RARE DISEASE
X-linked Charcot-Marie-Tooth disease type 2
X-linked Charcot-Marie-Tooth disease type 2
X-linked Charcot-Marie-Tooth disease type 2
Synonyms: CMTX2
Synonyms: CMTX2
Synonyms: CMTX2
Drug discovery
0
drugs
With orphan designations
Overview
X-linked Charcot-Marie-Tooth disease type 2 (CMTX2) is a rare genetic peripheral neuropathy characterized by X-linked recessive inheritance and childhood-onset progressive distal muscle weakness/atrophy (predominantly lower extremities), pes cavus, areflexia, and variable sensory deficits. Some patients develop intellectual disability [1][2][13]. Males exhibit more severe manifestations due to hemizygous mutations [9][17]. Nerve conduction studies typically show axonal neuropathy patterns [15].
Therapies
Supportive care: Orthoses (ankle-foot braces), physiotherapy, and surgical correction of foot deformities (osteotomy/arthrodesis) [7][8]
Neuropathic pain management: Gabapentinoids or tricyclic antidepressants [3][16]
Emerging therapies: Neurotrophin-3 trials showing modest efficacy [3][14]; muscle-targeted gene therapy under investigation [14][11]
Categories: rare developmental anomalies during embryogenesis, rare genetic diseases, rare neurological diseases, rare otorhinolaryngological diseases
Research Papers
90 drug discovery papers about X-linked Charcot-Marie-Tooth disease type 2, with 1 first-in-class emerging drug candidates forecasted to outperform the average preclinical success rate. Recent publications:
90 drug discovery papers about X-linked Charcot-Marie-Tooth disease type 2, with 1 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.
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.
2023-03-08 | Knock-in mouse models for CMTX1 show a loss of function phenotype in the peripheral nervous system.
The X-linked form of Charcot-Marie-Tooth disease (CMTX1) is the second most common form of CMT. In this study we used CRISPR/Cas9 to develop new "knock-in" models of CMTX1 that are more representative of the spectrum of mutations seen with CMTX1 than the Cx32 knockout (KO) mouse model used previously. We compared mice of four genotypes - wild-type, Cx32KO, p.T55I, and p.R75W. Sciatic motor conduction velocity slowing was the most robust electrophysiologic indicator of neuropathy, showing reductions in the Cx32KO by 3 months and in the p.T55I and p.R75W mice by 6 months. At both 6 and 12 months, all three mutant genotypes showed reduced four limb and hind limb grip strength compared to WT mice. Performance on 6 and 12 mm width balance beams revealed deficits that were most pronounced at on the 6 mm balance beam at 6 months of age. There were pathological changes of myelinated axons in the femoral motor nerve in all three mutant lines by 3 months of age, and these became more pronounced at 6 and 12 months of age; sensory nerves (femoral sensory and the caudal nerve of the tail) appeared normal at all ages examined. Our results demonstrate that mice can be used to show the pathogenicity of human GJB1 mutations, and these new models for CMTX1 should facilitate the preclinical work for developing treatments for CMTX1.
2022-02-14 | Effects of early crush on aging wild type and Connexin 32 knockout mice: Evidence for a neuroprotective state in CMT1X mouse nerve.
The long-term sequelae of nerve injury as well as age-related neurodegeneration have been documented in numerous studies, however the role of Cx32 in these processes is not well understood. There is a need for better understanding of the molecular mechanisms that underlie long-term suboptimal nerve function and for approaches to prevent or improve it. In this communication we describe our studies using whole animal electrophysiology to examine the long-term sequelae of sciatic nerve crush in both WT and Cx32KO mice, a model of X-linked Charcot Marie Tooth disease, a subtype of inherited peripheral neuropathies. We present results from electrical nerve recordings done 14 to 27 days and 18 to 20 months after a unilateral sciatic nerve crush performed on 35 to 37-day old mice. Contrary to expectations, we find that whereas crush injury leads to a degradation of WT nerve function relative to uninjured nerves at 18 to 20 months, previously crushed Cx32KO nerves perform at the same level as their uninjured counterparts. Thus, 18 to 20 months after injury, WT nerves perform below the level of normal (uninjured) WT nerves in both motor and sensory nerve function. In contrast, measures of nerve function in Cx32KO mice are degraded for sensory axons but exhibit no additional dysfunction in motor axons. Early nerve injury has no negative electrophysiologic effect on the Cx32 KO motor nerves. Based on our prior demonstration that the transcriptomic profile of uninjured Cx32KO and injured WT sciatic nerves are very similar, the lack of an additional effect of crush on Cx32KO motor nerve parameters suggests that Cx32 knockout may implement a form of neuroprotection that limits the effects of subsequent injury.
