RESUMO
The cytoskeleton mediates fundamental cellular processes by organizing inter-organelle interactions. Pathogenic variants of inverted formin 2 (INF2) CAAX isoform, an actin assembly factor that is predominantly expressed in the endoplasmic reticulum (ER), are linked to focal segmental glomerulosclerosis (FSGS) and Charcot-Marie-Tooth (CMT) neuropathy. To investigate how pathogenic INF2 variants alter ER integrity, we used high-resolution live imaging of HeLa cells. Cells expressing wild-type (WT) INF2 showed a predominant tubular ER with perinuclear clustering. Cells expressing INF2 FSGS variants that cause mild and intermediate disease induced more sheet-like ER, a pattern similar to that seen for cells expressing WT-INF2 that were treated with actin and microtubule (MT) inhibitors. Dual CMT-FSGS INF2 variants led to more severe ER dysmorphism, with a diffuse, fragmented ER and coarse INF2 aggregates. Proper organization of both F-actin and MT was needed to modulate the tubule vs. sheet conformation balance, while MT arrays regulated spatial expansion of tubular ER in the cell periphery. Pathogenic INF2 variants also induced mitochondria fragmentation and dysregulated mitochondria distribution. Such mitochondrial abnormalities were more prominent for cells expressing CMT-FSGS compared to those with FSGS variants, indicating that the severity of the dysfunction is linked to the degree of cytoskeletal disorganization. Our observations suggest that pathogenic INF2 variants disrupt ER continuity by altering interactions between the ER and the cytoskeleton that in turn impairs inter-organelle communication, especially at ER-mitochondria contact sites. ER continuity defects may be a common disease mechanism involved in both peripheral neuropathy and glomerulopathy.
Assuntos
Doença de Charcot-Marie-Tooth , Retículo Endoplasmático , Forminas , Mitocôndrias , Humanos , Retículo Endoplasmático/metabolismo , Forminas/metabolismo , Forminas/genética , Células HeLa , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/patologia , Mitocôndrias/metabolismo , Glomerulosclerose Segmentar e Focal/metabolismo , Glomerulosclerose Segmentar e Focal/patologia , Glomerulosclerose Segmentar e Focal/genética , Actinas/metabolismo , Microtúbulos/metabolismo , Citoesqueleto/metabolismoRESUMO
Mutations targeting distinct domains of the neuron-specific kinesin KIF5A associate with different neurodegenerative/neurodevelopmental disorders, but the molecular bases of this clinical heterogeneity are unknown. We characterised five key mutants covering the whole spectrum of KIF5A-related phenotypes: spastic paraplegia (SPG, R17Q and R280C), Charcot-Marie-Tooth disease (CMT, R864*), amyotrophic lateral sclerosis (ALS, N999Vfs*40), and neonatal intractable myoclonus (NEIMY, C975Vfs*73) KIF5A mutants. CMT-R864*-KIF5A and ALS-N999Vfs*40-KIF5A showed impaired autoinhibition and peripheral localisation accompanied by altered mitochondrial distribution, suggesting transport competence disruption. ALS-N999Vfs*40-KIF5A formed SQSTM1/p62-positive inclusions sequestering WT-KIF5A, indicating a gain of toxic function. SPG-R17Q-KIF5A and ALS-N999Vfs*40-KIF5A evidenced a shorter half-life compared to WT-KIF5A, and proteasomal blockage determined their accumulation into detergent-insoluble inclusions. Interestingly, SPG-R280C-KIF5A and ALS-N999Vfs*40-KIF5A both competed for degradation with proteasomal substrates. Finally, NEIMY-C975Vfs*73-KIF5A displayed a similar, but more severe aberrant behaviour compared to ALS-N999Vfs*40-KIF5A; these two mutants share an abnormal tail but cause disorders on the opposite end of KIF5A-linked phenotypic spectrum. Thus, our observations support the pathogenicity of novel KIF5A mutants, highlight abnormalities of recurrent variants, and demonstrate that both unique and shared mechanisms underpin KIF5A-related diseases.
