Interplay between Parkinson's disease proteins and chaperone-meditated autophagy

Abstract

Chaperone-mediated autophagy (CMA) is a selective form of autophagy by which cytosolic soluble proteins are transported one by one across the lysosomal membrane for degradation. CMA performs a variety of key cellular functions including the maintenance of homeostasis, quality control, and stress response. Dysfunctional CMA has been shown to occur in aging and has been linked to several neurodegenerative disorders. A fraction of cellular alphasynuclein (a-syn), a protein associated with Parkinson's disease (PD), is usually degraded by CMA, but pathogenic forms of a-syn exhibit impaired CMA and interfere with normal CMA of other substrates.;The goal of this thesis research was to determine whether other PD-associated proteins might also be CMA substrates, and whether mutated forms of these proteins would also impair CMA and by what mechanism.;In the first part of this study, we have analyzed the degradation by CMA of wild-type (WT) leucine-rich repeat kinase II (LRRK2), the protein most frequently mutated in familial forms of PD. We found that this PD-associated protein contains 8 putative CMA targeting motifs in its amino acid sequence, and using in vitro assays with isolated lysosomes, we demonstrated that LRRK2 meets the criteria of a bona fide CMA substrate. In fact, part of this protein is degraded via CMA in neuronal cells and in different brain regions but, in contrast to typical CMA substrates, lysosomal binding of LRRK2 proteins is enhanced in the presence of other CMA substrates.;Additionally, we have shown that various PD-associated mutations in LRRK2 impair CMA and we discovered that the mechanism behind this impairment was the ability of the mutant proteins to abrogate the formation of the translocation complex at the lysosomal membrane. We confirmed that cells respond to LRRK2-mediated CMA compromise by increasing levels of the III LRRK2 and CMA CMA lysosomal receptor. In fact, we detected a similar reactive upregulation of this receptor in neurons isolated from LRRK2 mutant mouse brains as well as in brains and neuronal differentiated iPSC derived from LRRK2 PD patients. Using these different experimental models, we have analyzed the consequences of this CMA inhibitory effect of LRRK2 in the context of other pathogenic proteins. We have found that the concurrence of LRRK2 and a-syn directly at the lysosomal membrane promotes abnormal oligomerization and blockage of degradation of a-syn whenever one of these proteins is mutated, thereby linking these two PD associated proteins directly at lysosomes.;In the second part of this work, we have utilized cultured cells knocked-down for LRRK2 as well as LRRK2 knock-out and transgenic mouse models to investigate the consequences of changes in LRRK2 levels in two subpopulations of lysosomes. We have found that LRRK2 is preferentially enriched in those lysosomes engaged in macroautophagy, but that are capable of maturing into CMA competent lysosomes upon stimulation of the CMA pathway. Based on our preliminary data, we propose that LRRK2 exerts an inhibitory effect on these lysosomes by preventing their conversion into the CMA competent form. At the same time, our data also suggest that LRRK2 acts as a positive modulator of CMA, likely by phosphorylating a key regulator of CMA activity directly at the lysosomal membrane. We propose that LRRK2 levels at lysosomes must be tightly regulated in order to maintain proper functioning of the lysosomal system, and thus, mutations in LRRK2 which alter its interactions with the lysosomal system will have devastating consequences for CMA and PD.;Overall, this work has garnered support for our theory that PD is, in part, a CMA disorder. We have expanded our current understanding of the relationship between CMA and PD-associated proteins to include LRRK2, the most commonly mutated protein in familial PD, and have identified, for the first time, the mechanism by which PD-associated proteins exert their toxic effect on CMA. Additionally, our studies link the two most prominent PD-associated proteins, LRRK2 and a-syn, directly at lysosomes by proposing a novel LRRK2 self- IV Abstract perpetuating inhibitory effect on CMA which, by compromising the degradation of a-syn, could underlie toxicity in PD affected neurons. Based on our findings, we propose that interventions aimed at enhancing CMA activity, or at preventing its functional decrease with age, may prove to be valuable in the treatment of PD and other age-related disorders resulting from alterations in cellular proteostasis.