Targeting the APOE-Succinylation Axis for Therapeutic Intervention in Alzheimer's Disease - PROJECT SUMMARY Succinylation is a recently discovered post-translational modification (PTM) that occurs spontaneously on proteins due to the high reactivity of succinyl-CoA. The four carbon succinyl- group forms a covalent bond to positively-charged lysine residues, converting them to a negative charge. Thus, succinylation dramatically alters the structure and function of the modified protein. TCA cycle activity and succinyl-CoA levels fluctuate rapidly within the cell depending on its metabolic state. These changes help coordinate a cell’s phenotypic response to its environment but in neurological diseases the mitochondria are damaged or dysfunctional, impairing homeostatic mechanisms. The ε4 allele of apolipoprotein E (APOE) is the strongest genetic risk factor for late- onset Alzheimer’s disease (AD). Previous work has shown that mitochondrial function and TCA cycle metabolism are compromised in ε4 microglia and astrocytes, contributing to AD risk. However, it is unknown whether these TCA cycle disturbances in ε4 might disrupt the regulation of PTMs linked to cellular metabolism, such as succinylation. To address this knowledge gap, we analyzed the succinylome of mice homozygous for human APOE alleles. We found a dramatic increase in succinylation in ε4 hippocampi, including modifications on tau and glial fibrillary acidic protein (GFAP), a marker of astrocyte activation and inflammation. To test whether succinylation might also be involved in amyloid-β (Aβ) pathology, we analyzed the succinylome of 5x familial AD (5xFAD) mice. Strikingly, we observed a more than 100-fold increase in succinylation of APOE in the 5xFAD hippocampus. We uncovered three modified lysines (K105, K173, and K252). Remarkably, K252 is located in the lipid binding domain, within a region critical for its interaction with Aβ. We hypothesize that succinylation of APOE alters its structure, disrupting its binding to Aβ and lipids. Further, the succinylation of APOE itself and substrates are modulated by Aβ and APOE genotype. We hypothesize that inhibiting succinylation through its primary physiological regulator, sirtuin-5 (SIRT5), will restore metabolic function and reduce inflammation in ε4 microglia and astrocytes. In Aim 1, we will use mass spectrometry and fluorescence microscopy to map the landscape of protein succinylation in the brains of EFAD mice (human APOE mice crossed to 5xFAD), followed by validation in AD human tissue. In Aim 2, we will synthesize recombinant succinyl-APOE for in vitro assays in microglia and astrocytes that will investigate how succinylation of APOE impacts Aβ phagocytosis, lipid droplet accumulation, and inflammatory cytokine secretion. Finally, in Aim 3, we will test whether inhibiting succinylation through overexpression of sirtuin-5 (SIRT5) or by using a small molecule inhibitor (succinyl phosphonate) will improve Aβ phagocytosis, lower lipid droplet accumulation, and reduce inflammation. Our studies will elucidate the role of succinylation in AD. This novel PTM modifies key AD proteins and is regulated by APOE. Modulation of succinylation and SIRT5 represents a therapeutic approach which has not yet been tested in AD.
