- Email: [email protected]
Does autophagy worsen or improve the survival of dopaminergic neurons?
Parkinsonism and Related Disorders 15S (2009) S24–S27
Does autophagy worsen or improve the survival of dopaminergic neurons? Livia Pasquali a , Stefano Ruggieri b , Luigi Murri a , Antonio Paparelli c , Francesco Fornai b,c, * a
Department of Neuroscience, Clinical Neurology, University of Pisa, Pisa, Italy Laboratory of Neurobiology of Movement Disorders INM, IRCCS Neuromed, Pozzilli, Isernia, Italy c Department of Human Morphology and Applied Biology, University of Pisa, Pisa, Italy b
A R T I C L E
I N F O
Keywords: Parkinson’s disease Methamphetamine Misfolded proteins Mitochondria Inherited parkinsonism Parkin Alpha-synuclein Autophagoproteasome Phagophore Substantia nigra Cell survival
A B S T R A C T In eukaryotic cells intracellular components are mainly degraded by autophagy and the ubiquitin-proteasome system. Autophagy is more flexible compared with the ubiquitin-proteasome system and it is involved in the degradation of long-lived proteins and organelles, such as mitochondria, which cannot be degraded by the ubiquitin-proteasome. Although autophagy is able to compensate for ubiquitin-proteasome dysfunction, the opposite does not occur. Autophagy is frequently involved in neurodegeneration; however, there is no consensus on its role in cell survival, as it can be either neuroprotective or neurotoxic. With respect to dopaminergic neurons, there is evidence that autophagy occurs during damage to substantia nigra neurons such as in Parkinson’s disease. Moreover, a variety of inherited forms of Parkinson’s disease are characterized by mutated proteins that belong to the autophagy pathway. Inhibition of autophagy precipitates dopaminergic cell death, whereas autophagy activation rescues the death of nigral dopaminergic neurons induced by proteasome inhibitors. Taken together, this evidence suggests that autophagy improves the survival of dopaminergic cells. © 2009 Elsevier Ltd. All rights reserved.
1. Proteasomes and autophagy are both involved in protein degradation Degradation of intracellular components in eukaryotic cells occurs through two different systems: autophagy (ATG) and the ubiquitin-proteasome (UP) system (Fig. 1). The UP system mainly serves to degrade short-lived misfolded proteins [1], which are targeted by the polyubiquitin conjugation cascade and further degraded by the proteolytic activities placed within the proteasome core. In fact, the proteasome is composed of a cylindrical 20 S core particle and two 19 S (PA700) regulatory particles at each end of the core cylinder. To enter the proteasome the protein substrates must be unfolded, since the pore located in the proteasome core is small. The 19 S particle regulates the entry and degradation of proteins in the proteolytic cavity of the core particle by recognizing and unravelling the polyubiquitin-conjugated substrates and perhaps by controlling the opening of the core particle. The 19 S particle binds to one or both ends of the 20 S core, where proteins transported by adenosine triphosphatases (ATPases) of the 19 S particle undergo proteolysis. ATG is involved in the degradation of long-lived proteins, protein aggregates and organelles, such as mitochondria, which are
* Correspondence to: Francesco Fornai, Department of Human Morphology and Applied Biology, Via Roma 55, I-56100 Pisa, Italy. Tel.: +39 050 2218601; fax: +39 050 2218606. E-mail address: [email protected] (F. Fornai). 1353-8020/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.
delivered to lysosomes for digestion [2]. There are three different types of ATG: 1. Macroautophagy, simply referred to as ATG, involves the “in bulk” degradation of complete regions of the cytosol, which contains long-lived proteins or organelles. ATG starts from discrete areas of the trans-Golgi network, which enucleates to form a phagophore. This piece of membrane forms a vacuole, which further develops a double membrane, the autophagosome, or immature autophagic vacuole (AVi). Following the merger of the endosomal compartment with the AV, an intermediate organelle – the amphisome – is formed. Although the AV itself is able to merge directly with lysosomes, a more efficient ATG is produced by the merging of the amphisome and lysosome to form a mature autophagolysosome, which provides all the enzymes necessary for protein/organelle degradation. 2. Microautophagy, in which lysosomes invaginate and directly sequester cytosolic components, without segregation into a phagosome. 3. Chaperone-mediated autophagy (CMA), where cytosolic proteins are tagged by a specific pentapeptide signalling sequence and so are identified for lysosomal degradation. ATG and CMA degrade a variety of misfolded proteins, including alpha-synuclein [3] and altered mitochondria [4]. Baseline ATG is normally active in the cell but it is enhanced when nutrient and energy depletion occurs. On the other hand, such activation also follows various insults, thus allowing the degradation of mutated or aggregated proteins, and the removal of damaged organelles such as mitochondria [4]. Although ATG is frequently involved
L. Pasquali et al. / Parkinsonism and Related Disorders 15S (2009) S24–S27
S25
Fig. 1. Autophagy and genetic Parkinson’s disease. The simplified cartoon shows the convergence between the ubiquitin-proteasome system and autophagy and their interaction with specific proteins that are mutated in inherited parkinsonism. The ubiquitin-proteasome system degrades the substrate depending on its size and conformation. In fact, the proteasome is composed of a cylindrical 20 S core particle and two 19 S (PA700) regulatory particles at each end of the core cylinder. To enter the proteasome the protein substrates must be unfolded, since the pore located in the proteasome core is small. Autophagy is based on intralysosomal degradation of substrates. Macroautophagy derives from the fusion between the endosome and autophagosome. The autophagosome fuses with the multivesicular body, derived from the endosome, to build up the amphisome. Then amphisome and lysosome fuse to form the autophagolysosome. The ubiquitin-proteasome system and autophagy might converge in the autophagoproteasome, a multilamellar peripheral structure that contains alpha-synuclein as well as autophagy and ubiquitin-proteasome components. The proteins that are mutated in familial Parkinson’s disease are connected to autophagy. These proteins regulate autophagy at different steps. Abbreviations: ATPase, adenosine triphosphatase; CMA, chaperone-mediated autophagy.
