RARE DISEASE
X-linked Charcot-Marie-Tooth disease type 4
X-linked Charcot-Marie-Tooth disease type 4
X-linked Charcot-Marie-Tooth disease type 4
Synonyms: CMT4X, CMTX4, Cowchock syndrome
Synonyms: CMT4X, CMTX4, Cowchock syndrome
Synonyms: CMT4X, CMTX4, Cowchock syndrome
Drug discovery
0
drugs
With orphan designations
Overview
X-linked Charcot-Marie-Tooth disease type 4 (CMTX4) is a rare axonal peripheral neuropathy caused by AIFM1 gene mutations on Xq26.1 [1][4][11]. It presents neonatally/early childhood with severe progressive distal muscle weakness (peroneal predominance), sensory deficits, foot deformities (pes cavus, hammertoes), and generalized areflexia [1][2]. Males exhibit full severity due to X-linked recessive inheritance, while female carriers remain asymptomatic [1][4]. Associated features include sensorineural hearing loss and cognitive impairment [2][4].
Burden
Categories: rare developmental anomalies during embryogenesis, rare genetic diseases, rare neurological diseases, rare otorhinolaryngological diseases
Research Papers
56 drug discovery papers about X-linked Charcot-Marie-Tooth disease type 4, with 1 first-in-class emerging drug candidates forecasted to outperform the average preclinical success rate. Recent publications:
56 drug discovery papers about X-linked Charcot-Marie-Tooth disease type 4, with 1 first-in-class emerging drug candidates forecasted to outperform the average preclinical success rate. Recent publications:
categories:
Small molecules
gene therapies
2026-04-10 | Clinical and molecular characterization of a novel pathogenic AIFM1 E336K mutation connecting mitochondrial dysfunction and neurodegeneration.
Mutations in the AIFM1 gene, encoding the apoptosis-inducing factor (AIF), have been associated with a spectrum of neurometabolic disorders. However, the mechanistic basis underlying their pathogenicity remains poorly understood. In this work, we identified and comprehensively characterized a novel hemizygous AIFM1 mutation c.1006G > A (E336K) in a male patient presenting with a progressive hereditary axonal sensorimotor polyneuropathy with childhood onset, inherited in an X-linked recessive pattern, associated with sensorineural hearing loss and without cognitive impairment. The clinical phenotype was consistent with Charcot-Marie-Tooth disease type 4 (CMTX4). Patient-derived fibroblasts exhibited reduced AIF protein stability despite preserved mRNA expression, impaired growth in OXPHOS-dependent conditions, decreased basal respiration, and altered assembly of mitochondrial respiratory supercomplexes. These defects were accompanied by reduced CHCHD4 protein levels and mitochondrial content. The purified E336K protein exhibited compromised FAD retention, decreased thermal stability, impaired NADH affinity, destabilization of the charge-transfer complex crucial for sustaining the AIF: CHCHD4 interaction, and a shift in coenzyme preference toward NADPH. Structurally, the substitution of Glu336 with Lys remodels the electrostatic environment of the NADH-binding cleft, thereby compromising redox function and weakening CHCHD4 binding. Despite these defects, the protein with the E336K mutation retained DNA binding, nuclease activity, and binding to nuclear partners, although parthanatos induction was attenuated in patient fibroblasts. Collectively, these molecular alterations converge on defective mitochondrial bioenergetics and dynamics, providing a direct mechanistic link to the patient’s clinical evolution.These findings provide a framework for understanding AIFM1-related disorders and pave the way for the development of future personalized molecular 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.
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.
