuclein aggregates andincreased ER stress were observed to coin-cide with growth arrest and loss of cellviability. ER and proteasome-specific sub-strates were used to show that the expres-sion of �-synuclein does not affect thegeneral proteasome activity. However, it sig-nificantly impairs the turnover of substratesfor which degradation requires traffickingfrom the ER to the Golgi as well as the trans-port of proteins that traffic through thispathway. Detailed dissection of the earlyevents occurring during the first 4 h demon-strated that the first detectable defects incell growth coincide with the impairment ofvesicular transport from the ER to the Golgiand occur before the induction of ER stress.

Rescuing Defective Vesicular Trafficking.Next, Lindquist and colleagues performed acomplementation screen for modifiers of�-synuclein toxicity, which identified 34genes that suppressed �-synuclein toxicityand 20 genes that increased it. Many sup-pressor genes that were specific for �-syn-uclein toxicity encode proteins that are alsoinvolved in ER-to-Golgi transport. If toxicityfrom increased �-synuclein levels and/oraggregation occurs through disruption of theER-to-Golgi transport machinery, then pro-moting the forward transport from the ER to

the Golgi should reverse �-synuclein toxic-ity. Indeed, this was the case. This was par-ticularly striking with Ypt1p and Rab1 (thehuman homologue of Ypt1p). In yeast, Dro-sophila, C. elegans, and rat DA neurons, theoverexpression of Ypt1p/Rab1 resulted insignificant reduction of �-synuclein-inducedneurodegeneration.

The small GTPases of the Rab family playessential roles in vesicle docking and fusion(Figure 2, panel c) (11). Rab1 has been

shown to be specifically involved in ER-to-Golgi trafficking and the docking of ER-derived transport vesicles at the Golgi mem-brane. This suggests that the transport stepaffected by �-synuclein toxicity is vesicledocking/fusion at the Golgi membrane. Thecolocalization of Ypt1p with �-synuclein incytosolic inclusion suggests that �-syn-uclein toxicity may involve the sequestrationof proteins that play a critical role in theER-to-Golgi transport and, eventually, the

Figure 2. Potential toxic mechanisms linking protein aggregation, defective trafficking, andselective degeneration of DA neurons in PD. a) Schematic depiction of the current understandingof the aggregation pathway of �-synuclein based on in vitro biophysical studies. b) DA oxidationresults in the production of reactive oxygen species and quinone and semiquinone intermedi-ates, all of which are highly cytotoxic. To protect against the toxic properties of DA metabolites,the majority of DA is stored in vesicles before its release. c) The study by Cooper et al. showsthat �-synuclein aggregates interfere with a Rab1-dependent step of ER-to-Golgi transport. Thiscould lead to fragmentation of the Golgi due to an imbalance of incoming and outgoing vesicles,reduced targeting of DA transporters to synaptic vesicles in the presynaptic terminal, and, as aconsequence, reduced uptake of DA into vesicles and an accumulation of DA in the cytosol.Formation of toxic DAQ intermediates has been shown to covalently modify �-synuclein andenhance �-synuclein toxicity through the kinetic stabilization of toxic prefibrillar aggregates.

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Colocalization of Ypt1p with �-synuclein suggests that

�-synuclein toxicity may involve the sequestration of

proteins that play a critical role in the ER-to-Golgi transport.

disruption of the secretory pathway. Furtherbiochemical and biophysical characteriza-tion of the �-synuclein aggregates couldprovide important insights into the mecha-nism of �-synuclein toxicity in yeast. Whether�-synuclein has a physiological role inER-to-Golgi trafficking or transport of syn-aptic vesicles remains to be determined.

�-Synuclein-Induced Disruption of ER-to-Golgi Transport. Given the essential roleof the Golgi apparatus in the processing andtargeting of proteins through the secretorypathway, any disruption at this level is likelyto have detrimental consequences for thefunction of the cell. Furthermore, a delicatebalance between anterograde and retro-grade membrane traffic through the Golgi iscritical to avoid its fragmentation. A linkbetween �-synuclein aggregation and Golgifragmentation is supported by previous find-ings demonstrating that formation of�-synuclein aggregates, particularly prefi-brillar aggregates that precede LB formation(Figure 2, panel a), causes fragmentation ofthe Golgi in cellular models of synucleinopa-thies (12) as well as in nigral neurons of PDpatients (13). Further studies are required toelucidate the exact mechanisms by which�-synuclein aggregation disrupts ER-to-Golgitransport and cause Golgi fragmentation.