2021-03-10 | AAV9-mediated Schwann cell-targeted gene therapy rescues a model of demyelinating neuropathy
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.
small molecules
2026-07-06 | Activity-dependent CO2 production in the axon triggers opening of Connexin32 in the Schwann cell paranode.
Loss of function mutations of Cx32, which is expressed in Schwann cells, cause X-linked Charcot-Marie-Tooth disease, a slowly progressive peripheral neuropathy. Action potential propagation causes Cx32 hemichannels in the Schwann cell paranode to open. As Cx32 hemichannels are directly sensitive to CO2, we have tested whether CO2 produced in the axon, as a consequence of the energetic demands of action potential propagation, might gate Cx32 hemichannels. Using isolated sciatic nerve from the mouse, we found that the critical components required for intercellular CO2 signaling are present (nodal mitochondria, the source of CO2; a CO2-permeable aquaporin, AQP1; paranodal Cx32; and carbonic anhydrase). We have used a membrane impermeant fluorescent dye, FITC, to demonstrate the opening of Cx32 in Schwann cells in response to an external CO2 stimulus or during action potential propagation in the isolated nerve. Pharmacological manipulations of AQP1 or carbonic anhydrase activity altered Cx32 gating during action potential firing. Expression of a modified Cx32 subunit, Cx32DN, that coassembles with Cx32WT, revealed that the activity-dependent dye loading of Schwann cells depended upon CO2 binding to Cx32. CO2 can, therefore, mediate neuron-to-glia signaling via connexins. CO2 permeable aquaporins and carbonic anhydrase are key components of this signaling mechanism.
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.
2024-03-14 | Gp78 regulates PMP22 and causes ER stress and autophagy in EV71-VP1-overexpressing mouse Schwann cells
Background: During Enterovirus type 71 (EV71) infection, the structural viral protein 1 (VP1) activates endoplasmic reticulum (ER) stress associated with peripheral myelin protein 22 (PMP22) accumulation and induces autophagy.However, the specific mechanism behind this process remains elusive.Methods: In this research, we used the VP1-overexpressing mouse Schwann cells (SCs) models co-transfected with a PMP22 silencing or Autocrine motility factor receptor (AMFR/gp78) overexpressing vector to explore the regulation of gp78 on PMP22 and its relationship with autophagy and apoptosis.Results: The activity of gp78 could be influenced by EV71-VP1, leading to a decrease in the ubiquitination and degradation of PMP22, resulting in PMP22 accumulation in ER.In VP1overexpressing mouse SCs, all three ER stress sensors, including pancreatic endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6) and inositol-requiring enzyme 1 (IRE1) and the related downstream signals (C/EBP-homologous protein (CHOP) and Caspase 12) were activated, as well as the ER-resident chaperone Glucoseregulated protein 78 (GRP78).In addition, VP1 upregulated the autophagy marker Microtubule-associated protein 1 light chain 3 beta (LC3B), while PMP22 silencing or gp78 overexpression reversed the phenomenon.Meanwhile, PMP22 silencing or gp78 overexpression increased proliferation of EV71-VP1-transfected mouse SCs.Conclusion:Gp78 could regulate PMP22 accumulation through ubiquitination degradation and cause ER stress and autophagy in EV71-VP1-overexpressing mouse SCs.Therefore, the gp78/PMP22/ER stress axis might emerge as a promising therapeutic target for myelin and neuronal damage induced by EV71 infection.
2023-07-21 | Amlexanox: Readthrough Induction and Nonsense-Mediated mRNA Decay Inhibition in a Charcot–Marie–Tooth Model of hiPSCs-Derived Neuronal Cells Harboring a Nonsense Mutation in GDAP1 Gene
Nonsense mutations are involved in multiple peripheral neuropathies. These mutations induce the presence of a premature termination codon (PTC) at the mRNA level. As a result, a dysfunctional or truncated protein is synthesized, or even absent linked to nonsense-mediated mRNA degradation (NMD) system activation. Readthrough molecules or NMD inhibitors could be innovative therapies in these hereditary neuropathies, particularly molecules harboring the dual activity as amlexanox. Charcot–Marie–Tooth (CMT) is the most common inherited pathology of the peripheral nervous system, affecting 1 in 2500 people worldwide. Nonsense mutations in the GDAP1 gene have been associated with a severe form of CMT, prompting us to investigate the effect of readthrough and NMD inhibitor molecules. Although not clearly defined, GDAP1 could be involved in mitochondrial functions, such as mitophagy. We focused on the homozygous c.581C>G (p.Ser194*) mutation inducing CMT2H using patient human induced pluripotent stem cell (hiPSC)-derived neuronal cells. Treatment during 20 h with 100 µM of amlexanox on this cell model stabilized GDAP1 mRNAs carrying UGA-PTC and induced a restoration of the mitochondrial morphology. These results highlight the potential of readthrough molecules associated to NMD inhibitors for the treatment of genetic alterations in CMT, opening the way for future investigations and a potential therapy.
