Dopamine-modified α-synuclein blocks chaperone-mediated autophagy
Marta Martinez-Vicente, … , David Sulzer, Ana Maria Cuervo
Marta Martinez-Vicente, … , David Sulzer, Ana Maria Cuervo
Published January 2, 2008
Citation Information: J Clin Invest. 2008;118(2):777-788. https://doi.org/10.1172/JCI32806.
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Research Article Neuroscience

Dopamine-modified α-synuclein blocks chaperone-mediated autophagy

Abstract

Altered degradation of α-synuclein (α-syn) has been implicated in the pathogenesis of Parkinson disease (PD). We have shown that α-syn can be degraded via chaperone-mediated autophagy (CMA), a selective lysosomal mechanism for degradation of cytosolic proteins. Pathogenic mutants of α-syn block lysosomal translocation, impairing their own degradation along with that of other CMA substrates. While pathogenic α-syn mutations are rare, α-syn undergoes posttranslational modifications, which may underlie its accumulation in cytosolic aggregates in most forms of PD. Using mouse ventral medial neuron cultures, SH-SY5Y cells in culture, and isolated mouse lysosomes, we have found that most of these posttranslational modifications of α-syn impair degradation of this protein by CMA but do not affect degradation of other substrates. Dopamine-modified α-syn, however, is not only poorly degraded by CMA but also blocks degradation of other substrates by this pathway. As blockage of CMA increases cellular vulnerability to stressors, we propose that dopamine-induced autophagic inhibition could explain the selective degeneration of PD dopaminergic neurons.

Authors

Marta Martinez-Vicente, Zsolt Talloczy, Susmita Kaushik, Ashish C. Massey, Joseph Mazzulli, Eugene V. Mosharov, Roberto Hodara, Ross Fredenburg, Du-Chu Wu, Antonia Follenzi, William Dauer, Serge Przedborski, Harry Ischiropoulos, Peter T. Lansbury, David Sulzer, Ana Maria Cuervo

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Figure 6

Increased intracellular levels of dopamine enhance the association of modified α-syn to lysosomes and block CMA.

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Increased intracellular levels of dopamine enhance the association of mo...
SH-SY5Y cells stably expressing wild-type (WT-syn) or a dopamine-insensitive mutant form of α-syn (DI–α-syn) were infected with an empty plasmid (none) or a plasmid encoding for mutant human TH1. (A) Protein degradation sensitive (lysosomal) or insensitive (nonlysosomal) to ammonium chloride (left) and protein degradation sensitive (macroautophagy) or insensitive (CMA) to 3-methyladenine. (B) Degradation via CMA of GAPDH by intact lysosomes isolated from the 4 different groups of cells. (C) Association of endogenous monomeric α-syn to lysosomes isolated from the same cells. Upper panel: representative immunoblot for α-syn and LAMP-1; lower panel: densitometric quantification of the amount of lysosome-associated α-syn (corrected per amount of lysosomes). (D) Left panels: Immunogold for α-syn of lysosomes isolated from cells stably expressing wild-type α-syn and an empty vector (none: upper panel) or a vector coding for TH1 (+TH: lower panel). The contribution of nonlysosomal structures to this fraction was less than 0.1%, and the percentage of total lysosomes active for CMA was 65%. Arrows: clusters (>5 particles) of gold particles. Insets: cluster-containing individual lysosomes at higher magnification. Scale bars: 0.5 μm. Right panels: number of gold particles associated with lysosomes per field (upper panel) and number of lysosome-associated clusters of more or less than 5 gold particles per field (lower panel) quantified in 4 different fields (≥50 lysosomes/field). (E) Presence of monomeric (α-syn m) and oligomeric (α-syn olig) forms of α-syn in total cellular homogenate (Hom) and in lysosomes (Lysosom) isolated from cells stably expressing wild-type or dopamine-insensitive mutant (DI–α-syn) and mutant TH1. All values are mean + SEM of cells cultured in 3 separate dishes or of triplicate samples. *P < 0.05.