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An In Vitro Model of Charcot-Marie-Tooth Disease Type 4B2 Provides Insight Into the Roles of MTMR13 and MTMR2 in Schwann Cell Myelination.

ABSTRACT: Charcot-Marie-Tooth Disorder Type 4B (CMT4B) is a demyelinating peripheral neuropathy caused by mutations in myotubularin-related (MTMR) proteins 2, 13, or 5 (CMT4B1/2/3), which regulate phosphoinositide turnover and endosomal trafficking. Although mouse models of CMT4B2 exist, an in vitro model would make possible pharmacological and reverse genetic experiments needed to clarify the role of MTMR13 in myelination. We have generated such a model using Schwann cell-dorsal root ganglion (SC-DRG) explants from Mtmr13-/- mice. Myelin sheaths in mutant cultures contain outfoldings highly reminiscent of those observed in the nerves of Mtmr13-/- mice and CMT4B2 patients. Mtmr13-/- SC-DRG explants also contain reduced Mtmr2, further supporting a role of Mtmr13 in stabilizing Mtmr2. Elevated PI(3,5)P2 has been implicated as a cause of myelin outfoldings in Mtmr2-/- models. In contrast, the role of elevated PI3P or PI(3,5)P2 in promoting outfoldings in Mtmr13-/- models is unclear. We found that over-expression of MTMR2 in Mtmr13-/- SC-DRGs moderately reduced the prevalence of myelin outfoldings. Thus, a manipulation predicted to lower PI3P and PI(3,5)P2 partially suppressed the phenotype caused by Mtmr13 deficiency. We also explored the relationship between CMT4B2-like myelin outfoldings and kinases that produce PI3P and PI(3,5)P2 by analyzing nerve pathology in mice lacking both Mtmr13 and one of two specific PI 3-kinases. Intriguingly, the loss of vacuolar protein sorting 34 or PI3K-C2? in Mtmr13-/- mice had no impact on the prevalence of myelin outfoldings. In aggregate, our findings suggest that the MTMR13 scaffold protein likely has critical functions other than stabilizing MTMR2 to achieve an adequate level of PI 3-phosphatase activity.

SUBMITTER: Robinson DC

PROVIDER: S-EPMC6236487 | biostudies-literature | 2018 Jan-Dec

REPOSITORIES: biostudies-literature

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An In Vitro Model of Charcot-Marie-Tooth Disease Type 4B2 Provides Insight Into the Roles of MTMR13 and MTMR2 in Schwann Cell Myelination.

Robinson Danielle C DC Mammel Anna E AE Logan Anne M AM Larson Aubree A AA Schmidt Eric J EJ Condon Alec F AF Robinson Fred L FL

ASN neuro 20180101

Charcot-Marie-Tooth Disorder Type 4B (CMT4B) is a demyelinating peripheral neuropathy caused by mutations in myotubularin-related (MTMR) proteins 2, 13, or 5 (CMT4B1/2/3), which regulate phosphoinositide turnover and endosomal trafficking. Although mouse models of CMT4B2 exist, an in vitro model would make possible pharmacological and reverse genetic experiments needed to clarify the role of MTMR13 in myelination. We have generated such a model using Schwann cell-dorsal root ganglion (SC-DRG) ex ...[more]

