A dysfunctional endolysosomal pathway common to two sub-types of demyelinating Charcot–Marie–Tooth disease

Abstract

Autosomal dominant mutations in LITAF are responsible for the rare demyelinating peripheral neuropathy, Charcot–Marie–Tooth disease type 1C (CMT1C). The LITAF protein is expressed in many human cell types and we have investigated the consequences of two different LITAF mutations in primary fibroblasts from CMT1C patients using confocal and electron microscopy. We observed the appearance of vacuolation/enlargement of late endocytic compartments (late endosomes and lysosomes). This vacuolation was also observed after knocking out LITAF from either control human fibroblasts or from the CMT1C patient-derived cells, consistent with it being the result of loss-of-function mutations in the CMT1C fibroblasts. The vacuolation was similar to that previously observed in fibroblasts from CMT4J patients, which have autosomal recessive mutations in FIG4. The FIG4 protein is a component of a phosphoinositide kinase complex that synthesises phosphatidylinositol 3,5-bisphosphate on the limiting membrane of late endosomes. Phosphatidylinositol 3,5-bisphosphate activates the release of lysosomal Ca2+ through the cation channel TRPML1, which is required to maintain the homeostasis of endosomes and lysosomes in mammalian cells. We observed that a small molecule activator of TRPML1, ML-SA1, was able to rescue the vacuolation phenotype of LITAF knockout, FIG4 knockout and CMT1C patient fibroblasts. Our data describe the first cellular phenotype common to two different subtypes of demyelinating CMT and are consistent with LITAF and FIG4 functioning on a common endolysosomal pathway that is required to maintain the homeostasis of late endosomes and lysosomes. Although our experiments were on human fibroblasts, they have implications for our understanding of the molecular pathogenesis and approaches to therapy in two subtypes of demyelinating Charcot–Marie–Tooth disease.

Held Up in Traffic—Defects in the Trafficking Machinery in Charcot-Marie-Tooth Disease

“…This lack of recruitment has been shown to decrease EGFR degradation leading to prolonged ERK1/2 signaling. Late endosomes/lysosomes are enlarged in patients' fibroblasts, similarly to the phenotype reported for mutant FIG4 in CMT4J showing a defect in the degradative pathway (Edgar et al, 2020). By activating cation channel TRPML1, a homeostasis regulator of lysosomes in mammalian cells, the authors were able to rescue the vacuolar phenotype of both LITAF or FIG4 knockout cells demonstrating a common pathway.…”

Rare among Rare: Phenotypes of Uncommon CMT Genotypes

“…Lipopolysaccharide-induced tumor necrosis factor (LITAF) is involved in an endolysosomal pathway that is required to maintain the homeostasis of late endosomes and lysosomes [ 34 ]. LITAF pathogenic variants cause an autosomal dominant chronic demyelinating polyneuropathy, classified as CMT1C [ 35 ] ( Table 1 ), frequently but not exclusively presenting in young age [ 36 ].…”

Endosomal recycling tubule scission and integrin recycling involve the membrane curvature-supporting protein LITAF

“…In other cells, such as A2780 cells, reduction in membrane scission appears to manifest simply as a slowing of cargo exit from the endosome. Impaired recycling would also lead to the build-up of membrane within the endolysosomal system and thus could contribute towards endosomal vacuolation, a further morphological manifestation of impaired LITAF function ( Edgar et al, 2020 ; Lee et al, 2012 ; Zhu et al, 2013 ).…”

“…Of note, myotubularins support the hydrolysis of PtdIns3P to promote exit from the endosome ( Wallroth and Haucke, 2018 ), and MTMR2, MTMR5 and MTMR13 are mutated in CMT4B1– CMT4B3, respectively ( Azzedine et al, 2003 ; Bolino et al, 2000 ; Nakhro et al, 2013 ; Senderek et al, 2003 ). Fig4, a component of the PIKfyve complex required for generating the late endosomal lipid PtdIns3,5P 2 , is mutated in individuals with CMT4J ( Chow et al, 2007 ), which shares morphological similarities to CMT1C ( Edgar et al, 2020 ). Furthermore, similar to our finding that CMT1C disease mutations impair the partitioning of LITAF into recycling endosomes, mutations in SH3CT2 that mislocalise this protein from the recycling endosome to the sorting endosome cause CMT4B ( Roberts et al, 2010 ).…”

“…Mutations in LITAF give rise to the peripheral neuropathy, Charcot Marie Tooth disease type 1C (CMT1C) ( Azzedine et al, 2012 ; Street et al, 2003 ). However, although LITAF disease mutations induce endosomal vacuolation ( Edgar et al, 2020 ; Lee et al, 2012 ; Zhu et al, 2013 ), the mechanisms underlying CMT1C remain elusive. Here, we identify LITAF as a new membrane curvature protein that regulates the morphogenesis of recycling endosomes and integrin trafficking by supporting the scission of recycling tubules.…”