Assuntos
Cinesinas , Mutação , Transtornos do Neurodesenvolvimento , Cinesinas/metabolismo , Cinesinas/genética , Humanos , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/metabolismo , Transtornos do Neurodesenvolvimento/patologia , Mutação/genética , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Animais , Mitocôndrias/metabolismo , Mitocôndrias/genética , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologiaRESUMO
Charcot-Marie-Tooth type 1B (CMT1B) is a peripheral neuropathy caused by mutations in the gene encoding myelin protein zero (MPZ), a key component of the myelin sheath in Schwann cells. Mutations in the MPZ gene can lead to protein misfolding, unfolded protein response (UPR), endoplasmic reticulum (ER) stress, or protein mistrafficking. Despite significant progress in understanding the disease mechanisms, there is currently no effective treatment for CMT1B, with therapeutic strategies primarily focused on supportive care. Gene therapy represents a promising therapeutic approach for treating CMT1B. To develop a treatment and better design preclinical studies, an in-depth understanding of the pathophysiological mechanisms and animal models is essential. In this review, we present a comprehensive overview of the disease mechanisms, preclinical models, and recent advancements in therapeutic research for CMT1B, while also addressing the existing challenges in the field. This review aims to deepen the understanding of CMT1B and to encourage further research towards the development of effective treatments for CMT1B patients.
Assuntos
Doença de Charcot-Marie-Tooth , Modelos Animais de Doenças , Terapia Genética , Doença de Charcot-Marie-Tooth/terapia , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Humanos , Animais , Terapia Genética/métodos , Mutação , Proteína P0 da Mielina/genética , Proteína P0 da Mielina/metabolismo , Resposta a Proteínas não Dobradas/genética , Estresse do Retículo Endoplasmático/genéticaRESUMO
Charcot-Marie-Tooth (CMT) neuropathy is a polygenic disorder of peripheral nerves with no effective cure. Thiamine (vitamin B1) is a neurotropic compound that improves neuropathies. Our pilot study characterizes therapeutic potential of daily oral administration of thiamine (100 mg) in CMT neuropathy and its molecular mechanisms. The patient hand grip strength was determined before and after thiamine administration along with the blood levels of the thiamine coenzyme form (thiamine diphosphate, ThDP), activities of endogenous holo-transketolase (without ThDP in the assay medium) and total transketolase (with ThDP in the assay medium), and transketolase activation by ThDP [1 - (holo-transketolase/total transketolase),%], corresponding to the fraction of ThDP-free apo-transketolase. Single cases of administration of sulbutiamine (200 mg) or benfotiamine (150 mg) reveal their effects on the assayed parameters within those of thiamine. Administration of thiamine or its pharmacological forms increased the hand grip strength in the CMT patients. Comparison of the thiamin status in patients with different forms of CMT disease to that of control subjects without diagnosed pathologies revealed no significant differences in the average levels of ThDP, holo-transketolase, or relative content of holo and apo forms of transketolase. However, the regulation of transketolase by thiamine/ThDP differed in the control and CMT groups: in the assay, ThDP activated transketolase from the control individuals, but not from CMT patients. Thiamine administration paradoxically decreased endogenous holo-transketolase in CMT patients; this effect was not observed in the control group. Correlation analysis revealed sex-specific differences in the relationship between the parameters of thiamine status in both the control subjects and patients with the CMT disease. Thus, our findings link physiological benefits of thiamine administration in CMT patients to changes in their thiamine status, in particular, the blood levels of ThDP and transketolase regulation.
Assuntos
Doença de Charcot-Marie-Tooth , Tiamina Pirofosfato , Tiamina , Transcetolase , Humanos , Doença de Charcot-Marie-Tooth/tratamento farmacológico , Doença de Charcot-Marie-Tooth/metabolismo , Tiamina/uso terapêutico , Tiamina/análogos & derivados , Tiamina/administração & dosagem , Tiamina/metabolismo , Tiamina Pirofosfato/metabolismo , Tiamina Pirofosfato/uso terapêutico , Transcetolase/metabolismo , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Força da Mão , Projetos Piloto , IdosoRESUMO
Charcot-Marie-Tooth type 2A (CMT2A) is a single-gene motor sensory neuropathy caused by Mfn2 mutation. It is generally believed that CMT2A involves mitochondrial fusion disruption. However, how Mfn2 mutation mediates the mitochondrial membrane fusion loss and its further pathogenic mechanisms remain unclear. Here, in vivo and in vitro mouse models harboring the Mfn2R364W, Mfn2G176S and Mfn2H165R mutations were constructed. Mitochondrial membrane fusion and fission proteins analysis showed that Mfn2R364W, Mfn2G176S, and Mfn2H165R/+ mutations maintain the expression of Mfn2, but promote Drp1 upregulation and Opa1 hydrolytic cleavage. In Mfn2H165R/H165R mutation, Mfn2, Drp1, and Opa1 all play a role in inducing mitochondrial fragmentation, and the mitochondrial aggregation is affected by Mfn2 loss. Further research into the pathogenesis of CMT2A showed these three mutations all induce mitochondria-mediated apoptosis, and mitochondrial oxidative phosphorylation damage. Overall, loss of overall fusion activity affects mitochondrial DNA (mtDNA) stability and causes mitochondrial loss and dysfunction, ultimately leading to CMT2A disease. Interestingly, the differences in the pathogenesis of CMT2A between Mfn2R364W, Mfn2G176S, Mfn2H165R/+ and Mfn2H165R/H165R mutations, including the distribution of Mfn2 and mitochondria, the expression of mitochondrial outer membrane-associated proteins (Bax, VDAC1 and AIF), and the enzyme activity of mitochondrial complex I, are related to the expression of Mfn2.