2023-07-01 | Rapid degeneration of iPSC-derived motor neurons lacking Gdap1 engages a mitochondrial-sustained innate immune response
Charcot-Marie-Tooth disease is a chronic hereditary motor and sensory polyneuropathy targeting Schwann cells and/or motor neurons. Its multifactorial and polygenic origin portrays a complex clinical phenotype of the disease with a wide range of genetic inheritance patterns. The disease-associated gene GDAP1 encodes for a mitochondrial outer membrane protein. Mouse and insect models with mutations in Gdap1 have reproduced several traits of the human disease. However, the precise function in the cell types affected by the disease remains unknown. Here, we use induced-pluripotent stem cells derived from a Gdap1 knockout mouse model to better understand the molecular and cellular phenotypes of the disease caused by the loss-of-function of this gene. Gdap1-null motor neurons display a fragile cell phenotype prone to early degeneration showing (1) altered mitochondrial morphology, with an increase in the fragmentation of these organelles, (2) activation of autophagy and mitophagy, (3) abnormal metabolism, characterized by a downregulation of Hexokinase 2 and ATP5b proteins, (4) increased reactive oxygen species and elevated mitochondrial membrane potential, and (5) increased innate immune response and p38 MAP kinase activation. Our data reveals the existence of an underlying Redox-inflammatory axis fueled by altered mitochondrial metabolism in the absence of Gdap1. As this biochemical axis encompasses a wide variety of druggable targets, our results may have implications for developing therapies using combinatorial pharmacological approaches and improving therefore human welfare. A Redox-immune axis underlying motor neuron degeneration caused by the absence of Gdap1. Our results show that Gdap1-/- motor neurons have a fragile cellular phenotype that is prone to degeneration. Gdap1-/- iPSCs differentiated into motor neurons showed an altered metabolic state: decreased glycolysis and increased OXPHOS. These alterations may lead to hyperpolarization of mitochondria and increased ROS levels. Excessive amounts of ROS might be the cause of increased mitophagy, p38 activation and inflammation as a cellular response to oxidative stress. The p38 MAPK pathway and the immune response may, in turn, have feedback mechanisms, leading to the induction of apoptosis and senescence, respectively. CAC, citric acid cycle; ETC, electronic transport chain; Glc, glucose; Lac, lactate; Pyr, pyruvate.
proteins
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.
2023-03-16 | Boosting peripheral BDNF rescues impaired in vivo axonal transport in CMT2D mice
Gain-of-function mutations in the housekeeping gene GARS1, which lead to the expression of toxic versions of glycyl-tRNA synthetase (GlyRS), cause the selective motor and sensory pathology characterizing Charcot-Marie-Tooth disease (CMT). Aberrant interactions between GlyRS mutants and different proteins, including neurotrophin receptor tropomyosin receptor kinase receptor B (TrkB), underlie CMT type 2D (CMT2D); however, our pathomechanistic understanding of this untreatable peripheral neuropathy remains incomplete. Through intravital imaging of the sciatic nerve, we show that CMT2D mice displayed early and persistent disturbances in axonal transport of neurotrophin-containing signaling endosomes in vivo. We discovered that brain-derived neurotrophic factor (BDNF)/TrkB impairments correlated with transport disruption and overall CMT2D neuropathology and that inhibition of this pathway at the nerve-muscle interface perturbed endosome transport in wild-type axons. Accordingly, supplementation of muscles with BDNF, but not other neurotrophins, completely restored physiological axonal transport in neuropathic mice. Together, these findings suggest that selectively targeting muscles with BDNF-boosting therapies could represent a viable therapeutic strategy for CMT2D.