in neurodegeneration, its significance in cell survival remains debated as it has been shown to be both neuroprotective [5,6] and neurotoxic [7]. Regarding the role of ATG in the survival of dopaminergic neurons, it is critical to establish whether ATG occurs during damage to dopaminergic neurons and, specifically, whether ATG is recruited consistently in Parkinson’s disease (PD). If this is the case, then inherited forms of PD are expected to be characterized by mutated proteins that belong to ATG. If there was convincing evidence for these issues, the critical question of whether ATG plays a beneficial or detrimental role could be tentatively addressed by using a mechanistic approach. Of the clearing systems, ATG is more flexible compared with the UP system. In fact, ATG is able to compensate for UP dysfunction, whereas the opposite does not occur. For instance, in Drosophila ATG rescues toxicity caused by UP inhibition as demonstrated both using rapamycin (a pharmacological ATG activator) or by overexpressing HDAC6 (genetic enhancement of ATG) [8]. With respect to the dopamine system, a recent study by Pan et al. [9] demonstrated that ATG activation reduced apoptosis in PC12 cells treated with a UP inhibitor [9]. This protection was partially reversed by the ATG inhibitor 3-methyladenine and it was reproduced in vivo, where ATG rescued the loss of nigral dopaminergic neurons and striatal dopamine levels induced by UP inhibition [9]. These data show that ATG enhancement exerts a protective role against neuronal damage during UP dysfunction and that ATG compensates for the UP system as emphasized by Rubinsztein [10]. Such an interaction between ATG and the UP system is not reciprocal since long-term ATG inhibition activates
the UP system, but this system is not able to compensate for the loss of ATG activity. Very recent studies by Korolchuk et al. [11,12] have clearly shown that long-term ATG inhibition impairs the UP system, which does not compensate. 2. Convergence between ATG and the UP system: the autophagoproteasome (Fig. 1) Although ATG and UP pathways have traditionally been considered as distinct, they share common sites of activity and converge functionally. In fact, they both require ubiquitination of their substrate [13]: with either mono- or polyubiquitination for ATG, and only polyubiquitination for UP. ATG and UP converge at the ultrastructural level, within a specific organelle observed in diseased dopamine-containing neurons [14]. This organelle can be termed an “autophagoproteasome” as it contains both UP [15] and ATG [16,17] components (Fig. 1). The autophagoproteasome is not present in the cell in baseline conditions, and it is generated following increased production of misfolded proteins. The autophagoproteasome consists of a multilamellar peripheral structure that surrounds altered mitochondria and contains alpha-synuclein as well as components of ATG (such as beclin and LC3-II) and UP (the 19 S and the 20 S proteasome subunit) [18] (Fig. 1). ATG components were not originally found in this organelle using transmission electron microscopy [15], and only further studies indicated the co-existence of ATG and UP proteins [16–18]. Taken together, this demonstrates that ATG and UP cooperate in the removal of proteins/organelles and they converge both functionally and structurally, with ATG being more prevalent than UP activity.
S26
L. Pasquali et al. / Parkinsonism and Related Disorders 15S (2009) S24–S27
3. Is there evidence that ATG occurs during damage to dopaminergic neurons? When examining dopaminergic neurons after exposure to toxic compounds, ATG is massively recruited. This is described very clearly for methamphetamine (METH) exposure both in vitro [15, 18,19] and in vivo [15]. Following METH, an extraordinary amount of ATG vacuoles occur, which express a number of ATG-related proteins [18,19]. Electron microscopy has given strong confirmation to these findings, providing evidence for double-layered subcellular structures within dopaminergic neurons both in vitro and in vivo in the substantia nigra following METH exposure [15]. 4. Is there pathological evidence that ATG is recruited in PD? To our knowledge, the first demonstration that ATG occurs as a neuropathological marker within the substantia nigra of PD patients was obtained more than a decade ago by Anglade et al. [20]. These investigators found the coexistence of AVs and apoptotic cells, which led them to suggest that dual neuronal death occurred in the process of PD: apoptosis and autophagic degeneration within melanized neurons of the human substantia nigra. The neurons undergoing ATG showed condensation of chromatin, moderate vacuolation of endoplasmic reticulum, and lysosome-like vacuoles containing cytoplasmic material. The existence of dual neuronal death suggests the occurrence of both ATG and apoptosis at different time intervals during the termination process of dopaminergic cells, with apoptosis as the final stage. This is in keeping with evidence that in dopaminergic cells dysfunctional ATG triggers apoptosis [18].
are related to parkin gene (PARK2) mutations. PINK1 mutations are the second most frequent cause of autosomal recessive PD (about 5% of cases). Interestingly, patients with parkin mutations display a phenotype similar to those with PINK mutations. In fact, recent studies have shown that PINK1 and parkin participate in the regulation of mitochondrial morphology [27]. PINK1 localizes in the mitochondria [28] and is involved in mitochondrial dynamics. Specifically, overexpression of PINK1 promotes fission, whereas inhibition of PINK1 leads to mitochondrial fusion [27]. Null mutations for PINK1 produce functional and structural impairment of mitochondria such as fragmented cristae, loss of the outer membrane and ATP depletion [29]. Parkin is primarily a cytosolic ubiquitin E3 ligase that also localizes in the mitochondria [30]. Loss of parkin is associated with mitochondrial alterations. PINK1 acts upstream of parkin in regulating mitochondrial integrity, and parkin overexpression suppresses the PINK1 null phenotype, such as mitochondrial defects. 5.3. PINK, parkin and ATG Recent studies suggest a role of parkin and PINK1 in regulating ATG of the mitochondria (mitophagy). In cell cultures, parkin is selectively recruited by depolarized mitochondria. These are then selectively removed by parkin, which is involved in the formation of the autophagosome, thereby inducing ATG. Parkin as well as PINK overexpression restores mitochondrial morphology and reverses cell death. These findings suggest that PINK1 and parkin cooperate in modifying mitochondrial morphology and inducing mitophagy. 5.4. UCH-L1 (PARK 5)