2019-08-06 | Gene replacement therapy after neuropathy onset provides therapeutic benefit in a model of CMT1X
Abstract X-linked Charcot-Marie-Tooth disease (CMT1X), one of the commonest forms of inherited demyelinating neuropathy, results from GJB1 gene mutations causing loss of function of the gap junction protein connexin32 (Cx32). The aim of this study was to examine whether delayed gene replacement therapy after the onset of peripheral neuropathy can provide a therapeutic benefit in the Gjb1-null/Cx32 knockout model of CMT1X. After delivery of the LV-Mpz.GJB1 lentiviral vector by a single lumbar intrathecal injection into 6-month-old Gjb1-null mice, we confirmed expression of Cx32 in lumbar roots and sciatic nerves correctly localized at the paranodal myelin areas. Gjb1-null mice treated with LV-Mpz.GJB1 compared with LV-Mpz.Egfp (mock) vector at the age of 6 months showed improved motor performance at 8 and 10 months. Furthermore, treated mice showed increased sciatic nerve conduction velocities, improvement of myelination and reduced inflammation in lumbar roots and peripheral nerves at 10 months of age, along with enhanced quadriceps muscle innervation. Plasma neurofilament light (NEFL) levels, a clinically relevant biomarker, were also ameliorated in fully treated mice. Intrathecal gene delivery after the onset of peripheral neuropathy offers a significant therapeutic benefit in this disease model, providing a proof of principle for treating patients with CMT1X at different ages.
2019-03-01 | Role of Connexin-Based Gap Junction Channels in Communication of Myelin Sheath in Schwann Cells
The capacity of peripheral nerves to conduct action potentials along great distances and quickly recover following damage is mainly due to Schwann cells (SCs), the most abundant glial cells of the peripheral nervous system. SCs wrap multiple times around an axonal segment, forming a myelin sheath, allowing a significant increase in action potentials conduction by insulating the axons. Mature myelin consists of compact and non-compact (or cytoplasmic) myelin zones. Non-compact myelin is found in paranodal loops bordering nodes of Ranvier, and in innermost and outermost cytoplasmic tongues, and is the region in which Schmidt-Lanterman incisures (continuous spirals of overlapping cytoplasmic expansions within areas of compact myelin) are located. Using different technologies, it was shown that the layers of non-compact myelin could be connected to each other by gap junction channels (GJCs), formed by connexin 32 (Cx32), and their relative abundance allows transfer of ions and different small molecules. Likewise, Cx29 is expressed in the innermost layer of myelin sheath where it does not form GJCs but it colocalizes with Kv1, which implies that the SCs play an active role in the electrical condition in mammals. The critical role of GJCs in the functioning of myelinating SCs is evident in Charcot-Marie-Tooth disease (CMT), X-linked form 1 (CMTX1), which is caused by mutations in the gap junction protein beta 1 gene that codes for Cx32. Although the management of CMT symptoms is currently supportive, recently a method for targeted gene delivery to myelinating cells rescues the phenotype in KO-Cx32 mice, a model of CMTX1. In this mini-review, we show the current knowledge about the role of Cxs in myelin-forming SCs and summarize the recent discoveries that may become a real possibility for patients with disorders such as CMT.
small molecules
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.
2023-03-23 | Intermolecular And Dynamic Investigation of The Mechanism of Action of Reldesemtiv on Fast Skeletal Muscle Troponin Complex Toward the Treatment of Impaired Muscle Function
Muscle weakness as a secondary feature of attenuated neuronal input often leads to disability and sometimes death in patients with neurogenic neuromuscular diseases. These impaired muscle function has been observed in several diseases including amyotrophic lateral sclerosis, Charcot-Marie-Tooth, spinal muscular atrophy and Myasthenia gravis. This has spurred the search for small molecules which could activate fast skeletal muscle troponin complex as a means to increase muscle strength. Discovered small molecules have however been punctuated by off-target and side effects leading to the development of the second-generation small molecule, Reldesemtiv. In this study, we investigated the impact of Reldesemtiv binding to the fast skeletal troponin complex and the molecular determinants that condition the therapeutic prowess of Redesemtiv through computational techniques. It was revealed that Reldesemtiv binding possibly potentiates troponin C compacting characterized by reduced exposure to solvent molecules which could favor the slow release of calcium ions and the resultant sensitization of the subunit to calcium. These conformational changes were underscored by conventional and carbon hydrogen bonds, pi-alkyl, pi-sulfur and halogen interactions between Reldesemtiv the binding site residues. Arg113 (-3.96 kcal/mol), Met116 (-2.23 kcal/mol), Val114 (-1.28 kcal/mol) and Met121 (-0.63 kcal/mol) of the switch region of the inhibitory subunit were among the residues that contributed the most to the total free binding energy of Reldesemtiv highlighting their importance. These findings present useful insights which could lay the foundation for the development of fast skeletal muscle small molecule activators with high specificity and potency.