2022-10-28 | GDAP1 binds 4-hydroxynonenal, the toxic end-product of lipid peroxidation, using its GST-like binding pocket
Abstract GDAP1 (Ganglioside-induced differentiation-associated protein 1) is a novel member of the GST superfamily of detoxifying enzymes that is anchored to the outer mitochondrial membrane. GDAP1 mutations and changes in expression levels result in the inherited neuropathy Charcot-Marie-Tooth (CMT) disease, types 2K, 4A and 4H. GDAP1 activity has been associated with many mitochondrial functions however direct molecular interactions underpinning these connections have remained elusive. Here we establish that GDAP1 can bind 4-hydroxynonenal (4HNE), a toxic end-product of lipid peroxidation. 4HNE binding requires the α-loop, a large sequence motif that is inserted within the substrate recognition domain and is unique to GDAP1. In human cells, GDAP1 overexpression plays a cytoprotective role against oxidative stress. This effect is lost upon deletion of the α-loop. Lastly, we demonstrate that a CMT-causing mutant that destabilizes α-loop positioning also results in a decrease in 4HNE binding affinity. Together these results establish 4HNE as the biological ligand for GDAP1, provide mechanistic insight into 4HNE binding, and demonstrate that altered 4HNE recognition is the likely mechanism underlying CMT-causing mutants such as T157P near the 4HNE binding site.
2020-04-03 | Defective nucleotide-dependent assembly and membrane fusion in Mfn2 CMT2A variants improved by Bax
Mitofusins are members of the dynamin-related protein family of large GTPases that harness the energy from nucleotide hydrolysis to remodel membranes. Mitofusins possess four structural domains, including a GTPase domain, two extended helical bundles (HB1 and HB2), and a transmembrane region. We have characterized four Charcot-Marie-Tooth type 2A–associated variants with amino acid substitutions in Mfn2 that are proximal to the hinge that connects HB1 and HB2. A functional defect was not apparent in cells as the mitochondrial morphology of Mfn2-null cells was restored by expression of any of these variants. However, a significant fusion deficiency was observed in vitro, which was improved by the addition of crude cytosol extract or soluble Bax. All four variants had reduced nucleotide-dependent assembly in cis, but not trans, and this was also improved by the addition of Bax. Together, our data demonstrate an important role for this region in Mfn2 GTP-dependent oligomerization and membrane fusion and is consistent with a model where cytosolic factors such as Bax are masking molecular defects associated with Mfn2 disease variants in cells.
2016-07-03 | Structural studies of N-terminal mutants of Connexin 26 and Connexin 32 using 1H NMR spectroscopy
Alterations in gap junctions underlie the etiologies of syndromic deafness (KID) and Charcot-Marie Tooth disease (CMTX). Functional gap junctions are composed of connexin molecules with N-termini containing a flexible turn around G12, inserting the N-termini into the channel pore allowing voltage gating. The loss of this turn correlates with loss of Connexin 32 (Cx32) function by impaired trafficking to the cell membrane. Using (1)H NMR we show the N-terminus of a syndromic deafness mutation Cx26G12R, producing "leaky channels", contains a turn around G12 which is less structured and more flexible than wild-type. In contrast, the N-terminal structure of the same mutation in Cx32 chimera, Cx32*43E1G12R shows a larger constricted turn and no membrane current expression but forms membrane inserted hemichannels. Their function was rescued by formation of heteromeric channels with wild type subunits. We suggest the inflexible Cx32G12R N-terminus blocks ion conduction in homomeric channels and this channel block is relieved by incorporation of wild type subunits. In contrast, the increased open probability of Cx26G12R hemichannels is likely due to the addition of positive charge in the channel pore changing pore electrostatics and impairing hemichannel regulation by Ca(2+). These results provide mechanistic information on aberrant channel activity observed in disease.
cell therapies
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.
2016-06-14 | Enhanced Immune Response in Immunodeficient Mice Improves Peripheral Nerve Regeneration Following Axotomy
Injuries to peripheral nerves cause loss of motor and sensory function, greatly affecting life quality. Successful repair of the lesioned nerve requires efficient cell debris removal, followed by axon regeneration and reinnervation of target organs. Such process is orchestrated by several cellular and molecular events in which glial and immune cells actively participate. It is known that tissue clearance is largely improved by macrophages, which activation is potentiated by cells and molecules of the acquired immune system, such as T helper lymphocytes and antibodies, respectively. In the present work, we evaluated the contribution of lymphocytes in the regenerative process of crushed sciatic nerves of immunocompetent (wild-type, WT) and T and B-deficient (RAG-KO) mice. In Knockout animals, we found increased amount of macrophages under basal conditions and during the initial phase of the regenerative process, that was evaluated at 2, 4, and 8 weeks after lesion (wal). That parallels with faster axonal regeneration evidenced by the quantification of neurofilament and a growth associated protein immunolabeling. The motor function, evaluated by the sciatic function index, was fully recovered in both mouse strains within 4 wal, either in a progressive fashion, as observed for RAG-KO mice, or presenting a subtle regression, as seen in WT mice between 2 and 3 wal. Interestingly, boosting the immune response by early adoptive transference of activated WT lymphocytes at 3 days after lesion improved motor recovery in WT and RAG-KO mice, which was not ameliorated when cells were transferred at 2 wal. When monitoring lymphocytes by in vivo imaging, in both mouse strains, cells migrated to the lesion site shortly after transference, remaining in the injured limb up to its complete motor recovery. Moreover, a first peak of hyperalgesia, determined by von-Frey test, was coincident with increased lymphocyte infiltration in the damaged paw. Overall, the present results suggest that a wave of immune cell infiltration takes place during subacute phase of axonal regeneration, resulting in transient set back of motor recovery following peripheral axonal injury. Moreover, modulation of the immune response can be an efficient approach to speed up nerve regeneration.
