Quantitative proteomic analysis reveals apoE4-dependent phosphorylation of the actin-regulating protein VASP

Abstract

Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimers Disease (AD). While neurons generally produce a minority of the apoE in the central nervous system, neuronal expression of apoE increases dramatically in response to stress and is sufficient to drive pathology. Currently, the molecular mechanisms of how apoE4 expression may regulate pathology are not fully understood. Here we expand upon our previous studies measuring the impact of apoE4 on protein abundance to include the analysis of protein phosphorylation and ubiquitylation signaling in isogenic Neuro-2a cells expressing apoE3 or apoE4. ApoE4 expression resulted in a dramatic increase in VASP S235 phosphorylation in a PKA-dependent manner. This phosphorylation disrupted VASP interactions with numerous actin cytoskeletal and microtubular proteins. Reduction of VASP S235 phosphorylation via PKA inhibition resulted in a significant increase in filopodia formation and neurite outgrowth in apoE4-expressing cells, exceeding levels observed in apoE3-expressing cells. Our results highlight the pronounced and diverse impact of apoE4 on multiple modes of protein regulation and identify potential therapeutic targets to restore apoE4-related cytoskeletal defects.

A foundational atlas of autism protein interactions reveals molecular convergence

“…Previously, we have used PPI mapping to gain insight into a variety of different disease areas, including cancer 26,27 , infectious diseases 35,36,[38][39][40] , neurodegeneration 34,148,149 and heart disease 30,32 . In this study, we built a PPI map to study ASD, the largest such dataset focused on a neuropsychiatric disorder, which can be used as a resource for the identification of ASD biomarkers and therapeutic targets.…”

“…Previously, we have used PPI mapping to gain insight into a variety of different disease areas, including cancer 26,27 , infectious diseases 35,36,38–40 , neurodegeneration 34,148,149 and heart disease 30,32 In this study, we built a PPI map to study autism, the largest such dataset focused on a neuropsychiatric disorder, which can be used as a resource for the identification of ASD biomarkers and therapeutic targets. Importantly, in this study, we describe a pipeline that combines the PPI data (wild-type and mutant) with AF2, which allowed us to narrow in on specific interaction interfaces regulated by individual mutations that we analyzed in greater detail using a suite of tools including CRISPR-based genetics, stem cell differentiation and organoid generation.…”

Lysosomal proteomics reveals mechanisms of neuronal apoE4-associated lysosomal dysfunction

“…The subsequent affinity purification was performed as previously described. 28 For each sample, two 15 cm dishes were each rinsed with PBS and scraped in 500 μL NP40 lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA pH 8.0, 0.5% Nonidet P40 Substitute, complete protease inhibitor tablet, phosSTOP phosphatase inhibitor tablet) and combined before freezing on dry ice for 20 minutes.…”

“…Cells were harvested, affinity-enriched for GFP, and interacting proteins eluted for mass spectrometric analysis as previously described. 28 Within a respective apoE genotype cell line, proteins found to be consistently detected in all Lgals3bp-GFP replicates and significantly enriched over GFP-transfected controls were considered Lgals3bp interaction partners. The obtained datasets could be readily clustered based on the overexpressed protein, indicating a distinct Lgals3bp-GFP interactome not governed by its GFP affinity tag (Suppl.…”