RESUMO
Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common inherited peripheral neuropathy caused by a 1.5 megabase tandem duplication of chromosome 17 harboring the PMP22 gene. This dose-dependent overexpression of PMP22 results in disrupted Schwann cell myelination of peripheral nerves. To get better insights into the underlying pathogenic mechanisms in CMT1A, we investigated the role of PMP22 duplication on 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 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 dose-dependently downregulated 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 signaling. 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 due to an alteration in the lipid composition, which ultimately may lead to impaired axo-glial interactions. Moreover, targeting lipid handling and metabolism could hold promise for the treatment of CMT1A patients.
RESUMO
Charcot-Marie-Tooth disease is the most common inherited disorder of the PNS. CMT1A accounts for 40-50% of all cases and is caused by a duplication of the PMP22 gene on chromosome 17, leading to dysmyelination in the PNS. Patient-derived models to study such myelination defects are lacking as the in vitro generation of human myelinating Schwann cells has proved to be particularly challenging. Here, we present an induced pluripotent stem cell-derived organoid culture, containing various cell types of the PNS, including myelinating human Schwann cells, which mimics the human PNS. Single-cell analysis confirmed the PNS-like cellular composition and provides insight into the developmental trajectory. We used this organoid model to study disease signatures of CMT1A, revealing early ultrastructural myelin alterations, including increased myelin periodic line distance and hypermyelination of small axons. Furthermore, we observed the presence of onion-bulb-like formations in a later developmental stage. These hallmarks were not present in the CMT1A-corrected isogenic line or in a CMT2A iPSC line, supporting the notion that these alterations are specific to CMT1A. Downregulation of PMP22 expression using short-hairpin RNAs or a combinatorial drug consisting of baclofen, naltrexone hydrochloride and D-sorbitol was able to ameliorate the myelin defects in CMT1A-organoids. In summary, this self-organizing organoid model can capture biologically meaningful features of the disease and capture the physiological complexity, forms an excellent model for studying demyelinating diseases and supports the therapeutic approach of reducing PMP22 expression.
Assuntos
Doença de Charcot-Marie-Tooth , Células-Tronco Pluripotentes Induzidas , Humanos , Bainha de Mielina/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Regulação para Baixo , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Organoides/metabolismo , Células de SchwannRESUMO
Axonal Charcot-Marie-Tooth neuropathies (CMT type 2) are caused by inherited mutations in various genes functioning in different pathways. The types of genes and multiplicity of mutations reflect the clinical and genetic heterogeneity in CMT2 disease, which complicates its diagnosis and has inhibited the development of therapies. Here, we used CMT2 patient-derived pluripotent stem cells (iPSCs) to identify common hallmarks of axonal degeneration shared by different CMT2 subtypes. We compared the cellular phenotypes of neurons differentiated from CMT2 patient iPSCs with those from healthy controls and a CRISPR/Cas9-corrected isogenic line. Our results demonstrated neurite network alterations along with extracellular electrophysiological abnormalities in the differentiated motor neurons. Progressive deficits in mitochondrial and lysosomal trafficking, as well as in mitochondrial morphology, were observed in all CMT2 patient lines. Differentiation of the same CMT2 iPSC lines into peripheral sensory neurons only gave rise to cellular phenotypes in subtypes with sensory involvement, supporting the notion that some gene mutations predominantly affect motor neurons. We revealed a common mitochondrial dysfunction in CMT2-derived motor neurons, supported by alterations in the expression pattern and oxidative phosphorylation, which could be recapitulated in the sciatic nerve tissue of a symptomatic mouse model. Inhibition of a dual leucine zipper kinase could partially ameliorate the mitochondrial disease phenotypes in CMT2 subtypes. Altogether, our data reveal shared cellular phenotypes across different CMT2 subtypes and suggests that targeting such common pathomechanisms could allow the development of a uniform treatment for CMT2.