2022-03-23 | The autophagy inducer SMER28 attenuates microtubule dynamics mediating neuroprotection
Abstract SMER28 was originally identified in a screen for small-molecules that act as modulators of autophagy. SMER28 enhanced the clearance of autophagic substrates such as mutant huntingtin, an effect that was independent of rapamycin-induced autophagy. Thus, SMER 28 was established as a positive regulator of autophagy acting independently of the mTOR pathway, increasing autophagosome biosynthesis and attenuating mutant huntingtin-fragment toxicity in cellular- and fruit fly Huntington’s disease models, suggesting therapeutic potential. Despite a wealth of previous studies, molecular mechanisms mediating SMER28 activities and its direct targets have remained elusive. Here we analyzed the effects of SMER28 on cells and found that it not only induces autophagy, but also significantly stabilizes microtubules and decelerates microtubule dynamics. Moreover, we report that SMER28 displays neurotrophic and neuroprotective effects at the cellular level by inducing neurite outgrowth and by protecting from excitotoxin-induced axon degeneration. Finally, we compare the effects of SMER28 with other autophagy-inducing or microtubule-stabilizing drugs: Whereas SMER28 and rapamycin both induce autophagy, the latter does not stabilize microtubules, and whereas both SMER28 and epothilone B stabilize microtubules, epothilone does not provoke increased autophagy. Thus, the effect of SMER28 on cells in general and neurons in particular is based on its unique spectrum of bioactivities distinct from other known microtubule-stabilizing or autophagy-inducing drugs.
2022-03-10 | Emerging Role of HDACs in Regeneration and Ageing in the Peripheral Nervous System: Repair Schwann Cells as Pivotal Targets
The peripheral nervous system (PNS) has a remarkable regenerative capacity in comparison to the central nervous system (CNS), a phenomenon that is impaired during ageing. The ability of PNS axons to regenerate after injury is due to Schwann cells (SC) being reprogrammed into a repair phenotype called Repair Schwann cells. These repair SCs are crucial for supporting axonal growth after injury, myelin degradation in a process known as myelinophagy, neurotropic factor secretion, and axonal growth guidance through the formation of Büngner bands. After regeneration, repair SCs can remyelinate newly regenerated axons and support nonmyelinated axons. Increasing evidence points to an epigenetic component in the regulation of repair SC gene expression changes, which is necessary for SC reprogramming and regeneration. One of these epigenetic regulations is histone acetylation by histone acetyl transferases (HATs) or histone deacetylation by histone deacetylases (HDACs). In this review, we have focused particularly on three HDAC classes (I, II, and IV) that are Zn2+-dependent deacetylases. These HDACs are important in repair SC biology and remyelination after PNS injury. Another key aspect explored in this review is HDAC genetic compensation in SCs and novel HDAC inhibitors that are being studied to improve nerve regeneration.
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.
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.
2012-07-30 | Faculty Opinions recommendation of Mitofusin2 mutations disrupt axonal mitochondrial positioning and promote axon degeneration.
Alterations in mitochondrial dynamics (fission, fusion, and movement) are implicated in many neurodegenerative diseases, from rare genetic disorders such as Charcot-Marie-Tooth disease, to common conditions including Alzheimer's disease.However, the relationship between altered mitochondrial dynamics and neurodegeneration is incompletely understood.Here we show that disease associated MFN2 proteins suppressed both mitochondrial fusion and transport, and produced classic features of segmental axonal degeneration without cell body death, including neurofilament filled swellings, loss of calcium homeostasis, and accumulation of reactive oxygen species.By contrast, depletion of Opa1 suppressed mitochondrial fusion while sparing transport, and did not induce axonal degeneration.Axon degeneration induced by mutant MFN2 proteins correlated with the disruption of the proper mitochondrial positioning within axons, rather than loss of overall mitochondrial movement, or global mitochondrial dysfunction.We also found that augmenting expression of MFN1 rescued the axonal degeneration caused by MFN2 mutants, suggesting a possible therapeutic strategy for Charcot-Marie-Tooth disease.These experiments provide evidence that the ability of mitochondria to sense energy requirements and localize properly within axons is key to maintaining axonal integrity, and may be a common pathway by which disruptions in axonal transport contribute to neurodegeneration.
