RARE DISEASE
X-linked Charcot-Marie-Tooth disease type 1
X-linked Charcot-Marie-Tooth disease type 1
X-linked Charcot-Marie-Tooth disease type 1
Synonyms: CMT1X, CMTX1
Synonyms: CMT1X, CMTX1
Synonyms: CMT1X, CMTX1
Drug discovery
0
drugs
With orphan designations
Overview
X-linked Charcot-Marie-Tooth disease type 1 (CMTX1) is the second most common hereditary neuropathy, caused by GJB1 gene mutations impairing connexin 32 gap junction function [1][5][13]. It manifests as progressive distal muscle weakness/atrophy, sensory loss, and areflexia, with males showing earlier/severe symptoms due to X-linked dominant inheritance [5][13][17]. Nerve conduction studies reveal intermediate slowing (25-45 m/s) with axonal degeneration [1][9]. Central nervous system involvement (transient white matter lesions, dysarthria) occurs rarely [9][13].
Burden
Progressive ambulatory decline: 34% of males require assistive devices by adulthood [13][18]
Chronic pain (68% patients) and fatigue directly correlate with disability severity [4][16]
Family planning challenges: 100% transmission risk from affected males to daughters [2][17]
Neurophysiological hallmark: Non-uniform conduction slowing (30-40 m/s in males) with temporal dispersion [5][13]
Therapies
Supportive care: Orthotics, physical therapy, and neuropathic pain management (gabapentin/pregabalin) [7][16]
Emerging therapies: Gene replacement (AAV-mediated GJB1 delivery) and neurotrophin-3 (AAV1.tMCK.NT3) in clinical trials [14][15]
Experimental targets: CSF-1 receptor inhibitors and connexin 32 chaperones in preclinical studies [1][3]
Categories: rare developmental anomalies during embryogenesis, rare genetic diseases, rare neurological diseases, rare otorhinolaryngological diseases
Research Papers
134 drug discovery papers about X-linked Charcot-Marie-Tooth disease type 1, with 2 first-in-class emerging drug candidates forecasted to outperform the average preclinical success rate. Recent publications:
134 drug discovery papers about X-linked Charcot-Marie-Tooth disease type 1, with 2 first-in-class emerging drug candidates forecasted to outperform the average preclinical success rate. Recent publications:
categories:
Small molecules
gene therapies
2025-03-09 | A dose escalation and safety study of AAVrh10-mediated Schwann cell-targeted gene therapy for CMT1X.
X-linked Charcot-Marie-Tooth disease (CMT1X) is an inherited demyelinating neuropathy caused by loss-of-function mutations in the GJB1 gene, encoding the gap junction protein connexin32 (Cx32). Cx32 plays a critical role in Schwann cell function and myelin formation in the peripheral nervous system. We have developed a gene replacement therapeutic approach using a humanized AAVrh10 vector construct expressing GJB1 under the control of the Schwann cell-specific human myelin protein zero (MPZ) promoter. Lumbar intrathecal injection of increasing AAVrh10-hMPZ.GJB1 doses (low: 1 × 1011 vg, standard: 2 × 1011 vg and high: 1 × 1012 vg) into Gjb1-null mice resulted in adequate, dose-dependent biodistribution of the vector in anterior lumbar roots and peripheral nerves, as well as high rates of Schwann cell-specific Cx32 expression in the standard- and high-dose groups. Both standard and high vector doses provided significant therapeutic benefit evaluated by behavioural, electrophysiological and morphological outcomes. Intrathecal delivery of AAVrh10-hMPZ.GJB1 induced the production of anti-AAVrh10 antibodies at 6 weeks post-injection. However, no histopathological or inflammatory changes were observed in neural or peripheral tissues, besides a mild increase in inflammatory cell numbers in sciatic nerves of mice treated with the high dose only. This study provides proof of concept for a clinically translatable AAVrh10-mediated gene therapy approach for CMT1X.
2024-01-05 | Episodic Neurological Dysfunction in X-Linked Charcot-Marie-Tooth Disease: Expansion of the Phenotypic and Genetic Spectrum.
X-linked Charcot-Marie-Tooth disease type 1 (CMTX1) is characterized by peripheral neuropathy with or without episodic neurological dysfunction. We performed clinical, neuropathological, and genetic investigations of a series of patients with mutations of the gap-junction beta-1 gene (GJB1) to extend the phenotypic and genetic description of CMTX1. Detailed clinical evaluations, sural nerve biopsy, and genetic analysis were applied to patients with CMTX1. We collected 27 patients with CMTX1 with GJB1 mutations from 14 unrelated families. The age at onset (AAO) was 20.9±12.2 years (mean±standard deviation; range, 2-45 years). Walking difficulties, weakness in the legs, and pes cavus were common initial symptoms. Compared with female patients, males tended to have a younger AAO (males vs. females=15.4±9.6 vs. 32.0±8.8 years, p=0.002), a longer disease course (16.8±16.1 vs. 5.5±3.8 years, p=0.034), and more-severe electrophysiological results. Besides peripheral neuropathy, six of the patients had special episodic central nervous system (CNS) evidence from symptoms, signs, and/or reversible white-matter lesions. Neuropathology revealed the loss of large myelinated fibers, increased number of regenerated axon clusters with abnormally thin myelin sheaths, and excessively folded myelin. Genetic analysis identified 14 GJB1 variants, 6 of which were novel. These findings expand the phenotypic and genetic spectrum of CMTX1. Although CMTX1 was found to have high phenotypic and CNS involvement variabilities, detailed neurological examinations and nerve conduction studies will provide critical clues for accurate diagnoses. Further exploration of the underlying mechanisms of connexin 32 involvement in neuropathy or CNS dysfunction is warranted to develop promising therapies.
2023-12-21 | X-linked Charcot Marie Tooth mutations alter CO2 sensitivity of connexin32 hemichannels
Connexin32 (Cx32) is expressed in myelinating Schwann cells. It forms both reflexive gap junctions, to facilitate transfer of molecules from the outer to the inner myelin layers and hemichannels at the paranode to permit action potential-evoked release of ATP into the extracellular space. Loss of function mutations in Cx32 cause X-linked Charcot Marie Tooth disease (CMTX), a slowly developing peripheral neuropathy. The mechanistic links between Cx32 mutations and CMTX are not well understood. As Cx32 hemichannels can be opened by increases in PCO 2 , we have examined whether CMTX mutations alter this CO 2 sensitivity. By using Ca 2+ imaging, dye loading and genetically encoded ATP sensors to measure ATP release, we have found 5 CMTX mutations that abolish the CO 2 sensitivity of Cx32 hemichannels (A88D, 111–116 Del, C179Y, E102G, V139M). Others cause a partial loss (L56F, R220Stop, and R15W). Some CMTX mutations have no apparent effect on CO 2 sensitivity (R15Q, L9F, G12S, V13L, V84I, W133R). The mutation R15W alters multiple additional aspects of hemichannel function including Ca 2+ and ATP permeability. The mutations that abolish CO 2 sensitivity are transdominant and abolish CO 2 sensitivity of co-expressed Cx32 WT . We have shown that Schwannoma RT4 D6P2T cells can release ATP in response to elevated PCO 2 via the opening of Cx32. This is consistent with the hypothesis that the CO 2 sensitivity of Cx32 may be important for maintenance of healthy myelin. Our data, showing a transdominant effect of certain CMTX mutations on CO 2 sensitivity, may need to be taken into account in any future gene therapies for this condition.
2023-09-22 | Clinical trials in Charcot-Marie-Tooth disorders: a retrospective and preclinical assessment
This study aimed to evaluate the progression of clinical and preclinical trials in Charcot-Marie-Tooth (CMT) disorders.CMT has historically been managed symptomatically and with genetic counseling. The evolution of molecular and pathologic understanding holds a therapeutic promise in gene-targeted therapies.ClinicalTrials.gov from December 1999 to June 2022 was data extracted for CMT with preclinical animal gene therapy trials also reviewed by PubMed search.The number of active trials was 1 in 1999 and 286 in 2022. Academic settings accounted for 91% and pharmaceutical companies 9%. Of the pharmaceutical and academic trials, 38% and 28%, respectively, were controlled, randomized, and double-blinded. Thirty-two countries participated: the United States accounted for 26% (75/286). In total, 86% of the trials were classified as therapeutic: 50% procedural (21% wrist/elbow surgery; 22% shock wave and hydrodissection therapy), 23% investigational drugs, 15% devices, and 11% physical therapy. Sixty-seven therapeutic trials (49%) were designated phases 1-2 and 51% phases 3-4. The remaining 14% represent non-therapeutic trials: diagnostic testing (3%), functional outcomes (4%), natural history (4%), and standard of care (3%). One-hundred and three (36%) resulted in publications. Phase I human pharmaceutical trials are focusing on the safety of small molecule therapies (n = 8) and AAV and non-viral gene therapy (n = 3). Preclinical animal gene therapy studies include 11 different CMT forms including viral, CRISPR-Cas9, and nanoparticle delivery.Current CMT trials are exploring procedural and molecular therapeutic options with substantial participation of the pharmaceutical industry worldwide. Emerging drug therapies directed at molecular pathogenesis are being advanced in human clinical trials; however, the majority remain within animal investigations.
2023-08-30 | Gene replacement therapy in two Golgi-retained CMT1X mutants before and after the onset of demyelinating neuropathy.
X-linked Charcot-Marie-Tooth disease type 1 (CMT1X) is a demyelinating neuropathy resulting from loss-of-function mutations affecting the GJB1/connexin 32 (Cx32) gene. We previously showed functional and morphological improvement in Gjb1-null mice following AAV9-mediated delivery of human Cx32 driven by the myelin protein zero (Mpz) promoter in Schwann cells. However, CMT1X mutants may interfere with virally delivered wild-type (WT) Cx32. To confirm the efficacy of this vector also in the presence of CMT1X mutants, we delivered AAV9-Mpz-GJB1 by lumbar intrathecal injection in R75W/Gjb1-null and N175D/Gjb1-null transgenic lines expressing Golgi-retained mutations, before and after the onset of the neuropathy. Widespread expression of virally delivered Cx32 was demonstrated in both genotypes. Re-establishment of WT Cx32 function resulted in improved muscle strength and increased sciatic nerve motor conduction velocities in all treated groups from both mutant lines when treated before as well as after the onset of the neuropathy. Furthermore, morphological analysis showed improvement of myelination and reduction of inflammation in lumbar motor roots and peripheral nerves. In conclusion, this study provides proof of principle for a clinically translatable gene therapy approach to treat CMT1X before and after the onset of the neuropathy, even in the presence of endogenously expressed Golgi-retained Cx32 mutants.
small molecules
2026-05-19 | Neuromuscular junction dysfunction in a subset of Charcot-Marie Tooth and related peripheral neuropathies mouse models.
