RESUMO
Charcot-Marie-Tooth disease type 2D (CMT2D) is a peripheral nerve disorder caused by dominant, toxic, gain-of-function mutations in the widely expressed, housekeeping gene, GARS The mechanisms underlying selective nerve pathology in CMT2D remain unresolved, as does the cause of the mild-to-moderate sensory involvement that distinguishes CMT2D from the allelic disorder distal spinal muscular atrophy type V. To elucidate the mechanism responsible for the underlying afferent nerve pathology, we examined the sensory nervous system of CMT2D mice. We show that the equilibrium between functional subtypes of sensory neuron in dorsal root ganglia is distorted by Gars mutations, leading to sensory defects in peripheral tissues and correlating with overall disease severity. CMT2D mice display changes in sensory behavior concordant with the afferent imbalance, which is present at birth and nonprogressive, indicating that sensory neuron identity is prenatally perturbed and that a critical developmental insult is key to the afferent pathology. Through in vitro experiments, mutant, but not wild-type, GlyRS was shown to aberrantly interact with the Trk receptors and cause misactivation of Trk signaling, which is essential for sensory neuron differentiation and development. Together, this work suggests that both neurodevelopmental and neurodegenerative mechanisms contribute to CMT2D pathogenesis, and thus has profound implications for the timing of future therapeutic treatments.
Assuntos
Doença de Charcot-Marie-Tooth/patologia , Glicina-tRNA Ligase/fisiologia , Mutação , Receptor trkA/metabolismo , Células Receptoras Sensoriais/patologia , Animais , Células Cultivadas , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Receptor trkA/genética , Células Receptoras Sensoriais/metabolismoRESUMO
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 collectively the most common hereditary neurological condition and a major health burden for society. Dominant mutations in the gene GARS, encoding the ubiquitous enzyme, glycyl-tRNA synthetase (GlyRS), cause peripheral nerve degeneration and lead to CMT disease type 2D. This genetic disorder exemplifies a recurring motif in neurodegeneration, whereby mutations in essential, widely expressed genes have selective deleterious consequences for the nervous system. Here, using novel Drosophila models, we show a potential solution to this phenomenon. Ubiquitous expression of mutant GlyRS leads to motor deficits, progressive neuromuscular junction (NMJ) denervation and pre-synaptic build-up of mutant GlyRS. Intriguingly, neuronal toxicity is, at least in part, non-cell autonomous, as expression of mutant GlyRS in mesoderm or muscle alone results in similar pathology. This mutant GlyRS toxic gain-of-function, which is WHEP domain-dependent, coincides with abnormal NMJ assembly, leading to synaptic degeneration, and, ultimately, reduced viability. Our findings suggest that mutant GlyRS gains access to ectopic sub-compartments of the motor neuron, providing a possible explanation for the selective neuropathology caused by mutations in a widely expressed gene.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Glicina-tRNA Ligase/genética , Degeneração Neural/genética , Junção Neuromuscular/genética , Animais , Doença de Charcot-Marie-Tooth/patologia , Modelos Animais de Doenças , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Glicina-tRNA Ligase/biossíntese , Humanos , Corpos Pedunculados/patologia , Mutação , Degeneração Neural/patologia , Junção Neuromuscular/crescimento & desenvolvimento , Junção Neuromuscular/patologia , Bulbo Olfatório/patologia , Nervos Periféricos/patologiaRESUMO
Dominant mutations in GARS, encoding the essential enzyme glycyl-tRNA synthetase (GlyRS), result in a form of Charcot-Marie-Tooth disease, type 2D (CMT2D), predominantly characterized by lower motor nerve degeneration. GlyRS charges the amino acid glycine with its cognate tRNA and is therefore essential for protein translation. However, the underlying mechanisms linking toxic gain-of-function GARS mutations to lower motor neuron degeneration remain unidentified. The neuromuscular junction (NMJ) appears to be an early target for pathology in a number of peripheral nerve diseases and becomes denervated at later stages in two mouse models of CMT2D. We therefore performed a detailed longitudinal examination of NMJs in the distal lumbrical muscles and the proximal transversus abdominis (TVA) muscles of wild-type and Gars mutant mice. We determined that mutant lumbrical NMJs display a persistent defect in maturation that precedes a progressive, age-dependent degeneration. Conversely, the TVA remains relatively unaffected, with only a subtle, short-lived impairment in pre- and post-synaptic development and no reduction in lower motor neuron connectivity to muscle. Together, these observations suggest that mutant Gars is associated with compromised development of the NMJ prior to synaptic degeneration and highlight the neuromuscular synapse as an important site of early, selective pathology in CMT2D mice.