5. Are proteins mutated in inherited PD connected to ATG? (Fig. 1) This paradigm is represented by alpha-synuclein, which is the hallmark of Lewy bodies. Previous findings indicate that alphasynuclein (coded by the PARK1 and PARK4 loci) is a preferential UP substrate, while it can be metabolized by UP and ATG [3]. It is known that enzymes responsible for genetic PD, such as parkin (coded by the PARK2 locus), or ubiquitin carboxy-terminal hydrolase type 1 (UCH-L1) (coded by the PARK5 locus) are not solely related to a deficiency of the UP system, but are primarily involved in early steps of the ATG pathway. Moreover, additional mutations in PD are involved in ATG but not in the UP system. 5.1. Alpha-synuclein (PARK 1, PARK 4) Missense mutations [21] and multiplications [22] are responsible for PARK1 and PARK4 autosomal dominant PD, respectively. Alphasynuclein is also linked to sporadic PD, where it is abundant in Lewy bodies in the phosphorylated or monoubiquitinated and nitrated form. Initially, it was reported that alpha-synuclein was degraded by the proteasome. Now we know, however, that alpha-synuclein can be degraded by ATG and CMA [23]. When alpha-synuclein is degraded by CMA, it binds to the lysosomal-associated membrane protein type 2A (LAMP-2A), a CMA receptor at the lysosomal membrane. When alpha-synuclein is mutated, it blocks the function of CMA. 5.2. Parkin (PARK2), PINK1 (PARK 6) and mitochondrial fission/fusion Mutations in the parkin (PARK2) [24], PINK1 (PARK6) (phosphatase and tensin [PTEN] homologue-induced putative kinase 1) [25] and DJ-1 (PARK7) [26] genes are responsible for early-onset autosomal recessive PD. About 50% of autosomal recessive PD cases
UCH-L1 is expressed abundantly in brain neurons, where it has several activities, including the role of ubiquitin ligase for monoubiquitinated alpha-synuclein. The role of the UCH-L1 gene in determining PD was first reported in a German family with autosomal dominant PD due to a missense mutation in the UCH-L1 gene [31]. UCH-L1, which is a component of Lewy bodies, interacts with the CMA pathway at the level of the lysosomal receptor for CMA, LAMP-2A, and Hsc70 and Hsp90 [32]. These interactions are aberrant in the UCH-L1 mutation, which leads to an increase in alpha-synuclein. These observations led to the hypothesis that the mechanism underlying PD associated with UCH-L1 mutation consists of the inhibition of alpha-synuclein degradation by CMA. Remarkably, structural changes in UCH-L1 also occur in sporadic PD. In the brains of these patients, the interaction of UCH-L1 with LAMP-2A, Hsc70 and Hsp90 is altered, leading to accumulation of CMA substrates, including alpha-synuclein [32]. 5.5. DJ-1 (PARK7) Of its several physiological effects, DJ-1, which is responsible for inherited PD when undergoing a point mutation, triggers the processing of ATG proteins, including LC3 [33]. 5.6. LRRK2/dardarin (PARK8) Mutations in the LRRK2 gene produce autosomal dominant PD (PARK8) [34]. Recently, it has been demonstrated that LRRK2 is involved in ATG-mediated neurite remodelling [35]. Moreover, LRRK2-transfected cells feature a considerable increase in number and size of AVs in both neuritic and somatic compartments, as demonstrated by electron microscopy and LC3 immunofluorescence, and Western blot analysis. Pharmacological treatment with rapamycin of LRRK2-transfected cells causes a further increase in neuritic AVs.
L. Pasquali et al. / Parkinsonism and Related Disorders 15S (2009) S24–S27
5.7. ATP13A2 (PARK9) Loss of functional mutations in the ATP13A2 gene have been linked to Kufor-Rakeb syndrome (PARK9) [36]. ATP13A2 is a predominantly neuronal P-type ATPase gene, which encodes lysosomal ATPase [36]. The involvement of lysosomes was originally described in experimental parkinsonism [37]. This is further confirmed by the association between Gaucher disease and PD. In fact, this lysosomal lipid storage disease is a risk factor for parkinsonism [38]. The clinical signs in these PD patients are different, including early onset, as well as marked olfactory dysfunction, hallucinations and symptoms of cognitive decline or dementia [38].
[17]
[18]
[19]
[20]
[21]
5.8. Synphilin (PARK11) [22]
Synphilin is a susceptibility factor for PD [39]; the protein was identified in the core of Lewy bodies of PD patients and interacts with parkin and alpha-synuclein in the formation of Lewy-bodylike cytosolic inclusions. One of the several physiological roles of synphilin is to prime aggresomes for autophagic clearance [40].
[23]
[24]
Conflict of interest
[25]
Each author declares no conflict of interest related to the present manuscript.
[26]
References
[27]
[1] Ciechanover A. The ubiquitin proteolytic system: from a vague idea, through basic mechanisms, and onto human diseases and drug targeting. Neurology 2006;66(2 Suppl 1):S7–19. [2] Mariño G, López-Otín C. Autophagy: molecular mechanisms, physiological functions and relevance in human pathology. Cell Mol Life Sci 2004;61:1439– 54. [3] Webb JL, Ravikumar B, Atkins J, Skepper JN, Rubinsztein DC. AlphaSynuclein is degraded by both autophagy and the proteasome. J Biol Chem 2003;278:25009–13. [4] Klionsky DJ, Emr SD. Autophagy as a regulated pathway of cellular degradation. Science 2000;290:1717–21. [5] Hara T, Nakamura K, Matsui M, Yamamoto A, Nakahara Y, Suzuki-Migishima R, et al. Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice. Nature 2006;441:885–9. [6] Komatsu M, Waguri S, Chiba T, Murata S, Iwata J, Tanida I, et al. Loss of autophagy in the central nervous system causes neurodegeneration in mice. Nature 2006;441(7095):880–4. [7] Tsujimoto Y, Shimizu S. Another way to die: autophagic programmed cell death. Cell Death Differ 2005;12 Suppl 2:1528–34. [8] Pandey UB, Nie Z, Batlevi Y, McCray BA, Ritson GP, Nedelsky NB, et al. HDAC6 rescues neurodegeneration and provides an essential link between autophagy and the UPS. Nature 2007;447:859–63. [9] Pan T, Kondo S, Zhu W, Xie W, Jankovic J, Le W. Neuroprotection of rapamycin in lactacystin-induced neurodegeneration via autophagy enhancement. Neurobiol Dis 2008;32:16–25. [10] Rubinsztein DC. Autophagy induction rescues toxicity mediated by proteasome inhibition. Neuron 2007;54:854–6. [11] Korolchuk VI, Mansilla A, Menzies FM, Rubinsztein DC. Autophagy inhibition compromises degradation of ubiquitin-proteasome pathway substrates. Mol Cell 2009;33:517–27. [12] Korolchuk VI, Menzies FM, Rubinsztein DC. A novel link between autophagy and the ubiquitin-proteasome system. Autophagy 2009;5:862–3. [13] Kim PK, Hailey DW, Mullen RT, Lippincott-Schwartz J. Ubiquitin signals autophagic degradation of cytosolic proteins and peroxisomes. Proc Natl Acad Sci USA 2008;105:20567–74. [14] Ferrucci M, Pasquali L, Ruggieri S, Paparelli A, Fornai F. Alpha-synuclein and autophagy as common steps in neurodegeneration. Parkinsonism Relat Disord 2008;14 Suppl 2:S180–4. [15] Fornai F, Lenzi P, Gesi M, Soldani P, Ferrucci M, Lazzeri G, et al. Methamphetamine produces neuronal inclusions in the nigrostriatal system and in PC12 cells. J Neurochem 2004;88:114–23. [16] Lazzeri G, Lenzi P, Busceti CL, Ferrucci M, Falleni A, Bruno V, et al.
