Defining the degradative and inflammatory responses to mitochondrial damage in astrocytes.

About This Grant

PROJECT ABSTRACT (SUMMARY) Astrocytes, the most abundant cell type in the brain, contribute to neuroinflammation seen in neurodegenerative diseases like Parkinson’s disease (PD). Mitochondrial dysfunction is also a hallmark of PD, and occurs in both neurons and astrocytes. However, the consequences of mitochondrial dysfunction in astrocytes are not well understood. As accumulating data link mitochondrial damage to onset of inflammation, it is essential to our understanding of PD to elucidate astrocytic responses to mitochondrial damage. Here, I will (1) define a pathway astrocytes use to clear damaged mitochondria (PINK1/Parkin mitophagy), and (2) identify inflammatory signaling cascades resulting from mitochondrial damage in primary astrocytes. In PINK1/Parkin mitophagy, PTEN-induced kinase 1 (PINK1) and ubiquitin E3 ligase Parkin coordinate assembly and phosphorylation of ubiquitin chains on damaged mitochondria. Autophagy receptors, which facilitate autophagosome biogenesis, are recruited to this ubiquitin. Our lab demonstrated that the NF-κB essential modulator (NEMO) protein is also recruited to Parkin- assembled ubiquitin chains. This is a novel mechanism for initiation of NF-κB signaling, a major innate immunity pathway. Using oxidative phosphorylation (OXPHOS) inhibitors as mitochondrial damaging agents, I found that NEMO recruitment to damaged mitochondria occurs in cortical murine astrocytes ex vivo. Prolonged treatment results in heightened transcription of NF-κB-associated cytokines TNF-α and Il6, and TNF-α upregulation is ameliorated by NF-κB inhibition. Studies in HeLa cells suggest the autophagy receptor p62 is recruited to mitochondria alongside NEMO. I observe both p62 upregulation and recruitment to damaged mitochondria in astrocytes, suggesting p62 participates in astrocytic mitophagy. About 50% of p62 localizes to phospho-ubiquitin, indicating phosphorylation of ubiquitin may drive p62/NEMO recruitment. However, it is unclear if p62 is necessary for mitophagy in astrocytes, how it influences NEMO recruitment, and whether NF-κB is the major pathway activated. Based on my initial data, I hypothesize p62 is required for mitophagy and promotes NEMO recruitment to ubiquitin chains (Aim 1), and that NF-κB is the major inflammatory pathway activated (Aim 2a,b), resulting in secretion of neurotoxic signals that promote neurodegeneration (Aim 2c). In Aim 1, I will test whether knocking down p62 hinders mitophagy in astrocytes. I will also perform in vitro reconstitution assays with purified protein to determine if the efficiency of NEMO recruitment to damaged mitochondria is increased by p62 and phosphorylation of ubiquitin. In Aim 2, I will analyze bulk RNA-sequencing data from wild-type (WT) and PINK1 knockout astrocytes treated with OXPHOS inhibitors or a vehicle control. I will look at differential gene expression related to inflammatory signaling and NF-κB, and validate sequencing results via an NF-κB inhibitor and qPCR. Finally, I will compare the neurotoxicity of WT and PINK1 knockout astrocytes with and without OXPHOS inhibitor treatment. This project will define how astrocytes clear damaged mitochondria and the inflammatory signaling activated by this mitochondrial damage, which is critical to understand the role of astrocytes in PD pathology.