2010-02-02 | Interleukin-6 upregulates the expression of PMP22 in cultured rat Schwann cells via a JAK2-dependent pathway
The interleukin-6 (IL-6) family of cytokines is thought to be involved in the development and regeneration of peripheral nerves; however, their roles in myelination remain unclear. In this study, we examined the effects of IL-6 on the expression of genes for compact myelin proteins using Schwann cell cultures prepared by multiple explantation of adult rat sciatic nerves. In semi-quantitative reverse transcription-polymerase chain reaction analysis, stimulation of Schwann cells with IL-6 significantly increased the mRNA level of peripheral myelin protein 22 (PMP22), but not those of myelin protein zero and myelin basic protein. The increase in PMP22 mRNA was markedly suppressed by AG490, a Janus kinase 2 (JAK2) inhibitor, but not significantly by PD098059, a mitogen-activated protein kinase inhibitor. Immunocytochemical staining revealed that IL-6 enhanced immunoreactivities for the phosphorylated forms of both JAK2 and signal transducer and activator of transcription 3 (STAT3), as well as that for PMP22. These results indicate that IL-6 can enhance PMP22 production in Schwann cells via a JAK2-dependent pathway by probably activating STAT3 and thus may contribute to myelination.
2005-08-03 | Prenylation-Defective Human Connexin32 Mutants Are Normally Localized and Function Equivalently to Wild-Type Connexin32 in Myelinating Schwann Cells
Mutations in GJB1, the gene encoding the gap junction protein connexin32 (Cx32), cause the X-linked form of Charcot-Marie-Tooth disease, an inherited demyelinating neuropathy. The C terminus of human Cx32 contains a putative prenylation motif that is conserved in Cx32 orthologs. Using [ 3 H]mevalonolactone ([ 3 H]MVA) incorporation, we demonstrated that wild-type human connexin32 can be prenylated in COS7 cells, in contrast to disease-associated mutations that are predicted to disrupt the prenylation motif. We generated transgenic mice that express these mutants in myelinating Schwann cells. Male mice expressing a transgene were crossed with female Gjb1 -null mice; the male offspring were all Gjb1 -null, and one-half were transgene positive; in these mice, all Cx32 was derived from expression of the transgene. The mutant human protein was properly localized in myelinating Schwann cells in multiple transgenic lines and did not alter the localization of other components of paranodes and incisures. Finally, both the C280G and the S281x mutants appeared to “rescue” the phenotype of Gjb1 -null mice, because transgene-positive male mice had significantly fewer abnormally myelinated axons than did their transgene-negative male littermates. These results indicate that Cx32 is prenylated, but that prenylation is not required for proper trafficking of Cx32 and perhaps not even for certain aspects of its function, in myelinating Schwann cells.
gene therapies
2026-04-10 | Clinical and molecular characterization of a novel pathogenic AIFM1 E336K mutation connecting mitochondrial dysfunction and neurodegeneration.
Mutations in the AIFM1 gene, encoding the apoptosis-inducing factor (AIF), have been associated with a spectrum of neurometabolic disorders. However, the mechanistic basis underlying their pathogenicity remains poorly understood. In this work, we identified and comprehensively characterized a novel hemizygous AIFM1 mutation c.1006G > A (E336K) in a male patient presenting with a progressive hereditary axonal sensorimotor polyneuropathy with childhood onset, inherited in an X-linked recessive pattern, associated with sensorineural hearing loss and without cognitive impairment. The clinical phenotype was consistent with Charcot-Marie-Tooth disease type 4 (CMTX4). Patient-derived fibroblasts exhibited reduced AIF protein stability despite preserved mRNA expression, impaired growth in OXPHOS-dependent conditions, decreased basal respiration, and altered assembly of mitochondrial respiratory supercomplexes. These defects were accompanied by reduced CHCHD4 protein levels and mitochondrial content. The purified E336K protein exhibited compromised FAD retention, decreased thermal stability, impaired NADH affinity, destabilization of the charge-transfer complex crucial for sustaining the AIF: CHCHD4 interaction, and a shift in coenzyme preference toward NADPH. Structurally, the substitution of Glu336 with Lys remodels the electrostatic environment of the NADH-binding cleft, thereby compromising redox function and weakening CHCHD4 binding. Despite these defects, the protein with the E336K mutation retained DNA binding, nuclease activity, and binding to nuclear partners, although parthanatos induction was attenuated in patient fibroblasts. Collectively, these molecular alterations converge on defective mitochondrial bioenergetics and dynamics, providing a direct mechanistic link to the patient’s clinical evolution.These findings provide a framework for understanding AIFM1-related disorders and pave the way for the development of future personalized molecular 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.
