Microglia play a pivotal role in neurodegenerative disease pathogenesis, but the mechanisms underlying microglia dysfunction and toxicity remain to be fully 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 lipid dysmetabolism and deficits in phagocytosis, a critical microglia function. Our cumulative data implicate an effect of ALS-linked PFN1 on the autophagy pathway, including enhanced binding of mutant PFN1 to the autophagy signaling molecule PI3P, as an underlying cause of defective phagocytosis in ALS-PFN1 iMGs. Indeed, phagocytic processing was restored in ALS-PFN1 iMGs with Rapamycin, an inducer of autophagic flux. These outcomes demonstrate the utility of iMGs for neurodegenerative disease research and highlight microglia vesicular degradation pathways as potential therapeutic targets for these disorders.
<p>Supplemental Figures 1-6. Figure S1: BRCA-deficient cancer cells fail to restrain replication in the presence of stress and ssDNA gaps develop. Figure S2: Replication restraint, depletion, and PDX controls. Figure S3: Depletion of SMARCAL1 or inhibition of MRE11 restores FP, but do not predict patient response and do not suppress ssDNA gaps, which are distinct from fork degradation. Figure S4: Fanconi Anemia Patient Fibroblasts with a RAD51 T131P Mutant Allele (HR Proficient, FP Deficient) Generate ssDNA Gaps, and FP is restored by RADX depletion. Figure S5: Apoptosis and Z-VAD-FMK Controls. Figure S6: Prediction Table of BRCAness and Chemoresponse.</p>
<div>Abstract<p>Defects in DNA repair and the protection of stalled DNA replication forks are thought to underlie the chemosensitivity of tumors deficient in the hereditary breast cancer genes <i>BRCA1</i> and <i>BRCA2</i> (BRCA). Challenging this assumption are recent findings that indicate chemotherapies, such as cisplatin used to treat BRCA-deficient tumors, do not initially cause DNA double-strand breaks (DSB). Here, we show that ssDNA replication gaps underlie the hypersensitivity of BRCA-deficient cancer and that defects in homologous recombination (HR) or fork protection (FP) do not. In BRCA-deficient cells, ssDNA gaps developed because replication was not effectively restrained in response to stress. Gap suppression by either restoration of fork restraint or gap filling conferred therapy resistance in tissue culture and BRCA patient tumors. In contrast, restored FP and HR could be uncoupled from therapy resistance when gaps were present. Moreover, DSBs were not detected after therapy when apoptosis was inhibited, supporting a framework in which DSBs are not directly induced by genotoxic agents, but rather are induced from cell death nucleases and are not fundamental to the mechanism of action of genotoxic agents. Together, these data indicate that ssDNA replication gaps underlie the BRCA cancer phenotype, “BRCAness,” and we propose they are fundamental to the mechanism of action of genotoxic chemotherapies.</p>Significance:<p>This study suggests that ssDNA replication gaps are fundamental to the toxicity of genotoxic agents and underlie the BRCA-cancer phenotype “BRCAness,” yielding promising biomarkers, targets, and opportunities to resensitize refractory disease.</p><p><i>See related commentary by Canman, p. 1214</i></p></div>
<p>Supplemental Figures 1-6. Figure S1: BRCA-deficient cancer cells fail to restrain replication in the presence of stress and ssDNA gaps develop. Figure S2: Replication restraint, depletion, and PDX controls. Figure S3: Depletion of SMARCAL1 or inhibition of MRE11 restores FP, but do not predict patient response and do not suppress ssDNA gaps, which are distinct from fork degradation. Figure S4: Fanconi Anemia Patient Fibroblasts with a RAD51 T131P Mutant Allele (HR Proficient, FP Deficient) Generate ssDNA Gaps, and FP is restored by RADX depletion. Figure S5: Apoptosis and Z-VAD-FMK Controls. Figure S6: Prediction Table of BRCAness and Chemoresponse.</p>
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