Assuntos
Doença de Charcot-Marie-Tooth/metabolismo , Mitocôndrias/metabolismo , Neurônios Motores/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Genótipo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Mitocôndrias/patologia , Neurônios Motores/patologia , Mutação , LinhagemRESUMO
Peripheral nerve axons require a well-organized axonal microtubule network for efficient transport to ensure the constant crosstalk between soma and synapse. Mutations in more than 80 different genes cause Charcot-Marie-Tooth disease, which is the most common inherited disorder affecting peripheral nerves. This genetic heterogeneity has hampered the development of therapeutics for Charcot-Marie-Tooth disease. The aim of this study was to explore whether histone deacetylase 6 (HDAC6) can serve as a therapeutic target focusing on the mutant glycyl-tRNA synthetase (GlyRS/GARS)-induced peripheral neuropathy. Peripheral nerves and dorsal root ganglia from the C201R mutant Gars mouse model showed reduced acetylated α-tubulin levels. In primary dorsal root ganglion neurons, mutant GlyRS affected neurite length and disrupted normal mitochondrial transport. We demonstrated that GlyRS co-immunoprecipitated with HDAC6 and that this interaction was blocked by tubastatin A, a selective inhibitor of the deacetylating function of HDAC6. Moreover, HDAC6 inhibition restored mitochondrial axonal transport in mutant GlyRS-expressing neurons. Systemic delivery of a specific HDAC6 inhibitor increased α-tubulin acetylation in peripheral nerves and partially restored nerve conduction and motor behaviour in mutant Gars mice. Our study demonstrates that α-tubulin deacetylation and disrupted axonal transport may represent a common pathogenic mechanism underlying Charcot-Marie-Tooth disease and it broadens the therapeutic potential of selective HDAC6 inhibition to other genetic forms of axonal Charcot-Marie-Tooth disease.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/terapia , Glicina-tRNA Ligase/genética , Desacetilase 6 de Histona/metabolismo , Mutação/genética , Animais , Transporte Axonal/genética , Células Cultivadas , Doença de Charcot-Marie-Tooth/fisiopatologia , Modelos Animais de Doenças , Inibidores Enzimáticos/uso terapêutico , Gânglios Espinais/citologia , Desacetilase 6 de Histona/genética , Ácidos Hidroxâmicos/uso terapêutico , Indóis/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Condução Nervosa/genética , Junção Neuromuscular/patologia , Junção Neuromuscular/fisiopatologia , Desempenho Psicomotor/fisiologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
Charcot-Marie-Tooth (CMT) neuropathies are common disorders of the peripheral nervous system caused by demyelination or axonal degeneration, or a combination of both features. We previously assigned the locus for autosomal dominant intermediate CMT neuropathy type C (DI-CMTC) to chromosome 1p34-p35. Here we identify two heterozygous missense mutations (G41R and E196K) and one de novo deletion (153-156delVKQV) in tyrosyl-tRNA synthetase (YARS) in three unrelated families affected with DI-CMTC. Biochemical experiments and genetic complementation in yeast show partial loss of aminoacylation activity of the mutant proteins, and mutations in YARS, or in its yeast ortholog TYS1, reduce yeast growth. YARS localizes to axonal termini in differentiating primary motor neuron and neuroblastoma cultures. This specific distribution is significantly reduced in cells expressing mutant YARS proteins. YARS is the second aminoacyl-tRNA synthetase found to be involved in CMT, thereby linking protein-synthesizing complexes with neurodegeneration.
