Unraveling the role of DJ-1 in cellular RNA dynamics Relevance for neurodegeneration and Parkinson's Disease

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IntroductionDJ-1 protein, encoded by the PARK7 (Parkinson disease protein 7) gene, is a conserved protein composed of 189 amino acids, with a crucial role within cells in the cell's protection from oxidative stress, in mitochondrial health and, regulation of autophagy. DJ-1 mutations have been linked to autosomal recessive Parkinson's disease. Indeed, several mutations of DJ-1 have been identified as disease causative in PD patients: L166P, M26I, D149A, E163K and E46D. However, the mechanisms by which DJ-1 initiates neurodegeneration are poorly identified and further studies are needed to elucidate its role in PD pathogenesis.Stress granules (SGs) are membraneless, cytoplasmic aggregates of translationally stalled mRNAs, associated translation initiation factors and multiple RNA-binding proteins (RBPs) that represent the morphological consequence of a RNA triage process induced by various stress stimuli such as heat, UV radiation, viral infections. They have been recognized as precursors of pathological protein aggregates in certain neurodegenerative disorders. The first link between DJ-1 and SG (Stress granules) was the co-localization of the yeast DJ-1 homologs Hsp31 with SG and P-bodies after glucose starvation. Intriguingly, DJ-1 was found to interact with several SGs components in mammalian cells upon induction of hyperosmotic shock and oxidative stress, including eIF4A in an RNA-dependent manner.Remarkably, this study similarly demonstrated an interaction between DJ-1 and several hnRNPs (hnRNPM, hnRNPA2/B1, hnRNPA1, hnRNPV, hnRNP). HnRNPs are considered among the main SGs components, they are RBPs and are intricately linked to RNA metabolism. According to the studies mentioned above, we hypothesize a potential novel role for DJ-1 in SG dynamics and translational control upon stress conditions, which we aim to investigate within this project. Aims and ObjectivesThis research projects aims at investigating the role of DJ-1 in stress granule dynamics to better understand its role in the pathogenesis of Parkinson's Disease.This will be achieved by exploring DJ-1/RNA interactome in different stress conditions, studying the compartmentalization of DJ-1 within stress granules and determining the mRNA populations targeted to SGs in presence or absence of DJ-1. Ultimately, such analyses will further elucidate how DJ-1 loss of function leads to PD, providing important insight into the molecular pathogenesis of this disorder and potentially informing novel therapeutic strategies.MethodologyHuman neuroblastoma SH-SY5Y cells will be exposed to hyperosmotic shock, oxidative stress, or parkinsonian toxins (rotenone, MPP+), and will be lysed in polysome lysis buffer. RNAs interacting with DJ-1 will be purified by co-immunoprecipitation as in Repici et al. (2019), and RNA sequencing will be carried out. Gene ontology and related bioinformatics approaches will be employed to identify functional groups arising from the various stimuli, which will inform the signaling pathways regulated by DJ-1. U-2 OS cells expressing a GFP-G3BP1 fusion protein will be used to isolate SG cores after different types of stress. Immunoblotting will be performed on the SG cores to look for total DJ-1, as well as oxidized forms of DJ-1. This will clarify whether DJ-1 acts as an RNA shuttle protein in the outer shell of SGs or as a core SG component. Mass spectrometry will be used for the analysis of SG components to identify DJ-1 oxidation state and post translational modifications.Wild-type or CRISPR knockout SH-SY5Y cells will be used to study the mRNA populations targeted to SGs in presence or absence of DJ-1.