The role of selective autophagy on tau clearance
Caballero Bendixsen, Benjamin
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The major cellular systems for protein degradation are the ubiquitin-proteasome system (UPS) and autophagy (lysosomal degradation). Two autophagic pathways, macroautophagy (MA) and chaperone-mediated autophagy (CMA) have been extensively characterized for their ability to degrade pathogenic proteins related to neurodegeneration, whereas the role of microautophagy (Ml) in this clearance remains poorly described. We are interested in the microtubule-associated protein tau, the major component of neurofibrillary tangles in neurons of patients affected by Alzheimer's disease (AD) that also accumulates in all tauopathies. Posttranslational modifications of tau (phosphorylation, acetylation, ubiquitination) modulate its aggregation and turnover. Failure to degrade tau leads to accumulation of the protein, which act as seeds for oligomerization and aggregation contributing to further dysfunction of the cellular quality control systems.;The goal of this thesis research was to characterize the contribution of different autophagy pathways to the degradation of normal and pathogenic forms of tau and to identify the impact of toxic tau proteins on these clearance mechanisms.;In the first part of this study we analyzed the degradation of unmodified and acetvlated tau. We found that endogenous tau associates with all autophagic compartments but with different relative abundance depending on the tau species. Thus, despite their usual degradation by macroautophagy, a fraction of acetylated tau variants associates with CMA related lysosomes whereas oligomeric acetylated forms of tau are enriched in late endosomes (LE). We have additionally isolated and interrogated these same fractions from healthy human brains for tau degradation, confirming our findings in control mouse models. When we analyzed the same fractions from human brains Tau and autophagy isolated from patients presenting with AD, we observed an increase in total acetylated tau levels, a redistribution of acetylated tau between lysosomal populations with different CMA activity, and an increase of oligomeric acetylated forms of tau in LE. Strikingly, these changes in acetylate tau resemble those that we previously observed in brains of mice with impaired CMA. In fact, our analysis of CMA activity in lysosomes isolated from human AD brain tissue confirmed aberrant CMA function in these patients.;Using cell lines expressing wild-type and acetyled mimetics of tau, we have found that acetylated species of tau inhibit CMA activity. To elucidate the molecular mechanism behind this inhibition, we isolated lysosomes and observed in an in vitro system an unusually rapid translocation of tau across the lysosomal membrane when compared to other CMA substrates. This observation is due in part to an increased interaction between tau and Hsc70 at lower pH, a phenomenon not observed to occur to acetylated tau. Exposure of lysosomes to acetylated forms of tau resulted in higher steady state levels of LAMP-2A to be organized into translocation complexes, which we attributed to the longer translocation time required for acetylated tau to cross the lysosomal membrane due to its reduced ability to interact with lumina) Hsc70. The differences in pH-dependent binding of acetylated tau to Hsc70 could potentially explain why tau is a very efficient substrate for CMA and why when it is acetylated its potential as a CMA substrate is diminished.;I presented in this thesis, we analyzed the interplay between autophagy and several tau variants and mutants. We engaged in a similar experimental approach as to those performed in the first part of this work in which we compare the efficiency of tau degradation via different autophagic pathways (CMA, MA and MI) utilizing physiological tau variants and two disease-related mutations. We found that mutations in tau result in a failure to be degraded by some autophagic pathways and in turn alter the activity of these clearance mechanisms. Additionally, we observe a compensatory upregulation of these autophagic pathways in the presence of specific forms of tau which fails to be sustained when additional cellular stressors occur, affecting cellular systems.;Overall, our data is consistent with a complex, bi-directional interplay between different forms of tau and the three autophagic pathways (CMA, MA, and MI). Understanding the intricacies of this cross-talk and the impact that posttranslational modifications and mutations have in the degradation of tau could help to direct the development of putative therapeutic approaches for tauopathies through the manipulation of autophagic pathways.