RESUMEN
Charcot-Marie-Tooth disease is an inherited peripheral neuropathy that is clinically and genetically heterogenous. Mutations in IGHMBP2, a ubiquitously expressed DNA/RNA helicase, have been shown to cause the infantile motor neuron disease spinal muscular atrophy with respiratory distress type 1 (SMARD1), and, more recently, juvenile-onset Charcot-Marie-Tooth disease type 2S (CMT2S). Using CRISPR-cas9 mutagenesis, we developed the first mouse models of CMT2S [p.Glu365del (E365del) and p.Tyr918Cys (Y918C)]. E365del is the first CMT2S mouse model to be discovered and Y918C is the first human CMT2S allele knock-in model. Phenotypic characterization of the homozygous models found progressive peripheral motor and sensory axonal degeneration. Neuromuscular and locomotor assays indicate that both E365del and Y918C mice have motor deficits, while neurobehavioral characterization of sensory function found that E365del mutants have mechanical allodynia. Analysis of femoral motor and sensory nerves identified axonal degeneration, which does not impact nerve conduction velocities in E365del mice, but it does so in the Y918C model. Based on these results, the E365del mutant mouse, and the human allele knock-in, Y918C, represent mouse models with the hallmark phenotypes of CMT2S, which will be critical for understanding the pathogenic mechanisms of IGHMBP2. These mice will complement existing Ighmbp2 alleles modeling SMARD1 to help understand the complex phenotypic and genotypic heterogeneity that is observed in patients with IGHMBP2 variants.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth , Proteínas de Unión al ADN , Modelos Animales de Enfermedad , Factores de Transcripción , Animales , Humanos , Ratones , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/patología , Proteínas de Unión al ADN/genética , Técnicas de Sustitución del Gen , Ratones Endogámicos C57BL , Debilidad Muscular/patología , Atrofia Muscular/patología , Fenotipo , Factores de Transcripción/genéticaRESUMEN
Clinical presentation of spinal muscular atrophy (SMA) ranges from a neonatal-onset, very severe disease to an adult-onset, milder form. SMA is caused by the mutation of the Survival Motor Neuron 1 (SMN1) gene, and prognosis inversely correlates with the number of copies of the SMN2 gene, a human-specific homolog of SMN1. Despite progress in identifying potential therapies for the treatment of SMA, many questions remain including how late after onset treatments can still be effective and what the target tissues should be. These questions can be addressed in part with preclinical animal models; however, modeling the array of SMA severities in the mouse, which lacks SMN2, has proven challenging. We created a new mouse model for the intermediate forms of SMA presenting with a delay in neuromuscular junction maturation and a decrease in the number of functional motor units, all relevant to the clinical presentation of the disease. Using this new model, in combination with clinical electrophysiology methods, we found that administering systemically SMN-restoring antisense oligonucleotides (ASOs) at the age of onset can extend survival and rescue the neurological phenotypes. Furthermore, these effects were also achieved by administration of the ASOs late after onset, independent of the restoration of SMN in the spinal cord. Thus, by adding to the limited repertoire of existing mouse models for type II/III SMA, we demonstrate that ASO therapy can be effective even when administered after onset of the neurological symptoms, in young adult mice, and without being delivered into the central nervous system.
Asunto(s)
Atrofia Muscular Espinal/fisiopatología , Oligonucleótidos Antisentido/farmacología , Animales , Modelos Animales de Enfermedad , Ratones , FenotipoRESUMEN
ALS therapy development has been hindered by the lack of rodent animal models. The discovery of TDP-43, a transcription factor that accumulates in the cytoplasm of motor neurons (MNs) in most cases of ALS, prompted attempts to develop TDP-43-based models of the disease. The current study sought to examine, in extensive detail, the emerging disease phenotype of a transgenic mouse model that overexpresses a mutant human TDP-43 (hTDP-43) gene under mouse prion promoter control. Careful attention was given to ALS-like characteristics to determine the appropriateness of this model for testing therapies for ALS. In light of previous reports that gastrointestinal (GI) dysfunction is responsible for early death in these mice, gut immunohistochemistry (IHC) and longitudinal gut motility assays were used to identify the onset and the progression of these defects. IHC studies revealed that site-specific overexpression of the hTDP-43 transgene in colonic myenteric plexes resulted in progressive neurodegeneration in this region. This change was associated with progressively reduced GI motility, culminating in frank stasis that was primarily responsible for decreasing longevity in these mice. The disease phenotype was gender- and genetic background-dependent, with congenic C57BL/6J male mice exhibiting the most aggressive form of the disease. Spinal cord IHC revealed ubiquitin-positive inclusions, but not TDP-43 aggregates, in the cytoplasm of MNs. Neither gender exhibited compelling ALS-like neuromuscular deficits, irrespective of age. While this model may be useful for studying GI tract neurodegeneration, in its present state it does not display a phenotype suitable for testing ALS therapeutics.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Colon/patología , Proteínas de Unión al ADN/metabolismo , Plexo Mientérico/patología , Animales , Colon/inervación , Colon/metabolismo , Proteínas de Unión al ADN/genética , Femenino , Motilidad Gastrointestinal , Tracto Gastrointestinal/patología , Proteína Ácida Fibrilar de la Glía , Humanos , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/patología , Masculino , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Plexo Mientérico/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Factores Sexuales , Médula Espinal/metabolismo , Médula Espinal/patología , Ubiquitina/metabolismoRESUMEN
Charcot-Marie-Tooth disease (CMT) is a clinically and genetically heterogeneous condition characterized by peripheral axon degeneration with subsequent motor and sensory deficits. Several CMT gene products function in endosomal sorting and trafficking to the lysosome, suggesting that defects in this cellular pathway might present a common pathogenic mechanism for these conditions. LRSAM1 is an E3 ubiquitin ligase that is implicated in this process, and mutations in LRSAM1 have recently been shown to cause CMT. We have generated mouse mutations in Lrsam1 to create an animal model of this form of CMT (CMT2P). Mouse Lrsam1 is abundantly expressed in the motor and sensory neurons of the peripheral nervous system. Both homozygous and heterozygous mice have largely normal neuromuscular performance and only a very mild neuropathy phenotype with age. However, Lrsam1 mutant mice are more sensitive to challenge with acrylamide, a neurotoxic agent that causes axon degeneration, indicating that the axons in the mutant mice are indeed compromised. In transfected cells, LRSAM1 primarily localizes in a perinuclear compartment immediately beyond the Golgi and shows little colocalization with components of the endosome to lysosome trafficking pathway, suggesting that other cellular mechanisms also merit consideration.