PMID: 30419760

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Project description:BackgroundCharcot-Marie-Tooth (CMT) disease is the most common neuromuscular disorder in humans affecting 40 out of 100,000 individuals. In 2008, we described the clinical, electrophysiological and pathological findings of a demyelinating motor and sensory neuropathy in Miniature Schnauzer dogs, with a suspected autosomal recessive mode of inheritance based on pedigree analysis. The discovery of additional cases has followed this work and led to a genome-wide association mapping approach to search for the underlying genetic cause of the disease.MethodsFor genome wide association screening, genomic DNA samples from affected and unaffected dogs were genotyped using the Illumina CanineHD SNP genotyping array. SBF2 and its variant were sequenced using primers and PCRs. RNA was extracted from muscle of an unaffected and an affected dog and RT-PCR performed. Immunohistochemistry for myelin basic protein was performed on peripheral nerve section specimens.ResultsThe genome-wide association study gave an indicative signal on canine chromosome 21. Although the signal was not of genome-wide significance due to the small number of cases, the SBF2 (also known as MTMR13) gene within the region of shared case homozygosity was a strong positional candidate, as 22 genetic variants in the gene have been associated with demyelinating forms of Charcot-Marie-Tooth disease in humans. Sequencing of SBF2 in cases revealed a splice donor site genetic variant, resulting in cryptic splicing and predicted early termination of the protein based on RNA sequencing results.ConclusionsThis study reports the first genetic variant in Miniature Schnauzer dogs responsible for the occurrence of a demyelinating peripheral neuropathy with abnormally folded myelin. This discovery establishes a genotype/phenotype correlation in affected Miniature Schnauzers that can be used for the diagnosis of these dogs. It further supports the dog as a natural model of a human disease; in this instance, Charcot-Marie-Tooth disease. It opens avenues to search the biological mechanisms responsible for the disease and to test new therapies in a non-rodent large animal model. In particular, recent gene editing methods that led to the restoration of dystrophin expression in a canine model of muscular dystrophy could be applied to other canine models such as this before translation to humans.

Project description:Charcot-Marie-Tooth type 4 (CMT4) is an autosomal recessive severe form of neuropathy with genetic heterogeneity. CMT4B1 is caused by mutations in the myotubularin-related 2 (MTMR2) gene and as a member of the myotubularin family, the MTMR2 protein is crucial for the modulation of membrane trafficking. To enable future clinical trials, we performed a detailed review of the published cases with MTMR2 mutations and describe four novel cases identified through whole-exome sequencing (WES). The four unrelated families harbor novel homozygous mutations in MTMR2 (NM_016156, Family 1: c.1490dupC; p.Phe498IlefsTer2; Family 2: c.1479+1G>A; Family 3: c.1090C>T; p.Arg364Ter; Family 4: c.883C>T; p.Arg295Ter) and present with CMT4B1-related severe early-onset motor and sensory neuropathy, generalized muscle atrophy, facial and bulbar weakness, and pes cavus deformity. The clinical description of the new mutations reported here overlap with previously reported CMT4B1 phenotypes caused by mutations in the phosphatase domain of MTMR2, suggesting that nonsense MTMR2 mutations, which are predicted to result in loss or disruption of the phosphatase domain, are associated with a severe phenotype and loss of independent ambulation by the early twenties. Whereas the few reported missense mutations and also those truncating mutations occurring at the C-terminus after the phosphatase domain cause a rather mild phenotype and patients were still ambulatory above the age 30 years. Charcot-Marie-Tooth neuropathy and Centronuclear Myopathy causing mutations have been shown to occur in proteins involved in membrane remodeling and trafficking pathway mediated by phosphoinositides. Earlier studies have showing the rescue of MTM1 myopathy by MTMR2 overexpression, emphasize the importance of maintaining the phosphoinositides equilibrium and highlight a potential compensatory mechanism amongst members of this pathway. This proved that the regulation of expression of these proteins involved in the membrane remodeling pathway may compensate each other's loss- or gain-of-function mutations by restoring the phosphoinositides equilibrium. This provides a potential therapeutic strategy for neuromuscular diseases resulting from mutations in the membrane remodeling pathway.

Dataset Information

An In Vitro Model of Charcot-Marie-Tooth Disease Type 4B2 Provides Insight Into the Roles of MTMR13 and MTMR2 in Schwann Cell Myelination.

Publications

An In Vitro Model of Charcot-Marie-Tooth Disease Type 4B2 Provides Insight Into the Roles of MTMR13 and MTMR2 in Schwann Cell Myelination.

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