Charcot-Marie Tooth (CMT) disease is a clinically and genetically heterogeneous inherited peripheral neuropathy for which there is no treatment. CMT patients often present with weakness, fatigue, and muscle atrophy in the distal limbs. Improving function at the neuromuscular junction (NMJ) may improve function in some CMT patients. Using mouse models, we investigated eight CMT subtypes for NMJ phenotypes by morphology and functional deficits assessed by electromyography (EMG). We did not find NMJ abnormalities in mice with mutations in Gjb1Y/Δ2 (CMT1X), or Yars1E196K/E196K (diCMTC). Mice with mutations in Ighmbp2Y918S/Y918S (CMT2S) and Pla2g6M1J/M1J (Infantile Neuroaxonal Dystrophy) have neuromuscular phenotypes that could imply NMJ dysfunction, but we did not find defects in synaptic transmission or anatomy. A transgenic model of PMP22 overexpression (CMT1A) had EMG deficits with high frequency stimulation that are consistent with NMJ involvement. Three models showed indications of altered NMJ morphology and/or function. Gars+/ΔETAQ mice, modeling CMT2D, displayed robust synaptic deficits morphologically and by EMG. Nadk2S330P/S330P mice, modeling an ultrarare neuromuscular disease, had an EMG phenotype coinciding with symptom onset. Nefl+/N98S mice, modeling CMT2E, had normal EMG; but pre-synaptic axon terminals were dysmorphic, with large varicosities, which were more pronounced in proximal muscles. Across multiple models, we found that the extensor digitorum longus was resistant to disease phenotypes based on NMJ innervation status and/or muscle weight and atrophy. Our results indicate that some subtypes of CMT have NMJ deficits, and that assessing neuromuscular disease patients for NMJ dysfunction may reveal a population that could benefit from therapies that enhance transmission.
2025-08-13 | Genetic Deletion of Sarm1 in Mouse Models of Three Neurological Diseases.
Degeneration of peripheral motor and sensory axons is a key aspect of the pathophysiology of Charcot-Marie-Tooth disease and related inherited neurodegenerative conditions. Given that mutations in many (> 100) genes can cause these disorders, it is unclear if a generalized therapeutic strategy can be identified that will apply across these disease subtypes; however, strategies to prevent or slow axon degeneration are attractive candidates. Wallerian axon degeneration is an active process following insults such as nerve injury, and SARM1 is a central mediator of this process. When SARM1 is inhibited, axons distal to the site of injury persist for weeks rather than degenerating. In addition, SARM1 inhibition or genetic deletion has been shown to provide benefit in acquired neuropathies such as diabetic/metabolic neuropathy and chemotherapy-induced neuropathy in animal models. Here we examined the effects of genetically deleting Sarm1 in mouse models of CMT. We bred knockout mice lacking Sarm1 to three different mouse models of CMT or related disorders. These include mice lacking Gjb1, modeling CMT1X, mice with mutations in Kif1a, modeling hereditary sensory neuropathy IIC and spastic paraplegia type 30, and mice lacking Fig4, modeling CMT4J and Yunis-Varon syndrome. Clinically relevant outcomes measures including survival (Kif1a and Fig4), grip strength and motor behavior, peripheral neurophysiology, molecular biomarkers, and nerve histopathology were assessed for each model with and without Sarm1 expression. No improvement in the mutant phenotype was found for any model, although elevated levels of circulating neurofilament light chain levels were delayed in the Fig4 mice. Kif1a mice showed deficits slightly earlier in the absence of Sarm1. While we found no benefit from deleting Sarm1 in these mouse models, they were chosen for their human disease relevance and not for biochemical indicators that SARM1 may be a good target. Thus, SARM1 inhibition may still be effective in other forms of inherited neuropathy, but additional research will be required to identify those candidate subtypes.
2025-04-17 | Therapeutic sphingosine-1-phosphate receptor modulation by repurposing fingolimod (FTY720) leads to mitigated neuropathy and improved clinical outcome in a mouse model for Charcot-Marie-Tooth 1X disease.
Previous studies have shown that both the innate and adaptive immune systems foster progression of neuropathy and clinical symptoms in a mouse model for Charcot-Marie-Tooth 1X disease. Here we demonstrate a possible therapeutic translation of these findings using the clinically approved sphingosine-1-phosphate receptor modulator fingolimod (FTY720) in connexin32-deficient mice mimicking Charcot-Marie-Tooth 1X disease. Treatment with FTY720 prevented an increase of CD8+ and CD4+ T-lymphocyte numbers in both femoral quadriceps nerve as well as in ventral spinal roots. While macrophages of ventral spinal roots show a similar, albeit non-significant trend, macrophages from quadriceps nerve are not reduced upon treatment. On the histopathological level, axonopathic changes were reduced in ventral spinal roots, but not in quadriceps nerves upon treatment. Electrophysiological recordings displayed improved nerve conduction parameters upon FTY720 treatment, while clinically, FTY720 treatment ameliorated distinct parameters of motor performance and grip strength. We suggest that targeting the adaptive immune system might be a pharmacological treatment option for mitigating disease burden particularly in severe cases of Charcot-Marie-Tooth 1X.
2025-01-16 | Cemdomespib Therapy Slows the Progression of Neuromuscular Weakness and Demyelination in the R75W-Connexin 32 Animal Model of Charcot-Marie-Tooth 1X Disease.
Mutations in connexin 32 (Cx32) are a common cause of Charcot-Marie-Tooth 1X (CMT1X) disease, an inherited peripheral neuropathy characterized by progressive neuromuscular weakness and demyelination. There are no approved pharmacologic therapies for CMT1X, and identifying new treatments that slow the onset and severity of neuromuscular decline may aid disease management. Cemdomespib is an orally bioavailable small molecule that improved demyelination and neuromuscular junction (NMJ) morphology in mice lacking Cx32 expression. However, whether a similar efficacy may manifest in models of CMT1X arising from Cx32 mutations that cause the organellar accumulation of the protein was unclear. Additionally, it was unclear whether cemdomespib therapy slowed the rate of demyelination/NMJ degeneration or stabilized nerve and NMJ morphology to levels present at the initiation of drug therapy. To address these issues, 4-month-old R75W-Cx32 mice, which accumulate the mutant Cx32 in golgi, were treated for 0, 10, or 20 weeks with 0 or 3 mg/kg cemdomespib. Grip strength, motor nerve conduction velocity (MNCV), femoral nerve myelination, and NMJ morphology were quantified. Daily drug therapy significantly slowed the decline in grip strength over the course of treatment, while 20 weeks of drug treatment significantly improved MNCV and decreased the g-ratio and the number of thinly myelinated femoral nerve axons. Similarly, 20 weeks of cemdomespib therapy improved the NMJ morphology and the overlap between presynaptic (synaptophysin) and postsynaptic (α-bungarotoxin) markers. These data show that cemdomespib therapy slows the rate of neuromuscular decline and demyelination and may present a disease-modifying approach for patients with gain-of-function Cx32 mutations.
2024-12-31 | Mechanism of connexin channel inhibition by mefloquine and 2-aminoethoxydiphenyl borate.
Gap junction intercellular communication (GJIC) between two adjacent cells involves direct exchange of cytosolic ions and small molecules via connexin gap junction channels (GJCs). Connexin GJCs have emerged as drug targets, with small molecule connexin inhibitors considered a viable therapeutic strategy in several diseases. The molecular mechanisms of GJC inhibition by known small molecule connexin inhibitors remain unknown, preventing the development of more potent and connexin-specific therapeutics. Here we show that two GJC inhibitors, mefloquine (MFQ) and 2-aminoethoxydiphenyl borate (2APB) bind to Cx32 and block dye permeation across Cx32 hemichannels (HCs) and GJCs. Cryo-EM analysis shows that 2APB binds to "site A", close to the N-terminal gating helix of Cx32 GJC, restricting the entrance to the channel pore. In contrast, MFQ binds to a distinct "site M", deeply buried within the pore. MFQ binding to this site modifies the electrostatic properties of Cx32 pore. Mutagenesis of V37, a key residue located in the site M, renders Cx32 HCs and GJCs insensitive to MFQ-mediated inhibition. Moreover, our cryo-EM analysis, mutagenesis and activity assays show that MFQ targets the M site in Cx43 GJC similarly to Cx32. Taken together, our results point to a conserved inhibitor binding site in connexin channels, opening a new route for development of specific drugs targeting connexins.
proteins
2026-06-10 | Reversible area postrema syndrome in a patient with GJB1 mutation mimicking neuromyelitis optica spectrum disorder.
Area postrema syndrome (APS) is a core feature of neuromyelitis optica spectrum disorder (NMOSD). Charcot-Marie-Tooth disease type 1X (CMT1X), caused by gap junction protein beta-1 (GJB1) mutations, can rarely involve the central nervous system (CNS). To report a case of CMT1X presenting with APS mimicking NMOSD. Clinical, electrophysiological, radiological, and genetic evaluations were performed. A 43-year-old woman experienced intractable hiccups, nausea, and a dorsal medullary lesion. Genetic testing revealed a pathogenic GJB1 p.Arg22Gln variant. The lesion resolved, and she remained relapse-free without maintenance immunotherapy. GJB1-related disorders should be considered in patients with episodic CNS symptoms, particularly when accompanied by features of hereditary neuropathy, to avoid misdiagnosis.