Asunto(s)
Axones/metabolismo , Enfermedad de Charcot-Marie-Tooth/enzimología , Degeneración Nerviosa/patología , Sistema Nervioso Periférico/patología , Ubiquitina-Proteína Ligasas/metabolismo , Acrilamida/farmacología , Animales , Axones/efectos de los fármacos , Conducta Animal/efectos de los fármacos , Línea Celular , Enfermedad de Charcot-Marie-Tooth/complicaciones , Enfermedad de Charcot-Marie-Tooth/patología , Modelos Animales de Enfermedad , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Ratones , Ratones Mutantes , Mutagénesis/genética , Mutación/genética , Degeneración Nerviosa/complicaciones , Degeneración Nerviosa/enzimología , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/metabolismo , Unión Neuromuscular/patología , Sistema Nervioso Periférico/efectos de los fármacos , Sistema Nervioso Periférico/enzimología , Fenotipo , Transporte de Proteínas/efectos de los fármacos , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/enzimología , TransfecciónRESUMEN
Glycine is the major inhibitory neurotransmitter in the spinal cord and some brain regions. The presynaptic glycine transporter, GlyT2, is required for sustained glycinergic transmission through presynaptic reuptake and recycling of glycine. Mutations in SLC6A5, encoding GlyT2, cause hereditary hyperekplexia in humans, and similar phenotypes in knock-out mice, and variants are associated with schizophrenia. We identified a spontaneous mutation in mouse Slc6a5, caused by a MusD retrotransposon insertion. The GlyT2 protein is undetectable in homozygous mutants, indicating a null allele. Homozygous mutant mice are normal at birth, but develop handling-induced spasms at five days of age, and only survive for two weeks, but allow the study of early activity-regulated developmental processes. At the neuromuscular junction, synapse elimination and the switch from embryonic to adult acetylcholine receptor subunits are hastened, consistent with a presumed increase in motor neuron activity, and transcription of acetylcholine receptors is elevated. Heterozygous mice, which show no reduction in lifespan but nonetheless have reduced levels of GlyT2, have a normal thermal sensitivity with the hot-plate test, but differences in repetitive grooming and decreased sleep time with home-cage monitoring. Open-field and elevated plus-maze tests did not detect anxiety-like behaviors; however, the latter showed a hyperactivity phenotype. Importantly, grooming and hyperactivity are observed in mouse schizophrenia models. Thus, mutations in Slc6a5 show changes in neuromuscular junction development as homozygotes, and behavioral phenotypes as heterozygotes, indicating their usefulness for studies related to glycinergic dysfunction.
Asunto(s)
Proteínas de Transporte de Glicina en la Membrana Plasmática/genética , Mutagénesis Insercional , Unión Neuromuscular/fisiología , Desempeño Psicomotor/fisiología , Retroelementos/genética , Animales , Ansiedad , Western Blotting , Mapeo Cromosómico , Cromosomas de los Mamíferos/genética , Femenino , Estudios de Asociación Genética , Proteínas de Transporte de Glicina en la Membrana Plasmática/metabolismo , Aseo Animal , Humanos , Masculino , Aprendizaje por Laberinto , Ratones , Ratones Endogámicos DBA , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Actividad Motora , Mutación , Inhibición Neural/genética , Inhibición Neural/fisiología , Unión Neuromuscular/genética , Médula Espinal/metabolismo , Médula Espinal/fisiopatologíaRESUMEN
Congenital myasthenic syndromes (CMS) are inherited diseases affecting the neuromuscular junction (NMJ). Mutations in AGRIN (AGRN) and other genes in the AGRIN signaling pathway cause CMS, and gene targeting studies in mice confirm the importance of this pathway for NMJ formation. However, these mouse mutations are complete loss-of-function alleles that result in an embryonic failure of NMJ formation, and homozygous mice do not survive postpartum. Therefore, mouse models of AGRIN-related CMS that would allow preclinical testing or studies of postnatal disease progression are lacking. Using chemical mutagenesis in mice, we identified a point mutation in Agrn that results in a partial loss-of-function allele, creating a valid model of CMS. The mutation changes phenylalanine 1061 to serine in the SEA domain of AGRIN, a poorly characterized motif shared by other extracellular proteoglycans. NMJs in homozygous mice progressively degrade postnataly. Severity differs with genetic background, in different muscles, and in different regions within a muscle in a pattern matching mouse models of motor neuron disease. Mutant NMJs have decreased acetylcholine receptor density and an increased subsynaptic reticulum, evident by electron microscopy. Synapses eventually denervate and the muscles atrophy. Molecularly, several factors contribute to the partial loss of AGRIN's function. The mutant protein is found at NMJs, but is processed differently than wild-type, with decreased glycosylation, changes in sensitivity to the protease neurotrypsin and other proteolysis, and less efficient externalization and secretion. Therefore, the Agrn point mutation is a model for CMS caused by Agrn mutations and potentially other related neuromuscular diseases.