Defective Vesicular Trafficking and DANeurons. Although �-synuclein is an abun-dant protein in different parts of the brain(up to 1% of total proteins), �-synucleinaggregation in PD occurs primarily in DAneurons of the SN. The wide distribution of�-synuclein in the brain suggests that�-synuclein on its own cannot explain theselective degeneration of DA neurons in PD.

Overexpression of human �-synuclein intransgenic flies (14) or specifically in the SNof rats (15) and primates (16) results inselective DA neuronal death and in the for-mation of �-synuclein-containing inclusions.Overexpression of PD-linked �-synucleinmutations in human mesencephalic cell linesleads to an impaired storage and secretion ofDA, causing an increase in cytosolic DA and

enhanced oxidative stress (17, 18). Xu et al.(19) reported that blocking DA synthesis incultured DA neurons prevents �-synucleintoxicity, consistent with toxicity being medi-ated by interactions between the two mol-ecules. These observations are consistentwith the known hypersensitivity of DAneurons that express high levels of cytoplas-mic DA (e.g., the SN rather than the ventraltegmental area) to cell death in PD.

The selective vulnerability of DA neuronsof the SN to �-synuclein toxicity in PD maybe related to the toxicity and increased con-centration of cytoplasmic DA in these cells;this suggests that improper packaging,secretion, and/or oxidation of DA mightexplain the selective degeneration of DAneurons (17, 18). DA oxidation and forma-tion of DA orthoquinone (DAQ) in vitrocovalently modifies �-synuclein and resultsin kinetic stabilization of toxic �-synucleinaggregates (20). Therefore, the simulta-neous contribution of several factors, includ-ing �-synuclein oligomerization, DA metabo-lism, and oxidative stress, might be requiredfor selective degeneration of DA in the SN ofPD brains. Disruption of vesicular traffickingis likely to hasten cell death by simulta-neously increasing the levels of cytosolic DAand enhancing �-synuclein aggregation.

The findings by Cooper and colleaguesoffer new insight into the mechanisms bywhich �-synuclein overexpression and/oraggregation interferes with normal functionand viability of neurons and reveal newtargets for therapeutic intervention in PDand related synucleinopathies. In additionto being a good model with which to studygenetic diseases, the yeast system is alsodemonstrated by these researchers to offeran excellent platform on which to screen fordruglike molecules as modifiers of �-synu-clein function(s), aggregation, and toxicity.The identification of specific pharmacologi-cal agents that suppress �-synuclein-inducedER-to-Golgi trafficking defects and prevent orreverse neurodegeneration in mouse modelsof PD as well as in clinical studies is the ulti-

mate proof of the therapeutic potential ofthese findings.

Acknowledgment: We thank Ruth Luthi-Carterfor critical reading of the article.

REFERENCES1. Cohen, F. E., and Kelly, J. W. (2003) Therapeutic ap-

proaches to protein-misfolding diseases, Nature426, 905–909.

2. Lee, M. C., Miller, E. A., Goldberg, J., Orci, L., andSchekman, R. (2004) Bi-directional protein trans-port between the ER and Golgi, Annu. Rev. Cell Dev.Biol. 20, 87–123.

3. Ross, C. A., and Poirier, M. A. (2005) Opinion: Whatis the role of protein aggregation in neurodegenera-tion? Nat. Rev. Mol. Cell Biol. 6, 891–898.

4. Cooper, A. A., Gitler, A. D., Cashikar, A., Haynes,C. M., Hill, K. J., Bhullar, B., Liu, K., Xu, K., Strath-earn, K. E., Liu, F., Cao, S., Caldwell, K. A., Caldwell,G. A., Marsischky, G., Kolodner, R. D., Labaer, J.,Rochet, J. C., Bonini, N. M., and Lindquist, S. (2006)Alpha-synuclein blocks ER-Golgi traffic and Rab1rescues neuron loss in Parkinson’s models, Science313, 324–328.

5. Outeiro, T. F., and Lindquist, S. (2003) Yeast cellsprovide insight into alpha-synuclein biology andpathobiology, Science 302, 1772–1775.

6. Polymeropoulos, M. H., Lavedan, C., Leroy, E., Ide,S. E., Dehejia, A., Dutra, A., Pike, B., Root, H., Ruben-stein, J., Boyer, R., Stenroos, E. S., Chan-drasekharappa, S., Athanassiadou, A., Papapetro-poulos, T., Johnson, W. G., Lazzarini, A. M., Duvoi-sin, R. C., Di Iorio, G., Golbe, L. I., and Nussbaum,R. L. (1997) Mutation in the alpha-synuclein geneidentified in families with Parkinson’s disease, Sci-ence 276, 2045–2047.

