2010/03/04 by Sally K. Mak, Alison L. McCormack, Amy Manning-Bog +4 · 27 citations
Medicine · #Parkinson's Disease Mechanisms and Treatments #Lysosomal Storage Disorders Research #Alzheimer's disease research and treatments
paper · pdf · doi:10.1074/jbc.m109.074617
Pathologic accumulation of α-synuclein is a feature of human parkinsonism and other neurodegenerative diseases. This accumulation may be counteracted by mechanisms of protein degradation that have been investigated in vitro but remain to be elucidated in animal models. In this study, lysosomal clearance of α-synuclein in vivo was indicated by the detection of α-synuclein in the lumen of lysosomes isolated from the mouse midbrain. When neuronal α-synuclein expression was enhanced as a result of toxic injury (i.e. treatment of mice with the herbicide paraquat) or transgenic protein overexpression, the intralysosomal content of α-synuclein was also significantly increased. This effect was paralleled by a marked elevation of the lysosome-associated membrane protein type 2A (LAMP-2A) and the lysosomal heat shock cognate protein of 70 kDa (hsc70), two essential components of chaperone-mediated autophagy (CMA). Immunofluorescence microscopy revealed an increase in punctate (lysosomal) LAMP-2A staining that co-localized with α-synuclein within nigral dopaminergic neurons of paraquat-treated and α-synuclein-overexpressing animals. The data provide in vivo evidence of lysosomal degradation of α-synuclein under normal conditions and, quite importantly, under conditions of enhanced protein burden. In the latter, increased lysosomal clearance of α-synuclein was mediated, at least in part, by CMA induction. It is conceivable that these neuronal mechanisms of protein clearance play an important role in neurodegenerative processes characterized by abnormal α-synuclein buildup. Pathologic accumulation of α-synuclein is a feature of human parkinsonism and other neurodegenerative diseases. This accumulation may be counteracted by mechanisms of protein degradation that have been investigated in vitro but remain to be elucidated in animal models. In this study, lysosomal clearance of α-synuclein in vivo was indicated by the detection of α-synuclein in the lumen of lysosomes isolated from the mouse midbrain. When neuronal α-synuclein expression was enhanced as a result of toxic injury (i.e. treatment of mice with the herbicide paraquat) or transgenic protein overexpression, the intralysosomal content of α-synuclein was also significantly increased. This effect was paralleled by a marked elevation of the lysosome-associated membrane protein type 2A (LAMP-2A) and the lysosomal heat shock cognate protein of 70 kDa (hsc70), two essential components of chaperone-mediated autophagy (CMA). Immunofluorescence microscopy revealed an increase in punctate (lysosomal) LAMP-2A staining that co-localized with α-synuclein within nigral dopaminergic neurons of paraquat-treated and α-synuclein-overexpressing animals. The data provide in vivo evidence of lysosomal degradation of α-synuclein under normal conditions and, quite importantly, under conditions of enhanced protein burden. In the latter, increased lysosomal clearance of α-synuclein was mediated, at least in part, by CMA induction. It is conceivable that these neuronal mechanisms of protein clearance play an important role in neurodegenerative processes characterized by abnormal α-synuclein buildup. IntroductionSeveral lines of clinical and experimental evidence support a pathogenetic role of α-synuclein in Parkinson disease (PD) 2The abbreviations used are: PDParkinson diseaseLAMPlysosome-associated membrane proteinhsc70heat shock cognate protein of 70 kDaCMAchaperone-mediated autophagyqPCRquantitative