Expression of ALS-PFN1 impairs vesicular degradation in iPSC-derived microglia.

Microglia play a pivotal role in neurodegenerative disease pathogenesis, but the mechanisms underlying microglia dysfunction and toxicity remain to be elucidated. To investigate the effect of neurodegenerative disease-linked genes on the intrinsic properties of microglia, we studied microglia-like cells derived from human induced pluripotent stem cells (iPSCs), termed iMGs, harboring mutations in profilin-1 (PFN1) that are causative for amyotrophic lateral sclerosis (ALS). ALS-PFN1 iMGs exhibited evidence of lipid dysmetabolism, autophagy dysregulation and deficient phagocytosis, a canonical microglia function. Mutant PFN1 also displayed enhanced binding affinity for PI3P, a critical signaling molecule involved in autophagic and endocytic processing. Our cumulative data implicate a gain-of-toxic function for mutant PFN1 within the autophagic and endo-lysosomal pathways, as administration of rapamycin rescued phagocytic dysfunction in ALS-PFN1 iMGs. These outcomes demonstrate the utility of iMGs for neurodegenerative disease research and implicate microglial vesicular degradation pathways in the pathogenesis of these disorders.

Anti-SOD1 Nanobodies That Stabilize Misfolded SOD1 Proteins Also Promote Neurite Outgrowth in Mutant SOD1 Human Neurons.

ALS-linked mutations induce aberrant conformations within the SOD1 protein that are thought to underlie the pathogenic mechanism of SOD1-mediated ALS. Although clinical trials are underway for gene silencing of SOD1, these approaches reduce both wild-type and mutated forms of SOD1. Here, we sought to develop anti-SOD1 nanobodies with selectivity for mutant and misfolded forms of human SOD1 over wild-type SOD1. Characterization of two anti-SOD1 nanobodies revealed that these biologics stabilize mutant SOD1 in vitro. Further, SOD1 expression levels were enhanced and the physiological subcellular localization of mutant SOD1 was restored upon co-expression of anti-SOD1 nanobodies in immortalized cells. In human motor neurons harboring the SOD1 A4V mutation, anti-SOD1 nanobody expression promoted neurite outgrowth, demonstrating a protective effect of anti-SOD1 nanobodies in otherwise unhealthy cells. In vitro assays revealed that an anti-SOD1 nanobody exhibited selectivity for human mutant SOD1 over endogenous murine SOD1, thus supporting the preclinical utility of anti-SOD1 nanobodies for testing in animal models of ALS. In sum, the anti-SOD1 nanobodies developed and presented herein represent viable biologics for further preclinical testing in human and mouse models of ALS.