7. Kruger, R., Kuhn, W., Muller, T., Woitalla, D., Grae-ber, M., Kosel, S., Przuntek, H., Epplen, J. T., Schols,L., and Riess, O. (1998) Ala30Pro mutation in thegene encoding alpha-synuclein in Parkinson’s dis-ease, Nat. Genet. 18, 106–108.

8. Singleton, A. B., 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,M. R., Muenter, M., Baptista, M., Miller, D., Blan-cato, J., Hardy, J., and Gwinn-Hardy, K. (2003) Alpha-synuclein locus triplication causes Parkinson’s dis-ease, Science 302, 841

9. Zarranz, J. J., Alegre, J., Gomez-Esteban, J. C., Lez-cano, E., Ros, R., Ampuero, I., Vidal, L., Hoenicka, J.,Rodriguez, O., Atares, B., Llorens, V., Tortosa, E. G.,Del Ser, T., Munoz, D. G., and De Yebenes, J. G.(2004) The new mutation, E46K, of alpha-synuclein causes parkinson and Lewy body demen-tia, Ann. Neurol. 55, 164–173.

10. Trojanowski, J. Q., and Lee, V. M. (2003) Parkin-son’s disease and related alpha-synucleinopathiesare brain amyloidoses, Ann. N.Y. Acad. Sci. 991,107–110.

11. Zerial, M., and McBride, H. (2001) Rab proteins asmembrane organizers, Nat. Rev. Mol. Cell Biol. 2,107–117.

www.acschemicalbiology.org VOL.1 NO.7 • 420–424 • 2006 423

Point ofVIEW

12. Gosavi, N., Lee, H. J., Lee, J. S., Patel, S., and Lee, S. J.(2002) Golgi fragmentation occurs in the cells withprefibrillar alpha-synuclein aggregates and precedesthe formation of fibrillar inclusion, J. Biol. Chem.277, 48984–48992.

13. Fujita, Y., Ohama, E., Takatama, M., Al-Sarraj, S., andOkamoto, K. (2006) Fragmentation of Golgi appara-tus of nigral neurons with alpha-synuclein-positiveinclusions in patients with Parkinson’s disease, ActaNeuropathol. DOI: 10.1007/s00401-006-0114-4.

14. Feany, M. B., and Bender, W. W. (2000) A Drosophi-la model of Parkinson’s disease. Nature 404,394–398.

15. Lo Bianco, C., Ridet, J. L., Schneider, B. L., Deglon,N., and Aebischer, P. (2002) Alpha-synucleinopathyand selective dopaminergic neuron loss in a ratlentiviral-based model of Parkinson’s disease, Proc.Natl. Acad. Sci. U.S.A. 99, 10813–10818.

16. Kirik, D., Annett, L. E., Burger, C., Muzyczka, N.,Mandel, R. J., and Bjorklund, A. (2003) Nigrostriatalalpha-synucleinopathy induced by viral vector-mediated overexpression of human alpha-synuclein: a new primate model of Parkinson’s dis-ease, Proc. Natl. Acad. Sci. U.S.A. 100, 2884–2889.

17. Lotharius, J., and Brundin, P. (2002) Pathogenesisof Parkinson’s disease: dopamine, vesicles andalpha-synuclein, Nat. Rev. Neurosci. 3, 932–942.

18. Lotharius, J., and Brundin, P. (2002) Impaired do-pamine storage resulting from alpha-synuclein mu-tations may contribute to the pathogenesis of Par-kinson’s disease, Hum. Mol. Genet. 11, 2395–2407.

19. Xu, J., Kao, S. Y., Lee, F. J., Song, W., Jin, L. W., andYankner, B. A. (2002) Dopamine-dependent neuro-toxicity of alpha-synuclein: a mechanism for se-lective neurodegeneration in Parkinson disease, Nat.Med. 8, 600–606.

20. Conway, K. A., Rochet, J. C., Bieganski, R. M., andLansbury, P. T., Jr. (2001) Kinetic stabilization of thealpha-synuclein protofibril by a dopamine-alpha-synuclein adduct, Science 294, 1346–1349.

424 VOL.1 NO.7 • 420–424 • 2006 www.acschemicalbiology.orgLASHUEL AND HIRLING