Assuntos
Apoptose , Doença de Charcot-Marie-Tooth , GTP Fosfo-Hidrolases , Mitocôndrias , Mutação , Fosforilação Oxidativa , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Animais , Mitocôndrias/metabolismo , Mitocôndrias/genética , Camundongos , Apoptose/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Dinâmica Mitocondrial/genética , Humanos , Modelos Animais de DoençasRESUMO
Charcot-Marie-Tooth disease type 1E (CMT1E) is an inherited autosomal dominant peripheral neuropathy caused by mutations in the peripheral myelin protein 22 (PMP22) gene. The identical leucine-to-proline (L16P) amino acid substitution in PMP22 is carried by the Trembler J (TrJ) mouse and is found in CMT1E patients presenting with early-onset disease. Peripheral nerves of patients diagnosed with CMT1E display a complex and varied histopathology, including Schwann cell hyperproliferation, abnormally thin myelin, axonal degeneration, and subaxonal morphological changes. Here, we have taken an unbiased data-independent analysis (DIA) mass spectrometry (MS) approach to quantify proteins from nerves of 3-week-old, age and genetic strain-matched wild-type (Wt) and heterozygous TrJ mice. Nerve proteins were dissolved in lysis buffer and digested into peptide fragments, and protein groups were quantified by liquid chromatography-mass spectrometry (LC-MS). A linear model determined statistically significant differences between the study groups, and proteins with an adjusted p-value of less than 0.05 were deemed significant. This untargeted proteomics approach identified 3759 quality-controlled protein groups, of which 884 demonstrated differential expression between the two genotypes. Gene ontology (GO) terms related to myelin and myelin maintenance confirm published data while revealing a previously undetected prominent decrease in peripheral myelin protein 2. The dataset corroborates the described pathophysiology of TrJ nerves, including elevated activity in the proteasome-lysosomal pathways, alterations in protein trafficking, and an increase in three macrophage-associated proteins. Previously unrecognized perturbations in RNA processing pathways and GO terms were also discovered. Proteomic abnormalities that overlap with other human neurological disorders besides CMT include Lafora Disease and Amyotrophic Lateral Sclerosis. Overall, this study confirms and extends current knowledge on the cellular pathophysiology in TrJ neuropathic nerves and provides novel insights for future examinations. Recognition of shared pathomechanisms across discrete neurological disorders offers opportunities for innovative disease-modifying therapeutics that could be effective for distinct neuropathies.
Assuntos
Proteômica , Animais , Proteômica/métodos , Camundongos , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo , Nervos Periféricos/metabolismo , Nervos Periféricos/patologia , Camundongos Endogâmicos C57BL , Masculino , Doenças do Sistema Nervoso Periférico/genética , Doenças do Sistema Nervoso Periférico/metabolismo , Doenças do Sistema Nervoso Periférico/patologia , FemininoRESUMO
Underexpression, overexpression, and point mutations in peripheral myelin protein 22 (PMP22) cause most cases of Charcot-Marie-Tooth disease (CMTD). While its exact functions remain unclear, PMP22 is clearly essential for formation and maintenance of healthy myelin in the peripheral nervous system. This review explores emerging evidence for roles of PMP22 in cholesterol homeostasis. First, we highlight dysregulation of lipid metabolism in PMP22-based forms of CMTD and recently-discovered interactions between PMP22 and cholesterol biosynthesis machinery. We then examine data that demonstrates PMP22 and cholesterol co-traffic in cells and co-localize in lipid rafts, including how disease-causing PMP22 mutations result in aberrations in cholesterol localization. Finally, we examine roles for interactions between PMP22 and ABCA1 in cholesterol efflux. Together, this emerging body of evidence suggests that PMP22 plays a role in facilitating enhanced cholesterol synthesis and trafficking necessary for production and maintenance of healthy myelin.