Assuntos
Doença de Charcot-Marie-Tooth/patologia , Neurônios Motores/patologia , Junção Neuromuscular/patologia , Músculos Abdominais/inervação , Animais , Doença de Charcot-Marie-Tooth/genética , Glicina-tRNA Ligase/genética , Membro Posterior/inervação , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação de Sentido IncorretoRESUMO
Charcot-Marie-Tooth disease type 2D (CMT2D) is a dominantly inherited peripheral neuropathy caused by missense mutations in the glycyl-tRNA synthetase gene (GARS). In addition to GARS, mutations in three other tRNA synthetase genes cause similar neuropathies, although the underlying mechanisms are not fully understood. To address this, we generated transgenic mice that ubiquitously over-express wild-type GARS and crossed them to two dominant mouse models of CMT2D to distinguish loss-of-function and gain-of-function mechanisms. Over-expression of wild-type GARS does not improve the neuropathy phenotype in heterozygous Gars mutant mice, as determined by histological, functional, and behavioral tests. Transgenic GARS is able to rescue a pathological point mutation as a homozygote or in complementation tests with a Gars null allele, demonstrating the functionality of the transgene and revealing a recessive loss-of-function component of the point mutation. Missense mutations as transgene-rescued homozygotes or compound heterozygotes have a more severe neuropathy than heterozygotes, indicating that increased dosage of the disease-causing alleles results in a more severe neurological phenotype, even in the presence of a wild-type transgene. We conclude that, although missense mutations of Gars may cause some loss of function, the dominant neuropathy phenotype observed in mice is caused by a dose-dependent gain of function that is not mitigated by over-expression of functional wild-type protein.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Glicina-tRNA Ligase/genética , Sistema Nervoso Periférico/metabolismo , Animais , Axônios/metabolismo , Modelos Animais de Doenças , Glicina-tRNA Ligase/metabolismo , Heterozigoto , Homozigoto , Humanos , Camundongos , Camundongos Transgênicos , Mutação de Sentido Incorreto/genética , Neurônios/metabolismo , Neurônios/patologia , Sistema Nervoso Periférico/patologia , Células de Schwann/metabolismo , Nervo Isquiático/metabolismoRESUMO
Charcot-Marie-Tooth (CMT) disease comprises a genetically and clinically heterogeneous group of peripheral nerve disorders characterized by impaired distal motor and sensory function. Mutations in three genes encoding aminoacyl-tRNA synthetases (ARSs) have been implicated in CMT disease primarily associated with an axonal pathology. ARSs are ubiquitously expressed, essential enzymes responsible for charging tRNA molecules with their cognate amino acids. To further explore the role of ARSs in CMT disease, we performed a large-scale mutation screen of the 37 human ARS genes in a cohort of 355 patients with a phenotype consistent with CMT. Here we describe three variants (p.Leu133His, p.Tyr173SerfsX7, and p.Ile302Met) in the lysyl-tRNA synthetase (KARS) gene in two patients from this cohort. Functional analyses revealed that two of these mutations (p.Leu133His and p.Tyr173SerfsX7) severely affect enzyme activity. Interestingly, both functional variants were found in a single patient with CMT disease and additional neurological and non-neurological sequelae. Based on these data, KARS becomes the fourth ARS gene associated with CMT disease, indicating that this family of enzymes is specifically critical for axon function.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Heterozigoto , Lisina-tRNA Ligase/genética , Modelos Moleculares , Doenças do Sistema Nervoso Periférico/genética , Conformação Proteica , Sequência de Aminoácidos , Sequência de Bases , Estudos de Coortes , Análise Mutacional de DNA , Humanos , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genéticaRESUMO
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
Charcot-Marie-Tooth (CMT) disease comprises a heterogeneous group of peripheral neuropathies characterized by muscle weakness and wasting, and impaired sensation in the extremities. Four genes encoding an aminoacyl-tRNA synthetase (ARS) have been implicated in CMT disease. ARSs are ubiquitously expressed, essential enzymes that ligate amino acids to cognate tRNA molecules. Recently, a p.Arg329His variant in the alanyl-tRNA synthetase (AARS) gene was found to segregate with dominant axonal CMT type 2N (CMT2N) in two French families; however, the functional consequence of this mutation has not been determined. To investigate the role of AARS in CMT, we performed a mutation screen of the AARS gene in patients with peripheral neuropathy. Our results showed that p.Arg329His AARS also segregated with CMT disease in a large Australian family. Aminoacylation and yeast viability assays showed that p.Arg329His AARS severely reduces enzyme activity. Genotyping analysis indicated that this mutation arose on three distinct haplotypes, and the results of bisulfite sequencing suggested that methylation-mediated deamination of a CpG dinucleotide gives rise to the recurrent p.Arg329His AARS mutation. Together, our data suggest that impaired tRNA charging plays a role in the molecular pathology of CMT2N, and that patients with CMT should be directly tested for the p.Arg329His AARS mutation.
