Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by loss of motor neurons. Cumulative evidence shows that microglia contribute to disease progression, but the underlying mechanisms are unclear. Several ALS-related genes are highly expressed in microglia compared to neurons, including profilin-1 (PFN1). This raises the possibility that ALS-linked PFN1 mutations could induce microglia cell-autonomous dysfunction. Here, I sought to interrogate this possibility by differentiating human pluripotent stem cells (iPSCs) into microglia-like cells (iMGs). My work uncovered that ALS-PFN1 iMGs accumulate undegraded phagocytosed cargo in endo-lysosomal compartments which is recapitulated in vivo. ALS-PFN1 iMGs also exhibit dysregulation in the expression and cellular localization of crucial components of the endo-lysosomal pathway, impairments in the autophagy flux, and accumulation of lipid droplets. Intriguingly, rapamycin treatment ameliorates the accumulation of phagocytosed material in ALS-PFN1 iMGs and rescues the defects in the autophagy pathway, suggesting that an impaired autophagy flux contributes to ALS-PFN1-linked defects in microglial phagocytosis. In vitro experimentation uncovered that PFN1 interacts with phosphatidylinositol-3phosphate, a signaling molecule essential for autophagy and phagocytosis, and that this interaction is altered when PFN1 is mutated in ALS. Collectively, these findings implicate that ALS-PFN1 causes microglia dysfunction by hindering the autophagy flux, perturbing the endo-lysosomal pathway, and, in turn, causing delays in the degradation process during phagocytosis and inducing lipid dysmetabolism. These alterations may be partially driven by ALS-PFN1 distorted interactions with phosphoinositides. My work provides insight into PFN1 biology and opens new perspectives regarding microglia cell-autonomous defects in ALS that may contribute to neurodegeneration.

Aberrant translational repression is a feature of multiple neurodegenerative diseases. The association between disease-linked proteins and stress granules further implicates impaired stress responses in neurodegeneration. However, our knowledge of the proteins that evade translational repression is incomplete. It is also unclear whether disease-linked proteins influence the proteome under conditions of translational repression. To address these questions, a quantitative proteomics approach was used to identify proteins that evade stress-induced translational repression in arsenite-treated cells expressing either wild-type or amyotrophic lateral sclerosis (ALS)-linked mutant FUS. This study revealed hundreds of proteins that are actively synthesized during stress-induced translational repression, irrespective of FUS genotype. In addition to proteins involved in RNA- and protein-processing, proteins associated with neurodegenerative diseases such as ALS were also actively synthesized during stress. Protein synthesis under stress was largely unperturbed by mutant FUS, although several proteins were found to be differentially expressed between mutant and control cells. One protein in particular, COPBI, was downregulated in mutant FUS-expressing cells under stress. COPBI is the beta subunit of the coat protein I (COPI), which is involved in Golgi to endoplasmic reticulum (ER) retrograde transport. Further investigation revealed reduced levels of other COPI subunit proteins and defects in COPBI-relatedprocesses in cells expressing mutant FUS. Even in the absence of stress, COPBI localization was altered in primary and human stem cell-derived neurons expressing ALS-linked FUS variants. Our results suggest that Golgi to ER retrograde transport may be important under conditions of stress and is perturbed upon the expression of disease-linked proteins such as FUS.