Assuntos
Doença de Charcot-Marie-Tooth , Colesterol , Homeostase , Proteínas da Mielina , Células de Schwann , Colesterol/metabolismo , Humanos , Células de Schwann/metabolismo , Proteínas da Mielina/metabolismo , Proteínas da Mielina/genética , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/genética , Animais , Bainha de Mielina/metabolismo , Metabolismo dos Lipídeos , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Transportador 1 de Cassete de Ligação de ATP/genética , MutaçãoRESUMO
Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common inherited peripheral neuropathy caused by a 1.5 Mb tandem duplication of chromosome 17 harbouring the PMP22 gene. This dose-dependent overexpression of PMP22 results in disrupted Schwann cell myelination of peripheral nerves. To obtain better insights into the underlying pathogenic mechanisms in CMT1A, we investigated the role of PMP22 duplication in cellular homeostasis in CMT1A mouse models and in patient-derived induced pluripotent stem cells differentiated into Schwann cell precursors (iPSC-SCPs). We performed lipidomic profiling and bulk RNA sequencing (RNA-seq) on sciatic nerves of two developing CMT1A mouse models and on CMT1A patient-derived iPSC-SCPs. For the sciatic nerves of the CMT1A mice, cholesterol and lipid metabolism was downregulated in a dose-dependent manner throughout development. For the CMT1A iPSC-SCPs, transcriptional analysis unveiled a strong suppression of genes related to autophagy and lipid metabolism. Gene ontology enrichment analysis identified disturbances in pathways related to plasma membrane components and cell receptor signalling. Lipidomic analysis confirmed the severe dysregulation in plasma membrane lipids, particularly sphingolipids, in CMT1A iPSC-SCPs. Furthermore, we identified reduced lipid raft dynamics, disturbed plasma membrane fluidity and impaired cholesterol incorporation and storage, all of which could result from altered lipid storage homeostasis in the patient-derived CMT1A iPSC-SCPs. Importantly, this phenotype could be rescued by stimulating autophagy and lipolysis. We conclude that PMP22 duplication disturbs intracellular lipid storage and leads to a more disordered plasma membrane owing to an alteration in the lipid composition, which might ultimately lead to impaired axo-glial interactions. Moreover, targeting lipid handling and metabolism could hold promise for the treatment of patients with CMT1A.
Assuntos
Membrana Celular , Doença de Charcot-Marie-Tooth , Homeostase , Células-Tronco Pluripotentes Induzidas , Metabolismo dos Lipídeos , Proteínas da Mielina , Células de Schwann , Animais , Humanos , Camundongos , Membrana Celular/metabolismo , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Duplicação Gênica , Homeostase/fisiologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Metabolismo dos Lipídeos/fisiologia , Proteínas da Mielina/metabolismo , Proteínas da Mielina/genética , Células de Schwann/metabolismo , Nervo Isquiático/metabolismoRESUMO
Charcot-Marie-Tooth type 2A (CMT2A) is an inherited sensorimotor neuropathy associated with mutations within the Mitofusin 2 (MFN2) gene. These mutations impair normal mitochondrial functioning via different mechanisms, disturbing the equilibrium between mitochondrial fusion and fission, of mitophagy and mitochondrial axonal transport. Although CMT2A disease causes a significant disability, no resolutive treatment for CMT2A patients to date. In this context, reliable experimental models are essential to precisely dissect the molecular mechanisms of disease and to devise effective therapeutic strategies. The most commonly used models are either in vitro or in vivo, and among the latter murine models are by far the most versatile and popular. Here, we critically revised the most relevant literature focused on the experimental models, providing an update on the mammalian models of CMT2A developed to date. We highlighted the different phenotypic, histopathological and molecular characteristics, and their use in translational studies for bringing potential therapies from the bench to the bedside. In addition, we discussed limitations of these models and perspectives for future improvement.