[28] [29]
[30]
[31] [32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
S27
Mechanisms involved in the formation of dopamine-induced intracellular bodies within striatal neurons. J Neurochem 2007;101:1414–27. Lenzi P, Fulceri F, Lazzeri G, Casini A, Ruggieri S, Paparelli A, et al. Analysis of single, purified inclusions as a novel approach to understand methamphetamine neurotoxicity. Ann N Y Acad Sci 2008;1139:186–90. Castino R, Lazzeri G, Lenzi P, Bellio N, Follo C, Ferrucci M, et al. Suppression of autophagy precipitates neuronal cell death following low doses of methamphetamine. J Neurochem 2008;106:1426–39. Larsen KE, Fon EA, Hastings TG, Edwards RH, Sulzer D. Methamphetamineinduced degeneration of dopaminergic neurons involves autophagy and upregulation of dopamine synthesis. J Neurosci 2002;22:895160. Anglade P, Vyas S, Hirsch EC, Agid Y. Apoptosis in dopaminergic neurons of the human substantia nigra during normal aging. Histol Histopathol 1997;12:603–10. Polymeropoulos MH, Lavedan C, Leroy E, Ide SE, Dehejia A, Dutra A, et al. Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease. Science 1997;276:2045–7. Singleton AB, Farrer M, Johnson J, Singleton A, Hague S, Kachergus J, et al. alpha-Synuclein locus triplication causes Parkinson’s disease. Science 2003;302:841. Cuervo AM, Stefanis L, Fredenburg R, Lansbury PT, Sulzer D. Impaired degradation of mutant alpha-synuclein by chaperone-mediated autophagy. Science 2004;305:1292–5. Kitada T, Asakawa S, Hattori N, Matsumine H, Yamamura Y, Minoshima S, et al. Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature 1998;392:605–8. Valente EM, Abou-Sleiman PM, Caputo V, Muqit MM, Harvey K, Gispert S, et al. Hereditary early-onset Parkinson’s disease caused by mutations in PINK1. Science 2004;304:1158–60. Bonifati V, Breedveld GJ, Squitieri F, Vanacore N, Brustenghi P, Harhangi BS, et al. Localization of autosomal recessive early-onset parkinsonism to chromosome 1p36 (PARK7) in an independent dataset. Ann Neurol 2002;51:253–6. Yang Y, Ouyang Y, Yang L, Beal MF, McQuibban A, Vogel H, et al. Pink1 regulates mitochondrial dynamics through interaction with the fission/fusion machinery. Proc Natl Acad Sci USA 2008;105:7070–5. Lin W, Kang UJ. Characterization of PINK1 processing, stability, and subcellular localization. J Neurochem 2008;106:464–74. Yang Y, Gehrke S, Imai Y, Huang Z, Ouyang Y, Wang JW, et al. Mitochondrial pathology and muscle and dopaminergic neuron degeneration caused by inactivation of Drosophila Pink1 is rescued by Parkin. Proc Natl Acad Sci USA 2006;103:10793–8. Shimura H, Hattori N, Kubo S, Yoshikawa M, Kitada T, Matsumine H et al. Immunohistochemical and subcellular localization of Parkin protein: absence of protein in autosomal recessive juvenile parkinsonism patients. Ann Neurol 1999;45:668–72. Leroy E, Boyer R, Auburger G, Leube B, Ulm G, Mezey E, et al. The ubiquitin pathway in Parkinson’s disease. Nature 1998;395:451–2. Kabuta T, Wada K. Insights into links between familial and sporadic Parkinson’s disease: physical relationship between UCH-L1 variants and chaperonemediated autophagy. Autophagy 2008;4:827–9. González-Polo R, Niso-Santano M, Morán JM, Ortiz-Ortiz MA, Bravo-San Pedro JM, Soler G, et al. Silencing DJ-1 reveals its contribution in paraquat-induced autophagy. J Neurochem 2009;109:889–98. Zimprich A, Biskup S, Leitner P, Lichtner P, Farrer M, Lincoln S, et al. Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology. Neuron 2004;44:601–7. Plowey ED, Cherra SJ 3rd, Liu YJ, Chu CT. Role of autophagy in G2019S-LRRK2associated neurite shortening in differentiated SH-SY5Y cells. J Neurochem 2008;105:1048–56. Ramirez A, Heimbach A, Gründemann J, Stiller B, Hampshire D, Cid LP, et al. Hereditary parkinsonism with dementia is caused by mutations in ATP13A2, encoding a lysosomal type 5 P-type ATPase. Nat Genet 2006;38:1184–91. Stefanis L, Larsen KE, Rideout HJ, Sulzer D, Greene LA. Expression of A53T mutant but not wild-type alpha-synuclein in PC12 cells induces alterations of the ubiquitin-dependent degradation system, loss of dopamine release, and autophagic cell death. J Neurosci 2001;21:9549–60. Goker-Alpan O, Lopez G, Vithayathil J, Davis J, Hallett M, Sidransky E. The spectrum of parkinsonian manifestations associated with glucocerebrosidase mutations. Arch Neurol 2008;65:1353–7. Engelender S, Kaminsky Z, Guo X, Sharp AH, Amaravi RK, Kleiderlein JJ, et al. Synphilin-1 associates with alpha-synuclein and promotes the formation of cytosolic inclusions. Nat Genet 1999;22:110–4. Zaarur N, Meriin AB, Gabai VL, Sherman MY. Triggering aggresome formation. Dissecting aggresome-targeting and aggregation signals in synphilin 1. J Biol Chem 2008;283:27575–84.