2023-06-01 | Cytokine-Induced Neurogenesis in Charcot-Marie-Tooth neuropathy with Connexin 32 Gene Mutation
Charcot-Marie-Tooth (CMT) neuropathy is one of the most common neuromuscular disorders.Despite the identification of more than 100 causative genes, there are few therapeutic options.We present the case of a 66-year-old man carrying a CMT mutation in the connexin 32 gene with the APOE genotype ε4/ε3 that had exhibited symptoms of slowly progressive muscle weakness and atrophy in the hands and feet and recently developed memory impairment.The muscle weakness, motor dysfunction, and memory impairment symptoms were significantly improved by cytokine-induced neurogenesis combined with physical wave therapy using an arbitrary waveform generator (AWG).Here, we discuss the possible mechanistic basis of how this new treatment ameliorates the symptoms of CMT and propose a novel clinical treatment strategy for CMT.
2023-05-05 | TGFβ4 alleviates the phenotype of Charcot–Marie–Tooth disease type 1A
The duplication of the peripheral myelin protein 22 (PMP22) gene causes a demyelinating type of neuropathy, commonly known as Charcot-Marie-Tooth disease type 1A (CMT1A). Development of effective drugs for CMT1A still remains as an unmet medical need. In the present study, we assessed the role of the transforming growth factor beta 4 (TGFβ4)/Nodal axis in the pathogenesis of CMT1A. First, we identified PMP22 overexpression-induced Nodal expression in Schwann cells, which might be one of the downstream effectors in CMT1A. Administration of Nodal protein at the developmental stage of peripheral nerves induced the demyelinating phenotype in vivo. Second, we further isolated TGFβ4 as an antagonist that could abolish Nodal-induced demyelination. Finally, we developed a recombinant TGFβ4-fragment crystallizable (Fc) fusion protein, CX201, and demonstrated that its application had promyelinating efficacy in Schwann cells. CX201 administration improved the demyelinating phenotypes of CMT1A mouse models at both pre-symptomatic and post-symptomatic stages. These results suggest that the TGFβ4/Nodal axis plays a crucial role in the pathogenesis of CMT1A and might be a potential therapeutic target for CMT1A.
2017-11-06 | Graded Elevation of c-Jun in Schwann Cells In Vivo: Gene Dosage Determines Effects on Development, Remyelination, Tumorigenesis, and Hypomyelination.
Schwann cell c-Jun is implicated in adaptive and maladaptive functions in peripheral nerves. In injured nerves, this transcription factor promotes the repair Schwann cell phenotype and regeneration and promotes Schwann-cell-mediated neurotrophic support in models of peripheral neuropathies. However, c-Jun is associated with tumor formation in some systems, potentially suppresses myelin genes, and has been implicated in demyelinating neuropathies. To clarify these issues and to determine how c-Jun levels determine its function, we have generated c-Jun OE/+ and c-Jun OE/OE mice with graded expression of c-Jun in Schwann cells and examined these lines during development, in adulthood, and after injury using RNA sequencing analysis, quantitative electron microscopic morphometry, Western blotting, and functional tests. Schwann cells are remarkably tolerant of elevated c-Jun because the nerves of c-Jun OE/+ mice, in which c-Jun is elevated ∼6-fold, are normal with the exception of modestly reduced myelin thickness. The stronger elevation of c-Jun in c-Jun OE/OE mice is, however, sufficient to induce significant hypomyelination pathology, implicating c-Jun as a potential player in demyelinating neuropathies. The tumor suppressor P19 ARF is strongly activated in the nerves of these mice and, even in aged c-Jun OE/OE mice, there is no evidence of tumors. This is consistent with the fact that tumors do not form in injured nerves, although they contain proliferating Schwann cells with strikingly elevated c-Jun. Furthermore, in crushed nerves of c-Jun OE/+ mice, where c-Jun levels are overexpressed sufficiently to accelerate axonal regeneration, myelination and function are restored after injury. SIGNIFICANCE STATEMENT In injured and diseased nerves, the transcription factor c-Jun in Schwann cells is elevated and variously implicated in controlling beneficial or adverse functions, including trophic Schwann cell support for neurons, promotion of regeneration, tumorigenesis, and suppression of myelination. To analyze the functions of c-Jun, we have used transgenic mice with graded elevation of Schwann cell c-Jun. We show that high c-Jun elevation is a potential pathogenic mechanism because it inhibits myelination. Conversely, we did not find a link between c-Jun elevation and tumorigenesis. Modest c-Jun elevation, which is beneficial for regeneration, is well tolerated during Schwann cell development and in the adult and is compatible with restoration of myelination and nerve function after injury.
2017-10-25 | Cx32 hemichannel opening by cytosolic Ca2+ is inhibited by the R220X mutation that causes Charcot-Marie-Tooth disease
Mutations of the GJB1 gene encoding connexin 32 (Cx32) cause the X-linked form of Charcot–Marie–Tooth disease (CMTX1), a demyelinating peripheral neuropathy for which there is no cure. A growing body of evidence indicates that ATP release through Cx32 hemichannels in Schwann cells could be critical for nerve myelination, but it is unknown if CMTX1 mutations alter the cytosolic Ca2+-dependent gating mechanism that controls Cx32 hemichannel opening and ATP release. The current study uncovered that loss of the C-terminus in Cx32 (R220X mutation), which causes a severe CMTX1 phenotype, inhibits hemichannel opening during a canonical IP3-mediated increase in cytosolic Ca2+ in HeLa cells. Interestingly, the gating function of R220X hemichannels was completely restored by both the intracellular and extracellular application of a peptide that mimics the Cx32 cytoplasmic loop. All-atom molecular dynamics simulations suggest that loss of the C-terminus in the mutant hemichannel triggers abnormal fluctuations of the cytoplasmic loop which are prevented by binding to the mimetic peptide. Experiments that stimulated R220X hemichannel opening by cell depolarization displayed reduced voltage sensitivity with respect to wild-type hemichannels which was explained by loss of subconductance states at the single channel level. Finally, experiments of intercellular diffusion mediated by wild-type or R220X gap junction channels revealed similar unitary permeabilities to ions, signalling molecules (cAMP) or larger solutes (Lucifer yellow). Taken together, our findings support the hypothesis that paracrine signalling alteration due to Cx32 hemichannel dysfunction underlies CMTX1 pathogenesis and suggest a candidate molecule for novel studies investigating a therapeutic approach.
antibodies
2025-09-29 | [A case of X-linked Charcot-Marie-Tooth disease type 1 (CMTX1) diagnosed based on recurrent brain lesions despite peripheral neuropathy responsive to immunotherapy].
The patient is a 17-year-old male. He had a history of hospitalization for influenza at the age of 11, and Brain MRI at that time showed reversible brain lesions in the splenium of the corpus callosum and cerebral white matter. Fifteen months ago, he visited the pediatrics department due to dysphagia, dysarthria, facial paralysis, and muscle weakness. Brain MRI revealed lesions similar to those observed here, and nerve conduction study revealed demyelinating neuropathy. He was treated with intravenous immunoglobulin (IVIg) and intravenous methylprednisolone, and his symptoms disappeared within a few days and Brain MRI 5 weeks after treatment revealed that the lesions had disappeared. Three months ago, while walking, the patient developed a knee strain, which was thought to be a recurrence of the immune-mediated neuropathy. His subjective symptom disappeared after administration of IVIg. The patient was diagnosed with X-linked Charcot-Marie-Tooth disease (CMTX1) based on genetic testing, which revealed a pathological variant of GJB1, c.124A>T (p.Ser42Cys). Peripheral neuropathy in CMTX1 may present with fluctuating symptoms and can be responsive to IVIg treatment. Recurrent brain lesions should also be considered in the diagnosis of CMTX1.
2024-12-06 | A fully human IgG1 antibody targeting connexin 32 extracellular domain blocks CMTX1 hemichannel dysfunction in an in vitro model.
Connexins (Cxs) are fundamental in cell-cell communication, functioning as gap junction channels (GJCs) that facilitate solute exchange between adjacent cells and as hemichannels (HCs) that mediate solute exchange between the cytoplasm and the extracellular environment. Mutations in the GJB1 gene, which encodes Cx32, lead to X-linked Charcot-Marie-Tooth type 1 (CMTX1), a rare hereditary demyelinating disorder of the peripheral nervous system (PNS) without an effective cure or treatment. In Schwann cells, Cx32 HCs are thought to play a role in myelination by enhancing intracellular and intercellular Ca2+ signaling, which is crucial for proper PNS myelination. Single-point mutations (p.S85C, p.D178Y, p.F235C) generate pathological Cx32 HCs characterized by increased permeability ("leaky") or excessive activity ("hyperactive").We investigated the effects of abEC1.1-hIgG1, a fully human immunoglobulin G1 (hIgG1) monoclonal antibody, on wild-type (WT) and mutant Cx32D178Y HCs. Using HeLa DH cells conditionally co-expressing Cx and a genetically encoded Ca2+ biosensor (GCaMP6s), we demonstrated that mutant HCs facilitated 58% greater Ca2+ uptake in response to elevated extracellular Ca2+ concentrations ([Ca2+]ex) compared to WT HCs. abEC1.1-hIgG1 dose-dependently inhibited Ca2+ uptake, achieving a 50% inhibitory concentration (EC50) of ~ 10 nM for WT HCs and ~ 80 nM for mutant HCs. Additionally, the antibody suppressed DAPI uptake and ATP release. An atomistic computational model revealed that serine 56 (S56) of the antibody interacts with aspartate 178 (D178) of WT Cx32 HCs, contributing to binding affinity. Despite the p.D178Y mutation weakening this interaction, the antibody maintained binding to the mutant HC epitope at sub-micromolar concentrations.In conclusion, our study shows that abEC1.1-hIgG1 effectively inhibits both WT and mutant Cx32 HCs, highlighting its potential as a therapeutic approach for CMTX1. These findings expand the antibody's applicability for treating diseases associated with Cx HCs and inform the rational design of next-generation antibodies with enhanced affinity and efficacy against mutant HCs.