Assuntos
Doença de Charcot-Marie-Tooth , Modelos Animais de Doenças , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/patologia , Doença de Charcot-Marie-Tooth/terapia , Doença de Charcot-Marie-Tooth/metabolismo , Animais , Humanos , Mutação , Mitocôndrias/metabolismo , Mitocôndrias/genética , Mitocôndrias/patologia , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Camundongos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Dinâmica Mitocondrial/genéticaRESUMO
Charcot-Marie-Tooth disease (CMT) is a genetic peripheral neuropathy caused by mutations in many functionally diverse genes. The aminoacyl-tRNA synthetase (ARS) enzymes, which transfer amino acids to partner tRNAs for protein synthesis, represent the largest protein family genetically linked to CMT aetiology, suggesting pathomechanistic commonalities. Dominant intermediate CMT type C (DI-CMTC) is caused by YARS1 mutations driving a toxic gain-of-function in the encoded tyrosyl-tRNA synthetase (TyrRS), which is mediated by exposure of consensus neomorphic surfaces through conformational changes of the mutant protein. In this study, we first showed that human DI-CMTC-causing TyrRSE196K mis-interacts with the extracellular domain of the BDNF receptor TrkB, an aberrant association we have previously characterised for several mutant glycyl-tRNA synthetases linked to CMT type 2D (CMT2D). We then performed temporal neuromuscular assessments of YarsE196K mice modelling DI-CMT. We determined that YarsE196K homozygotes display a selective, age-dependent impairment in in vivo axonal transport of neurotrophin-containing signalling endosomes, phenocopying CMT2D mice. This impairment is replicated by injection of recombinant TyrRSE196K, but not TyrRSWT, into muscles of wild-type mice. Augmenting BDNF in DI-CMTC muscles, through injection of recombinant protein or muscle-specific gene therapy, resulted in complete axonal transport correction. Therefore, this work identifies a non-cell autonomous pathomechanism common to ARS-related neuropathies, and highlights the potential of boosting BDNF levels in muscles as a therapeutic strategy.
Assuntos
Transporte Axonal , Fator Neurotrófico Derivado do Encéfalo , Doença de Charcot-Marie-Tooth , Modelos Animais de Doenças , Animais , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/genética , Camundongos , Tirosina-tRNA Ligase/genética , Tirosina-tRNA Ligase/metabolismo , Humanos , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Receptor trkB/metabolismo , Receptor trkB/genética , MutaçãoRESUMO
Demyelinating Charcot-Marie-Tooth 4G (CMT4G) results from a recessive mutation in the 5'UTR region of the Hexokinase 1 (HK1) gene. HK participates in mitochondrial calcium homeostasis by binding to the Voltage-Dependent Anion Channel (VDAC), through its N-terminal porin-binding domain. Our hypothesis is that CMT4G mutation results in a broken interaction between mutant HK1 and VDAC, disturbing mitochondrial calcium homeostasis. We studied a cohort of 25 CMT4G patients recruited in the French gypsy population. The disease was characterized by a childhood onset, an intermediate demyelinating pattern, and a significant phenotype leading to becoming wheelchair-bound by the fifth decade of life. Co-IP and PLA studies indicated a strong decreased interaction between VDAC and HK1 in the patients' PBMCs and sural nerve. We observed that either wild-type HK1 expression or a peptide comprising the 15 aa of the N-terminal wild-type HK1 administration decreased mitochondrial calcium release in HEK293 cells. However, mutated CMT4G HK1 or the 15 aa of the mutated HK1 was unable to block mitochondrial calcium release. Taken together, these data show that the CMT4G-induced modification of the HK1 N-terminus disrupts HK1-VDAC interaction. This alters mitochondrial calcium buffering that has been shown to be critical for myelin sheath maintenance.