Assuntos
Axônios/enzimologia , Doença de Charcot-Marie-Tooth/enzimologia , Doença de Charcot-Marie-Tooth/genética , Genes Dominantes/genética , Mutação/genética , Tirosina-tRNA Ligase/genética , Tirosina-tRNA Ligase/metabolismo , Sequência de Aminoácidos , Animais , Axônios/metabolismo , Axônios/patologia , Bioensaio , Células COS , Linhagem Celular Tumoral , Células Cultivadas , Doença de Charcot-Marie-Tooth/metabolismo , Chlorocebus aethiops , Teste de Complementação Genética , Heterozigoto , Humanos , Camundongos , Dados de Sequência Molecular , Transporte Proteico , Proteínas Recombinantes , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Alinhamento de Sequência , Tirosina-tRNA Ligase/químicaRESUMO
Charcot-Marie-Tooth disease (CMT) is the most common inherited neuromuscular disease and is characterized by considerable clinical and genetic heterogeneity. We previously reported a Russian family with autosomal dominant axonal CMT and assigned the locus underlying the disease (CMT2F; OMIM 606595) to chromosome 7q11-q21 (ref. 2). Here we report a missense mutation in the gene encoding 27-kDa small heat-shock protein B1 (HSPB1, also called HSP27) that segregates in the family with CMT2F. Screening for mutations in HSPB1 in 301 individuals with CMT and 115 individuals with distal hereditary motor neuropathies (distal HMNs) confirmed the previously observed mutation and identified four additional missense mutations. We observed the additional HSPB1 mutations in four families with distal HMN and in one individual with CMT neuropathy. Four mutations are located in the Hsp20-alpha-crystallin domain, and one mutation is in the C-terminal part of the HSP27 protein. Neuronal cells transfected with mutated HSPB1 were less viable than cells expressing the wild-type protein. Cotransfection of neurofilament light chain (NEFL) and mutant HSPB1 resulted in altered neurofilament assembly in cells devoid of cytoplasmic intermediate filaments.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Proteínas de Choque Térmico/genética , Neuropatia Hereditária Motora e Sensorial/genética , Mutação de Sentido Incorreto , Proteínas de Neoplasias/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , DNA Complementar/genética , Feminino , Proteínas de Choque Térmico HSP27 , Humanos , Masculino , Camundongos , Chaperonas Moleculares , Dados de Sequência Molecular , Degeneração Neural/genética , Proteínas Recombinantes/genética , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
Type 1 Charcot-Marie-Tooth disease (CMT1) is the most common demyelinating peripheral neuropathy. Patients suffer from progressive muscle weakness and sensory problems. The underlying disease mechanisms of CMT1 are still unclear and no therapy is currently available, hence patients completely rely on supportive care. Balancing protein levels is a complex multistep process fundamental to maintain cells in their healthy state and a disrupted proteostasis is a hallmark of several neurodegenerative diseases. When protein misfolding occurs, protein quality control systems are activated such as chaperones, the lysosomal-autophagy system and proteasomal degradation to ensure proper degradation. However, in pathological circumstances, these mechanisms are overloaded and thereby become inefficient to clear the load of misfolded proteins. Recent evidence strongly indicates that a disbalance in proteostasis plays an important role in several forms of CMT1. In this review, we present an overview of the protein quality control systems, their role in CMT1, and potential treatment strategies to restore proteostasis.
Assuntos
Doença de Charcot-Marie-Tooth , Humanos , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , ProteostaseRESUMO
Mutations in the small heat shock protein HSPB1 (HSP27) are causative for Charcot-Marie-Tooth (CMT) neuropathy. We previously showed that a subset of these mutations displays higher chaperone activity and enhanced affinity to client proteins. We hypothesized that this excessive binding property might cause the HSPB1 mutant proteins to disturb the function of proteins essential for the maintenance or survival of peripheral neurons. In the present work, we explored this hypothesis further and compared the protein complexes formed by wild-type and mutant HSPB1. Tubulin came out as the most striking differential interacting protein, with hyperactive mutants binding more strongly to both tubulin and microtubules. This anomalous binding leads to a stabilization of the microtubule network in a microtubule-associated protein-like manner as reflected by resistance to cold depolymerization, faster network recovery after nocodazole treatment, and decreased rescue and catastrophe rates of individual microtubules. In a transgenic mouse model for mutant HSPB1 that recapitulates all features of CMT, we could confirm the enhanced interaction of mutant HSPB1 with tubulin. Increased stability of the microtubule network was also clear in neurons isolated from these mice. Since neuronal cells are particularly vulnerable to disturbances in microtubule dynamics, this mechanism might explain the neuron-specific CMT phenotype caused by HSPB1 mutations.