2016-02-10 | Cell-Surface and Secreted Isoforms of CSF-1 Exert Opposing Roles in Macrophage-Mediated Neural Damage in Cx32-Deficient Mice
Previous studies in myelin-mutant mouse models of the inherited and incurable nerve disorder, Charcot-Marie-Tooth (CMT) neuropathy, have demonstrated that low-grade secondary inflammation implicating phagocytosing macrophages amplifies demyelination, Schwann cell dedifferentiation and perturbation of axons. The cytokine colony stimulating factor-1 (CSF-1) acts as an important regulator of these macrophage-related disease mechanisms, as genetic and pharmacologic approaches to block the CSF-1/CSF-1R signaling result in a significant alleviation of pathological alterations in mutant peripheral nerves. In mouse models of CMT1A and CMT1X, as well as in human biopsies, CSF-1 is predominantly expressed by endoneurial fibroblasts, which are closely associated with macrophages, suggesting local stimulatory mechanisms. Here we investigated the impact of cell-surface and secreted isoforms of CSF-1 on macrophage-related disease in connexin32-deficient ( Cx32def ) mice, a mouse model of CMT1X. Our present observations suggest that the secreted proteoglycan isoform (spCSF-1) is predominantly expressed by fibroblasts, whereas the membrane-spanning cell-surface isoform (csCSF-1) is expressed by macrophages. Using crossbreeding approaches to selectively restore or overexpress distinct isoforms in CSF-1-deficient (osteopetrotic) Cx32def mice, we demonstrate that both isoforms equally regulate macrophage numbers dose-dependently. However, spCSF-1 mediates macrophage activation and macrophage-related neural damage, whereas csCSF-1 inhibits macrophage activation and attenuates neuropathy. These results further corroborate the important role of secondary inflammation in mouse models of CMT1 and might identify specific targets for therapeutic approaches to modulate innate immune reactions. SIGNIFICANCE STATEMENT Mouse models of Charcot-Marie-Tooth neuropathy have indicated that low-grade secondary inflammation involving phagocytosing macrophages amplifies demyelination, Schwann cell dedifferentiation, and perturbation of axons. The recruitment and pathogenic activation of detrimental macrophages is regulated by CSF-1, a cytokine that is mostly expressed by fibroblasts in the diseased nerve and exists in three isoforms. We show that the cell-surface and secreted isoforms of CSF-1 have opposing effects on macrophage activation and disease progression in a mouse model of CMT1X. These insights into opposing functions of disease-modulating cytokine isoforms might enable the development of specific therapeutic approaches.
2015-05-02 | Desert hedgehog is a mediator of demyelination in compression neuropathies
The secreted protein desert hedgehog (dhh) controls the formation of the nerve perineurium during development and is a key component of Schwann cells that ensures peripheral nerve survival. We postulated that dhh may play a critical role in maintaining myelination and investigated its role in demyelination-induced compression neuropathies by using a post-natal model of a chronic nerve injury in wildtype and dhh(-/-) mice. We evaluated demyelination using electrophysiological, morphological, and molecular approaches. dhh transcripts and protein are down-regulated early after injury in wild-type mice, suggesting an intimate relationship between the hedgehog pathway and demyelination. In dhh(-/-) mice, nerve injury induced more prominent and severe demyelination relative to their wild-type counterparts, suggesting a protective role of dhh. Alterations in nerve fiber characteristics included significant decreases in nerve conduction velocity, increased myelin debris, and substantial decreases in internodal length. Furthermore, in vitro studies showed that dhh blockade via either adenovirus-mediated (shRNA) or pharmacological inhibition both resulted in severe demyelination, which could be rescued by exogenous Dhh. Exogenous Dhh was protective against this demyelination and maintained myelination at baseline levels in a custom in vitro bioreactor to applied biophysical forces to myelinated DRG/Schwann cell co-cultures. Together, these results demonstrate a pivotal role for dhh in maintaining myelination. Furthermore, dhh signaling reveals a potential target for therapeutic intervention to prevent and treat demyelination of peripheral nerves in compression neuropathies.
2015-01-27 | CSF-1-activated macrophages are target-directed and essential mediators of Schwann cell dedifferentiation and dysfunction in Cx32-deficient mice.
We investigated connexin 32 (Cx32)‐deficient mice, a model for the X‐linked form of Charcot‐Marie‐Tooth neuropathy (CMT1X), regarding the impact of low‐grade inflammation on Schwann cell phenotype. Whereas we previously identified macrophages as amplifiers of the neuropathy, we now explicitly focus on the impact of the phagocytes on Schwann cell dedifferentiation, a so far not‐yet addressed disease‐related mechanism for CMT1X. Using mice heterozygously deficient for Cx32 and displaying both Cx32‐positive and ‐negative Schwann cells in one and the same nerve, we could demonstrate that macrophage clusters rather than single macrophages precisely associate with mutant but not with Cx32‐positive Schwann cells. Similarly, in an advanced stage of Schwann cell perturbation, macrophage clusters were strongly associated with NCAM‐ and L1‐positive, dedifferentiated Schwann cells. To clarify the role of macrophages regarding Schwann cell dedifferentiation, we generated Cx32‐deficient mice additionally deficient for the macrophage‐directed cytokine colony‐stimulating factor (CSF)−1. In the absence of CSF‐1, Cx32‐deficient Schwann cells not only showed the expected amelioration in myelin preservation but also failed to upregulate the Schwann cell dedifferentiation markers NCAM and L1. Another novel and unexpected finding in the double mutants was the retained activation of ERK signaling, a pathway which is detrimental for Schwann cell homeostasis in myelin mutant models. Our findings demonstrate that increased ERK signaling can be compatible with the maintenance of Schwann cell differentiation and homeostasis in vivo and identifies CSF‐1‐activated macrophages as crucial mediators of detrimental Schwann cell dedifferentiation in Cx32‐deficient mice. GLIA 2015;63:977–986
other
2026-02-03 | Patient-derived neural organoids reveal developmental impairments associated with a novel GJB1 mutation in X-linked Charcot-Marie-Tooth disease.
Charcot-Marie-Tooth disease (CMT) is one of the most prevalent inherited peripheral neuropathies. CMT type X1 (CMTX1), caused by mutations in the GJB1 gene, represents the most common X-linked subtype with central nervous system (CNS) involvement. Here, we report the identification and functional characterization of a novel GJB1 variant (c.554C > T, p.Thr185Ile) in a CMTX1-affected family and its pathogenic impact using patient-derived induced pluripotent stem cells (iPSCs) and three-dimensional (3D) neural organoid models. The GJB1 gene encodes connexin 32 (Cx32), a gap junction protein. Immunofluorescent analysis revealed aberrant intracellular reduction and aggregation of the mutant Cx32 protein, suggesting impaired gap junction function. iPSC-derived neural organoids carrying the GJB1 mutation exhibited significant delay in neural differentiation and disrupted neural rosette organization. These findings underscore the critical role of Cx32 in neural development and provide a physiologically relevant platform for underlying CMTX1 pathological mechanisms on central nervous system. The established GJB1-variant organoid model holds promise for investigating genotype-phenotype correlations and facilitating the development of targeted therapeutic strategies for CMTX1.
2023-12-17 | Preclinical Efficacy of Peripheral Nerve Regeneration by Schwann Cell-like Cells Differentiated from Human Tonsil-Derived Mesenchymal Stem Cells in C22 Mice
Charcot-Marie-Tooth disease (CMT) is a hereditary disease with heterogeneous phenotypes and genetic causes. CMT type 1A (CMT1A) is a type of disease affecting the peripheral nerves and is caused by the duplication of the peripheral myelin protein 22 (PMP22) gene. Human tonsil-derived mesenchymal stem cells (TMSCs) are useful for stem cell therapy in various diseases and can be differentiated into Schwann cell-like cells (TMSC-SCs). We investigated the potential of TMSC-SCs called neuronal regeneration-promoting cells (NRPCs) for peripheral nerve and muscle regeneration in C22 mice, a model for CMT1A. We transplanted NRPCs manufactured in a good manufacturing practice facility into the bilateral thigh muscles of C22 mice and performed behavior and nerve conduction tests and histological and ultrastructural analyses. Significantly, the motor function was much improved, the ratio of myelinated axons was increased, and the G-ratio was reduced by the transplantation of NRPCs. The sciatic nerve and gastrocnemius muscle regeneration of C22 mice following the transplantation of NRPCs downregulated PMP22 overexpression, which was observed in a dose-dependent manner. These results suggest that NRPCs are feasible for clinical research for the treatment of CMT1A patients. Research applying NRPCs to other peripheral nerve diseases is also needed.
2023-08-07 | The Current State of Charcot-Marie-Tooth Disease Treatment.
Charcot-Marie-Tooth disease (CMT) and associated neuropathies are the most predominant genetically transmitted neuromuscular conditions; however, effective pharmacological treatments have not established. The extensive genetic heterogeneity of CMT, which impacts the peripheral nerves and causes lifelong disability, presents a significant barrier to the development of comprehensive treatments. An estimated 100 loci within the human genome are linked to various forms of CMT and its related inherited neuropathies. This review delves into prospective therapeutic strategies used for the most frequently encountered CMT variants, namely CMT1A, CMT1B, CMTX1, and CMT2A. Compounds such as PXT3003, which are being clinically and preclinically investigated, and a broad array of therapeutic agents and their corresponding mechanisms are discussed. Furthermore, the progress in established gene therapy techniques, including gene replacement via viral vectors, exon skipping using antisense oligonucleotides, splicing modification, and gene knockdown, are appraised. Each of these gene therapies has the potential for substantial advancements in future research.
2019-08-27 | Downregulation of the human peripheral myelin protein 22 gene by miR-29a in cellular models of Charcot–Marie–Tooth disease
The majority of hereditary neuropathies are caused by duplication of the peripheral myelin protein 22 (PMP22) gene. Therefore, mechanisms to suppress the expression of the PMP22 gene have high therapeutic significance. Here we asked whether the human PMP22 gene is a target for regulation by microRNA 29a (miR-29a). Using bioinformatics, we determined that the human PMP22 gene contains the conserved seed sequence of the miR-29a binding site and this regulatory motif is included in the duplicated region in neuropathic patients. Using luciferase reporter assays in HEK293 cells, we demonstrated that transient transfection of a miR-29a mimic is associated with reduction in PMP22 3′UTR reporter activity. Transfecting normal and humanized transgenic neuropathic mouse Schwann cells with a miR-29a expression plasmid effectively lowered both the endogenous mouse and the transgenic human PMP22 transcripts compared with control vector. In dermal fibroblasts derived from neuropathic patients, ectopic expression of miR-29a led to ~50% reduction in PMP22 mRNA, which corresponded to ~20% decrease in PMP22 protein levels. Significantly, miR-29a-mediated reduction in PMP22 mitigated the reduced mitotic capacity of the neuropathic cells. Together, these results support further testing of miR-29a and/or PMP22-targeting siRNAs as therapeutic agents for correcting the aberrant expression of PMP22 in neuropathic patients.
