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Abstract

Lysosomal PI(3,5)P2 levels are controlled by the FIG4 phosphoinositide 5-phosphatase, functioning in complex with PIKFYVE and VAC14. Inherited FIG4 mutations are causative for Charcot-Marie-Tooth neuropathy type 4J (CMT4J), which manifests in progressive muscle weakness due to abnormal lysosomal storage in neurons. We isolated primary human skin fibroblasts from three CMT4J patients, all carrying the founder mutation c.122T>C, p.Ile41Thr and a second compound heterozygous mutation, and reprogrammed those into induced pluripotent stem cells (iPSCs) which expressed pluripotency markers and differentiated into cells of all germ layers in vitro. Primary fibroblasts as well as iPSC-derived cortical neurons (iCNs) were subjected to image-based autophagy assessments using the autophagy marker WIPI2, an important PI3P effector functioning in autophagy initiation, and p62, an autophagy receptor for ubiquitinated cargo, functioning in selective autophagy. Significantly less WIPI2-positive autophagosomes were detected in CMT4J patient-derived cells with dysfunctional FIG4, possibly due to a decrease in cellular PI3P levels. Further, less WIPI2-positive autophagosomes were also positive for p62, suggesting that selective cargo degradation may be compromised in CMT4J. These results were also achieved by using FIG4 knockout HAP1 cells. In summary, we provide evidence that compromised function of FIG4 affects selective autophagy, which may contribute to the pathology of CMT4J.