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.
2019-08-06 | Gene replacement therapy after neuropathy onset provides therapeutic benefit in a model of CMT1X
Abstract X-linked Charcot-Marie-Tooth disease (CMT1X), one of the commonest forms of inherited demyelinating neuropathy, results from GJB1 gene mutations causing loss of function of the gap junction protein connexin32 (Cx32). The aim of this study was to examine whether delayed gene replacement therapy after the onset of peripheral neuropathy can provide a therapeutic benefit in the Gjb1-null/Cx32 knockout model of CMT1X. After delivery of the LV-Mpz.GJB1 lentiviral vector by a single lumbar intrathecal injection into 6-month-old Gjb1-null mice, we confirmed expression of Cx32 in lumbar roots and sciatic nerves correctly localized at the paranodal myelin areas. Gjb1-null mice treated with LV-Mpz.GJB1 compared with LV-Mpz.Egfp (mock) vector at the age of 6 months showed improved motor performance at 8 and 10 months. Furthermore, treated mice showed increased sciatic nerve conduction velocities, improvement of myelination and reduced inflammation in lumbar roots and peripheral nerves at 10 months of age, along with enhanced quadriceps muscle innervation. Plasma neurofilament light (NEFL) levels, a clinically relevant biomarker, were also ameliorated in fully treated mice. Intrathecal gene delivery after the onset of peripheral neuropathy offers a significant therapeutic benefit in this disease model, providing a proof of principle for treating patients with CMT1X at different ages.
2019-03-01 | Role of Connexin-Based Gap Junction Channels in Communication of Myelin Sheath in Schwann Cells
The capacity of peripheral nerves to conduct action potentials along great distances and quickly recover following damage is mainly due to Schwann cells (SCs), the most abundant glial cells of the peripheral nervous system. SCs wrap multiple times around an axonal segment, forming a myelin sheath, allowing a significant increase in action potentials conduction by insulating the axons. Mature myelin consists of compact and non-compact (or cytoplasmic) myelin zones. Non-compact myelin is found in paranodal loops bordering nodes of Ranvier, and in innermost and outermost cytoplasmic tongues, and is the region in which Schmidt-Lanterman incisures (continuous spirals of overlapping cytoplasmic expansions within areas of compact myelin) are located. Using different technologies, it was shown that the layers of non-compact myelin could be connected to each other by gap junction channels (GJCs), formed by connexin 32 (Cx32), and their relative abundance allows transfer of ions and different small molecules. Likewise, Cx29 is expressed in the innermost layer of myelin sheath where it does not form GJCs but it colocalizes with Kv1, which implies that the SCs play an active role in the electrical condition in mammals. The critical role of GJCs in the functioning of myelinating SCs is evident in Charcot-Marie-Tooth disease (CMT), X-linked form 1 (CMTX1), which is caused by mutations in the gap junction protein beta 1 gene that codes for Cx32. Although the management of CMT symptoms is currently supportive, recently a method for targeted gene delivery to myelinating cells rescues the phenotype in KO-Cx32 mice, a model of CMTX1. In this mini-review, we show the current knowledge about the role of Cxs in myelin-forming SCs and summarize the recent discoveries that may become a real possibility for patients with disorders such as CMT.
small molecules
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.