Assuntos
Cálcio , Doença de Charcot-Marie-Tooth , Hexoquinase , Mitocôndrias , Canal de Ânion 1 Dependente de Voltagem , Adolescente , Adulto , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Regiões 5' não Traduzidas/genética , Cálcio/metabolismo , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Células HEK293 , Hexoquinase/genética , Hexoquinase/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/genética , Mutação , Ligação Proteica , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Canal de Ânion 1 Dependente de Voltagem/genéticaRESUMO
Mutations in the gene encoding MFN2 have been identified as associated with Charcot-Marie-Tooth disease type 2A (CMT2A), a neurological disorder characterized by a broad clinical phenotype involving the entire nervous system. MFN2, a dynamin-like GTPase protein located on the outer mitochondrial membrane, is well-known for its involvement in mitochondrial fusion. Numerous studies have demonstrated its participation in a network crucial for various other mitochondrial functions, including mitophagy, axonal transport, and its controversial role in endoplasmic reticulum (ER)-mitochondria contacts. Considerable progress has been made in the last three decades in elucidating the disease pathogenesis, aided by the generation of animal and cellular models that have been instrumental in studying disease physiology. A review of the literature reveals that, up to now, no definitive pharmacological treatment for any CMT2A variant has been established; nonetheless, recent years have witnessed substantial progress. Many treatment approaches, especially concerning molecular therapy, such as histone deacetylase inhibitors, peptide therapy to increase mitochondrial fusion, the new therapeutic strategies based on MF1/MF2 balance, and SARM1 inhibitors, are currently in preclinical testing. The literature on gene silencing and gene replacement therapies is still limited, except for a recent study by Rizzo et al.(Rizzo et al., 2023), which recently first achieved encouraging results in in vitro and in vivo models of the disease. The near-future goal for these promising therapies is to progress to the stage of clinical translation.
Assuntos
Doença de Charcot-Marie-Tooth , Animais , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/terapia , Doença de Charcot-Marie-Tooth/metabolismo , Mitocôndrias/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Fenótipo , Proteínas Mitocondriais/metabolismo , MutaçãoRESUMO
Mutations in the Microrchidia CW-type zinc finger 2 (MORC2) GHKL ATPase module cause a broad range of neuropathies, such as Charcot-Marie-Tooth disease type 2Z; however, the aetiology and therapeutic strategy are not fully understood. Previously, we reported that the Morc2a p.S87L mouse model exhibited neuropathy and muscular dysfunction through DNA damage accumulation. In the present study, we analysed the gene expression of Morc2a p.S87L mice and designated the primary causing factor. We investigated the pathological pathway using Morc2a p.S87L mouse embryonic fibroblasts and human fibroblasts harbouring MORC2 p.R252W. We subsequently assessed the therapeutic effect of gene therapy administered to Morc2a p.S87L mice. This study revealed that Morc2a p.S87L causes a protein synthesis defect, resulting in the loss of function of Morc2a and high cellular apoptosis induced by high hydroxyl radical levels. We considered the Morc2a GHKL ATPase domain as a therapeutic target because it simultaneously complements hydroxyl radical scavenging and ATPase activity. We used the adeno-associated virus (AAV)-PHP.eB serotype, which has a high CNS transduction efficiency, to express Morc2a or Morc2a GHKL ATPase domain protein in vivo. Notably, AAV gene therapy ameliorated neuropathy and muscular dysfunction with a single treatment. Loss-of-function characteristics due to protein synthesis defects in Morc2a p.S87L were also noted in human MORC2 p.S87L or p.R252W variants, indicating the correlation between mouse and human pathogenesis. In summary, CMT2Z is known as an incurable genetic disorder, but the present study demonstrated its mechanisms and treatments based on established animal models. This study demonstrates that the Morc2a p.S87L variant causes hydroxyl radical-mediated neuropathy, which can be rescued through AAV-based gene therapy.
Assuntos
Terapia Genética , Animais , Humanos , Camundongos , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/terapia , Dependovirus/genética , Fibroblastos/metabolismo , Terapia Genética/métodos , Radical Hidroxila/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Mutations in Mitofusin2 (MFN2) associated with the pathology of the debilitating neuropathy Charcot-Marie-Tooth type 2A (CMT2A) are known to alter mitochondrial morphology. Previously, such mutations have been shown to elicit two diametrically opposite phenotypes - while some mutations have been causally linked to enhanced mitochondrial fragmentation, others have been shown to induce hyperfusion. Our study identifies one such MFN2 mutant, T206I that causes mitochondrial hyperfusion. Cells expressing this MFN2 mutant have elongated and interconnected mitochondria. T206I-MFN2 mutation in the GTPase domain increases MFN2 stability and renders cells susceptible to stress. We show that cells expressing T206I-MFN2 have a higher predisposition towards mitophagy under conditions of serum starvation. We also detect increased DRP1 recruitment onto the outer mitochondrial membrane, though the total DRP1 protein level remains unchanged. Here we have characterized a lesser studied CMT2A-linked MFN2 mutant to show that its presence affects mitochondrial morphology and homeostasis.
