Severe Consequences of SAC3/FIG4 Phosphatase Deficiency to Phosphoinositides in Patients with Charcot-Marie-Tooth Disease Type-4J

Charcot-Marie-Tooth disease type-4J (CMT4J), an autosomal recessively inherited peripheral neuropathy characterized by neuronal degeneration, segmental demyelination, and limb muscle weakness, is caused by compound heterozygous mutations in the SAC3/FIG4 gene, resulting in SAC3/FIG4 protein deficiency. SAC3/FIG4 is a phosphatase that not only turns over PtdIns(3,5)P₂ to PtdIns3P but also promotes PtdIns(3,5)P₂ synthesis by activating the PIKFYVE kinase that also makes PtdIns5P. Whether CMT4J patients have alterations in PtdIns(3,5)P₂, PtdIns5P or in other phosphoinositides (PIs), and if yes, in what direction these changes might be, has never been examined. We performed PI profiling in primary fibroblasts from a cohort of CMT4J patients. Subsequent to myo-[2-³H]inositol cell labeling to equilibrium, steady-state levels of PIs were quantified by HPLC under conditions concurrently detecting PtdIns5P, PtdIns(3,5)P₂, and the other PIs. Immunoblotting verified SAC3/FIG4 depletion in CMT4J fibroblasts. Compared to normal human controls (n = 9), both PtdIns(3,5)P₂ and PtdIns5P levels were significantly decreased in CMT4J fibroblasts (n = 13) by 36.4 ± 3.6% and 43.1 ± 4.4%, respectively (p < 0.0001). These reductions were independent of patients' gender or disease onset. Although mean values for PtdIns3P in the CMT4J cohort remained unchanged, there were high variations in PtdIns3P among individual patients. Aberrant endolysosomal vacuoles, typically seen under PtdIns(3,5)P₂ reduction, were apparent but not in fibroblasts from all patients. The subset of patients without aberrant vacuoles exhibited especially low PtdIns3P levels. Concomitant decreases in PtdIns5P and PtdIns(3,5)P₂ and the link between PtdIns3P levels and cellular vacuolization are novel insights shedding further light into the molecular determinants in CMT4J polyneuropathy.