Assuntos
Proteínas de Choque Térmico HSP27/genética , Microtúbulos/metabolismo , Mutação/genética , Neurônios/metabolismo , Análise de Variância , Animais , Células Cultivadas , Chlorocebus aethiops , Gânglios Espinais/citologia , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Choque Térmico , Humanos , Gelo/efeitos adversos , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/genética , Chaperonas Moleculares , Neurônios/efeitos dos fármacos , Nocodazol/farmacologia , Ligação Proteica , Ressonância de Plasmônio de Superfície , Espectrometria de Massas em Tandem/métodos , Fatores de Tempo , Transfecção/métodos , Tubulina (Proteína)/genética , Tubulina (Proteína)/farmacologia , Moduladores de Tubulina/farmacologiaRESUMO
Missense mutations (K141N and K141E) in the alpha-crystallin domain of the small heat shock protein HSPB8 (HSP22) cause distal hereditary motor neuropathy (distal HMN) or Charcot-Marie-Tooth neuropathy type 2L (CMT2L). The mechanism through which mutant HSPB8 leads to a specific motor neuron disease phenotype is currently unknown. To address this question, we compared the effect of mutant HSPB8 in primary neuronal and glial cell cultures. In motor neurons, expression of both HSPB8 K141N and K141E mutations clearly resulted in neurite degeneration, as manifested by a reduction in number of neurites per cell, as well as in a reduction in average length of the neurites. Furthermore, expression of the K141E (and to a lesser extent, K141N) mutation also induced spheroids in the neurites. We did not detect any signs of apoptosis in motor neurons, showing that mutant HSPB8 resulted in neurite degeneration without inducing neuronal death. While overt in motor neurons, these phenotypes were only very mildly present in sensory neurons and completely absent in cortical neurons. Also glial cells did not show an altered phenotype upon expression of mutant HSPB8. These findings show that despite the ubiquitous presence of HSPB8, only motor neurons appear to be affected by the K141N and K141E mutations which explain the predominant motor neuron phenotype in distal HMN and CMT2L.
Assuntos
Proteínas de Choque Térmico HSP20/metabolismo , Neurônios Motores/metabolismo , Proteínas Musculares/metabolismo , Mutação , Neuritos/metabolismo , Substituição de Aminoácidos , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Apoptose , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Dano ao DNA , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Choque Térmico HSP20/genética , Proteínas de Choque Térmico , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Chaperonas Moleculares , Neurônios Motores/patologia , Proteínas Musculares/genética , Neuritos/patologia , Neuroglia/metabolismo , Ratos , Ratos Wistar , TransfecçãoRESUMO
Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy, with currently no effective treatment or cure. CMT1A is caused by a duplication of the PMP22 gene, which leads to Schwann cell differentiation defects and dysmyelination of the peripheral nerves. The epigenetic regulator histone deacetylase 3 (HDAC3) has been shown to negatively regulate myelination as well as its associated signaling pathways, PI3K-AKT and MAPK-ERK. We showed that these signaling pathways are indeed downregulated in the C3-PMP22 mouse model, similar to what has been shown in the CMT1A rat model. We confirmed that early postnatal defects are present in the peripheral nerves of the C3-PMP22 mouse model, which led to a progressive reduction in axon caliber size and myelination. The aim of this study was to investigate whether pharmacological HDAC3 inhibition could be a valuable therapeutic approach for this CMT1A mouse model. We demonstrated that early treatment of CMT1A mice with the selective HDAC3 inhibitor RGFP966 increased myelination and myelin g-ratios, which was associated with improved electrophysiological recordings. However, a high dose of RGFP966 caused a decline in rotarod performance and a decline in overall grip strength. Additionally, macrophage presence in peripheral nerves was increased in RGFP966 treated CMT1A mice. We conclude that HDAC3 does not only play a role in regulating myelination but is also important in the neuroimmune modulation. Overall, our results indicate that correct dosing of HDAC3 inhibitors is of crucial importance if translated to a clinical setting for demyelinating forms of CMT or other neurological disorders.