2017-12-03 | Antisense oligonucleotides offer hope to patients with Charcot-Marie-Tooth disease type 1A
Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common heritable peripheral neuropathy and results from a duplication on chromosome 17 that results in an extra copy and increased dosage of peripheral myelin protein 22 (PMP22). Zhao et al., in this issue of the JCI, successfully utilized antisense oligonucleotides (ASOs) to reduce PMP22 and ameliorated neuropathy in both mouse and rat models of CMT1A. These data confirm that strategies to reduce PMP22 have potential as effective therapeutic approaches for CMT1A and lay the groundwork for clinical trials in humans afflicted with this chronic, debilitating neurodegenerative disease.
gene therapies
2025-03-09 | A dose escalation and safety study of AAVrh10-mediated Schwann cell-targeted gene therapy for CMT1X.
X-linked Charcot-Marie-Tooth disease (CMT1X) is an inherited demyelinating neuropathy caused by loss-of-function mutations in the GJB1 gene, encoding the gap junction protein connexin32 (Cx32). Cx32 plays a critical role in Schwann cell function and myelin formation in the peripheral nervous system. We have developed a gene replacement therapeutic approach using a humanized AAVrh10 vector construct expressing GJB1 under the control of the Schwann cell-specific human myelin protein zero (MPZ) promoter. Lumbar intrathecal injection of increasing AAVrh10-hMPZ.GJB1 doses (low: 1 × 1011 vg, standard: 2 × 1011 vg and high: 1 × 1012 vg) into Gjb1-null mice resulted in adequate, dose-dependent biodistribution of the vector in anterior lumbar roots and peripheral nerves, as well as high rates of Schwann cell-specific Cx32 expression in the standard- and high-dose groups. Both standard and high vector doses provided significant therapeutic benefit evaluated by behavioural, electrophysiological and morphological outcomes. Intrathecal delivery of AAVrh10-hMPZ.GJB1 induced the production of anti-AAVrh10 antibodies at 6 weeks post-injection. However, no histopathological or inflammatory changes were observed in neural or peripheral tissues, besides a mild increase in inflammatory cell numbers in sciatic nerves of mice treated with the high dose only. This study provides proof of concept for a clinically translatable AAVrh10-mediated gene therapy approach for CMT1X.
2024-01-05 | Episodic Neurological Dysfunction in X-Linked Charcot-Marie-Tooth Disease: Expansion of the Phenotypic and Genetic Spectrum.
X-linked Charcot-Marie-Tooth disease type 1 (CMTX1) is characterized by peripheral neuropathy with or without episodic neurological dysfunction. We performed clinical, neuropathological, and genetic investigations of a series of patients with mutations of the gap-junction beta-1 gene (GJB1) to extend the phenotypic and genetic description of CMTX1. Detailed clinical evaluations, sural nerve biopsy, and genetic analysis were applied to patients with CMTX1. We collected 27 patients with CMTX1 with GJB1 mutations from 14 unrelated families. The age at onset (AAO) was 20.9±12.2 years (mean±standard deviation; range, 2-45 years). Walking difficulties, weakness in the legs, and pes cavus were common initial symptoms. Compared with female patients, males tended to have a younger AAO (males vs. females=15.4±9.6 vs. 32.0±8.8 years, p=0.002), a longer disease course (16.8±16.1 vs. 5.5±3.8 years, p=0.034), and more-severe electrophysiological results. Besides peripheral neuropathy, six of the patients had special episodic central nervous system (CNS) evidence from symptoms, signs, and/or reversible white-matter lesions. Neuropathology revealed the loss of large myelinated fibers, increased number of regenerated axon clusters with abnormally thin myelin sheaths, and excessively folded myelin. Genetic analysis identified 14 GJB1 variants, 6 of which were novel. These findings expand the phenotypic and genetic spectrum of CMTX1. Although CMTX1 was found to have high phenotypic and CNS involvement variabilities, detailed neurological examinations and nerve conduction studies will provide critical clues for accurate diagnoses. Further exploration of the underlying mechanisms of connexin 32 involvement in neuropathy or CNS dysfunction is warranted to develop promising therapies.
2023-12-21 | X-linked Charcot Marie Tooth mutations alter CO2 sensitivity of connexin32 hemichannels
Connexin32 (Cx32) is expressed in myelinating Schwann cells. It forms both reflexive gap junctions, to facilitate transfer of molecules from the outer to the inner myelin layers and hemichannels at the paranode to permit action potential-evoked release of ATP into the extracellular space. Loss of function mutations in Cx32 cause X-linked Charcot Marie Tooth disease (CMTX), a slowly developing peripheral neuropathy. The mechanistic links between Cx32 mutations and CMTX are not well understood. As Cx32 hemichannels can be opened by increases in PCO 2 , we have examined whether CMTX mutations alter this CO 2 sensitivity. By using Ca 2+ imaging, dye loading and genetically encoded ATP sensors to measure ATP release, we have found 5 CMTX mutations that abolish the CO 2 sensitivity of Cx32 hemichannels (A88D, 111–116 Del, C179Y, E102G, V139M). Others cause a partial loss (L56F, R220Stop, and R15W). Some CMTX mutations have no apparent effect on CO 2 sensitivity (R15Q, L9F, G12S, V13L, V84I, W133R). The mutation R15W alters multiple additional aspects of hemichannel function including Ca 2+ and ATP permeability. The mutations that abolish CO 2 sensitivity are transdominant and abolish CO 2 sensitivity of co-expressed Cx32 WT . We have shown that Schwannoma RT4 D6P2T cells can release ATP in response to elevated PCO 2 via the opening of Cx32. This is consistent with the hypothesis that the CO 2 sensitivity of Cx32 may be important for maintenance of healthy myelin. Our data, showing a transdominant effect of certain CMTX mutations on CO 2 sensitivity, may need to be taken into account in any future gene therapies for this condition.
2023-09-22 | Clinical trials in Charcot-Marie-Tooth disorders: a retrospective and preclinical assessment
This study aimed to evaluate the progression of clinical and preclinical trials in Charcot-Marie-Tooth (CMT) disorders.CMT has historically been managed symptomatically and with genetic counseling. The evolution of molecular and pathologic understanding holds a therapeutic promise in gene-targeted therapies.ClinicalTrials.gov from December 1999 to June 2022 was data extracted for CMT with preclinical animal gene therapy trials also reviewed by PubMed search.The number of active trials was 1 in 1999 and 286 in 2022. Academic settings accounted for 91% and pharmaceutical companies 9%. Of the pharmaceutical and academic trials, 38% and 28%, respectively, were controlled, randomized, and double-blinded. Thirty-two countries participated: the United States accounted for 26% (75/286). In total, 86% of the trials were classified as therapeutic: 50% procedural (21% wrist/elbow surgery; 22% shock wave and hydrodissection therapy), 23% investigational drugs, 15% devices, and 11% physical therapy. Sixty-seven therapeutic trials (49%) were designated phases 1-2 and 51% phases 3-4. The remaining 14% represent non-therapeutic trials: diagnostic testing (3%), functional outcomes (4%), natural history (4%), and standard of care (3%). One-hundred and three (36%) resulted in publications. Phase I human pharmaceutical trials are focusing on the safety of small molecule therapies (n = 8) and AAV and non-viral gene therapy (n = 3). Preclinical animal gene therapy studies include 11 different CMT forms including viral, CRISPR-Cas9, and nanoparticle delivery.Current CMT trials are exploring procedural and molecular therapeutic options with substantial participation of the pharmaceutical industry worldwide. Emerging drug therapies directed at molecular pathogenesis are being advanced in human clinical trials; however, the majority remain within animal investigations.
2023-08-30 | Gene replacement therapy in two Golgi-retained CMT1X mutants before and after the onset of demyelinating neuropathy.
X-linked Charcot-Marie-Tooth disease type 1 (CMT1X) is a demyelinating neuropathy resulting from loss-of-function mutations affecting the GJB1/connexin 32 (Cx32) gene. We previously showed functional and morphological improvement in Gjb1-null mice following AAV9-mediated delivery of human Cx32 driven by the myelin protein zero (Mpz) promoter in Schwann cells. However, CMT1X mutants may interfere with virally delivered wild-type (WT) Cx32. To confirm the efficacy of this vector also in the presence of CMT1X mutants, we delivered AAV9-Mpz-GJB1 by lumbar intrathecal injection in R75W/Gjb1-null and N175D/Gjb1-null transgenic lines expressing Golgi-retained mutations, before and after the onset of the neuropathy. Widespread expression of virally delivered Cx32 was demonstrated in both genotypes. Re-establishment of WT Cx32 function resulted in improved muscle strength and increased sciatic nerve motor conduction velocities in all treated groups from both mutant lines when treated before as well as after the onset of the neuropathy. Furthermore, morphological analysis showed improvement of myelination and reduction of inflammation in lumbar motor roots and peripheral nerves. In conclusion, this study provides proof of principle for a clinically translatable gene therapy approach to treat CMT1X before and after the onset of the neuropathy, even in the presence of endogenously expressed Golgi-retained Cx32 mutants.
small molecules
2026-05-19 | Neuromuscular junction dysfunction in a subset of Charcot-Marie Tooth and related peripheral neuropathies mouse models.
