| Notes: | Advisor: James A. Mastrianni.
Thesis (Ph.D.)--The University of Chicago, Division of the Biological Sciences and the Pritzker School of Medicine, Committee on Cellular and Molecular Physiology, 2010.
Dissertation Abstracts International, Volume: 71-10, Section: B, page: 5955.
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| Summary: | Prion diseases are transmissible neurodegenerative disorders linked to the accumulation of a misfolded isoform (PrPSc) of the prion protein. Accumulating evidence suggests lysosomes might be a degradation endpoint and site of accumulation of PrPSc. We questioned whether this organelle plays a role in early quality control of misfolded PrP. I found that PrP carrying a disease-linked mutation (T183A) localized to lysosomes, despite the lack of trafficking to the plasmalemma and its retention in the ER. Chemical disruption of Golgi by brefeldin A did not prevent delivery to lysosomes, suggesting a direct ER-to-lysosome route. Time-lapse live cell imaging revealed rapid formation and early intracellular uptake of GFP-tagged PrP-T182A (murine equivalent of human T183A) aggregates into LysoTracker labeled vesicles. Compared with Wt-PrP, Mut-PrP was associated with accumulation of autophagy-related EM structures. Immunolabeling of these ultrastructures revealed colabeling of LAMP-1 or LC3 and PrP-T182A, confirming light microscopy results. PrP-T182A expressing cells also displayed a redistribution of LC3 positive vesicles towards sites of PrP expression, as well as an increased active LC3II to inactive LC3I ratio, a classical measure of autophagy induction. These cells also had an increase in autophagy-related protein levels such as LAMP-1, Beclin-1, Atg5 and Atg12. Colocalization of T182A-PrP with lysosomes was significantly reduced in the presence of the autophagy inhibitor 3-MA, or when expressed in autophagy-deficient ATG5-/- mouse embryonic fibroblasts (MEFs). Inhibition of autophagy increased the insoluble fraction of Mut-PrP and protease-resistant PrP, while autophagy activation by rapamycin reduced both. I also detected increased levels of CHOP and BiP chaperone transcripts, classical signs of ER stress in PrP-T182A expressing cells. These results support a link between early, pre-Golgi PrP misfolding/aggregation, leading to ER stress and activation of the Unfolded Protein Response (UPR) and the induction of autophagy, as a quality control mechanism to eliminate misfolded PrP, that contributes to the pathogenic pool of PrP.
To study the potential role of autophagy in vivo, the therapeutic effect of Rapamycin was tested in a mouse model of inherited prion disease. The Tg(PrP-A116V) mice express the murine homologue of PrP-A117V,129V, that causes GSS in humans. These mice recapitulate several findings of human GSS, including development of progressive ataxia, accumulation of insoluble PrP-A116V, and accumulation of plaques in cerebellum and hippocampus. Chronic Rapamycin injections of 20 mg/kg significantly improved survival (average age of death 173 +/- 3.7 days for vehicle group versus 189 +/- 3.8 days for Rapamycin treated group) in these mice, as well as delayed the appearance of ataxic symptoms (average age of onset of 134 +/- 2.6 days for vehicle group, compared to 159 + 4.2 days in Rapamycin group). Rapamycin-treated mice also had a significant reduction in the level of insoluble PrP-A116V relative to age-matched vehicle-treated mice. The significant delay in disease onset, increase in life span, and improvement in symptomatology of these mice support a beneficial effect of Rapamycin or similar analogues. This may be a useful therapeutic option for genetic prion disease, especially in light of its current availability for use in humans. This study represents the first evidence for a favorable effect on the treatment for genetic-based prion disease.
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