Study On The Alterative Characteristics And Relevant Molecular Mechanisms Of Autophagy During Prion Infections In Vitro And In Vivo

Prion diseases, also named transmissible spongiform encephalopathies (TSEs), are a kind of fatal neurodegenerative diseases affecting many mammalian hosts. Human prion diseases are classified as Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS), fatal familial insomnia (FFI) and Kuru. Animal prion diseases include scrapie in sheep, chronic wasting disease in deer and elk, bovine spongiform encephalopathy (BSE) in cattle and so on. All prion diseases share common neuropathological features, including neuron loss, spongiform degeneration and reactive gliosis. According to protein-only hypothesis, PrPSc with the ability to replicate itself, is the pathogen of prion diseases. Based on the dynamic study of PrPSc by Borchelt, the synthetic process of PrPSc is relatively slow, with a half-time of15h. Once PrPSc matures, it appears to be nondegradable and accumulatable. Therefore, we speculate that cellular protein degradation system probably plays critical roles in deposition of PrPSc and progression of prion diseases.Cellular autophagy existing widely in eukaryocytes is a lysosome-dependent degradation pathway. Many substrates can be degraded by autophagy, such as cellular macromolecule (proteins, glucides and lipids, etc.), cellular endogenous substrates (organelles) and exogenous substrates (bacteria and virus, etc.). Autophagy initiates as the formation of liposome-like membrane structure. Then it elongates and embraces substrates and form autophagosomes with double membranes. Subsequently, autophagosome is delivered to lysosome where it fuses with lysosome that can degrade substrates. Autophagy may function as double-blade in virus infections and neurodegenerative diseases. On one hand, autophagy can degrade virions and misfolded proteins to protect cells from apoptosis and death. On the other hand, over-stimulated autophagy can induce autophagy-associated cell death.To elucidate roles of autophagy in PrPSc deposition and development of prion diseases, we designed a series of in vitro and in vivo experiments. Our results illustrated the alterative characteristics and molecular mechanisms of autophagic system in prion diseases, which may help to establish novel therapeutic strategy based on autophagy for anti-prion treatment.The main results of this study are as follows:1. The numbers of autophagosomes and the autophagic flux were tested in a mouse brain cell line SMB-S15that is stably infected by scrapie agent Chandler strain, and its normal partner cell line SMB-PS derived from cured SMB-S15cells with pentosan sulfate. Our results showed that the level of LC3-II was up-regulated but that of p62was down-regulated in SMB-S15vs. SMB-PS cells. In autophagic flux assay, the number of autolysosomes also increased in SMB-S15cells after transfection of tfLC3reporter, suggesting that prion infection can increase the number of autophagosomes and activate autophagic flux.2. The mTOR signaling pathway was down-regulated in SMB-S15cells but Beclinl signaling pathway maintained almost unchanged compared with SMB-PS cells. Meanwhile, E3ligase FBXW7targeting mTOR was up-regulated and more prone to colocalize with mTOR after prion infection.3. To assess the exact role of endogenous autophagy in PrPSc deposition, we detected PrPSc after blocking autophagic flux by RNA interference technique targeting Atg5and several autophagy inhibitors in SMB-S15cells. Our results indicated that inhibition of autophagy could increase PrPSc deposition in the cells.4. To assess the exact roles of FBXW7in endogenous autophagy and PrPSc deposition, we observed the change of PrPSc deposit after RNA interference targeting FBXW7. Our results indicated that knockdown of FBXW7could inhibit autophagy and increase PrPSc deposition.5. To investigate the autophagy regulation in vivo, brain samples from263K-infected hamsters and two patients with CJD and FFI were analyzed. Our results showed severely down-regulated mTOR signaling pathway in the brains of different prion diseases, which consequently activated autophagy system.6. To test if PrPSc colocalized with autophagosome, SMB-S15cell line and brain tissue sections of263K-infected hamsters were subjected to immunoffuorescent assay. In the presence of Bafilomycin Al, obvious colocalization of PrPSc and autophagosome were observed in SMB-S15cell line but not in brain tissue sections of263K-infected hamsters.7. To address the relationship between autophagy and cell viability, the survival of SMB-S15cells were tested after treatment with autophagy inhibitor3MA. Simultaneously, the brain tissue sections of263K-infected hamsters were stained with Nissl dye and then the neuron loss were analyzed regionally. The results indicated that cell viability was increased after autophagy was blocked, suggesting that there was spatial correlation between autophagy and neuronal loss.Taking together, in the present study, we demonstrated that prion infection could activate autophagy in vitro and in vivo. Further, activated autophagy was induced by mTOR degradation that caused by FBXW7up-regulation. Activation of endogenous autophagy promoted PrPSc clearance and served as innate immune response. Meanwhile, activated autophagy may also contribute to neuronal death in prion infection.