PCRThy-1thymus cell antigen 1PKproteinase KPQparaquatPBSphosphate-buffered saline. and other neurodegenerative disorders. The precise mechanisms by which this endogenously expressed protein becomes involved in pathologic processes have yet to be fully elucidated. However, data from both clinical and laboratory studies indicate that enhanced α-synuclein expression is itself capable of triggering a parkinsonian syndrome in humans and PD-like pathology in animal models. Indeed, multiplication mutations of the α-synuclein gene that result in increased expression of the wild-type protein are causally associated with autosomal dominant parkinsonism (1Singleton 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. Blancato J. Hardy J. Gwinn-Hardy K. Science. 2003; 302: 841Crossref PubMed Scopus (3425) Google Scholar, 2Farrer M. Kachergus J. Forno L. Lincoln S. Wang D.S. Hulihan M. Maraganore D. Gwinn-Hardy K. Wszolek Z. Dickson D. Langston J.W. Ann. Neurol. 2004; 55: 174-179Crossref PubMed Scopus (583) Google Scholar). From the experimental standpoint, evidence of a gain of toxic function of elevated α-synuclein includes the observation of neurodegeneration and neuronal inclusions in the substantia nigra of rats and monkeys that overexpress α-synuclein after viral-mediated neuronal transduction (3Lo Bianco C. Ridet J.L. Schneider B.L. Deglon N. Aebischer P. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 10813-10818Crossref PubMed Scopus (423) Google Scholar, 4Eslamboli A. Romero-Ramos M. Burger C. Bjorklund T. Muzyczka N. Mandel R.J. Baker H. Ridley R.M. Kirik D. Brain. 2007; 130: 799-815Crossref PubMed Scopus (156) Google Scholar).An important corollary to the concept that enhanced α-synuclein may have deleterious consequences is that intraneuronal mechanisms of protein homeostasis, such as degradation pathways, could well play a key role in maintaining “non-toxic” levels of α-synuclein. Although α-synuclein clearance is likely to occur through different mechanisms, recent reports have underscored the important contribution of lysosomal pathways of protein degradation and, in particular, chaperone-mediated autophagy (CMA) (5Webb J.L. Ravikumar B. Atkins J. Skepper J.N. Rubinsztein D.C. J. Biol. Chem. 2003; 278: 25009-25013Abstract Full Text Full Text PDF PubMed Scopus (1129) Google Scholar, 6Cuervo A.M. Stefanis L. Fredenburg R. Lansbury P.T. Sulzer D. Science. 2004; 305: 1292-1295Crossref PubMed Scopus (1526) Google Scholar, 7Martinez-Vicente M. Talloczy Z. Kaushik S. Massey A.C. Mazzulli J. Mosharov E.V. Hodara R. Fredenburg R. Wu D.C. Follenzi A. Dauer W. Przedborski S. Ischiropoulos H. Lansbury P.T. Sulzer D. Cuervo A.M. J. Clin. Invest. 2008; 118: 777-788PubMed Google Scholar, 8Vogiatzi T. Xilouri M. Vekrellis K. Stefanis L. J. Biol. Chem. 2008; 283: 23542-23556Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar). CMA targets specific cytosolic proteins that are recognized by the heat shock cognate protein of 70 kDa (hsc70) and, after interacting with the lysosome-associated membrane protein type 2A (LAMP-2A), are translocated into the lysosomal lumen for rapid degradation (9Massey A.C. Zhang C. Cuervo A.M. Curr. Top. Dev. Biol. 2006; 73: 205-235Crossref PubMed Scopus (252) Google Scholar). The amino acid sequence of α-synuclein contains a pentapeptide succession consistent with a CMA recognition motif. Experiments in which purified α-synuclein was added to intact lysosomes revealed that this CMA motif is essential for the internalization of α-synuclein into the lysosomal lumen and degradation by lysosomal proteases (6Cuervo A.M. Stefanis L. Fredenburg R. Lansbury P.T. Sulzer D. Science. 