Does autophagy worsen or improve the survival of dopaminergic neurons? Livia Pasquali a , Stefano Ruggieri b , Luigi Murri a , Antonio Paparelli c , Francesco Fornai b,c, * a
Department of Neuroscience, Clinical Neurology, University of Pisa, Pisa, Italy Laboratory of Neurobiology of Movement Disorders INM, IRCCS Neuromed, Pozzilli, Isernia, Italy c Department of Human Morphology and Applied Biology, University of Pisa, Pisa, Italy b
A R T I C L E
I N F O
Keywords: Parkinson’s disease Methamphetamine Misfolded proteins Mitochondria Inherited parkinsonism Parkin Alpha-synuclein Autophagoproteasome Phagophore Substantia nigra Cell survival
A B S T R A C T In eukaryotic cells intracellular components are mainly degraded by autophagy and the ubiquitin-proteasome system. Autophagy is more flexible compared with the ubiquitin-proteasome system and it is involved in the degradation of long-lived proteins and organelles, such as mitochondria, which cannot be degraded by the ubiquitin-proteasome. Although autophagy is able to compensate for ubiquitin-proteasome dysfunction, the opposite does not occur. Autophagy is frequently involved in neurodegeneration; however, there is no consensus on its role in cell survival, as it can be either neuroprotective or neurotoxic. With respect to dopaminergic neurons, there is evidence that autophagy occurs during damage to substantia nigra neurons such as in Parkinson’s disease. Moreover, a variety of inherited forms of Parkinson’s disease are characterized by mutated proteins that belong to the autophagy pathway. Inhibition of autophagy precipitates dopaminergic cell death, whereas autophagy activation rescues the death of nigral dopaminergic neurons induced by proteasome inhibitors. Taken together, this evidence suggests that autophagy improves the survival of dopaminergic cells. © 2009 Elsevier Ltd. All rights reserved.
1. Proteasomes and autophagy are both involved in protein degradation Degradation of intracellular components in eukaryotic cells occurs through two different systems: autophagy (ATG) and the ubiquitin-proteasome (UP) system (Fig. 1). The UP system mainly serves to degrade short-lived misfolded proteins [1], which are targeted by the polyubiquitin conjugation cascade and further degraded by the proteolytic activities placed within the proteasome core. In fact, the proteasome is composed of a cylindrical 20 S core particle and two 19 S (PA700) regulatory particles at each end of the core cylinder. To enter the proteasome the protein substrates must be unfolded, since the pore located in the proteasome core is small. The 19 S particle regulates the entry and degradation of proteins in the proteolytic cavity of the core particle by recognizing and unravelling the polyubiquitin-conjugated substrates and perhaps by controlling the opening of the core particle. The 19 S particle binds to one or both ends of the 20 S core, where proteins transported by adenosine triphosphatases (ATPases) of the 19 S particle undergo proteolysis. ATG is involved in the degradation of long-lived proteins, protein aggregates and organelles, such as mitochondria, which are
* Correspondence to: Francesco Fornai, Department of Human Morphology and Applied Biology, Via Roma 55, I-56100 Pisa, Italy. Tel.: +39 050 2218601; fax: +39 050 2218606. E-mail address: [email protected] (F. Fornai). 1353-8020/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.
delivered to lysosomes for digestion [2]. There are three different types of ATG: 1. Macroautophagy, simply referred to as ATG, involves the “in bulk” degradation of complete regions of the cytosol, which contains long-lived proteins or organelles. ATG starts from discrete areas of the trans-Golgi network, which enucleates to form a phagophore. This piece of membrane forms a vacuole, which further develops a double membrane, the autophagosome, or immature autophagic vacuole (AVi). Following the merger of the endosomal compartment with the AV, an intermediate organelle – the amphisome – is formed. Although the AV itself is able to merge directly with lysosomes, a more efficient ATG is produced by the merging of the amphisome and lysosome to form a mature autophagolysosome, which provides all the enzymes necessary for protein/organelle degradation. 2. Microautophagy, in which lysosomes invaginate and directly sequester cytosolic components, without segregation into a phagosome. 3. Chaperone-mediated autophagy (CMA), where cytosolic proteins are tagged by a specific pentapeptide signalling sequence and so are identified for lysosomal degradation. ATG and CMA degrade a variety of misfolded proteins, including alpha-synuclein [3] and altered mitochondria [4]. Baseline ATG is normally active in the cell but it is enhanced when nutrient and energy depletion occurs. On the other hand, such activation also follows various insults, thus allowing the degradation of mutated or aggregated proteins, and the removal of damaged organelles such as mitochondria [4]. Although ATG is frequently involved
L. Pasquali et al. / Parkinsonism and Related Disorders 15S (2009) S24–S27
S25
Fig. 1. Autophagy and genetic Parkinson’s disease. The simplified cartoon shows the convergence between the ubiquitin-proteasome system and autophagy and their interaction with specific proteins that are mutated in inherited parkinsonism. The ubiquitin-proteasome system degrades the substrate depending on its size and conformation. In fact, the proteasome is composed of a cylindrical 20 S core particle and two 19 S (PA700) regulatory particles at each end of the core cylinder. To enter the proteasome the protein substrates must be unfolded, since the pore located in the proteasome core is small. Autophagy is based on intralysosomal degradation of substrates. Macroautophagy derives from the fusion between the endosome and autophagosome. The autophagosome fuses with the multivesicular body, derived from the endosome, to build up the amphisome. Then amphisome and lysosome fuse to form the autophagolysosome. The ubiquitin-proteasome system and autophagy might converge in the autophagoproteasome, a multilamellar peripheral structure that contains alpha-synuclein as well as autophagy and ubiquitin-proteasome components. The proteins that are mutated in familial Parkinson’s disease are connected to autophagy. These proteins regulate autophagy at different steps. Abbreviations: ATPase, adenosine triphosphatase; CMA, chaperone-mediated autophagy.