Assuntos
Doença de Charcot-Marie-Tooth , Doenças Desmielinizantes , Animais , Doença de Charcot-Marie-Tooth/genética , Doenças Desmielinizantes/metabolismo , Modelos Animais de Doenças , Histona Desacetilases/metabolismo , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Ratos , Células de Schwann/metabolismoRESUMO
Histone deacetylase 6 (HDAC6) is a promising therapeutic target for the treatment of neurodegenerative disorders. SW-100 (1a), a phenylhydroxamate-based HDAC6 inhibitor (HDAC6i) bearing a tetrahydroquinoline (THQ) capping group, is a highly potent and selective HDAC6i that was shown to be effective in mouse models of Fragile X syndrome and Charcot-Marie-Tooth disease type 2A (CMT2A). In this study, we report the discovery of a new THQ-capped HDAC6i, termed SW-101 (1s), that possesses excellent HDAC6 potency and selectivity, together with markedly improved metabolic stability and druglike properties compared to SW-100 (1a). X-ray crystallography data reveal the molecular basis of HDAC6 inhibition by SW-101 (1s). Importantly, we demonstrate that SW-101 (1s) treatment elevates the impaired level of acetylated α-tubulin in the distal sciatic nerve, counteracts progressive motor dysfunction, and ameliorates neuropathic symptoms in a CMT2A mouse model bearing mutant MFN2. Taken together, these results bode well for the further development of SW-101 (1s) as a disease-modifying HDAC6i.
Assuntos
Doença de Charcot-Marie-Tooth/tratamento farmacológico , Desacetilase 6 de Histona/antagonistas & inibidores , Inibidores de Histona Desacetilases/uso terapêutico , Quinolinas/química , Acetilação , Animais , Benzamidas/química , Benzamidas/metabolismo , Sítios de Ligação , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Cristalografia por Raios X , Modelos Animais de Doenças , Meia-Vida , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Fenótipo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Quinolinas/metabolismo , Quinolinas/uso terapêutico , Relação Estrutura-Atividade , Tubulina (Proteína)/metabolismoRESUMO
Histone deacetylase 6 (HDAC6) plays a central role in various processes that are key for neuronal survival. In this review, we summarize the current evidence related to disease pathways in the axonal form of Charcot-Marie-Tooth disease (CMT) and highlight the role of HDAC6 in these pathways. We hypothesize that HDAC6 might in fact actively contribute to the pathogenesis of certain forms of axonal CMT. HDAC6 plays a deacetylase activity-dependent, negative role in axonal transport and axonal regeneration, which are both processes implicated in axonal CMT. On the other hand, HDAC6 coordinates a protective response during elimination of toxic misfolded proteins, but this is mostly mediated independent of its deacetylase activity. The current mechanistic insights on these functions of HDAC6 in axonal CMT, along with the selective druggability against its deacetylase activity, make the targeting of HDAC6 particularly attractive. We elaborate on the preclinical studies that demonstrated beneficial effects of HDAC6 inhibitors in axonal CMT models and outline possible modes of action. Overall, this overview ultimately provides a rationale for the use of small-molecule HDAC6 inhibitors as a therapeutic strategy for this devastating disease.