2004; 305: 1292-1295Crossref PubMed Scopus (1526) Google Scholar). Furthermore, inhibition of lysosomal proteolysis and CMA activity has been found to decrease α-synuclein clearance in a variety of in vitro systems, including primary neuronal cultures (6Cuervo A.M. Stefanis L. Fredenburg R. Lansbury P.T. Sulzer D. Science. 2004; 305: 1292-1295Crossref PubMed Scopus (1526) Google Scholar, 7Martinez-Vicente M. Talloczy Z. Kaushik S. Massey A.C. Mazzulli J. Mosharov E.V. Hodara R. Fredenburg R. Wu D.C. Follenzi A. Dauer W. Przedborski S. Ischiropoulos H. Lansbury P.T. Sulzer D. Cuervo A.M. J. Clin. Invest. 2008; 118: 777-788PubMed Google Scholar, 8Vogiatzi T. Xilouri M. Vekrellis K. Stefanis L. J. Biol. Chem. 2008; 283: 23542-23556Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar).Despite these compelling in vitro data, the relationship between α-synuclein, lysosomes, and CMA remains unexplored in the brain in vivo. It is unknown if, in this setting, α-synuclein is translocated into lysosomes and if enhanced α-synuclein expression is associated with CMA induction and increased lysosomal levels of the protein. In this study, lysosome-α-synuclein interactions were investigated in normal mice as well as in mice in which increased α-synuclein expression was a consequence of either toxic injury or genetic manipulation. Lysosomes were isolated from midbrain tissue because neuronal cells within this region (i.e. dopaminergic neurons in the substantia nigra) are highly vulnerable to neurodegeneration and α-synuclein pathology in PD. In a first set of experiments, levels of α-synuclein and CMA markers were compared in midbrain lysosomes from control mice versus animals injected with the herbicide paraquat. Paraquat administration was used as a model of enhanced α-synuclein burden because it has previously been shown to induce a marked up-regulation of the protein within nigrostriatal dopaminergic neurons (10Manning-Bog A.B. McCormack A.L. Li J. Uversky V.N. Fink A.L. Di Monte D.A. J. Biol. Chem. 2002; 277: 1641-1644Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar, 11Manning-Bog A.B. McCormack A.L. Purisai M.G. Bolin L.M. Di Monte D.A. J. Neurosci. 2003; 23: 3095-3099Crossref PubMed Google Scholar). In a second set of experiments, the association between elevated α-synuclein and CMA induction was assessed in transgenic mice overexpressing α-synuclein. Taken together, results of these studies provide in vivo evidence of lysosomal clearance of α-synuclein that is enhanced under conditions of increased protein burden and is mediated, at least in part, by CMA activation.DISCUSSIONResults of this study provide evidence of lysosomal degradation of α-synuclein in vivo by showing internalization of the protein into lysosomes from the midbrain of control mice and mice injected with paraquat. In the latter, selective injury of nigrostriatal dopaminergic neurons is accompanied by an increased expression of α-synuclein (10Manning-Bog A.B. McCormack A.L. Li J. Uversky V.N. Fink A.L. Di Monte D.A. J. Biol. Chem. 2002; 277: 1641-1644Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar, 25McCormack A.L. Thiruchelvam M. Manning-Bog A.B. Thiffault C. Langston J.W. Cory-Slechta D.A. Di Monte D.A. Neurobiol. Dis. 2002; 10: 119-127Crossref PubMed Scopus (614) Google Scholar). Of note, levels of intralysosomal α-synuclein were also markedly enhanced after paraquat exposure, suggesting a relationship between cytosolic up-regulation and lysosomal translocation of the protein. One possible explanation for the increased lysosomal α-synuclein after paraquat exposure may be the formation of post-translationally modified forms of the protein resistant to degradation by lysosomal proteases. In particular, formation of oxidized α-synuclein could be hypothesized under our experimental conditions given the pro-oxidant effects of paraquat on nigral dopaminergic neurons (26McCormack A.L. Atienza J.G. Johnston L.C. Andersen J.K. Vu S. Di Monte D.A. J. Neurochem. 