2023-03-23 | Intermolecular And Dynamic Investigation of The Mechanism of Action of Reldesemtiv on Fast Skeletal Muscle Troponin Complex Toward the Treatment of Impaired Muscle Function
Muscle weakness as a secondary feature of attenuated neuronal input often leads to disability and sometimes death in patients with neurogenic neuromuscular diseases. These impaired muscle function has been observed in several diseases including amyotrophic lateral sclerosis, Charcot-Marie-Tooth, spinal muscular atrophy and Myasthenia gravis. This has spurred the search for small molecules which could activate fast skeletal muscle troponin complex as a means to increase muscle strength. Discovered small molecules have however been punctuated by off-target and side effects leading to the development of the second-generation small molecule, Reldesemtiv. In this study, we investigated the impact of Reldesemtiv binding to the fast skeletal troponin complex and the molecular determinants that condition the therapeutic prowess of Redesemtiv through computational techniques. It was revealed that Reldesemtiv binding possibly potentiates troponin C compacting characterized by reduced exposure to solvent molecules which could favor the slow release of calcium ions and the resultant sensitization of the subunit to calcium. These conformational changes were underscored by conventional and carbon hydrogen bonds, pi-alkyl, pi-sulfur and halogen interactions between Reldesemtiv the binding site residues. Arg113 (-3.96 kcal/mol), Met116 (-2.23 kcal/mol), Val114 (-1.28 kcal/mol) and Met121 (-0.63 kcal/mol) of the switch region of the inhibitory subunit were among the residues that contributed the most to the total free binding energy of Reldesemtiv highlighting their importance. These findings present useful insights which could lay the foundation for the development of fast skeletal muscle small molecule activators with high specificity and potency.
2022-03-23 | The autophagy inducer SMER28 attenuates microtubule dynamics mediating neuroprotection
Abstract SMER28 was originally identified in a screen for small-molecules that act as modulators of autophagy. SMER28 enhanced the clearance of autophagic substrates such as mutant huntingtin, an effect that was independent of rapamycin-induced autophagy. Thus, SMER 28 was established as a positive regulator of autophagy acting independently of the mTOR pathway, increasing autophagosome biosynthesis and attenuating mutant huntingtin-fragment toxicity in cellular- and fruit fly Huntington’s disease models, suggesting therapeutic potential. Despite a wealth of previous studies, molecular mechanisms mediating SMER28 activities and its direct targets have remained elusive. Here we analyzed the effects of SMER28 on cells and found that it not only induces autophagy, but also significantly stabilizes microtubules and decelerates microtubule dynamics. Moreover, we report that SMER28 displays neurotrophic and neuroprotective effects at the cellular level by inducing neurite outgrowth and by protecting from excitotoxin-induced axon degeneration. Finally, we compare the effects of SMER28 with other autophagy-inducing or microtubule-stabilizing drugs: Whereas SMER28 and rapamycin both induce autophagy, the latter does not stabilize microtubules, and whereas both SMER28 and epothilone B stabilize microtubules, epothilone does not provoke increased autophagy. Thus, the effect of SMER28 on cells in general and neurons in particular is based on its unique spectrum of bioactivities distinct from other known microtubule-stabilizing or autophagy-inducing drugs.
2022-03-10 | Emerging Role of HDACs in Regeneration and Ageing in the Peripheral Nervous System: Repair Schwann Cells as Pivotal Targets
The peripheral nervous system (PNS) has a remarkable regenerative capacity in comparison to the central nervous system (CNS), a phenomenon that is impaired during ageing. The ability of PNS axons to regenerate after injury is due to Schwann cells (SC) being reprogrammed into a repair phenotype called Repair Schwann cells. These repair SCs are crucial for supporting axonal growth after injury, myelin degradation in a process known as myelinophagy, neurotropic factor secretion, and axonal growth guidance through the formation of Büngner bands. After regeneration, repair SCs can remyelinate newly regenerated axons and support nonmyelinated axons. Increasing evidence points to an epigenetic component in the regulation of repair SC gene expression changes, which is necessary for SC reprogramming and regeneration. One of these epigenetic regulations is histone acetylation by histone acetyl transferases (HATs) or histone deacetylation by histone deacetylases (HDACs). In this review, we have focused particularly on three HDAC classes (I, II, and IV) that are Zn2+-dependent deacetylases. These HDACs are important in repair SC biology and remyelination after PNS injury. Another key aspect explored in this review is HDAC genetic compensation in SCs and novel HDAC inhibitors that are being studied to improve nerve regeneration.