Assuntos
Doença de Charcot-Marie-Tooth , Mitofagia , Humanos , Dinâmica Mitocondrial , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mitocôndrias/metabolismo , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Mutação , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismoRESUMO
Investigating the impact of disease-causing mutations, their affected pathways, and/or potential therapeutic strategies using disease modeling often requires the generation of different in vivo and in cellulo models. To date, several approaches have been established to induce transgene expression in a controlled manner in different model systems. Several rounds of subcloning are, however, required, depending on the model organism used, thus bringing labor-intensive experiments into the technical approach and analysis comparison. The GeneSwitch™ technology is an adapted version of the classical UAS-GAL4 inducible system, allowing the spatial and temporal modulation of transgene expression. It consists of three components: a plasmid encoding for the chimeric regulatory pSwitch protein, Mifepristone as an inducer, and an inducible plasmid. While the pSwitch-containing first plasmid can be used both in vivo and in cellulo, the inducible second plasmid can only be used in cellulo. This requires a specific subcloning strategy of the inducible plasmid tailored to the model organism used. To avoid this step and unify gene expression in the transgenic models generated, we replaced the backbone vector with standard pUAS-attB plasmid for both plasmids containing either the chimeric GeneSwitch™ cDNA sequence or the transgene cDNA sequence. We optimized this adapted system to regulate transgene expression in several mammalian cell lines. Moreover, we took advantage of this new system to generate unified cellular and fruit fly models for YARS1-induced Charco-Marie-Tooth neuropathy (CMT). These new models displayed the expected CMT-like phenotypes. In the N2a neuroblastoma cells expressing YARS1 transgenes, we observed the typical "teardrop" distribution of the synthetase that was perturbed when expressing the YARS1CMT mutation. In flies, the ubiquitous expression of YARS1CMT induced dose-dependent developmental lethality and pan-neuronal expression caused locomotor deficit, while expression of the wild-type allele was harmless. Our proof-of-concept disease modeling studies support the efficacy of the adapted transgenesis system as a powerful tool allowing the design of studies with optimal data comparability.
Assuntos
Doença de Charcot-Marie-Tooth , Tirosina-tRNA Ligase , Animais , DNA Complementar/metabolismo , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Drosophila/genética , Mutação , Neurônios/metabolismo , Tirosina-tRNA Ligase/metabolismo , Modelos Animais de Doenças , Mamíferos/genéticaRESUMO
Mitofusin-2 (MFN2) is an outer mitochondrial membrane protein essential for mitochondrial networking in most cells. Autosomal dominant mutations in the MFN2 gene cause Charcot-Marie-Tooth type 2A disease (CMT2A), a severe and disabling sensory-motor neuropathy that impacts the entire nervous system. Here, we propose a novel therapeutic strategy tailored to correcting the root genetic defect of CMT2A. Though mutant and wild-type MFN2 mRNA are inhibited by RNA interference (RNAi), the wild-type protein is restored by overexpressing cDNA encoding functional MFN2 modified to be resistant to RNAi. We tested this strategy in CMT2A patient-specific human induced pluripotent stem cell (iPSC)-differentiated motor neurons (MNs), demonstrating the correct silencing of endogenous MFN2 and replacement with an exogenous copy of the functional wild-type gene. This approach significantly rescues the CMT2A MN phenotype in vitro, stabilizing the altered axonal mitochondrial distribution and correcting abnormal mitophagic processes. The MFN2 molecular correction was also properly confirmed in vivo in the MitoCharc1 CMT2A transgenic mouse model after cerebrospinal fluid (CSF) delivery of the constructs into newborn mice using adeno-associated virus 9 (AAV9). Altogether, our data support the feasibility of a combined RNAi and gene therapy strategy for treating the broad spectrum of human diseases associated with MFN2 mutations.