Assuntos
Doença de Charcot-Marie-Tooth , Desacetilase 6 de Histona/antagonistas & inibidores , Inibidores de Histona Desacetilases/administração & dosagem , Animais , Axônios/efeitos dos fármacos , Axônios/patologia , Doença de Charcot-Marie-Tooth/tratamento farmacológico , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/patologia , Modelos Animais de Doenças , Humanos , Pesquisa Translacional BiomédicaRESUMO
Charcot-Marie-Tooth disease (CMT) is a group of inherited neurological disorders of the peripheral nervous system. CMT is subdivided into two main types: a demyelinating form, known as CMT1, and an axonal form, known as CMT2. Nearly 30 genes have been identified as a cause of CMT2. One of these is the 'dehydrogenase E1 and transketolase domain containing 1' (DHTKD1) gene. We previously demonstrated that a nonsense mutation [c.1455 T > G (p.Y485*)] in exon 8 of DHTKD1 is one of the disease-causing mutations in CMT2Q (MIM 615025). The aim of the current study was to investigate whether human disease-causing mutations in the Dhtkd1 gene cause CMT2Q phenotypes in a mouse model in order to investigate the physiological function and pathogenic mechanisms associated with mutations in the Dhtkd1 gene in vivo. Therefore, we generated a knock-in mouse model with the Dhtkd1Y486* point mutation. We observed that the Dhtkd1 expression level in sciatic nerve of knock-in mice was significantly lower than in wild-type mice. Moreover, a histopathological phenotype was observed, reminiscent of a peripheral neuropathy, including reduced large axon diameter and abnormal myelination in peripheral nerves. The knock-in mice also displayed clear sensory defects, while no abnormalities in the motor performance were observed. In addition, accumulation of mitochondria and an elevated energy metabolic state was observed in the knock-in mice. Taken together, our study indicates that the Dhtkd1Y486* knock-in mice partially recapitulate the clinical phenotypes of CMT2Q patients and we hypothesize that there might be a compensatory effect from the elevated metabolic state in the knock-in mice that enables them to maintain their normal locomotor function.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Modelos Animais de Doenças , Complexo Cetoglutarato Desidrogenase/genética , Camundongos , Mitocôndrias/patologia , Nervo Isquiático/metabolismo , Distúrbios Somatossensoriais/genética , Animais , Axônios/patologia , Axônios/ultraestrutura , Doença de Charcot-Marie-Tooth/patologia , Doença de Charcot-Marie-Tooth/fisiopatologia , Códon sem Sentido , Metabolismo Energético , Técnicas de Introdução de Genes , Complexo Cetoglutarato Desidrogenase/metabolismo , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/patologia , Mitocôndrias Musculares/ultraestrutura , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/ultraestrutura , Bainha de Mielina/patologia , Bainha de Mielina/ultraestrutura , Condução Nervosa , Degradação do RNAm Mediada por Códon sem Sentido/genética , Nervos Periféricos/patologia , Nervos Periféricos/ultraestrutura , Fenótipo , Mutação Puntual , Nervo Isquiático/patologia , Nervo Isquiático/ultraestrutura , Distúrbios Somatossensoriais/patologia , Distúrbios Somatossensoriais/fisiopatologiaRESUMO
Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy, with an estimated prevalence of 1 in 2500. The degeneration of motor and sensory nerve axons leads to motor and sensory symptoms that progress over time and have an important impact on the daily life of these patients. Currently, there is no curative treatment available. Recently, we identified histone deacetylase 6 (HDAC6), which deacetylates α-tubulin, as a potential therapeutic target in axonal CMT (CMT2). Pharmacological inhibition of the deacetylating function of HDAC6 reversed the motor and sensory deficits in a mouse model for mutant "small heat shock protein B1" (HSPB1)-induced CMT2 at the behavioral and electrophysiological level. In order to translate this potential therapeutic strategy into a clinical application, small drug-like molecules that are potent and selective HDAC6 inhibitors are essential. To screen for these, we developed a method that consisted of 3 distinct phases and that was based on the pathological findings in the mutant HSPB1-induced CMT2 mouse model. Three different inhibitors (ACY-738, ACY-775, and ACY-1215) were tested and demonstrated to be both potent and selective HDAC6 inhibitors. Moreover, these inhibitors increased the innervation of the neuromuscular junctions in the gastrocnemius muscle and improved the motor and sensory nerve conduction, confirming that HDAC6 inhibition is a potential therapeutic strategy in CMT2. Furthermore, ACY-1215 is an interesting lead molecule as it is currently tested in clinical trials for cancer. Taken together, these results may speed up the translation of pharmacological inhibition of HDAC6 into a therapy against CMT2.
