P1‐211: PRO‐APOPTOTIC PROTEIN PUMA REGULATES AUTOPHAGY IN AN ALZHEIMER'S DISEASE MODEL

Abstract

Selective synaptic loss followed by neuronal loss underlies the pathogenesis of Alzheimer's disease (AD). Two major cell death modalities which influence the fate of a cell, under normal or anomalous conditions like neurodegeneration, are apoptosis and autophagy. Literature suggests autophagy being critical for aberrant neuronal health in AD. Pro-apoptotic protein Puma is implicated in AD and is shown to play an essential role in mitophagy in cancer cells. Our aim is to understand the role of Puma in regulating the autophagic pathway in an AD model. We performed immunohistochemistry and western blotting for autophagy profiling in 5XFAD mice brains. For time kinetics and down-regulation studies, immunocytochemistry and western blotting was done. Survivability assays were performed by nuclear counting technique. Mechanistic studies were performed by RNAi, transfection and co-immunoprecipitation. Cellular studies were performed in primary cortical neurons and pheochromocytoma (PC12) cells under amyloid-β (Aβ) treatment. Our immunohistochemistry studies suggested an increase in the levels of autophagic markers, LC3 and LAMP1, in brain slices of six month old 5XFAD mice as compared to wild type mice which was substantiated in the tissue samples by western blot. We performed a time-kinetics analysis of occurrence of autophagy and apoptosis in cortical neurons and PC12 cells under Aβ treatment for a period of 24 h and found a gradual advent of both the processes simultaneously. Interestingly, we found that inhibiting autophagy by 3-methyladenine and apoptosis by pan-caspase inhibitor had a synergystic effect on the survival of neurons suggesting a vicious crosstalk. Also, when Puma and its regulator FoxO transcription factor (both induce apoptosis in Aβ-treated neurons) were down-regulated exclusively, there were a significant decrease in autophagic initiation (LC3) and increase in its flux (p62) as opposed to Aβ treatment. Finally, our co-immunoprecipitation data showing an interaction between Puma and Beclin1 under Aβ-treated condition suggest that there may be a crosstalk between apoptosis and autophagy via Puma in AD. An extensive study on the Puma-Beclin1 gateway is under process. We conclude that pro-apoptotic protein Puma plays a major role in the initiation of autophagy and maintaining its flux in AD.