SH3TC2/KIAA1985 protein is required for proper myelination and the integrity of the node of Ranvier in the peripheral nervous system

Abstract

Charcot–Marie–Tooth disease type 4C (CMT4C) is an early-onset, autosomal recessive form of demyelinating neuropathy. The clinical manifestations include progressive scoliosis, delayed age of walking, muscular atrophy, distal weakness, and reduced nerve conduction velocity. The gene mutated in CMT4C disease, SH3TC2 / KIAA1985 , was recently identified; however, the function of the protein it encodes remains unknown. We have generated knockout mice where the first exon of the Sh3tc2 gene is replaced with an enhanced GFP cassette. The Sh3tc2 Δ Ex1 /Δ Ex1 knockout animals develop progressive peripheral neuropathy manifested by decreased motor and sensory nerve conduction velocity and hypomyelination. We show that Sh3tc2 is specifically expressed in Schwann cells and localizes to the plasma membrane and to the perinuclear endocytic recycling compartment, concordant with its possible function in myelination and/or in regions of axoglial interactions. Concomitantly, transcriptional profiling performed on the endoneurial compartment of peripheral nerves isolated from control and Sh3tc2 Δ Ex1 /Δ Ex1 animals uncovered changes in transcripts encoding genes involved in myelination and cell adhesion. Finally, detailed analyses of the structures composed of compact and noncompact myelin in the peripheral nerve of Sh3tc2 Δ Ex1 /Δ Ex1 animals revealed abnormal organization of the node of Ranvier, a phenotype that we confirmed in CMT4C patient nerve biopsies. The generated Sh3tc2 knockout mice thus present a reliable model of CMT4C neuropathy that was instrumental in establishing a role for Sh3tc2 in myelination and in the integrity of the node of Ranvier, a morphological phenotype that can be used as an additional CMT4C diagnostic marker.

Altered Distribution of Juxtaparanodal Kv1.2 Subunits Mediates Peripheral Nerve Hyperexcitability in Type 2 Diabetes Mellitus

“…Morphometric analysis, including the g-ratio calculation, was performed as previously described (Arnaud et al, 2009).…”

“…RNA extraction, reverse transcription and qPCR conditions have been performed as previously described (Arnaud et al, 2009). The primers used were as follows: forward 5Ј-CTGGTACCCATCTGCA AG-3Ј, reverse 5Ј-GTGTGCTCTAGGACTGGATG-3Ј for K v 1.2; forward 5Ј-AAGGACGGGAAACGCGAGGG-3Ј, reverse 5Ј-ATCGATGGACGCTG GCGGG-3Ј for K v 1.1; forward 5Ј-AGACAGGCTCCCCCGGGATG-3Ј, reverse 5Ј-CATGGCCCGCACGGTCTCTTC-3Ј for K v ␤2; forward 5Ј-ACAC TAGTGGAAGAGCTGGA-3Ј, reverse 5Ј-ACGATCAGGTTCACAATC TC-3Ј for Na v 1.6; forward 5Ј-TTCACAAGTCTTCTAAGGACTCCTCG-3Ј, reverse 5Ј-GCACTGGCGTCTGCCG-3Ј for MPZ and forward 5Ј-TTGC TCTTCGTCTCCACCATC-3Ј, reverse 5Ј-TCGTGTGTCCATTGCCCA C-3Ј for PMP22.…”

“…For immunostainings, the following primary antibodies were used: K v 1.2 (1:200; NeuroMab, K14/16), pan-Na v (1: 100; Sigma, SP19 S6936), K v ␤2 (1:100, Alomone, APC-117), K v 1.1 (1:100; Alomone, APC-009) and MBP (1:100, Millipore Bioscience Research Reagents, MAB386) with the appropriate fluorescent secondary antibodies (Alexa Fluor 594 or 488 conjugated anti-rabbit, anti-mouse or anti-rat at a dilution of 1:200; Invitrogen). Nile Red staining on teased fibers was performed as previously described (Arnaud et al, 2009).…”

“…Mouse tissues were processed as described previously (Arnaud et al, 2009). Twenty-micrometer-thick sciatic nerve sections were prepared and fixed with Zamboni's fixative for 15 min at room temperature (RT).…”

A role of peripheral myelin protein 2 in lipid homeostasis of myelinating schwann cells

“…RNA extraction (pooled samples from three to five mice per condition were used) and reverse transcription have been described previously (Arnaud et al, 2009) …”