Assuntos
Doença de Charcot-Marie-Tooth/tratamento farmacológico , Doença de Charcot-Marie-Tooth/enzimologia , Avaliação Pré-Clínica de Medicamentos , Desacetilase 6 de Histona/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Pirimidinas/farmacologia , Animais , Transporte Axonal/efeitos dos fármacos , Gânglios Espinais/efeitos dos fármacos , Inibidores de Histona Desacetilases/uso terapêutico , Ácidos Hidroxâmicos/uso terapêutico , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/inervação , Junção Neuromuscular/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Pirimidinas/uso terapêutico , Células Tumorais CultivadasRESUMO
Charcot-Marie-Tooth (CMT) disease is a disorder of the peripheral nervous system where progressive degeneration of motor and sensory nerves leads to motor problems and sensory loss and for which no pharmacological treatment is available. Recently, it has been shown in a model for the axonal form of CMT that histone deacetylase 6 (HDAC6) can serve as a target for the development of a pharmacological therapy. Therefore, we aimed at developing new selective and activity-specific HDAC6 inhibitors with improved biochemical properties. By utilizing a bicyclic cap as the structural scaffold from which to build upon, we developed several analogues that showed improved potency compared to tubastatin A while maintaining excellent selectivity compared to HDAC1. Further screening in N2a cells examining both the acetylation of α-tubulin and histones narrowed down the library of compounds to three potent and selective HDAC6 inhibitors. In mutant HSPB1-expressing DRG neurons, serving as an in vitro model for CMT2, these inhibitors were able to restore the mitochondrial axonal transport deficits. Combining structure-based development of HDAC6 inhibitors, screening in N2a cells and in a neuronal model for CMT2F, and preliminary ADMET and pharmacokinetic profiles, resulted in the selection of compound 23d that possesses improved biochemical, functional, and druglike properties compared to tubastatin A.
Assuntos
Doença de Charcot-Marie-Tooth/tratamento farmacológico , Doença de Charcot-Marie-Tooth/enzimologia , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/metabolismo , Acetilação/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Células Cultivadas , Modelos Animais de Doenças , Gânglios Espinais/citologia , Desacetilase 6 de Histona , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Humanos , Interleucina-2/genética , Camundongos , Camundongos Transgênicos , Mutação/genética , Neuroblastoma/patologia , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
Charcot-Marie-Tooth disease is the most common inherited disorder of the peripheral nervous system. The disease is characterized by a progressive muscle weakness and atrophy, sensory loss, foot (and hand) deformities and steppage gait. While many of the genes associated with axonal CMT have been identified, to date it is unknown which mechanism(s) causes the disease. However, genetic findings indicate that the underlying mechanisms mainly converge to the axonal cytoskeleton. In this review, we will summarize the evidence for this pathogenic convergence. Furthermore, recent work with new transgenic mouse models has led to the identification of histone deacetylase 6 as a potential therapeutic target for inherited peripheral neuropathies. This enzyme deacetylates microtubules and plays a crucial role in the regulation of axonal transport. These findings offer new perspectives for a potential therapy to treat axonal Charcot-Marie-Tooth disease and other neurodegenerative disorders characterized by axonal transport defects.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/terapia , Predisposição Genética para Doença/genética , Histona Desacetilases/genética , Animais , Transporte Axonal/genética , Axônios/metabolismo , Axônios/patologia , Doença de Charcot-Marie-Tooth/patologia , Desacetilase 6 de Histona , Humanos , Camundongos , Modelos Genéticos , MutaçãoRESUMO
Charcot-Marie-Tooth disease (CMT) is the most common inherited disorder of the peripheral nervous system. Mutations in the 27-kDa small heat-shock protein gene (HSPB1) cause axonal CMT or distal hereditary motor neuropathy (distal HMN). We developed and characterized transgenic mice expressing two different HSPB1 mutations (S135F and P182L) in neurons only. These mice showed all features of CMT or distal HMN dependent on the mutation. Expression of mutant HSPB1 decreased acetylated α-tubulin abundance and induced severe axonal transport deficits. An increase of α-tubulin acetylation induced by pharmacological inhibition of histone deacetylase 6 (HDAC6) corrected the axonal transport defects caused by HSPB1 mutations and rescued the CMT phenotype of symptomatic mutant HSPB1 mice. Our findings demonstrate the pathogenic role of α-tubulin deacetylation in mutant HSPB1-induced neuropathies and offer perspectives for using HDAC6 inhibitors as a therapeutic strategy for hereditary axonopathies.