2005; 93: 1030-1037Crossref PubMed Scopus (200) Google Scholar, 27McCormack A.L. Atienza J.G. Langston J.W. Di Monte D.A. Neuroscience. 2006; 141: 929-937Crossref PubMed Scopus (58) Google Scholar). A recent report, however, has demonstrated that the rate of lysosomal clearance is not significantly different between unmodified and oxidized α-synuclein, making it unlikely that paraquat would cause a lysosomal accumulation of protease-resistant forms of the protein (7Martinez-Vicente M. Talloczy Z. Kaushik S. Massey A.C. Mazzulli J. Mosharov E.V. Hodara R. Fredenburg R. Wu D.C. Follenzi A. Dauer W. Przedborski S. Ischiropoulos H. Lansbury P.T. Sulzer D. Cuervo A.M. J. Clin. Invest. 2008; 118: 777-788PubMed Google Scholar). Furthermore, we found that ∼50% of α-synuclein was degraded during a 5-min incubation of lysosomal matrix extracts, regardless of whether they were obtained from control or paraquat-treated mice. Thus, lysosomal accumulation of α-synuclein following treatment with the herbicide cannot simply be explained by reduced clearance capability. Rather, it reflects an increase in lysosomal uptake triggered by the cytosolic buildup of the protein.To elucidate mechanisms involved in the enhanced translocation of α-synuclein into lysosomes after paraquat administration, we assessed changes in lysosomal hsc70 and LAMP-2A as markers of CMA induction. The rationale for investigating the relationship between α-synuclein clearance and CMA in the paraquat model was 2-fold. First, earlier in vitro data are consistent with α-synuclein being a CMA substrate and with CMA playing an important role in preventing neuronal accumulation of the protein (6Cuervo A.M. Stefanis L. Fredenburg R. Lansbury P.T. Sulzer D. Science. 2004; 305: 1292-1295Crossref PubMed Scopus (1526) Google Scholar, 7Martinez-Vicente M. Talloczy Z. Kaushik S. Massey A.C. Mazzulli J. Mosharov E.V. Hodara R. Fredenburg R. Wu D.C. Follenzi A. Dauer W. Przedborski S. Ischiropoulos H. Lansbury P.T. Sulzer D. Cuervo A.M. J. Clin. Invest. 2008; 118: 777-788PubMed Google Scholar, 8Vogiatzi T. Xilouri M. Vekrellis K. Stefanis L. J. Biol. Chem. 2008; 283: 23542-23556Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar). Second, paraquat administration has previously been shown to upregulate CMA activity in rat liver and cultured fibroblasts (16Massey A.C. Kaushik S. Sovak G. Kiffin R. Cuervo A.M. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 5805-5810Crossref PubMed Scopus (405) Google Scholar, 22Kiffin R. Christian C. Knecht E. Cuervo A.M. Mol. Biol. Cell. 2004; 15: 4829-4840Crossref PubMed Scopus (474) Google Scholar). Our current findings provide evidence in favor of a contribution of CMA to α-synuclein degradation in the midbrain of paraquat-exposed mice. In these animals, higher levels of lysosomal hsc70 and LAMP-2A paralleled the enhanced cytosolic expression and lysosomal internalization of α-synuclein. Moreover, a punctate α-synuclein and LAMP-2A co-immunoreactivity most likely reflected the lysosomal localization of both these proteins within nigral neurons. Finally, binding of α-synuclein to cytosolic hsc70 was augmented in the midbrain of paraquat-treated mice, further supporting a relationship between CMA induction and increased lysosomal clearance of α-synuclein.Quite interestingly, paraquat-induced up-regulation of LAMP-2A was achieved through de novo synthesis, as indicated by the dramatic elevation of LAMP-2A mRNA in the ventral mesencephalon of mice injected with the herbicide. At least two mechanisms have been described to increase LAMP-2A under conditions associated with CMA induction. During nutritional stress, the most extensively studied inducer of CMA, enhanced LAMP-2A results from a decrease in its degradation as well as a relocation of the receptor protein from the lysosomal lumen to the lysosomal membrane (18Cuervo A.M. Dice J.F. Traffic. 