Investigating aberrant vesicular degradation and lipid metabolism in neurodegeneration

BOSCO/MASSI – ABSTRACT Profilin-1 (PFN1) is an actin-binding protein that is best known for its role in regulating cytoskeletal dynamics. In 2012, we identified mutations in PFN1 that cause the uniformly lethal neurodegenerative disease amyotrophic lateral sclerosis (ALS), however, the mechanism underlying PFN1-mediated ALS remains poorly understood. To address this knowledge gap, our team has established and/or characterized multiple ALS-PFN1 models, including human induced pluripotent stem cells (iPSCs) and knock-in mice with an ALS-linked PFN1 mutation, both of which allow for investigation of ALS-linked PFN1 at physiological expression levels. Further, we have established methods for isolating recombinant PFN1 variants for structural studies using NMR and for examining the molecular dynamics of PFN1 in complex with relevant binding partners. Through our collaborative research, the Bosco and Massi laboratories uncovered defects in phagocytic vesicular degradation in iPSC-derived microglia cells. The phagocytosis, autophagy and endolysosomal pathways all entail vesicular degradation with a convergence on the lysosome. Notably, our study identified a novel interaction between PFN1 and phosphatidylinositol 3-phosphate (PI3P), a lipid that is essential for autophagy and that is involved throughout the vesicular degradation pathway. Based on these observations, we hypothesize that mutant PFN1 impairs autophagy and vesicular degradation through a gain-of-toxic interaction with PI3P and potentially other PIPs involved in vesicular degradation. The goals of this proposal are to interrogate this hypothesis using PIP-specific probes and modulators in multiple ALS-relevant cell types (Aim 1) and through biophysical binding and structural analyses of PIP/PFN1 complexes within biologically relevant membrane contexts (Aim 2). Our preliminary lipidomics data also revealed lipid dysregulation in ALS-PFN1 mice that may be relevant to disease pathogenesis. Intriguingly, several of these lipids also regulate autophagy. In Aim 3, we will examine potential links between autophagy impairment and specific classes of lipids in models of ALS-PFN1 and TDP-43 that are associated with both ALS and the related disorder frontotemporal dementia (FTD). Collectively, the outcomes from these Aims will provide novel insight into PFN1-mediated ALS and unprecedented structural information on PFN1/PIP complexes. We will also gain broad insight into mechanisms of lipid dyshomeostasis and their link to vesicular degradation in ALS and ALS/FTD, with the goal of identifying interventive strategies that will mitigate lipid dyshomeostasis in these disorders.