Search

Background Charcot–Marie–Tooth disease (CMT) is a genetically and clinically heterogeneous group of inherited neuropathies. Monoallelic pathogenic variants in ATP1A1 were associated with axonal and intermediate CMT. ATP1A1 encodes for the catalytic α1 subunit of the Na+/ K+ ATPase. Besides neuropathy, other associated phenotypes are spastic paraplegia, intellectual disability, and renal hypomagnesemia. We hereby report the first demyelinating CMT case due to a novel ATP1A1 variant. Methods Whole-exome sequencing on the patient’s genomic DNA and Sanger sequencing to validate and confirm the segregation of the identified p.P600R ATP1A1 variation were performed. To evaluate functional effects, blood-derived mRNA and protein levels of ATP1A1 and the auxiliary β1 subunit encoded by ATP1B1 were investigated. The ouabain-survival assay was performed in transfected HEK cells to assess cell viability, and two-electrode voltage clamp studies were performed in Xenopus oocytes. Results The variant was absent in the local and global control datasets, falls within a highly conserved protein position, and is in a missense-constrained region. The expression levels of ATP1A1 and ATP1B1 were significantly reduced in the patient compared to healthy controls. Electrophysiology indicated that ATP1A1p.P600R injected Xenopus oocytes have reduced Na+/ K+ ATPase function. Moreover, HEK cells transfected with a construct encoding ATP1A1p.P600R harbouring variants that confers ouabain insensitivity displayed a significant decrease in cell viability after ouabain treatment compared to the wild type, further supporting the pathogenicity of this variant. Conclusion Our results further confirm the causative role of ATP1A1 in peripheral neuropathy and broaden the mutational and phenotypic spectrum of ATP1A1-associated CMT.

Funder: The Republic of Cyprus through the Research and Innovation Foundation Foundation (Project: CULTURE/BR-NE/0416/07), Funder: Wallenberg Scholar and the fluid biomarker measurements in the lab of HZ and AJH were supported by the UK Dementia Research Institute at UCL, Mutations in the GJB1 gene, encoding the gap junction (GJ) protein connexin32 (Cx32), cause X-linked Charcot-Marie-Tooth disease (CMT1X), an inherited demyelinating neuropathy. We developed a gene therapy approach for CMT1X using an AAV9 vector to deliver the GJB1/Cx32 gene under the myelin protein zero (Mpz) promoter for targeted expression in Schwann cells. Lumbar intrathecal injection of the AAV9-Mpz.GJB1 resulted in widespread biodistribution in the peripheral nervous system including lumbar roots, sciatic and femoral nerves, as well as in Cx32 expression in the paranodal non-compact myelin areas of myelinated fibers. A pre-, as well as post-onset treatment trial in Gjb1-null mice, demonstrated improved motor performance and sciatic nerve conduction velocities along with improved myelination and reduced inflammation in peripheral nerve tissues. Blood biomarker levels were also significantly ameliorated in treated mice. This study provides evidence that a clinically translatable AAV9-mediated gene therapy approach targeting Schwann cells could potentially treat CMT1X.

Diseases of the nervous system; Myelin biology and repair; Peripheral nervous system Enfermedades del sistema nervioso; Biología y reparación de la mielina; Sistema nervioso periférico Malalties del sistema nerviós; Biologia i reparació de la mielina; Sistema nerviós perifèric To optimize gene delivery to myelinating Schwann cells we compared clinically relevant AAV serotypes and injection routes. AAV9 and AAVrh10 vectors expressing either EGFP or the neuropathy-associated gene GJB1/Connexin32 (Cx32) under a myelin specific promoter were injected intrathecally or intravenously in wild type and Gjb1-null mice, respectively. Vector biodistribution in lumbar roots and sciatic nerves was higher in AAVrh10 injected mice while EGFP and Cx32 expression rates and levels were similar between the two serotypes. A gradient of biodistribution away from the injection site was seen with both intrathecal and intravenous delivery, while similar expression rates were achieved despite higher vector amounts injected intravenously. Quantified immune cells in relevant tissues were similar to non-injected littermates. Overall, AAV9 and AAVrh10 efficiently transduce Schwann cells throughout the peripheral nervous system with both clinically relevant routes of administration, although AAV9 and intrathecal injection may offer a more efficient approach for treating demyelinating neuropathies. Generalitat de Catalunya, 2019FI_B2 00061, 2019FI_B2 00061, Fundació la Marató de TV3, 201607.10, Muscular Dystrophy Association, 603003.

Oligodendrocyte gap junctions (GJs) are vital for central nervous system myelination, but their involvement in multiple sclerosis (MS) pathology remains unknown. The aim of this study was to examine alterations of oligodendrocyte and related astrocyte GJs in MS lesions and normal-appearing white matter (NAWM). Post-mortem brain samples from 9 MS and 11 age-matched non-MS control patients were studied. Tissue sections that included both chronic active and inactive lesions were characterized neuropathologically with Luxol Fast Blue staining and immunostaining for myelin oligodendrocyte glycoprotein (MOG) and the microglial marker Iba1. We analyzed the expression of Cx32 and Cx47 in oligodendrocytes and of Cx43, the major astrocytic partner in oligodendrocyte-astrocyte (O/A) GJs by quantitative immunoblot and real-time PCR. Formation of GJ plaques was quantified by immunohistochemistry. Compared to control brains, both Cx32 and Cx47 GJ plaques and protein levels were reduced in and around MS lesions, while Cx43 was increased as part of astrogliosis. In the NAWM, Cx32 was significantly reduced along myelinated fibers whereas Cx47 showed increased expression mainly in oligodendrocyte precursor cells (OPCs). However, OPCs showed only limited connectivity to astrocytes. Cx43 showed modestly increased levels in MS NAWM compared to controls, while GJ plaque counts were unchanged. Our findings indicate that oligodendrocyte GJs are affected not only in chronic MS lesions but also in NAWM, where disruption of Cx32 GJs in myelinated fibers may impair myelin structure and function. Moreover, limited O/A GJ connectivity of recruited OPCs in the setting of persistent inflammation and astrogliosis may prevent differentiation and remyelination.

The X-linked form of Charcot-Marie-Tooth disease (CMT1X) is the second most common molecularly designated form of hereditary motor and sensory neuropathy. The clinical phenotype is characterized by progressive distal muscle atrophy and weakness, areflexia, and variable sensory abnormalities. Affected males have moderate-to-severe symptoms, whereas heterozygous females are usually mildly affected or even asymptomatic. Several patients also have manifestations of central nervous system involvement or hearing impairment. Electrophysiological and pathological studies of peripheral nerves show evidence of demyelinating neuropathy with prominent axonal degeneration. A large number of mutations in the GJB1 gene encoding the gap junction (GJ) protein connexin32 (Cx32) cause CMT1X. Cx32 is expressed by Schwann cells and oligodendrocytes, as well as by other tissues, and the GJ formed by Cx32 play an important role in the homeostasis of myelinated axons. The reported CMT1X mutations are diverse and affect both the promoter region as well as the coding region of GJB1. Many Cx32 mutants fail to form functional GJ, or form GJ with abnormal biophysical properties. Furthermore, Cx32 mutants are often retained intracellularly either in the endoplasmic reticulum or Golgi in which they could potentially have additional dominant-negative effects. Animal models of CMT1X demonstrate that loss of Cx32 in myelinating Schwann cells causes a demyelinating neuropathy. No definite phenotype-genotype correlation has yet been established for CMT1X and effective molecular based therapeutics for this disease, remain to be developed.