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.
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.
2023-03-08 | Knock-in mouse models for CMTX1 show a loss of function phenotype in the peripheral nervous system.
The X-linked form of Charcot-Marie-Tooth disease (CMTX1) is the second most common form of CMT. In this study we used CRISPR/Cas9 to develop new "knock-in" models of CMTX1 that are more representative of the spectrum of mutations seen with CMTX1 than the Cx32 knockout (KO) mouse model used previously. We compared mice of four genotypes - wild-type, Cx32KO, p.T55I, and p.R75W. Sciatic motor conduction velocity slowing was the most robust electrophysiologic indicator of neuropathy, showing reductions in the Cx32KO by 3 months and in the p.T55I and p.R75W mice by 6 months. At both 6 and 12 months, all three mutant genotypes showed reduced four limb and hind limb grip strength compared to WT mice. Performance on 6 and 12 mm width balance beams revealed deficits that were most pronounced at on the 6 mm balance beam at 6 months of age. There were pathological changes of myelinated axons in the femoral motor nerve in all three mutant lines by 3 months of age, and these became more pronounced at 6 and 12 months of age; sensory nerves (femoral sensory and the caudal nerve of the tail) appeared normal at all ages examined. Our results demonstrate that mice can be used to show the pathogenicity of human GJB1 mutations, and these new models for CMTX1 should facilitate the preclinical work for developing treatments for CMTX1.
2022-02-14 | Effects of early crush on aging wild type and Connexin 32 knockout mice: Evidence for a neuroprotective state in CMT1X mouse nerve.
The long-term sequelae of nerve injury as well as age-related neurodegeneration have been documented in numerous studies, however the role of Cx32 in these processes is not well understood. There is a need for better understanding of the molecular mechanisms that underlie long-term suboptimal nerve function and for approaches to prevent or improve it. In this communication we describe our studies using whole animal electrophysiology to examine the long-term sequelae of sciatic nerve crush in both WT and Cx32KO mice, a model of X-linked Charcot Marie Tooth disease, a subtype of inherited peripheral neuropathies. We present results from electrical nerve recordings done 14 to 27 days and 18 to 20 months after a unilateral sciatic nerve crush performed on 35 to 37-day old mice. Contrary to expectations, we find that whereas crush injury leads to a degradation of WT nerve function relative to uninjured nerves at 18 to 20 months, previously crushed Cx32KO nerves perform at the same level as their uninjured counterparts. Thus, 18 to 20 months after injury, WT nerves perform below the level of normal (uninjured) WT nerves in both motor and sensory nerve function. In contrast, measures of nerve function in Cx32KO mice are degraded for sensory axons but exhibit no additional dysfunction in motor axons. Early nerve injury has no negative electrophysiologic effect on the Cx32 KO motor nerves. Based on our prior demonstration that the transcriptomic profile of uninjured Cx32KO and injured WT sciatic nerves are very similar, the lack of an additional effect of crush on Cx32KO motor nerve parameters suggests that Cx32 knockout may implement a form of neuroprotection that limits the effects of subsequent injury.
2021-03-10 | AAV9-mediated Schwann cell-targeted gene therapy rescues a model of demyelinating neuropathy
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.
small molecules
2026-07-06 | Activity-dependent CO2 production in the axon triggers opening of Connexin32 in the Schwann cell paranode.