in neurodegeneration, its significance in cell survival remains debated as it has been shown to be both neuroprotective [5,6] and neurotoxic [7]. Regarding the role of ATG in the survival of dopaminergic neurons, it is critical to establish whether ATG occurs during damage to dopaminergic neurons and, specifically, whether ATG is recruited consistently in Parkinson’s disease (PD). If this is the case, then inherited forms of PD are expected to be characterized by mutated proteins that belong to ATG. If there was convincing evidence for these issues, the critical question of whether ATG plays a beneficial or detrimental role could be tentatively addressed by using a mechanistic approach. Of the clearing systems, ATG is more flexible compared with the UP system. In fact, ATG is able to compensate for UP dysfunction, whereas the opposite does not occur. For instance, in Drosophila ATG rescues toxicity caused by UP inhibition as demonstrated both using rapamycin (a pharmacological ATG activator) or by overexpressing HDAC6 (genetic enhancement of ATG) [8]. With respect to the dopamine system, a recent study by Pan et al. [9] demonstrated that ATG activation reduced apoptosis in PC12 cells treated with a UP inhibitor [9]. This protection was partially reversed by the ATG inhibitor 3-methyladenine and it was reproduced in vivo, where ATG rescued the loss of nigral dopaminergic neurons and striatal dopamine levels induced by UP inhibition [9]. These data show that ATG enhancement exerts a protective role against neuronal damage during UP dysfunction and that ATG compensates for the UP system as emphasized by Rubinsztein [10]. Such an interaction between ATG and the UP system is not reciprocal since long-term ATG inhibition activates
the UP system, but this system is not able to compensate for the loss of ATG activity. Very recent studies by Korolchuk et al. [11,12] have clearly shown that long-term ATG inhibition impairs the UP system, which does not compensate. 2. Convergence between ATG and the UP system: the autophagoproteasome (Fig. 1) Although ATG and UP pathways have traditionally been considered as distinct, they share common sites of activity and converge functionally. In fact, they both require ubiquitination of their substrate [13]: with either mono- or polyubiquitination for ATG, and only polyubiquitination for UP. ATG and UP converge at the ultrastructural level, within a specific organelle observed in diseased dopamine-containing neurons [14]. This organelle can be termed an “autophagoproteasome” as it contains both UP [15] and ATG [16,17] components (Fig. 1). The autophagoproteasome is not present in the cell in baseline conditions, and it is generated following increased production of misfolded proteins. The autophagoproteasome consists of a multilamellar peripheral structure that surrounds altered mitochondria and contains alpha-synuclein as well as components of ATG (such as beclin and LC3-II) and UP (the 19 S and the 20 S proteasome subunit) [18] (Fig. 1). ATG components were not originally found in this organelle using transmission electron microscopy [15], and only further studies indicated the co-existence of ATG and UP proteins [16–18]. Taken together, this demonstrates that ATG and UP cooperate in the removal of proteins/organelles and they converge both functionally and structurally, with ATG being more prevalent than UP activity.
S26
L. Pasquali et al. / Parkinsonism and Related Disorders 15S (2009) S24–S27
3. Is there evidence that ATG occurs during damage to dopaminergic neurons? When examining dopaminergic neurons after exposure to toxic compounds, ATG is massively recruited. This is described very clearly for methamphetamine (METH) exposure both in vitro [15, 18,19] and in vivo [15]. Following METH, an extraordinary amount of ATG vacuoles occur, which express a number of ATG-related proteins [18,19]. Electron microscopy has given strong confirmation to these findings, providing evidence for double-layered subcellular structures within dopaminergic neurons both in vitro and in vivo in the substantia nigra following METH exposure [15]. 4. Is there pathological evidence that ATG is recruited in PD? To our knowledge, the first demonstration that ATG occurs as a neuropathological marker within the substantia nigra of PD patients was obtained more than a decade ago by Anglade et al. [20]. These investigators found the coexistence of AVs and apoptotic cells, which led them to suggest that dual neuronal death occurred in the process of PD: apoptosis and autophagic degeneration within melanized neurons of the human substantia nigra. The neurons undergoing ATG showed condensation of chromatin, moderate vacuolation of endoplasmic reticulum, and lysosome-like vacuoles containing cytoplasmic material. The existence of dual neuronal death suggests the occurrence of both ATG and apoptosis at different time intervals during the termination process of dopaminergic cells, with apoptosis as the final stage. This is in keeping with evidence that in dopaminergic cells dysfunctional ATG triggers apoptosis [18].
are related to parkin gene (PARK2) mutations. PINK1 mutations are the second most frequent cause of autosomal recessive PD (about 5% of cases). Interestingly, patients with parkin mutations display a phenotype similar to those with PINK mutations. In fact, recent studies have shown that PINK1 and parkin participate in the regulation of mitochondrial morphology [27]. PINK1 localizes in the mitochondria [28] and is involved in mitochondrial dynamics. Specifically, overexpression of PINK1 promotes fission, whereas inhibition of PINK1 leads to mitochondrial fusion [27]. Null mutations for PINK1 produce functional and structural impairment of mitochondria such as fragmented cristae, loss of the outer membrane and ATP depletion [29]. Parkin is primarily a cytosolic ubiquitin E3 ligase that also localizes in the mitochondria [30]. Loss of parkin is associated with mitochondrial alterations. PINK1 acts upstream of parkin in regulating mitochondrial integrity, and parkin overexpression suppresses the PINK1 null phenotype, such as mitochondrial defects. 5.3. PINK, parkin and ATG Recent studies suggest a role of parkin and PINK1 in regulating ATG of the mitochondria (mitophagy). In cell cultures, parkin is selectively recruited by depolarized mitochondria. These are then selectively removed by parkin, which is involved in the formation of the autophagosome, thereby inducing ATG. Parkin as well as PINK overexpression restores mitochondrial morphology and reverses cell death. These findings suggest that PINK1 and parkin cooperate in modifying mitochondrial morphology and inducing mitophagy. 5.4. UCH-L1 (PARK 5)