2000; 1: 570-583Crossref PubMed Scopus (221) Google Scholar). In contrast, LAMP-2A changes associated with CMA activation during oxidative stress were found to be caused by an actual increase in LAMP-2A synthesis (22Kiffin R. Christian C. Knecht E. Cuervo A.M. Mol. Biol. Cell. 2004; 15: 4829-4840Crossref PubMed Scopus (474) Google Scholar). This latter mechanism could explain our present data: the oxidative damage caused by paraquat administration would induce new synthesis of LAMP-2A as a critical CMA factor involved in the lysosomal degradation of α-synuclein and other toxic/damaged proteins.A causal relationship between intraneuronal α-synuclein levels, CMA induction and enhanced translocation of α-synuclein into lysosomes may be difficult to demonstrate in the in vivo setting. It could be argued that the findings of increased lysosomal α-synuclein and CMA activation in the paraquat model are compatible with but do not necessarily indicate a direct link between these events. In particular, they do not address the issue of whether α-synuclein accumulation is itself a condition leading to CMA induction. To further investigate this possibility, a final set of experiments was carried out in mice in which increased α-synuclein levels resulted from transgenic overexpression of the protein. Data revealed a robust up-regulation of LAMP-2A in the brain of transgenic animals and supported a direct correlation between levels of α-synuclein overexpression and CMA activation. Indeed, the increase in LAMP-2A was greatest in the brain region with the highest α-synuclein level (ventral mesencephalon) and smallest in the area with the lowest protein expression (frontal cortex). CMA induction as a consequence of transgene expression was also indicated by enhanced hsc70 immunoreactivity within nigral neurons laden with α-synuclein.As suggested by earlier studies, the clearance of wild-type or mutated α-synuclein may involve a variety of mechanisms (5Webb J.L. Ravikumar B. Atkins J. Skepper J.N. Rubinsztein D.C. J. Biol. Chem. 2003; 278: 25009-25013Abstract Full Text Full Text PDF PubMed Scopus (1129) Google Scholar, 6Cuervo A.M. Stefanis L. Fredenburg R. Lansbury P.T. Sulzer D. Science. 2004; 305: 1292-1295Crossref PubMed Scopus (1526) Google Scholar, 8Vogiatzi T. Xilouri M. Vekrellis K. Stefanis L. J. Biol. Chem. 2008; 283: 23542-23556Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar, 28Bennett M.C. Bishop J.F. Leng Y. Chock P.B. Chase T.N. Mouradian M.M. J. Biol. Chem. 1999; 274: 33855-33858Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar, 29Lee H.J. Khoshaghideh F. Patel S. Lee S.J. J. Neurosci. 2004; 24: 1888-1896Crossref PubMed Scopus (342) Google Scholar, 30Sarkar S. Davies J.E. Huang Z. Tunnacliffe A. Rubinsztein D.C. J. Biol. Chem. 2007; 282: 5641-5652Abstract Full Text Full Text PDF PubMed Scopus (883) Google Scholar, 31Yu W.H. Dorado B. Figueroa H.Y. Wang L. Planel E. Cookson M.R. Clark L.N. Duff K.E. Am. J. Pathol. 2009; 175: 736-747Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The present findings do not rule out this possibility but clearly reveal the importance of lysosomal pathways and, in particular, CMA in the neuronal degradation of wild-type α-synuclein in vivo. The observation of CMA induction and enhanced lysosomal internalization of α-synuclein under conditions of increased protein expression (i.e. paraquat-induced up-regulation and transgenic overexpression of α-synuclein) bears significant implications. Both clinical and experimental data suggest that higher levels of neuronal α-synuclein may trigger or predispose to pathologic including formation and neurodegeneration M. Kachergus J. Forno L. Lincoln S. Wang D.S. Hulihan M. Maraganore D. Gwinn-Hardy K. Wszolek Z. Dickson D. Langston J.W. Ann. Neurol. 