Loss of function mutations of Cx32, which is expressed in Schwann cells, cause X-linked Charcot-Marie-Tooth disease, a slowly progressive peripheral neuropathy. Action potential propagation causes Cx32 hemichannels in the Schwann cell paranode to open. As Cx32 hemichannels are directly sensitive to CO2, we have tested whether CO2 produced in the axon, as a consequence of the energetic demands of action potential propagation, might gate Cx32 hemichannels. Using isolated sciatic nerve from the mouse, we found that the critical components required for intercellular CO2 signaling are present (nodal mitochondria, the source of CO2; a CO2-permeable aquaporin, AQP1; paranodal Cx32; and carbonic anhydrase). We have used a membrane impermeant fluorescent dye, FITC, to demonstrate the opening of Cx32 in Schwann cells in response to an external CO2 stimulus or during action potential propagation in the isolated nerve. Pharmacological manipulations of AQP1 or carbonic anhydrase activity altered Cx32 gating during action potential firing. Expression of a modified Cx32 subunit, Cx32DN, that coassembles with Cx32WT, revealed that the activity-dependent dye loading of Schwann cells depended upon CO2 binding to Cx32. CO2 can, therefore, mediate neuron-to-glia signaling via connexins. CO2 permeable aquaporins and carbonic anhydrase are key components of this signaling mechanism.
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.
2024-03-14 | Gp78 regulates PMP22 and causes ER stress and autophagy in EV71-VP1-overexpressing mouse Schwann cells
Background: During Enterovirus type 71 (EV71) infection, the structural viral protein 1 (VP1) activates endoplasmic reticulum (ER) stress associated with peripheral myelin protein 22 (PMP22) accumulation and induces autophagy.However, the specific mechanism behind this process remains elusive.Methods: In this research, we used the VP1-overexpressing mouse Schwann cells (SCs) models co-transfected with a PMP22 silencing or Autocrine motility factor receptor (AMFR/gp78) overexpressing vector to explore the regulation of gp78 on PMP22 and its relationship with autophagy and apoptosis.Results: The activity of gp78 could be influenced by EV71-VP1, leading to a decrease in the ubiquitination and degradation of PMP22, resulting in PMP22 accumulation in ER.In VP1overexpressing mouse SCs, all three ER stress sensors, including pancreatic endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6) and inositol-requiring enzyme 1 (IRE1) and the related downstream signals (C/EBP-homologous protein (CHOP) and Caspase 12) were activated, as well as the ER-resident chaperone Glucoseregulated protein 78 (GRP78).In addition, VP1 upregulated the autophagy marker Microtubule-associated protein 1 light chain 3 beta (LC3B), while PMP22 silencing or gp78 overexpression reversed the phenomenon.Meanwhile, PMP22 silencing or gp78 overexpression increased proliferation of EV71-VP1-transfected mouse SCs.Conclusion:Gp78 could regulate PMP22 accumulation through ubiquitination degradation and cause ER stress and autophagy in EV71-VP1-overexpressing mouse SCs.Therefore, the gp78/PMP22/ER stress axis might emerge as a promising therapeutic target for myelin and neuronal damage induced by EV71 infection.
2023-07-21 | Amlexanox: Readthrough Induction and Nonsense-Mediated mRNA Decay Inhibition in a Charcot–Marie–Tooth Model of hiPSCs-Derived Neuronal Cells Harboring a Nonsense Mutation in GDAP1 Gene
Nonsense mutations are involved in multiple peripheral neuropathies. These mutations induce the presence of a premature termination codon (PTC) at the mRNA level. As a result, a dysfunctional or truncated protein is synthesized, or even absent linked to nonsense-mediated mRNA degradation (NMD) system activation. Readthrough molecules or NMD inhibitors could be innovative therapies in these hereditary neuropathies, particularly molecules harboring the dual activity as amlexanox. Charcot–Marie–Tooth (CMT) is the most common inherited pathology of the peripheral nervous system, affecting 1 in 2500 people worldwide. Nonsense mutations in the GDAP1 gene have been associated with a severe form of CMT, prompting us to investigate the effect of readthrough and NMD inhibitor molecules. Although not clearly defined, GDAP1 could be involved in mitochondrial functions, such as mitophagy. We focused on the homozygous c.581C>G (p.Ser194*) mutation inducing CMT2H using patient human induced pluripotent stem cell (hiPSC)-derived neuronal cells. Treatment during 20 h with 100 µM of amlexanox on this cell model stabilized GDAP1 mRNAs carrying UGA-PTC and induced a restoration of the mitochondrial morphology. These results highlight the potential of readthrough molecules associated to NMD inhibitors for the treatment of genetic alterations in CMT, opening the way for future investigations and a potential therapy.
