In this study, we demonstrate that various posttranslational modifications of α-syn have different consequences for the ability of the protein to be degraded via CMA and different effects on CMA of other substrates. Only α-syn monomers and dimers, but not oligomers, are degraded via CMA. Oxidation and nitration of α-syn slightly inhibit degradation by CMA, while phosphorylation and exposure to dopamine almost completely prevent protein removal through this pathway. Notably, only DA–α-syn inhibited CMA activity in isolated lysosomes, cultured dopaminergic cell lines, and VM neurons.
There are a wide range of effects on CMA of α-syn depending on the type of protein modification. Although the reasons for their altered CMA remain elusive, it is possible that a low binding affinity of the native protein or a particular conformational change induced by its phosphorylation could be responsible for the poor lysosomal translocation of phosphorylated α-syn. Extensive oxidation of CMA substrates has been previously found to interfere with their translocation across the lysosomal membrane, likely as the result of aggregation once in the lipid environment of the membrane (40
). A combination of the lipid-induced conformational changes described for α-syn (41
) and those induced by the particular modifications could inhibit CMA of oxidized or nitrated α-syn. The failure of lysosomes to take up oligomeric α-syn could be in part due to an inability to disassemble into simpler α-syn forms compatible with membrane translocation or because the oligomers bind to the lysosomal membrane through CMA-independent mechanisms. In support of the latter hypothesis, membrane-bound nitrated oligomers could not be degraded by CMA or displaced by other CMA substrates but did not inhibit CMA more than the other forms. Unfortunately, aggregation of oligomers of α-syn under the conditions required for coimmunoprecipitation of CMA substrates with the lysosomal receptor prevented us from conclusively documenting the absence of binding of nitrated oligomers directly to the CMA receptor.
The inhibitory effect of DA–α-syn on CMA resembled that previously described for 2 of the mutations of α-syn identified in familial forms of PD (9
). High intracellular levels of wild-type α-syn also inhibit CMA, although to a lesser extent (ref. 9
and A.M. Cuervo, L. Stefanis, and D. Sulzer, unpublished results). Because α-syn is one of the most efficient CMA substrates identified to date, it is possible that high cytosolic levels of the protein could saturate CMA for other substrates. However, we cannot discard the possibility that increased absolute content of α-syn would also increase the size of the pool of the protein undergoing posttranslational modifications. These modified forms of α-syn, and in particular that mediated by dopamine, could be directly responsible for the inhibitory effect on CMA observed under these conditions.
Although most of the modifications of α-syn impair its own CMA degradation, intracellular DA–α-syn could be particularly detrimental for the cells due to its additional inhibitory effect on CMA of other substrates. We have recently shown that cells respond to blockage of CMA by upregulating macroautophagy (13
). This compensatory mechanism is sufficient to maintain normal rates of protein degradation to guarantee cell survival. Consistent with these previous findings, we found that blockage of CMA in SH-SY5Y cells with increased intracellular dopamine (Figure ) and in VM cultures after exposure to l
-DOPA resulting in increased cytosolic DA (Figure ) did not result in significant quantitative changes in the total degradation rates of long-lived proteins in lysosomes (as determined by sensitivity to ammonium chloride) due to compensatory macroautophagy. The observation that l
-DOPA had much less effect on CMA in VM cultures from α-syn–null mice strongly suggests that a reaction between dopamine and α-syn is responsible for CMA blockage. The upregulation of macroautophagy observed in the VM cultures from α-syn–null mice treated with l
-DOPA could be at least in part in response to intracellular protein aggregation (14
) or in part due to the remaining slight reduction in CMA, but in any case, the enhancement in macroautophagy in the cultures from α-syn–null mice is clearly lower than that observed in the wild-type VM neurons. Macroautophagy cannot, however, replace CMA under stress conditions, as CMA blockage increases cellular vulnerability to stressors, resulting in apoptosis and cell death (13
). The unique effect of DA–α-syn on CMA could be a cause of the massive loss of dopaminergic SN neurons in PD as well as norepinephrine-releasing neurons of the LC, which likewise possess cytosolic dopamine and produce neuromelanin, a product of dopamine modifications localized within macroautophagic organelles (33
While a variety of modifications of α-syn likely occurred following treatment of cell lines and neurons with l-DOPA, the observations that CMA inhibition was more pronounced in dopaminergic neurons than in cortical neurons (Figure B) and that a dopamine-insensitive mutant of α-syn (still susceptible to oxidation) was efficiently degraded by CMA (Supplemental Figure 4) and did not interfere with CMA (Figure , A and B) further support a critical role for dopamine-mediated modifications of α-syn in the impaired CMA activity. The inhibitory effect of DA–α-syn on CMA did not result from a direct effect of dopamine on lysosomes because it was not observed when GAPDH was incubated with lysosomes with dopamine but in the absence of α-syn (data not shown). In addition, the effect observed for DA–α-syn could be reproduced with α-syn treated with the fully oxidized dopamine product (dopaminochrome), which indicates that the effect is not due to the oxidation of the protein or due to the oxidants generated during the oxidation of dopamine during the incubation with lysosomes. Both exogenously added DA–α-syn (Figure ) and endogenous α-syn in cells with increased cytosolic dopamine (Figure ) bound with higher affinity to the lysosomal membrane than their unmodified counterparts.
Although there is no reliable method for the detection of DA–α-syn inside cells, we provide in this study independent evidence to support the belief that DA–α-syn is responsible for the inhibitory effect on CMA: (a) this effect is not observed in dopaminergic neuronal cultures lacking α-syn (Figure ), indicating that cellular presence of α-syn is necessary; (b) this effect is not observed in cells expressing a dopamine-insensitive mutant form of α-syn (Figure ), indicating that the presence of α-syn is not sufficient but that it has to be susceptible to dopamine modification; (c) this inhibitory effect can be reproduced when DA–α-syn is presented to isolated lysosomes (the most direct approach for measuring CMA, as the effect of other proteolytic systems or cellular factors is eliminated); (d) the inhibition of CMA activity in lysosomes isolated from cells with increased cytosolic levels of dopamine and which have associated with their membrane endogenous α-syn is remarkably similar to that observed in the in vitro system. Furthermore, this effect is only observed in cells expressing α-syn susceptible to dopamine modification.
The dopamine-modified exogenous and endogenous proteins were detected at the lysosomal membrane in oligomeric complexes (Figure D and Figure , B and C). In contrast to the covalently linked oligomeric forms of nitrated α-syn, oligomers formed from DA–α-syn inhibited CMA. The different consequences of the binding of both oligomeric structures to lysosomes could arise from the sequence of events that led to their association with the membranes: nitrated oligomers bound to the lysosomal membrane as oligomeric structures and, as we observed no competition, apparently to sites different from those of CMA substrates. In contrast, DA–α-syn oligomers appeared to form at the lysosomal membrane after binding as monomers to the CMA translocation complex. These results suggest that membrane-bound DA–α-syn monomers act as nucleation seeds, resulting in the formation of CMA-blocking multimeric complexes at the lysosomal membrane, visualized as slow migrating forms of α-syn by electrophoresis (Figure E and Figure A) and large-size clusters of gold particles by immunogold (Figure D and Figure , B and C). Future attempts to prevent the blockage of CMA by DA–α-syn should consider interventions aimed to diminish or revert oligomerization of the modified protein at the lysosomal membrane. Furthermore, in light of the pathogenic effect of the accumulation of α-syn in other neuronal populations and in glia in other synucleinopathies (4
), it would be interesting to determine in the future additional cell-type–dependent effects of the posttranslational modifications of α-syn on the autophagic system.