2004; 55: 174-179Crossref PubMed Scopus (583) Google Scholar, 4Eslamboli A. Romero-Ramos M. Burger C. Bjorklund T. Muzyczka N. Mandel R.J. Baker H. Ridley R.M. Kirik D. Brain. 2007; 130: 799-815Crossref PubMed Scopus (156) Google Scholar, A.L. M. Langston Forno Di Monte D.A. J. Neurol. 2008; PubMed Scopus Google Scholar). induction of lysosomal degradation pathways may an important mechanism that of this which may from normal C. Cuervo A.M. 2008; PubMed Scopus Google could play a role in the of and other of these pathways could a for in these human diseases. IntroductionSeveral lines of clinical and experimental evidence support a pathogenetic role of α-synuclein in Parkinson disease (PD) 2The abbreviations used are: PDParkinson diseaseLAMPlysosome-associated membrane proteinhsc70heat shock cognate protein of 70 kDaCMAchaperone-mediated autophagyqPCRquantitative PCRThy-1thymus cell antigen 1PKproteinase KPQparaquatPBSphosphate-buffered saline. and other neurodegenerative disorders. The precise mechanisms by which this endogenously expressed protein becomes involved in pathologic processes have yet to be fully elucidated. However, data from both clinical and laboratory studies indicate that enhanced α-synuclein expression is itself capable of triggering a parkinsonian syndrome in humans and PD-like pathology in animal models. Indeed, multiplication mutations of the α-synuclein gene that result in increased expression of the wild-type protein are causally associated with autosomal dominant parkinsonism (1Singleton 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. Blancato J. Hardy J. Gwinn-Hardy K. Science. 2003; 302: 841Crossref PubMed Scopus (3425) Google Scholar, 2Farrer M. Kachergus J. Forno L. Lincoln S. Wang D.S. Hulihan M. Maraganore D. Gwinn-Hardy K. Wszolek Z. Dickson D. Langston J.W. Ann. Neurol. 2004; 55: 174-179Crossref PubMed Scopus (583) Google Scholar). From the experimental standpoint, evidence of a gain of toxic function of elevated α-synuclein includes the observation of neurodegeneration and neuronal inclusions in the substantia nigra of rats and monkeys that overexpress α-synuclein after viral-mediated neuronal transduction (3Lo Bianco C. Ridet J.L. Schneider B.L. Deglon N. Aebischer P. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 10813-10818Crossref PubMed Scopus (423) Google Scholar, 4Eslamboli A. Romero-Ramos M. Burger C. Bjorklund T. Muzyczka N. Mandel R.J. Baker H. Ridley R.M. Kirik D. Brain. 2007; 130: 799-815Crossref PubMed Scopus (156) Google Scholar).An important corollary to the concept that enhanced α-synuclein may have deleterious consequences is that intraneuronal mechanisms of protein homeostasis, such as degradation pathways, could well play a key role in maintaining “non-toxic” levels of α-synuclein. Although α-synuclein clearance is likely to occur through different mechanisms, recent reports have underscored the important contribution of lysosomal pathways of protein degradation and, in particular, chaperone-mediated autophagy (CMA) (5Webb J.L. Ravikumar B. Atkins J. Skepper J.N. Rubinsztein D.C. J. Biol. Chem. 2003; 278: 25009-25013Abstract Full Text Full Text PDF PubMed Scopus (1129) Google Scholar, 6Cuervo A.M. Stefanis L. Fredenburg R. Lansbury P.T. Sulzer D. Science. 