2023-07-01 | Rapid degeneration of iPSC-derived motor neurons lacking Gdap1 engages a mitochondrial-sustained innate immune response
Charcot-Marie-Tooth disease is a chronic hereditary motor and sensory polyneuropathy targeting Schwann cells and/or motor neurons. Its multifactorial and polygenic origin portrays a complex clinical phenotype of the disease with a wide range of genetic inheritance patterns. The disease-associated gene GDAP1 encodes for a mitochondrial outer membrane protein. Mouse and insect models with mutations in Gdap1 have reproduced several traits of the human disease. However, the precise function in the cell types affected by the disease remains unknown. Here, we use induced-pluripotent stem cells derived from a Gdap1 knockout mouse model to better understand the molecular and cellular phenotypes of the disease caused by the loss-of-function of this gene. Gdap1-null motor neurons display a fragile cell phenotype prone to early degeneration showing (1) altered mitochondrial morphology, with an increase in the fragmentation of these organelles, (2) activation of autophagy and mitophagy, (3) abnormal metabolism, characterized by a downregulation of Hexokinase 2 and ATP5b proteins, (4) increased reactive oxygen species and elevated mitochondrial membrane potential, and (5) increased innate immune response and p38 MAP kinase activation. Our data reveals the existence of an underlying Redox-inflammatory axis fueled by altered mitochondrial metabolism in the absence of Gdap1. As this biochemical axis encompasses a wide variety of druggable targets, our results may have implications for developing therapies using combinatorial pharmacological approaches and improving therefore human welfare. A Redox-immune axis underlying motor neuron degeneration caused by the absence of Gdap1. Our results show that Gdap1-/- motor neurons have a fragile cellular phenotype that is prone to degeneration. Gdap1-/- iPSCs differentiated into motor neurons showed an altered metabolic state: decreased glycolysis and increased OXPHOS. These alterations may lead to hyperpolarization of mitochondria and increased ROS levels. Excessive amounts of ROS might be the cause of increased mitophagy, p38 activation and inflammation as a cellular response to oxidative stress. The p38 MAPK pathway and the immune response may, in turn, have feedback mechanisms, leading to the induction of apoptosis and senescence, respectively. CAC, citric acid cycle; ETC, electronic transport chain; Glc, glucose; Lac, lactate; Pyr, pyruvate.
proteins
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.
2023-03-16 | Boosting peripheral BDNF rescues impaired in vivo axonal transport in CMT2D mice
Gain-of-function mutations in the housekeeping gene GARS1, which lead to the expression of toxic versions of glycyl-tRNA synthetase (GlyRS), cause the selective motor and sensory pathology characterizing Charcot-Marie-Tooth disease (CMT). Aberrant interactions between GlyRS mutants and different proteins, including neurotrophin receptor tropomyosin receptor kinase receptor B (TrkB), underlie CMT type 2D (CMT2D); however, our pathomechanistic understanding of this untreatable peripheral neuropathy remains incomplete. Through intravital imaging of the sciatic nerve, we show that CMT2D mice displayed early and persistent disturbances in axonal transport of neurotrophin-containing signaling endosomes in vivo. We discovered that brain-derived neurotrophic factor (BDNF)/TrkB impairments correlated with transport disruption and overall CMT2D neuropathology and that inhibition of this pathway at the nerve-muscle interface perturbed endosome transport in wild-type axons. Accordingly, supplementation of muscles with BDNF, but not other neurotrophins, completely restored physiological axonal transport in neuropathic mice. Together, these findings suggest that selectively targeting muscles with BDNF-boosting therapies could represent a viable therapeutic strategy for CMT2D.
2022-10-28 | GDAP1 binds 4-hydroxynonenal, the toxic end-product of lipid peroxidation, using its GST-like binding pocket
Abstract GDAP1 (Ganglioside-induced differentiation-associated protein 1) is a novel member of the GST superfamily of detoxifying enzymes that is anchored to the outer mitochondrial membrane. GDAP1 mutations and changes in expression levels result in the inherited neuropathy Charcot-Marie-Tooth (CMT) disease, types 2K, 4A and 4H. GDAP1 activity has been associated with many mitochondrial functions however direct molecular interactions underpinning these connections have remained elusive. Here we establish that GDAP1 can bind 4-hydroxynonenal (4HNE), a toxic end-product of lipid peroxidation. 4HNE binding requires the α-loop, a large sequence motif that is inserted within the substrate recognition domain and is unique to GDAP1. In human cells, GDAP1 overexpression plays a cytoprotective role against oxidative stress. This effect is lost upon deletion of the α-loop. Lastly, we demonstrate that a CMT-causing mutant that destabilizes α-loop positioning also results in a decrease in 4HNE binding affinity. Together these results establish 4HNE as the biological ligand for GDAP1, provide mechanistic insight into 4HNE binding, and demonstrate that altered 4HNE recognition is the likely mechanism underlying CMT-causing mutants such as T157P near the 4HNE binding site.