Assuntos
Doença de Charcot-Marie-Tooth , Células-Tronco Pluripotentes Induzidas , Humanos , Camundongos , Animais , Interferência de RNA , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/terapia , Doença de Charcot-Marie-Tooth/metabolismo , Mutação , Hidrolases/genética , Camundongos TransgênicosRESUMO
The mitochondrial outer membrane creates a diffusion barrier between the cytosol and the mitochondrial intermembrane space, allowing the exchange of metabolic products, important for efficient mitochondrial function in neurons. The ganglioside-induced differentiation-associated protein 1 (GDAP1) is a mitochondrial outer membrane protein with a critical role in mitochondrial dynamics and metabolic balance in neurons. Missense mutations in the GDAP1 gene are linked to the most common human peripheral neuropathy, Charcot-Marie-Tooth disease (CMT). GDAP1 is a distant member of the glutathione-S-transferase (GST) superfamily, with unknown enzymatic properties or functions at the molecular level. The structure of the cytosol-facing GST-like domain has been described, but there is no consensus on how the protein interacts with the mitochondrial outer membrane. Here, we describe a model for GDAP1 assembly on the membrane using peptides vicinal to the GDAP1 transmembrane domain. We used oriented circular dichroism spectroscopy (OCD) with synchrotron radiation to study the secondary structure and orientation of GDAP1 segments at the outer and inner surfaces of the outer mitochondrial membrane. These experiments were complemented by small-angle X-ray scattering, providing the first experimental structural models for full-length human GDAP1. The results indicate that GDAP1 is bound into the membrane via a single transmembrane helix, flanked by two peripheral helices interacting with the outer and inner leaflets of the mitochondrial outer membrane in different orientations. Impairment of these interactions could be a mechanism for CMT in the case of missense mutations affecting these segments instead of the GST-like domain.
Assuntos
Doença de Charcot-Marie-Tooth , Membranas Mitocondriais , Humanos , Membranas Mitocondriais/metabolismo , Síncrotrons , Mitocôndrias/metabolismo , Neurônios/metabolismo , Mutação de Sentido Incorreto , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Proteínas de Membrana/metabolismoRESUMO
Small heat shock proteins (sHSPs) are ATP-independent molecular chaperones with the α-crystalline domain that is critical to their chaperone activity. Within the sHSP family, three (HSPB1, HSPB3, and HSPB8) proteins are linked with inherited peripheral neuropathies, including distal hereditary motor neuropathy (dHMN) and Charco-Marie-Tooth disease (CMT). In this study, we introduced the HSPB3 Y118H (HSPB3Y118H) mutant gene identified from the CMT2 family in Drosophila. With a missense mutation on its α-crystalline domain, this human HSPB3 mutant gene induced a loss of motor activity accompanied by reduced mitochondrial membrane potential in fly neuronal tissues. Moreover, mitophagy, a critical mechanism of mitochondrial quality control, is downregulated in fly motor neurons expressing HSPB3Y118H. Surprisingly, PINK1 and Parkin, the core regulators of mitophagy, successfully rescued these motor and mitochondrial abnormalities in HSPB3 mutant flies. Results from the first animal model of HSPB3 mutations suggest that mitochondrial dysfunction plays a critical role in HSPB3-associated human pathology.
Assuntos
Doença de Charcot-Marie-Tooth , Proteínas de Drosophila , Proteínas de Choque Térmico Pequenas , Animais , Humanos , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Drosophila , Proteínas de Drosophila/genética , Proteínas de Choque Térmico/genética , Mitocôndrias/metabolismo , Mutação , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Inter-organelle contact sites between mitochondria and lysosomes mediate the crosstalk and bidirectional regulation of their dynamics in health and disease. However, mitochondria-lysosome contact sites and their misregulation have not been investigated in peripheral sensory neurons. Charcot-Marie-Tooth type 2B disease is an autosomal dominant axonal neuropathy affecting peripheral sensory neurons caused by mutations in the GTPase Rab7. Using live super-resolution and confocal time-lapse microscopy, we showed that mitochondria-lysosome contact sites dynamically form in the soma and axons of peripheral sensory neurons. Interestingly, Charcot-Marie-Tooth type 2B mutant Rab7 led to prolonged mitochondria-lysosome contact site tethering preferentially in the axons of peripheral sensory neurons, due to impaired Rab7 GTP hydrolysis-mediated contact site untethering. We further generated a Charcot-Marie-Tooth type 2B mutant Rab7 knock-in mouse model which exhibited prolonged axonal mitochondria-lysosome contact site tethering and defective downstream axonal mitochondrial dynamics due to impaired Rab7 GTP hydrolysis as well as fragmented mitochondria in the axon of the sciatic nerve. Importantly, mutant Rab7 mice further demonstrated preferential sensory behavioral abnormalities and neuropathy, highlighting an important role for mutant Rab7 in driving degeneration of peripheral sensory neurons. Together, this study identifies an important role for mitochondria-lysosome contact sites in the pathogenesis of peripheral neuropathy.