5. Are proteins mutated in inherited PD connected to ATG? (Fig. 1) This paradigm is represented by alpha-synuclein, which is the hallmark of Lewy bodies. Previous findings indicate that alphasynuclein (coded by the PARK1 and PARK4 loci) is a preferential UP substrate, while it can be metabolized by UP and ATG [3]. It is known that enzymes responsible for genetic PD, such as parkin (coded by the PARK2 locus), or ubiquitin carboxy-terminal hydrolase type 1 (UCH-L1) (coded by the PARK5 locus) are not solely related to a deficiency of the UP system, but are primarily involved in early steps of the ATG pathway. Moreover, additional mutations in PD are involved in ATG but not in the UP system. 5.1. Alpha-synuclein (PARK 1, PARK 4) Missense mutations [21] and multiplications [22] are responsible for PARK1 and PARK4 autosomal dominant PD, respectively. Alphasynuclein is also linked to sporadic PD, where it is abundant in Lewy bodies in the phosphorylated or monoubiquitinated and nitrated form. Initially, it was reported that alpha-synuclein was degraded by the proteasome. Now we know, however, that alpha-synuclein can be degraded by ATG and CMA [23]. When alpha-synuclein is degraded by CMA, it binds to the lysosomal-associated membrane protein type 2A (LAMP-2A), a CMA receptor at the lysosomal membrane. When alpha-synuclein is mutated, it blocks the function of CMA. 5.2. Parkin (PARK2), PINK1 (PARK 6) and mitochondrial fission/fusion Mutations in the parkin (PARK2) [24], PINK1 (PARK6) (phosphatase and tensin [PTEN] homologue-induced putative kinase 1) [25] and DJ-1 (PARK7) [26] genes are responsible for early-onset autosomal recessive PD. About 50% of autosomal recessive PD cases
UCH-L1 is expressed abundantly in brain neurons, where it has several activities, including the role of ubiquitin ligase for monoubiquitinated alpha-synuclein. The role of the UCH-L1 gene in determining PD was first reported in a German family with autosomal dominant PD due to a missense mutation in the UCH-L1 gene [31]. UCH-L1, which is a component of Lewy bodies, interacts with the CMA pathway at the level of the lysosomal receptor for CMA, LAMP-2A, and Hsc70 and Hsp90 [32]. These interactions are aberrant in the UCH-L1 mutation, which leads to an increase in alpha-synuclein. These observations led to the hypothesis that the mechanism underlying PD associated with UCH-L1 mutation consists of the inhibition of alpha-synuclein degradation by CMA. Remarkably, structural changes in UCH-L1 also occur in sporadic PD. In the brains of these patients, the interaction of UCH-L1 with LAMP-2A, Hsc70 and Hsp90 is altered, leading to accumulation of CMA substrates, including alpha-synuclein [32]. 5.5. DJ-1 (PARK7) Of its several physiological effects, DJ-1, which is responsible for inherited PD when undergoing a point mutation, triggers the processing of ATG proteins, including LC3 [33]. 5.6. LRRK2/dardarin (PARK8) Mutations in the LRRK2 gene produce autosomal dominant PD (PARK8) [34]. Recently, it has been demonstrated that LRRK2 is involved in ATG-mediated neurite remodelling [35]. Moreover, LRRK2-transfected cells feature a considerable increase in number and size of AVs in both neuritic and somatic compartments, as demonstrated by electron microscopy and LC3 immunofluorescence, and Western blot analysis. Pharmacological treatment with rapamycin of LRRK2-transfected cells causes a further increase in neuritic AVs.
L. Pasquali et al. / Parkinsonism and Related Disorders 15S (2009) S24–S27
5.7. ATP13A2 (PARK9) Loss of functional mutations in the ATP13A2 gene have been linked to Kufor-Rakeb syndrome (PARK9) [36]. ATP13A2 is a predominantly neuronal P-type ATPase gene, which encodes lysosomal ATPase [36]. The involvement of lysosomes was originally described in experimental parkinsonism [37]. This is further confirmed by the association between Gaucher disease and PD. In fact, this lysosomal lipid storage disease is a risk factor for parkinsonism [38]. The clinical signs in these PD patients are different, including early onset, as well as marked olfactory dysfunction, hallucinations and symptoms of cognitive decline or dementia [38].
[17]
[18]
[19]
[20]
[21]
5.8. Synphilin (PARK11) [22]
Synphilin is a susceptibility factor for PD [39]; the protein was identified in the core of Lewy bodies of PD patients and interacts with parkin and alpha-synuclein in the formation of Lewy-bodylike cytosolic inclusions. One of the several physiological roles of synphilin is to prime aggresomes for autophagic clearance [40].
[23]
[24]
Conflict of interest
[25]
Each author declares no conflict of interest related to the present manuscript.
[26]
References
[27]
[1] Ciechanover A. The ubiquitin proteolytic system: from a vague idea, through basic mechanisms, and onto human diseases and drug targeting. Neurology 2006;66(2 Suppl 1):S7–19. [2] Mariño G, López-Otín C. Autophagy: molecular mechanisms, physiological functions and relevance in human pathology. Cell Mol Life Sci 2004;61:1439– 54. [3] Webb JL, Ravikumar B, Atkins J, Skepper JN, Rubinsztein DC. AlphaSynuclein is degraded by both autophagy and the proteasome. J Biol Chem 2003;278:25009–13. [4] Klionsky DJ, Emr SD. Autophagy as a regulated pathway of cellular degradation. Science 2000;290:1717–21. [5] Hara T, Nakamura K, Matsui M, Yamamoto A, Nakahara Y, Suzuki-Migishima R, et al. Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice. Nature 2006;441:885–9. [6] Komatsu M, Waguri S, Chiba T, Murata S, Iwata J, Tanida I, et al. Loss of autophagy in the central nervous system causes neurodegeneration in mice. Nature 2006;441(7095):880–4. [7] Tsujimoto Y, Shimizu S. Another way to die: autophagic programmed cell death. Cell Death Differ 2005;12 Suppl 2:1528–34. [8] Pandey UB, Nie Z, Batlevi Y, McCray BA, Ritson GP, Nedelsky NB, et al. HDAC6 rescues neurodegeneration and provides an essential link between autophagy and the UPS. Nature 2007;447:859–63. [9] Pan T, Kondo S, Zhu W, Xie W, Jankovic J, Le W. Neuroprotection of rapamycin in lactacystin-induced neurodegeneration via autophagy enhancement. Neurobiol Dis 2008;32:16–25. [10] Rubinsztein DC. Autophagy induction rescues toxicity mediated by proteasome inhibition. Neuron 2007;54:854–6. [11] Korolchuk VI, Mansilla A, Menzies FM, Rubinsztein DC. Autophagy inhibition compromises degradation of ubiquitin-proteasome pathway substrates. Mol Cell 2009;33:517–27. [12] Korolchuk VI, Menzies FM, Rubinsztein DC. A novel link between autophagy and the ubiquitin-proteasome system. Autophagy 2009;5:862–3. [13] Kim PK, Hailey DW, Mullen RT, Lippincott-Schwartz J. Ubiquitin signals autophagic degradation of cytosolic proteins and peroxisomes. Proc Natl Acad Sci USA 2008;105:20567–74. [14] Ferrucci M, Pasquali L, Ruggieri S, Paparelli A, Fornai F. Alpha-synuclein and autophagy as common steps in neurodegeneration. Parkinsonism Relat Disord 2008;14 Suppl 2:S180–4. [15] Fornai F, Lenzi P, Gesi M, Soldani P, Ferrucci M, Lazzeri G, et al. Methamphetamine produces neuronal inclusions in the nigrostriatal system and in PC12 cells. J Neurochem 2004;88:114–23. [16] Lazzeri G, Lenzi P, Busceti CL, Ferrucci M, Falleni A, Bruno V, et al.