2020-04-03 | Defective nucleotide-dependent assembly and membrane fusion in Mfn2 CMT2A variants improved by Bax
Mitofusins are members of the dynamin-related protein family of large GTPases that harness the energy from nucleotide hydrolysis to remodel membranes. Mitofusins possess four structural domains, including a GTPase domain, two extended helical bundles (HB1 and HB2), and a transmembrane region. We have characterized four Charcot-Marie-Tooth type 2A–associated variants with amino acid substitutions in Mfn2 that are proximal to the hinge that connects HB1 and HB2. A functional defect was not apparent in cells as the mitochondrial morphology of Mfn2-null cells was restored by expression of any of these variants. However, a significant fusion deficiency was observed in vitro, which was improved by the addition of crude cytosol extract or soluble Bax. All four variants had reduced nucleotide-dependent assembly in cis, but not trans, and this was also improved by the addition of Bax. Together, our data demonstrate an important role for this region in Mfn2 GTP-dependent oligomerization and membrane fusion and is consistent with a model where cytosolic factors such as Bax are masking molecular defects associated with Mfn2 disease variants in cells.
2016-07-03 | Structural studies of N-terminal mutants of Connexin 26 and Connexin 32 using 1H NMR spectroscopy
Alterations in gap junctions underlie the etiologies of syndromic deafness (KID) and Charcot-Marie Tooth disease (CMTX). Functional gap junctions are composed of connexin molecules with N-termini containing a flexible turn around G12, inserting the N-termini into the channel pore allowing voltage gating. The loss of this turn correlates with loss of Connexin 32 (Cx32) function by impaired trafficking to the cell membrane. Using (1)H NMR we show the N-terminus of a syndromic deafness mutation Cx26G12R, producing "leaky channels", contains a turn around G12 which is less structured and more flexible than wild-type. In contrast, the N-terminal structure of the same mutation in Cx32 chimera, Cx32*43E1G12R shows a larger constricted turn and no membrane current expression but forms membrane inserted hemichannels. Their function was rescued by formation of heteromeric channels with wild type subunits. We suggest the inflexible Cx32G12R N-terminus blocks ion conduction in homomeric channels and this channel block is relieved by incorporation of wild type subunits. In contrast, the increased open probability of Cx26G12R hemichannels is likely due to the addition of positive charge in the channel pore changing pore electrostatics and impairing hemichannel regulation by Ca(2+). These results provide mechanistic information on aberrant channel activity observed in disease.
cell therapies
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.
2016-06-14 | Enhanced Immune Response in Immunodeficient Mice Improves Peripheral Nerve Regeneration Following Axotomy
Injuries to peripheral nerves cause loss of motor and sensory function, greatly affecting life quality. Successful repair of the lesioned nerve requires efficient cell debris removal, followed by axon regeneration and reinnervation of target organs. Such process is orchestrated by several cellular and molecular events in which glial and immune cells actively participate. It is known that tissue clearance is largely improved by macrophages, which activation is potentiated by cells and molecules of the acquired immune system, such as T helper lymphocytes and antibodies, respectively. In the present work, we evaluated the contribution of lymphocytes in the regenerative process of crushed sciatic nerves of immunocompetent (wild-type, WT) and T and B-deficient (RAG-KO) mice. In Knockout animals, we found increased amount of macrophages under basal conditions and during the initial phase of the regenerative process, that was evaluated at 2, 4, and 8 weeks after lesion (wal). That parallels with faster axonal regeneration evidenced by the quantification of neurofilament and a growth associated protein immunolabeling. The motor function, evaluated by the sciatic function index, was fully recovered in both mouse strains within 4 wal, either in a progressive fashion, as observed for RAG-KO mice, or presenting a subtle regression, as seen in WT mice between 2 and 3 wal. Interestingly, boosting the immune response by early adoptive transference of activated WT lymphocytes at 3 days after lesion improved motor recovery in WT and RAG-KO mice, which was not ameliorated when cells were transferred at 2 wal. When monitoring lymphocytes by in vivo imaging, in both mouse strains, cells migrated to the lesion site shortly after transference, remaining in the injured limb up to its complete motor recovery. Moreover, a first peak of hyperalgesia, determined by von-Frey test, was coincident with increased lymphocyte infiltration in the damaged paw. Overall, the present results suggest that a wave of immune cell infiltration takes place during subacute phase of axonal regeneration, resulting in transient set back of motor recovery following peripheral axonal injury. Moreover, modulation of the immune response can be an efficient approach to speed up nerve regeneration.
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