Assuntos
Alanina-tRNA Ligase/genética , Doença de Charcot-Marie-Tooth/genética , Mutação , Aminoacilação de RNA de Transferência/genética , Alanina-tRNA Ligase/metabolismo , Aminoacilação , Arginina/genética , Arginina/metabolismo , Austrália , Axônios , Estudos de Casos e Controles , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Ilhas de CpG , Feminino , França , Genes Dominantes , Ligação Genética , Haplótipos , Histidina/genética , Histidina/metabolismo , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , LevedurasRESUMO
Charcot-Marie-Tooth disease type 2D, a hereditary axonal neuropathy, is caused by mutations in glycyl-tRNA synthetase (GARS). The mutations are distributed throughout the protein in multiple functional domains. In biochemical and cell culture experiments, some mutant forms of GARS have been indistinguishable from wild-type protein, suggesting that these in vitro tests might not adequately assess the aberrant activity responsible for axonal degeneration. Recently, mouse and fly models have offered new insights into the disease mechanism. There are still gaps in our understanding of how mutations in a ubiquitously expressed component of the translation machinery result in axonal neuropathy. Here, we review recent reports, weigh the evidence for and against possible mechanisms and suggest areas of focus for future work.
Assuntos
Glicina-tRNA Ligase/genética , Neuropatia Hereditária Motora e Sensorial/genética , Neuropatia Hereditária Motora e Sensorial/fisiopatologia , Mutação/genética , Animais , Modelos Animais de Doenças , Glicina-tRNA Ligase/deficiência , Neuropatia Hereditária Motora e Sensorial/epidemiologia , Humanos , Doenças Mitocondriais/genéticaRESUMO
Mutations in the enzyme glycyl-tRNA synthetase (GARS) cause motor and sensory axon loss in the peripheral nervous system in humans, described clinically as Charcot-Marie-Tooth type 2D or distal spinal muscular atrophy type V. Here, we characterise a new mouse mutant, Gars(C201R), with a point mutation that leads to a non-conservative substitution within GARS. Heterozygous mice with a C3H genetic background have loss of grip strength, decreased motor flexibility and disruption of fine motor control; this relatively mild phenotype is more severe on a C57BL/6 background. Homozygous mutants have a highly deleterious set of features, including movement difficulties and death before weaning. Heterozygous animals have a reduction in axon diameter in peripheral nerves, slowing of nerve conduction and an alteration in the recovery cycle of myelinated axons, as well as innervation defects. An assessment of GARS levels showed increased protein in 15-day-old mice compared with controls; however, this increase was not observed in 3-month-old animals, indicating that GARS function may be more crucial in younger animals. We found that enzyme activity was not reduced detectably in heterozygotes at any age, but was diminished greatly in homozygous mice compared with controls; thus, homozygous animals may suffer from a partial loss of function. The Gars(C201R) mutation described here is a contribution to our understanding of the mechanism by which mutations in tRNA synthetases, which are fundamentally important, ubiquitously expressed enzymes, cause axonopathy in specific sets of neurons.