Charcot-Marie Tooth (CMT) disease is a clinically and genetically heterogeneous inherited peripheral neuropathy for which there is no treatment. CMT patients often present with weakness, fatigue, and muscle atrophy in the distal limbs. Improving function at the neuromuscular junction (NMJ) may improve function in some CMT patients. Using mouse models, we investigated eight CMT subtypes for NMJ phenotypes by morphology and functional deficits assessed by electromyography (EMG). We did not find NMJ abnormalities in mice with mutations in Gjb1Y/Δ2 (CMT1X), or Yars1E196K/E196K (diCMTC). Mice with mutations in Ighmbp2Y918S/Y918S (CMT2S) and Pla2g6M1J/M1J (Infantile Neuroaxonal Dystrophy) have neuromuscular phenotypes that could imply NMJ dysfunction, but we did not find defects in synaptic transmission or anatomy. A transgenic model of PMP22 overexpression (CMT1A) had EMG deficits with high frequency stimulation that are consistent with NMJ involvement. Three models showed indications of altered NMJ morphology and/or function. Gars+/ΔETAQ mice, modeling CMT2D, displayed robust synaptic deficits morphologically and by EMG. Nadk2S330P/S330P mice, modeling an ultrarare neuromuscular disease, had an EMG phenotype coinciding with symptom onset. Nefl+/N98S mice, modeling CMT2E, had normal EMG; but pre-synaptic axon terminals were dysmorphic, with large varicosities, which were more pronounced in proximal muscles. Across multiple models, we found that the extensor digitorum longus was resistant to disease phenotypes based on NMJ innervation status and/or muscle weight and atrophy. Our results indicate that some subtypes of CMT have NMJ deficits, and that assessing neuromuscular disease patients for NMJ dysfunction may reveal a population that could benefit from therapies that enhance transmission.
2025-08-13 | Genetic Deletion of Sarm1 in Mouse Models of Three Neurological Diseases.
Degeneration of peripheral motor and sensory axons is a key aspect of the pathophysiology of Charcot-Marie-Tooth disease and related inherited neurodegenerative conditions. Given that mutations in many (> 100) genes can cause these disorders, it is unclear if a generalized therapeutic strategy can be identified that will apply across these disease subtypes; however, strategies to prevent or slow axon degeneration are attractive candidates. Wallerian axon degeneration is an active process following insults such as nerve injury, and SARM1 is a central mediator of this process. When SARM1 is inhibited, axons distal to the site of injury persist for weeks rather than degenerating. In addition, SARM1 inhibition or genetic deletion has been shown to provide benefit in acquired neuropathies such as diabetic/metabolic neuropathy and chemotherapy-induced neuropathy in animal models. Here we examined the effects of genetically deleting Sarm1 in mouse models of CMT. We bred knockout mice lacking Sarm1 to three different mouse models of CMT or related disorders. These include mice lacking Gjb1, modeling CMT1X, mice with mutations in Kif1a, modeling hereditary sensory neuropathy IIC and spastic paraplegia type 30, and mice lacking Fig4, modeling CMT4J and Yunis-Varon syndrome. Clinically relevant outcomes measures including survival (Kif1a and Fig4), grip strength and motor behavior, peripheral neurophysiology, molecular biomarkers, and nerve histopathology were assessed for each model with and without Sarm1 expression. No improvement in the mutant phenotype was found for any model, although elevated levels of circulating neurofilament light chain levels were delayed in the Fig4 mice. Kif1a mice showed deficits slightly earlier in the absence of Sarm1. While we found no benefit from deleting Sarm1 in these mouse models, they were chosen for their human disease relevance and not for biochemical indicators that SARM1 may be a good target. Thus, SARM1 inhibition may still be effective in other forms of inherited neuropathy, but additional research will be required to identify those candidate subtypes.
2025-04-17 | Therapeutic sphingosine-1-phosphate receptor modulation by repurposing fingolimod (FTY720) leads to mitigated neuropathy and improved clinical outcome in a mouse model for Charcot-Marie-Tooth 1X disease.
Previous studies have shown that both the innate and adaptive immune systems foster progression of neuropathy and clinical symptoms in a mouse model for Charcot-Marie-Tooth 1X disease. Here we demonstrate a possible therapeutic translation of these findings using the clinically approved sphingosine-1-phosphate receptor modulator fingolimod (FTY720) in connexin32-deficient mice mimicking Charcot-Marie-Tooth 1X disease. Treatment with FTY720 prevented an increase of CD8+ and CD4+ T-lymphocyte numbers in both femoral quadriceps nerve as well as in ventral spinal roots. While macrophages of ventral spinal roots show a similar, albeit non-significant trend, macrophages from quadriceps nerve are not reduced upon treatment. On the histopathological level, axonopathic changes were reduced in ventral spinal roots, but not in quadriceps nerves upon treatment. Electrophysiological recordings displayed improved nerve conduction parameters upon FTY720 treatment, while clinically, FTY720 treatment ameliorated distinct parameters of motor performance and grip strength. We suggest that targeting the adaptive immune system might be a pharmacological treatment option for mitigating disease burden particularly in severe cases of Charcot-Marie-Tooth 1X.
2025-01-16 | Cemdomespib Therapy Slows the Progression of Neuromuscular Weakness and Demyelination in the R75W-Connexin 32 Animal Model of Charcot-Marie-Tooth 1X Disease.
Mutations in connexin 32 (Cx32) are a common cause of Charcot-Marie-Tooth 1X (CMT1X) disease, an inherited peripheral neuropathy characterized by progressive neuromuscular weakness and demyelination. There are no approved pharmacologic therapies for CMT1X, and identifying new treatments that slow the onset and severity of neuromuscular decline may aid disease management. Cemdomespib is an orally bioavailable small molecule that improved demyelination and neuromuscular junction (NMJ) morphology in mice lacking Cx32 expression. However, whether a similar efficacy may manifest in models of CMT1X arising from Cx32 mutations that cause the organellar accumulation of the protein was unclear. Additionally, it was unclear whether cemdomespib therapy slowed the rate of demyelination/NMJ degeneration or stabilized nerve and NMJ morphology to levels present at the initiation of drug therapy. To address these issues, 4-month-old R75W-Cx32 mice, which accumulate the mutant Cx32 in golgi, were treated for 0, 10, or 20 weeks with 0 or 3 mg/kg cemdomespib. Grip strength, motor nerve conduction velocity (MNCV), femoral nerve myelination, and NMJ morphology were quantified. Daily drug therapy significantly slowed the decline in grip strength over the course of treatment, while 20 weeks of drug treatment significantly improved MNCV and decreased the g-ratio and the number of thinly myelinated femoral nerve axons. Similarly, 20 weeks of cemdomespib therapy improved the NMJ morphology and the overlap between presynaptic (synaptophysin) and postsynaptic (α-bungarotoxin) markers. These data show that cemdomespib therapy slows the rate of neuromuscular decline and demyelination and may present a disease-modifying approach for patients with gain-of-function Cx32 mutations.
2024-12-31 | Mechanism of connexin channel inhibition by mefloquine and 2-aminoethoxydiphenyl borate.
Gap junction intercellular communication (GJIC) between two adjacent cells involves direct exchange of cytosolic ions and small molecules via connexin gap junction channels (GJCs). Connexin GJCs have emerged as drug targets, with small molecule connexin inhibitors considered a viable therapeutic strategy in several diseases. The molecular mechanisms of GJC inhibition by known small molecule connexin inhibitors remain unknown, preventing the development of more potent and connexin-specific therapeutics. Here we show that two GJC inhibitors, mefloquine (MFQ) and 2-aminoethoxydiphenyl borate (2APB) bind to Cx32 and block dye permeation across Cx32 hemichannels (HCs) and GJCs. Cryo-EM analysis shows that 2APB binds to "site A", close to the N-terminal gating helix of Cx32 GJC, restricting the entrance to the channel pore. In contrast, MFQ binds to a distinct "site M", deeply buried within the pore. MFQ binding to this site modifies the electrostatic properties of Cx32 pore. Mutagenesis of V37, a key residue located in the site M, renders Cx32 HCs and GJCs insensitive to MFQ-mediated inhibition. Moreover, our cryo-EM analysis, mutagenesis and activity assays show that MFQ targets the M site in Cx43 GJC similarly to Cx32. Taken together, our results point to a conserved inhibitor binding site in connexin channels, opening a new route for development of specific drugs targeting connexins.
proteins
2026-06-10 | Reversible area postrema syndrome in a patient with GJB1 mutation mimicking neuromyelitis optica spectrum disorder.
Area postrema syndrome (APS) is a core feature of neuromyelitis optica spectrum disorder (NMOSD). Charcot-Marie-Tooth disease type 1X (CMT1X), caused by gap junction protein beta-1 (GJB1) mutations, can rarely involve the central nervous system (CNS). To report a case of CMT1X presenting with APS mimicking NMOSD. Clinical, electrophysiological, radiological, and genetic evaluations were performed. A 43-year-old woman experienced intractable hiccups, nausea, and a dorsal medullary lesion. Genetic testing revealed a pathogenic GJB1 p.Arg22Gln variant. The lesion resolved, and she remained relapse-free without maintenance immunotherapy. GJB1-related disorders should be considered in patients with episodic CNS symptoms, particularly when accompanied by features of hereditary neuropathy, to avoid misdiagnosis.
2023-06-01 | Cytokine-Induced Neurogenesis in Charcot-Marie-Tooth neuropathy with Connexin 32 Gene Mutation
Charcot-Marie-Tooth (CMT) neuropathy is one of the most common neuromuscular disorders.Despite the identification of more than 100 causative genes, there are few therapeutic options.We present the case of a 66-year-old man carrying a CMT mutation in the connexin 32 gene with the APOE genotype ε4/ε3 that had exhibited symptoms of slowly progressive muscle weakness and atrophy in the hands and feet and recently developed memory impairment.The muscle weakness, motor dysfunction, and memory impairment symptoms were significantly improved by cytokine-induced neurogenesis combined with physical wave therapy using an arbitrary waveform generator (AWG).Here, we discuss the possible mechanistic basis of how this new treatment ameliorates the symptoms of CMT and propose a novel clinical treatment strategy for CMT.