2004; 305: 1292-1295Crossref PubMed Scopus (1526) Google Scholar, 7Martinez-Vicente M. Talloczy Z. Kaushik S. Massey A.C. Mazzulli J. Mosharov E.V. Hodara R. Fredenburg R. Wu D.C. Follenzi A. Dauer W. Przedborski S. Ischiropoulos H. Lansbury P.T. Sulzer D. Cuervo A.M. J. Clin. Invest. 2008; 118: 777-788PubMed Google Scholar, 8Vogiatzi T. Xilouri M. Vekrellis K. Stefanis L. J. Biol. Chem. 2008; 283: 23542-23556Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar). CMA targets specific cytosolic proteins that are recognized by the heat shock cognate protein of 70 kDa (hsc70) and, after interacting with the lysosome-associated membrane protein type 2A (LAMP-2A), are translocated into the lysosomal lumen for rapid degradation (9Massey A.C. Zhang C. Cuervo A.M. Curr. Top. Dev. Biol. 2006; 73: 205-235Crossref PubMed Scopus (252) Google Scholar). The amino acid sequence of α-synuclein contains a pentapeptide succession consistent with a CMA recognition motif. Experiments in which purified α-synuclein was added to intact lysosomes revealed that this CMA motif is essential for the internalization of α-synuclein into the lysosomal lumen and degradation by lysosomal proteases (6Cuervo A.M. Stefanis L. Fredenburg R. Lansbury P.T. Sulzer D. Science. 2004; 305: 1292-1295Crossref PubMed Scopus (1526) Google Scholar). Furthermore, inhibition of lysosomal proteolysis and CMA activity has been found to decrease α-synuclein clearance in a variety of in vitro systems, including primary neuronal cultures (6Cuervo A.M. Stefanis L. Fredenburg R. Lansbury P.T. Sulzer D. Science. 2004; 305: 1292-1295Crossref PubMed Scopus (1526) Google Scholar, 7Martinez-Vicente M. Talloczy Z. Kaushik S. Massey A.C. Mazzulli J. Mosharov E.V. Hodara R. Fredenburg R. Wu D.C. Follenzi A. Dauer W. Przedborski S. Ischiropoulos H. Lansbury P.T. Sulzer D. Cuervo A.M. J. Clin. Invest. 2008; 118: 777-788PubMed Google Scholar, 8Vogiatzi T. Xilouri M. Vekrellis K. Stefanis L. J. Biol. Chem. 2008; 283: 23542-23556Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar).Despite these compelling in vitro data, the relationship between α-synuclein, lysosomes, and CMA remains unexplored in the brain in vivo. It is unknown if, in this setting, α-synuclein is translocated into lysosomes and if enhanced α-synuclein expression is associated with CMA induction and increased lysosomal levels of the protein. In this study, lysosome-α-synuclein interactions were investigated in normal mice as well as in mice in which increased α-synuclein expression was a consequence of either toxic injury or genetic manipulation. Lysosomes were isolated from midbrain tissue because neuronal cells within this region (i.e. dopaminergic neurons in the substantia nigra) are highly vulnerable to neurodegeneration and α-synuclein pathology in PD. In a first set of experiments, levels of α-synuclein and CMA markers were compared in midbrain lysosomes from control mice versus animals injected with the herbicide paraquat. Paraquat administration was used as a model of enhanced α-synuclein burden because it has previously been shown to induce a marked up-regulation of the protein within nigrostriatal dopaminergic neurons (10Manning-Bog A.B. McCormack A.L. Li J. Uversky V.N. Fink A.L. Di Monte D.A. J. Biol. Chem. 2002; 277: 1641-1644Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar, 11Manning-Bog A.B. McCormack A.L. Purisai M.G. Bolin L.M. Di Monte D.A. J. Neurosci. 2003; 23: 3095-3099Crossref PubMed Google Scholar). In a second set of experiments, the association between elevated α-synuclein and CMA induction was assessed in transgenic mice overexpressing α-synuclein. Taken together, results of these studies provide in vivo evidence of lysosomal clearance of α-synuclein that is enhanced under conditions of increased protein burden and is mediated, at least in part, by CMA activation.