[28] [29]
[30]
[31] [32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
S27
Mechanisms involved in the formation of dopamine-induced intracellular bodies within striatal neurons. J Neurochem 2007;101:1414–27. Lenzi P, Fulceri F, Lazzeri G, Casini A, Ruggieri S, Paparelli A, et al. Analysis of single, purified inclusions as a novel approach to understand methamphetamine neurotoxicity. Ann N Y Acad Sci 2008;1139:186–90. Castino R, Lazzeri G, Lenzi P, Bellio N, Follo C, Ferrucci M, et al. Suppression of autophagy precipitates neuronal cell death following low doses of methamphetamine. J Neurochem 2008;106:1426–39. Larsen KE, Fon EA, Hastings TG, Edwards RH, Sulzer D. Methamphetamineinduced degeneration of dopaminergic neurons involves autophagy and upregulation of dopamine synthesis. J Neurosci 2002;22:895160. Anglade P, Vyas S, Hirsch EC, Agid Y. Apoptosis in dopaminergic neurons of the human substantia nigra during normal aging. Histol Histopathol 1997;12:603–10. Polymeropoulos MH, Lavedan C, Leroy E, Ide SE, Dehejia A, Dutra A, et al. Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease. Science 1997;276:2045–7. Singleton AB, Farrer M, Johnson J, Singleton A, Hague S, Kachergus J, et al. alpha-Synuclein locus triplication causes Parkinson’s disease. Science 2003;302:841. Cuervo AM, Stefanis L, Fredenburg R, Lansbury PT, Sulzer D. Impaired degradation of mutant alpha-synuclein by chaperone-mediated autophagy. Science 2004;305:1292–5. Kitada T, Asakawa S, Hattori N, Matsumine H, Yamamura Y, Minoshima S, et al. Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature 1998;392:605–8. Valente EM, Abou-Sleiman PM, Caputo V, Muqit MM, Harvey K, Gispert S, et al. Hereditary early-onset Parkinson’s disease caused by mutations in PINK1. Science 2004;304:1158–60. Bonifati V, Breedveld GJ, Squitieri F, Vanacore N, Brustenghi P, Harhangi BS, et al. Localization of autosomal recessive early-onset parkinsonism to chromosome 1p36 (PARK7) in an independent dataset. Ann Neurol 2002;51:253–6. Yang Y, Ouyang Y, Yang L, Beal MF, McQuibban A, Vogel H, et al. Pink1 regulates mitochondrial dynamics through interaction with the fission/fusion machinery. Proc Natl Acad Sci USA 2008;105:7070–5. Lin W, Kang UJ. Characterization of PINK1 processing, stability, and subcellular localization. J Neurochem 2008;106:464–74. Yang Y, Gehrke S, Imai Y, Huang Z, Ouyang Y, Wang JW, et al. Mitochondrial pathology and muscle and dopaminergic neuron degeneration caused by inactivation of Drosophila Pink1 is rescued by Parkin. Proc Natl Acad Sci USA 2006;103:10793–8. Shimura H, Hattori N, Kubo S, Yoshikawa M, Kitada T, Matsumine H et al. Immunohistochemical and subcellular localization of Parkin protein: absence of protein in autosomal recessive juvenile parkinsonism patients. Ann Neurol 1999;45:668–72. Leroy E, Boyer R, Auburger G, Leube B, Ulm G, Mezey E, et al. The ubiquitin pathway in Parkinson’s disease. Nature 1998;395:451–2. Kabuta T, Wada K. Insights into links between familial and sporadic Parkinson’s disease: physical relationship between UCH-L1 variants and chaperonemediated autophagy. Autophagy 2008;4:827–9. González-Polo R, Niso-Santano M, Morán JM, Ortiz-Ortiz MA, Bravo-San Pedro JM, Soler G, et al. Silencing DJ-1 reveals its contribution in paraquat-induced autophagy. J Neurochem 2009;109:889–98. Zimprich A, Biskup S, Leitner P, Lichtner P, Farrer M, Lincoln S, et al. Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology. Neuron 2004;44:601–7. Plowey ED, Cherra SJ 3rd, Liu YJ, Chu CT. Role of autophagy in G2019S-LRRK2associated neurite shortening in differentiated SH-SY5Y cells. J Neurochem 2008;105:1048–56. Ramirez A, Heimbach A, Gründemann J, Stiller B, Hampshire D, Cid LP, et al. Hereditary parkinsonism with dementia is caused by mutations in ATP13A2, encoding a lysosomal type 5 P-type ATPase. Nat Genet 2006;38:1184–91. Stefanis L, Larsen KE, Rideout HJ, Sulzer D, Greene LA. Expression of A53T mutant but not wild-type alpha-synuclein in PC12 cells induces alterations of the ubiquitin-dependent degradation system, loss of dopamine release, and autophagic cell death. J Neurosci 2001;21:9549–60. Goker-Alpan O, Lopez G, Vithayathil J, Davis J, Hallett M, Sidransky E. The spectrum of parkinsonian manifestations associated with glucocerebrosidase mutations. Arch Neurol 2008;65:1353–7. Engelender S, Kaminsky Z, Guo X, Sharp AH, Amaravi RK, Kleiderlein JJ, et al. Synphilin-1 associates with alpha-synuclein and promotes the formation of cytosolic inclusions. Nat Genet 1999;22:110–4. Zaarur N, Meriin AB, Gabai VL, Sherman MY. Triggering aggresome formation. Dissecting aggresome-targeting and aggregation signals in synphilin 1. J Biol Chem 2008;283:27575–84.