2023-05-05 | TGFβ4 alleviates the phenotype of Charcot–Marie–Tooth disease type 1A
The duplication of the peripheral myelin protein 22 (PMP22) gene causes a demyelinating type of neuropathy, commonly known as Charcot-Marie-Tooth disease type 1A (CMT1A). Development of effective drugs for CMT1A still remains as an unmet medical need. In the present study, we assessed the role of the transforming growth factor beta 4 (TGFβ4)/Nodal axis in the pathogenesis of CMT1A. First, we identified PMP22 overexpression-induced Nodal expression in Schwann cells, which might be one of the downstream effectors in CMT1A. Administration of Nodal protein at the developmental stage of peripheral nerves induced the demyelinating phenotype in vivo. Second, we further isolated TGFβ4 as an antagonist that could abolish Nodal-induced demyelination. Finally, we developed a recombinant TGFβ4-fragment crystallizable (Fc) fusion protein, CX201, and demonstrated that its application had promyelinating efficacy in Schwann cells. CX201 administration improved the demyelinating phenotypes of CMT1A mouse models at both pre-symptomatic and post-symptomatic stages. These results suggest that the TGFβ4/Nodal axis plays a crucial role in the pathogenesis of CMT1A and might be a potential therapeutic target for CMT1A.
2017-11-06 | Graded Elevation of c-Jun in Schwann Cells In Vivo: Gene Dosage Determines Effects on Development, Remyelination, Tumorigenesis, and Hypomyelination.
Schwann cell c-Jun is implicated in adaptive and maladaptive functions in peripheral nerves. In injured nerves, this transcription factor promotes the repair Schwann cell phenotype and regeneration and promotes Schwann-cell-mediated neurotrophic support in models of peripheral neuropathies. However, c-Jun is associated with tumor formation in some systems, potentially suppresses myelin genes, and has been implicated in demyelinating neuropathies. To clarify these issues and to determine how c-Jun levels determine its function, we have generated c-Jun OE/+ and c-Jun OE/OE mice with graded expression of c-Jun in Schwann cells and examined these lines during development, in adulthood, and after injury using RNA sequencing analysis, quantitative electron microscopic morphometry, Western blotting, and functional tests. Schwann cells are remarkably tolerant of elevated c-Jun because the nerves of c-Jun OE/+ mice, in which c-Jun is elevated ∼6-fold, are normal with the exception of modestly reduced myelin thickness. The stronger elevation of c-Jun in c-Jun OE/OE mice is, however, sufficient to induce significant hypomyelination pathology, implicating c-Jun as a potential player in demyelinating neuropathies. The tumor suppressor P19 ARF is strongly activated in the nerves of these mice and, even in aged c-Jun OE/OE mice, there is no evidence of tumors. This is consistent with the fact that tumors do not form in injured nerves, although they contain proliferating Schwann cells with strikingly elevated c-Jun. Furthermore, in crushed nerves of c-Jun OE/+ mice, where c-Jun levels are overexpressed sufficiently to accelerate axonal regeneration, myelination and function are restored after injury. SIGNIFICANCE STATEMENT In injured and diseased nerves, the transcription factor c-Jun in Schwann cells is elevated and variously implicated in controlling beneficial or adverse functions, including trophic Schwann cell support for neurons, promotion of regeneration, tumorigenesis, and suppression of myelination. To analyze the functions of c-Jun, we have used transgenic mice with graded elevation of Schwann cell c-Jun. We show that high c-Jun elevation is a potential pathogenic mechanism because it inhibits myelination. Conversely, we did not find a link between c-Jun elevation and tumorigenesis. Modest c-Jun elevation, which is beneficial for regeneration, is well tolerated during Schwann cell development and in the adult and is compatible with restoration of myelination and nerve function after injury.
2017-10-25 | Cx32 hemichannel opening by cytosolic Ca2+ is inhibited by the R220X mutation that causes Charcot-Marie-Tooth disease
Mutations of the GJB1 gene encoding connexin 32 (Cx32) cause the X-linked form of Charcot–Marie–Tooth disease (CMTX1), a demyelinating peripheral neuropathy for which there is no cure. A growing body of evidence indicates that ATP release through Cx32 hemichannels in Schwann cells could be critical for nerve myelination, but it is unknown if CMTX1 mutations alter the cytosolic Ca2+-dependent gating mechanism that controls Cx32 hemichannel opening and ATP release. The current study uncovered that loss of the C-terminus in Cx32 (R220X mutation), which causes a severe CMTX1 phenotype, inhibits hemichannel opening during a canonical IP3-mediated increase in cytosolic Ca2+ in HeLa cells. Interestingly, the gating function of R220X hemichannels was completely restored by both the intracellular and extracellular application of a peptide that mimics the Cx32 cytoplasmic loop. All-atom molecular dynamics simulations suggest that loss of the C-terminus in the mutant hemichannel triggers abnormal fluctuations of the cytoplasmic loop which are prevented by binding to the mimetic peptide. Experiments that stimulated R220X hemichannel opening by cell depolarization displayed reduced voltage sensitivity with respect to wild-type hemichannels which was explained by loss of subconductance states at the single channel level. Finally, experiments of intercellular diffusion mediated by wild-type or R220X gap junction channels revealed similar unitary permeabilities to ions, signalling molecules (cAMP) or larger solutes (Lucifer yellow). Taken together, our findings support the hypothesis that paracrine signalling alteration due to Cx32 hemichannel dysfunction underlies CMTX1 pathogenesis and suggest a candidate molecule for novel studies investigating a therapeutic approach.
antibodies
2025-09-29 | [A case of X-linked Charcot-Marie-Tooth disease type 1 (CMTX1) diagnosed based on recurrent brain lesions despite peripheral neuropathy responsive to immunotherapy].
The patient is a 17-year-old male. He had a history of hospitalization for influenza at the age of 11, and Brain MRI at that time showed reversible brain lesions in the splenium of the corpus callosum and cerebral white matter. Fifteen months ago, he visited the pediatrics department due to dysphagia, dysarthria, facial paralysis, and muscle weakness. Brain MRI revealed lesions similar to those observed here, and nerve conduction study revealed demyelinating neuropathy. He was treated with intravenous immunoglobulin (IVIg) and intravenous methylprednisolone, and his symptoms disappeared within a few days and Brain MRI 5 weeks after treatment revealed that the lesions had disappeared. Three months ago, while walking, the patient developed a knee strain, which was thought to be a recurrence of the immune-mediated neuropathy. His subjective symptom disappeared after administration of IVIg. The patient was diagnosed with X-linked Charcot-Marie-Tooth disease (CMTX1) based on genetic testing, which revealed a pathological variant of GJB1, c.124A>T (p.Ser42Cys). Peripheral neuropathy in CMTX1 may present with fluctuating symptoms and can be responsive to IVIg treatment. Recurrent brain lesions should also be considered in the diagnosis of CMTX1.
2024-12-06 | A fully human IgG1 antibody targeting connexin 32 extracellular domain blocks CMTX1 hemichannel dysfunction in an in vitro model.
Connexins (Cxs) are fundamental in cell-cell communication, functioning as gap junction channels (GJCs) that facilitate solute exchange between adjacent cells and as hemichannels (HCs) that mediate solute exchange between the cytoplasm and the extracellular environment. Mutations in the GJB1 gene, which encodes Cx32, lead to X-linked Charcot-Marie-Tooth type 1 (CMTX1), a rare hereditary demyelinating disorder of the peripheral nervous system (PNS) without an effective cure or treatment. In Schwann cells, Cx32 HCs are thought to play a role in myelination by enhancing intracellular and intercellular Ca2+ signaling, which is crucial for proper PNS myelination. Single-point mutations (p.S85C, p.D178Y, p.F235C) generate pathological Cx32 HCs characterized by increased permeability ("leaky") or excessive activity ("hyperactive").We investigated the effects of abEC1.1-hIgG1, a fully human immunoglobulin G1 (hIgG1) monoclonal antibody, on wild-type (WT) and mutant Cx32D178Y HCs. Using HeLa DH cells conditionally co-expressing Cx and a genetically encoded Ca2+ biosensor (GCaMP6s), we demonstrated that mutant HCs facilitated 58% greater Ca2+ uptake in response to elevated extracellular Ca2+ concentrations ([Ca2+]ex) compared to WT HCs. abEC1.1-hIgG1 dose-dependently inhibited Ca2+ uptake, achieving a 50% inhibitory concentration (EC50) of ~ 10 nM for WT HCs and ~ 80 nM for mutant HCs. Additionally, the antibody suppressed DAPI uptake and ATP release. An atomistic computational model revealed that serine 56 (S56) of the antibody interacts with aspartate 178 (D178) of WT Cx32 HCs, contributing to binding affinity. Despite the p.D178Y mutation weakening this interaction, the antibody maintained binding to the mutant HC epitope at sub-micromolar concentrations.In conclusion, our study shows that abEC1.1-hIgG1 effectively inhibits both WT and mutant Cx32 HCs, highlighting its potential as a therapeutic approach for CMTX1. These findings expand the antibody's applicability for treating diseases associated with Cx HCs and inform the rational design of next-generation antibodies with enhanced affinity and efficacy against mutant HCs.
2016-02-10 | Cell-Surface and Secreted Isoforms of CSF-1 Exert Opposing Roles in Macrophage-Mediated Neural Damage in Cx32-Deficient Mice
Previous studies in myelin-mutant mouse models of the inherited and incurable nerve disorder, Charcot-Marie-Tooth (CMT) neuropathy, have demonstrated that low-grade secondary inflammation implicating phagocytosing macrophages amplifies demyelination, Schwann cell dedifferentiation and perturbation of axons. The cytokine colony stimulating factor-1 (CSF-1) acts as an important regulator of these macrophage-related disease mechanisms, as genetic and pharmacologic approaches to block the CSF-1/CSF-1R signaling result in a significant alleviation of pathological alterations in mutant peripheral nerves. In mouse models of CMT1A and CMT1X, as well as in human biopsies, CSF-1 is predominantly expressed by endoneurial fibroblasts, which are closely associated with macrophages, suggesting local stimulatory mechanisms. Here we investigated the impact of cell-surface and secreted isoforms of CSF-1 on macrophage-related disease in connexin32-deficient ( Cx32def ) mice, a mouse model of CMT1X. Our present observations suggest that the secreted proteoglycan isoform (spCSF-1) is predominantly expressed by fibroblasts, whereas the membrane-spanning cell-surface isoform (csCSF-1) is expressed by macrophages. Using crossbreeding approaches to selectively restore or overexpress distinct isoforms in CSF-1-deficient (osteopetrotic) Cx32def mice, we demonstrate that both isoforms equally regulate macrophage numbers dose-dependently. However, spCSF-1 mediates macrophage activation and macrophage-related neural damage, whereas csCSF-1 inhibits macrophage activation and attenuates neuropathy. These results further corroborate the important role of secondary inflammation in mouse models of CMT1 and might identify specific targets for therapeutic approaches to modulate innate immune reactions. SIGNIFICANCE STATEMENT Mouse models of Charcot-Marie-Tooth neuropathy have indicated that low-grade secondary inflammation involving phagocytosing macrophages amplifies demyelination, Schwann cell dedifferentiation, and perturbation of axons. The recruitment and pathogenic activation of detrimental macrophages is regulated by CSF-1, a cytokine that is mostly expressed by fibroblasts in the diseased nerve and exists in three isoforms. We show that the cell-surface and secreted isoforms of CSF-1 have opposing effects on macrophage activation and disease progression in a mouse model of CMT1X. These insights into opposing functions of disease-modulating cytokine isoforms might enable the development of specific therapeutic approaches.
