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

Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer’s 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 protein targets to restore apoE4-related cytoskeletal defects. ### Competing Interest Statement The NJK laboratory has received research support from Vir Biotechnology, F. Hoffmann-La Roche, and Rezo Therapeutics. NJK has financially compensated consulting agreements with the Icahn School of Medicine at Mount Sinai, New York, Maze Therapeutics, Interline Therapeutics, Rezo Therapeutics, GEn1E Lifesciences, Inc. and Twist Bioscience Corp. He is on the Board of Directors of Rezo Therapeutics and is a shareholder in Tenaya Therapeutics, Maze Therapeutics, Rezo Therapeutics, and Interline Therapeutics. DLS has a consulting agreement with Maze Therapeutics and Rezo Therapeutics. RWM is the co-founder and shareholder of Escape Bio, Inc. RWM is the CEO, CSO, and shareholder of the stem cell company GABAeron, Inc. AdC is an employee of GABAeron, Inc. * PTMs : Post-translational modifications apoE : Apolipoprotein E VASP : Vasodilator-stimulated phosphoprotein Neuro-2a KD : Knockdown WT : Wild type Aβ : Amyloid β NFT : Neurofibrillary Tangle PPIs : protein-protein interactions APMS : Affinity purification-mass spectrometry