Uncovering a role for S-nitrosylated phospholipase D3 in the microglial and neuronal pathophysiology of Alzheimer's disease

About This Grant

Project Summary/Abstract Alzheimer’s disease (AD) is an age-related neurodegenerative disease affecting millions of people worldwide. Its pathological hallmarks include extracellular plaques formed from amyloid beta (Aβ) peptide, dystrophic neurites containing hyperphosphorylated tau protein, oxidative and nitrosative stress, microglial activation, severe synapse loss, and neuronal death; the consequence of these changes is progressive dementia. Attempts to treat AD by targeting Aβ have so far fallen short of addressing the entire pathology, and more diverse strategies are critical to the pursuit of disease-altering therapies. A promising area of study is the cellular changes caused by redox processes that affect protein function; one such change is protein S- nitrosylation, a redox-mediated posttranslational modification whereby nitric oxide (NO)-related species react with a thiol group (or more properly thiolate anion) on a cysteine residue, a reaction discovered in our laboratory. S-Nitrosylated proteins (SNO-proteins) undergo structural and functional changes which can be disease-relevant, although the full import of these changes throughout the proteome is only now beginning to be explored. In a recent unbiased mass spectrometry study, our group found that S-nitrosylated phospholipase D3 (SNO-PLD3) is associated with AD in human brain samples compared to controls. This proposal seeks to elucidate mechanistically the pathological importance of this result to AD. PLD3, a lysosomal 5’ exonuclease found in many cell types, is involved in regulation of toll-like receptor (TLR)9 innate immune signaling in macrophages; it is also known to colocalize with Aβ plaque-associated inclusions within dystrophic neurites in AD mouse models and human AD patients, and single nucleotide polymorphisms in PLD3 confer genetic risk for AD. My preliminary data show that S-nitrosylation of PLD3 increases its enzymatic activity. In light of these results, recently-published crystal structure data, and the fact that S-nitrosylation of a cysteine residue can facilitate disulfide bond formation between vincinal cysteines, I propose to test the hypothesis that S- nitrosylation of PLD3 facilitates a reversible “signaling” disulfide bond that serves to increase PLD3 stability and enzymatic activity, thus downregulating TLR9 signaling—which has recently been linked to memory formation. I will elucidate the effect of S-nitrosylation on PLD3 structure, and then examine the cellular consequences, including TLR9 dysregulation, in human induced pluripotent stem cell (hiPSC)-derived models of microglia and neurons. Accordingly, my Specific Aims are to: 1. Determine the effect of S-nitrosylation on PLD3 structure and stability, and 2. Determine the impact of redox-mediated modulation of PLD3 exonuclease function on TLR9 signaling in microglia and neurons. Understanding the role of SNO-PLD3 in cellular pathologies may provide a new therapeutic target for AD involving redox modulation of protein function.