2015-05-02 | Desert hedgehog is a mediator of demyelination in compression neuropathies
The secreted protein desert hedgehog (dhh) controls the formation of the nerve perineurium during development and is a key component of Schwann cells that ensures peripheral nerve survival. We postulated that dhh may play a critical role in maintaining myelination and investigated its role in demyelination-induced compression neuropathies by using a post-natal model of a chronic nerve injury in wildtype and dhh(-/-) mice. We evaluated demyelination using electrophysiological, morphological, and molecular approaches. dhh transcripts and protein are down-regulated early after injury in wild-type mice, suggesting an intimate relationship between the hedgehog pathway and demyelination. In dhh(-/-) mice, nerve injury induced more prominent and severe demyelination relative to their wild-type counterparts, suggesting a protective role of dhh. Alterations in nerve fiber characteristics included significant decreases in nerve conduction velocity, increased myelin debris, and substantial decreases in internodal length. Furthermore, in vitro studies showed that dhh blockade via either adenovirus-mediated (shRNA) or pharmacological inhibition both resulted in severe demyelination, which could be rescued by exogenous Dhh. Exogenous Dhh was protective against this demyelination and maintained myelination at baseline levels in a custom in vitro bioreactor to applied biophysical forces to myelinated DRG/Schwann cell co-cultures. Together, these results demonstrate a pivotal role for dhh in maintaining myelination. Furthermore, dhh signaling reveals a potential target for therapeutic intervention to prevent and treat demyelination of peripheral nerves in compression neuropathies.
2015-01-27 | CSF-1-activated macrophages are target-directed and essential mediators of Schwann cell dedifferentiation and dysfunction in Cx32-deficient mice.
We investigated connexin 32 (Cx32)‐deficient mice, a model for the X‐linked form of Charcot‐Marie‐Tooth neuropathy (CMT1X), regarding the impact of low‐grade inflammation on Schwann cell phenotype. Whereas we previously identified macrophages as amplifiers of the neuropathy, we now explicitly focus on the impact of the phagocytes on Schwann cell dedifferentiation, a so far not‐yet addressed disease‐related mechanism for CMT1X. Using mice heterozygously deficient for Cx32 and displaying both Cx32‐positive and ‐negative Schwann cells in one and the same nerve, we could demonstrate that macrophage clusters rather than single macrophages precisely associate with mutant but not with Cx32‐positive Schwann cells. Similarly, in an advanced stage of Schwann cell perturbation, macrophage clusters were strongly associated with NCAM‐ and L1‐positive, dedifferentiated Schwann cells. To clarify the role of macrophages regarding Schwann cell dedifferentiation, we generated Cx32‐deficient mice additionally deficient for the macrophage‐directed cytokine colony‐stimulating factor (CSF)−1. In the absence of CSF‐1, Cx32‐deficient Schwann cells not only showed the expected amelioration in myelin preservation but also failed to upregulate the Schwann cell dedifferentiation markers NCAM and L1. Another novel and unexpected finding in the double mutants was the retained activation of ERK signaling, a pathway which is detrimental for Schwann cell homeostasis in myelin mutant models. Our findings demonstrate that increased ERK signaling can be compatible with the maintenance of Schwann cell differentiation and homeostasis in vivo and identifies CSF‐1‐activated macrophages as crucial mediators of detrimental Schwann cell dedifferentiation in Cx32‐deficient mice. GLIA 2015;63:977–986
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2026-02-03 | Patient-derived neural organoids reveal developmental impairments associated with a novel GJB1 mutation in X-linked Charcot-Marie-Tooth disease.
Charcot-Marie-Tooth disease (CMT) is one of the most prevalent inherited peripheral neuropathies. CMT type X1 (CMTX1), caused by mutations in the GJB1 gene, represents the most common X-linked subtype with central nervous system (CNS) involvement. Here, we report the identification and functional characterization of a novel GJB1 variant (c.554C > T, p.Thr185Ile) in a CMTX1-affected family and its pathogenic impact using patient-derived induced pluripotent stem cells (iPSCs) and three-dimensional (3D) neural organoid models. The GJB1 gene encodes connexin 32 (Cx32), a gap junction protein. Immunofluorescent analysis revealed aberrant intracellular reduction and aggregation of the mutant Cx32 protein, suggesting impaired gap junction function. iPSC-derived neural organoids carrying the GJB1 mutation exhibited significant delay in neural differentiation and disrupted neural rosette organization. These findings underscore the critical role of Cx32 in neural development and provide a physiologically relevant platform for underlying CMTX1 pathological mechanisms on central nervous system. The established GJB1-variant organoid model holds promise for investigating genotype-phenotype correlations and facilitating the development of targeted therapeutic strategies for CMTX1.
2023-12-17 | Preclinical Efficacy of Peripheral Nerve Regeneration by Schwann Cell-like Cells Differentiated from Human Tonsil-Derived Mesenchymal Stem Cells in C22 Mice
Charcot-Marie-Tooth disease (CMT) is a hereditary disease with heterogeneous phenotypes and genetic causes. CMT type 1A (CMT1A) is a type of disease affecting the peripheral nerves and is caused by the duplication of the peripheral myelin protein 22 (PMP22) gene. Human tonsil-derived mesenchymal stem cells (TMSCs) are useful for stem cell therapy in various diseases and can be differentiated into Schwann cell-like cells (TMSC-SCs). We investigated the potential of TMSC-SCs called neuronal regeneration-promoting cells (NRPCs) for peripheral nerve and muscle regeneration in C22 mice, a model for CMT1A. We transplanted NRPCs manufactured in a good manufacturing practice facility into the bilateral thigh muscles of C22 mice and performed behavior and nerve conduction tests and histological and ultrastructural analyses. Significantly, the motor function was much improved, the ratio of myelinated axons was increased, and the G-ratio was reduced by the transplantation of NRPCs. The sciatic nerve and gastrocnemius muscle regeneration of C22 mice following the transplantation of NRPCs downregulated PMP22 overexpression, which was observed in a dose-dependent manner. These results suggest that NRPCs are feasible for clinical research for the treatment of CMT1A patients. Research applying NRPCs to other peripheral nerve diseases is also needed.
2023-08-07 | The Current State of Charcot-Marie-Tooth Disease Treatment.
Charcot-Marie-Tooth disease (CMT) and associated neuropathies are the most predominant genetically transmitted neuromuscular conditions; however, effective pharmacological treatments have not established. The extensive genetic heterogeneity of CMT, which impacts the peripheral nerves and causes lifelong disability, presents a significant barrier to the development of comprehensive treatments. An estimated 100 loci within the human genome are linked to various forms of CMT and its related inherited neuropathies. This review delves into prospective therapeutic strategies used for the most frequently encountered CMT variants, namely CMT1A, CMT1B, CMTX1, and CMT2A. Compounds such as PXT3003, which are being clinically and preclinically investigated, and a broad array of therapeutic agents and their corresponding mechanisms are discussed. Furthermore, the progress in established gene therapy techniques, including gene replacement via viral vectors, exon skipping using antisense oligonucleotides, splicing modification, and gene knockdown, are appraised. Each of these gene therapies has the potential for substantial advancements in future research.
2019-08-27 | Downregulation of the human peripheral myelin protein 22 gene by miR-29a in cellular models of Charcot–Marie–Tooth disease
The majority of hereditary neuropathies are caused by duplication of the peripheral myelin protein 22 (PMP22) gene. Therefore, mechanisms to suppress the expression of the PMP22 gene have high therapeutic significance. Here we asked whether the human PMP22 gene is a target for regulation by microRNA 29a (miR-29a). Using bioinformatics, we determined that the human PMP22 gene contains the conserved seed sequence of the miR-29a binding site and this regulatory motif is included in the duplicated region in neuropathic patients. Using luciferase reporter assays in HEK293 cells, we demonstrated that transient transfection of a miR-29a mimic is associated with reduction in PMP22 3′UTR reporter activity. Transfecting normal and humanized transgenic neuropathic mouse Schwann cells with a miR-29a expression plasmid effectively lowered both the endogenous mouse and the transgenic human PMP22 transcripts compared with control vector. In dermal fibroblasts derived from neuropathic patients, ectopic expression of miR-29a led to ~50% reduction in PMP22 mRNA, which corresponded to ~20% decrease in PMP22 protein levels. Significantly, miR-29a-mediated reduction in PMP22 mitigated the reduced mitotic capacity of the neuropathic cells. Together, these results support further testing of miR-29a and/or PMP22-targeting siRNAs as therapeutic agents for correcting the aberrant expression of PMP22 in neuropathic patients.
2017-12-03 | Antisense oligonucleotides offer hope to patients with Charcot-Marie-Tooth disease type 1A
Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common heritable peripheral neuropathy and results from a duplication on chromosome 17 that results in an extra copy and increased dosage of peripheral myelin protein 22 (PMP22). Zhao et al., in this issue of the JCI, successfully utilized antisense oligonucleotides (ASOs) to reduce PMP22 and ameliorated neuropathy in both mouse and rat models of CMT1A. These data confirm that strategies to reduce PMP22 have potential as effective therapeutic approaches for CMT1A and lay the groundwork for clinical trials in humans afflicted with this chronic, debilitating neurodegenerative disease.
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