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.
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.
2012-07-30 | Faculty Opinions recommendation of Mitofusin2 mutations disrupt axonal mitochondrial positioning and promote axon degeneration.
Alterations in mitochondrial dynamics (fission, fusion, and movement) are implicated in many neurodegenerative diseases, from rare genetic disorders such as Charcot-Marie-Tooth disease, to common conditions including Alzheimer's disease.However, the relationship between altered mitochondrial dynamics and neurodegeneration is incompletely understood.Here we show that disease associated MFN2 proteins suppressed both mitochondrial fusion and transport, and produced classic features of segmental axonal degeneration without cell body death, including neurofilament filled swellings, loss of calcium homeostasis, and accumulation of reactive oxygen species.By contrast, depletion of Opa1 suppressed mitochondrial fusion while sparing transport, and did not induce axonal degeneration.Axon degeneration induced by mutant MFN2 proteins correlated with the disruption of the proper mitochondrial positioning within axons, rather than loss of overall mitochondrial movement, or global mitochondrial dysfunction.We also found that augmenting expression of MFN1 rescued the axonal degeneration caused by MFN2 mutants, suggesting a possible therapeutic strategy for Charcot-Marie-Tooth disease.These experiments provide evidence that the ability of mitochondria to sense energy requirements and localize properly within axons is key to maintaining axonal integrity, and may be a common pathway by which disruptions in axonal transport contribute to neurodegeneration.
2010-02-02 | Interleukin-6 upregulates the expression of PMP22 in cultured rat Schwann cells via a JAK2-dependent pathway
The interleukin-6 (IL-6) family of cytokines is thought to be involved in the development and regeneration of peripheral nerves; however, their roles in myelination remain unclear. In this study, we examined the effects of IL-6 on the expression of genes for compact myelin proteins using Schwann cell cultures prepared by multiple explantation of adult rat sciatic nerves. In semi-quantitative reverse transcription-polymerase chain reaction analysis, stimulation of Schwann cells with IL-6 significantly increased the mRNA level of peripheral myelin protein 22 (PMP22), but not those of myelin protein zero and myelin basic protein. The increase in PMP22 mRNA was markedly suppressed by AG490, a Janus kinase 2 (JAK2) inhibitor, but not significantly by PD098059, a mitogen-activated protein kinase inhibitor. Immunocytochemical staining revealed that IL-6 enhanced immunoreactivities for the phosphorylated forms of both JAK2 and signal transducer and activator of transcription 3 (STAT3), as well as that for PMP22. These results indicate that IL-6 can enhance PMP22 production in Schwann cells via a JAK2-dependent pathway by probably activating STAT3 and thus may contribute to myelination.
2005-08-03 | Prenylation-Defective Human Connexin32 Mutants Are Normally Localized and Function Equivalently to Wild-Type Connexin32 in Myelinating Schwann Cells
Mutations in GJB1, the gene encoding the gap junction protein connexin32 (Cx32), cause the X-linked form of Charcot-Marie-Tooth disease, an inherited demyelinating neuropathy. The C terminus of human Cx32 contains a putative prenylation motif that is conserved in Cx32 orthologs. Using [ 3 H]mevalonolactone ([ 3 H]MVA) incorporation, we demonstrated that wild-type human connexin32 can be prenylated in COS7 cells, in contrast to disease-associated mutations that are predicted to disrupt the prenylation motif. We generated transgenic mice that express these mutants in myelinating Schwann cells. Male mice expressing a transgene were crossed with female Gjb1 -null mice; the male offspring were all Gjb1 -null, and one-half were transgene positive; in these mice, all Cx32 was derived from expression of the transgene. The mutant human protein was properly localized in myelinating Schwann cells in multiple transgenic lines and did not alter the localization of other components of paranodes and incisures. Finally, both the C280G and the S281x mutants appeared to “rescue” the phenotype of Gjb1 -null mice, because transgene-positive male mice had significantly fewer abnormally myelinated axons than did their transgene-negative male littermates. These results indicate that Cx32 is prenylated, but that prenylation is not required for proper trafficking of Cx32 and perhaps not even for certain aspects of its function, in myelinating Schwann cells.
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