Zachary Augur

(he/him)

BWH Job Title:

Graduate student

Academic Rank:

N/A

Department/Division/Lab:

Neurology

Authors:

Zachary M. Augur, Garrett M. Fogo, Mason Arbery, Richard Zhu, Richard V. Pearse, Tracy L. Young-Pearse

Protein degradation in Alzheimer's disease: Insights from iPSC-derived neurons

Abstract

Alzheimer’s disease (AD) pathology is characterized by the aberrant aggregation of extracellular amyloid-beta plaques and intracellular phosphorylated tau tangles within the brain, leading to progressive cognitive decline. The protein degradation network, encompassing the autophagy-lysosomal system (autophagy) and the ubiquitin-proteasome system (UPS), is frequently implicated as deficient in AD, potentially influencing neurodegeneration and overall protein turnover in the brain. To explore selective protein degradation and turnover in neuronal proteostasis and AD, we utilized an induced pluripotent stem cell (iPSC)-derived neuronal system (iNs). Specifically, our lab has established over 50 human iN lines sourced from the Religious Order Study and Rush Memory and Aging Project (ROSMAP) cohorts, covering the entire clinical and neuropathological spectrum of late-onset AD. Across this genetically diverse iN population, we evaluated basal autophagy and UPS activity, revealing an inverse relationship between these two principal protein degradation pathways, potentially indicating compensatory mechanisms of basal protein degradation. We employed stable isotope labeling by amino acids in cell culture (SILAC) in a subset of ROSMAP lines and iNs with familial AD mutations, to create a resource for quantifying protein turnover at a single-protein level across different genetic backgrounds. This innovative approach allows for precise tracking of protein dynamics and alterations in response to genetic background or basal protein turnover. Proteomic analysis and western blotting of these iNs unveiled significant associations between phosphorylated tau species and proteins pertinent to selective autophagy of protein aggregates (aggrephagy). Notably, the ubiquitin-dependent aggrephagy/mitophagy adaptor protein, optineurin (OPTN), exhibited the most significant negative correlation with aggregated phospho-tau forms in our AD neurons. To investigate the role of OPTN further, we disrupted OPTN expression, via CRISPR-Cas9 mediated editing, in two iN genetic backgrounds and subsequently differentiated these cells into iNs. OPTN knockout (KO) altered tau levels without affecting total autophagic flux or basal mitochondrial respiration, despite its interaction with several mitochondrial proteins, as confirmed by co-immunoprecipitation (co-IP). The chaperone protein Clusterin (Clu), displayed upregulation upon OPTN deletion in iNs, suggesting it as a potential substrate of OPTN-mediated autophagy. To deepen our understanding of OPTN ablation in neurons, we plan to subject these cells to various stressors, including human AD brain extract, oxidative stress, and inhibitors of mitochondrial function, to assess stress-induced mitophagy, mitochondrial function, and neuronal activity.