RESUMEN
Huntington disease (HD) is a fatal neurodegenerative disease caused by a trinucleotide repeat expansion in exon 1 of the huntingtin gene (HTT) resulting in toxic gain-of-function and cell death. Despite its monogenic cause, the pathogenesis of HD is highly complex and increasing evidence indicates that, in addition to the full-length (FL) mutant HTT protein, the expanded exon 1 HTT (HTTexon1) protein that is translated from the HTT1a transcript generated by aberrant splicing is prone to aggregate and may contribute to HD pathology. This finding suggests that reducing the expression of HTT1a may achieve a greater therapeutic benefit than targeting only FL mutant HTT. Conversely, strategies that exclusively target FL HTT may not fully prevent the pathogenesis of HD. We have developed an engineered microRNA targeting the HTT exon 1 sequence (miHTT), delivered via adeno-associated virus serotype 5 (AAV5). The target sequence of miHTT is present in both FL HTT and HTT1a transcripts. Preclinical studies with AAV5-miHTT have demonstrated efficacy in several rodent and large animal models by reducing FL HTT mRNA and protein and rescuing HD-like phenotypes, and have been the rationale for phase I/II clinical studies now ongoing in the US and Europe. In the present study, we evaluated the ability of AAV5-miHTT to reduce the levels of aberrantly spliced HTT1a mRNA and the HTTexon1 protein in the brain of two mouse models of HD (heterozygous zQ175 knock-in mice and humanized Hu128/21 mice). Polyadenylated HTT1a mRNA and HTTexon1 protein were detected in the striatum and cortex of heterozygous zQ175 knock-in mice, but not in wild-type, littermate control mice. Intrastriatal administration of AAV5-miHTT resulted in dose-dependent expression of mature miHTT microRNA in cortical brain regions, accompanied by significant lowering of both FL HTT and HTT1a mRNA expression at two months post-injection. Mutant HTT and HTTexon1 protein levels were also significantly reduced in the striatum and cortex of heterozygous zQ175 knock-in at 2 months after AAV5-miHTT treatment and in humanized Hu128/21 mice 7 months post-treatment. The effects were confirmed in primary Hu128/21 neuronal cultures. These results demonstrate that AAV5-miHTT gene therapy is an effective approach to lower both FL HTT and the pathogenic HTTexon1 levels, which could potentially have an additive therapeutic benefit compared to other HTT-targeting modalities.
RESUMEN
Huntington's disease pathogenesis involves a genetic gain-of-function toxicity mechanism triggered by the expanded HTT CAG repeat. Current therapeutic efforts aim to suppress expression of total or mutant huntingtin, though the relationship of huntingtin's normal activities to the gain-of-function mechanism and what the effects of huntingtin-lowering might be are unclear. Here, we have re-investigated a rare family segregating two presumed HTT loss-of-function (LoF) variants associated with the developmental disorder, Lopes-Maciel-Rodan syndrome (LOMARS), using whole-genome sequencing of DNA from cell lines, in conjunction with analysis of mRNA and protein expression. Our findings correct the muddled annotation of these HTT variants, reaffirm they are the genetic cause of the LOMARS phenotype and demonstrate that each variant is a huntingtin hypomorphic mutation. The NM_002111.8: c.4469+1G>A splice donor variant results in aberrant (exon 34) splicing and severely reduced mRNA, whereas, surprisingly, the NM_002111.8: c.8157T>A NP_002102.4: Phe2719Leu missense variant results in abnormally rapid turnover of the Leu2719 huntingtin protein. Thus, although rare and subject to an as yet unknown LoF intolerance at the population level, bona fide HTT LoF variants can be transmitted by normal individuals leading to severe consequences in compound heterozygotes due to huntingtin deficiency.
Asunto(s)
Regulación de la Expresión Génica , Proteína Huntingtina/genética , Mutación , Trastornos del Neurodesarrollo/genética , Secuencia de Aminoácidos , Línea Celular , Niño , Preescolar , Femenino , Humanos , Proteína Huntingtina/química , Proteína Huntingtina/metabolismo , Mutación con Pérdida de Función , Masculino , Mutación Missense , Trastornos del Neurodesarrollo/metabolismo , Linaje , Fenotipo , Empalme del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Recent genome-wide association studies of age-at-onset in Huntington's disease (HD) point to distinct modes of potential disease modification: altering the rate of somatic expansion of the HTT CAG repeat or altering the resulting CAG threshold length-triggered toxicity process. Here, we evaluated the mouse orthologs of two HD age-at-onset modifier genes, FAN1 and RRM2B, for an influence on somatic instability of the expanded CAG repeat in Htt CAG knock-in mice. Fan1 knock-out increased somatic expansion of Htt CAG repeats, in the juvenile- and the adult-onset HD ranges, whereas knock-out of Rrm2b did not greatly alter somatic Htt CAG repeat instability. Simultaneous knock-out of Mlh1, the ortholog of a third HD age-at-onset modifier gene (MLH1), which suppresses somatic expansion of the Htt knock-in CAG repeat, blocked the Fan1 knock-out-induced acceleration of somatic CAG expansion. This genetic interaction indicates that functional MLH1 is required for the CAG repeat destabilizing effect of FAN1 loss. Thus, in HD, it is uncertain whether the RRM2B modifier effect on timing of onset may be due to a DNA instability mechanism. In contrast, the FAN1 modifier effects reveal that functional FAN1 acts to suppress somatic CAG repeat expansion, likely in genetic interaction with other DNA instability modifiers whose combined effects can hasten or delay onset and other CAG repeat length-driven phenotypes.
Asunto(s)
Proteínas de Ciclo Celular/genética , Endodesoxirribonucleasas/genética , Exodesoxirribonucleasas/genética , Proteína Huntingtina/genética , Enfermedad de Huntington/genética , Enzimas Multifuncionales/genética , Homólogo 1 de la Proteína MutL/genética , Ribonucleótido Reductasas/genética , Edad de Inicio , Animales , Modelos Animales de Enfermedad , Genes Modificadores/genética , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Enfermedad de Huntington/patología , Ratones , Ratones Noqueados , Fenotipo , Expansión de Repetición de Trinucleótido/genéticaRESUMEN
Regulator of G protein signaling z1 (RGSz1), a member of the RGS family of proteins, is present in several networks expressing mu opioid receptors (MOPRs). By using genetic mouse models for global or brain region-targeted manipulations of RGSz1 expression, we demonstrated that the suppression of RGSz1 function increases the analgesic efficacy of MOPR agonists in male and female mice and delays the development of morphine tolerance while decreasing the sensitivity to rewarding and locomotor activating effects. Using biochemical assays and next-generation RNA sequencing, we identified a key role of RGSz1 in the periaqueductal gray (PAG) in morphine tolerance. Chronic morphine administration promotes RGSz1 activity in the PAG, which in turn modulates transcription mediated by the Wnt/ß-catenin signaling pathway to promote analgesic tolerance to morphine. Conversely, the suppression of RGSz1 function stabilizes Axin2-Gαz complexes near the membrane and promotes ß-catenin activation, thereby delaying the development of analgesic tolerance. These data show that the regulation of RGS complexes, particularly those involving RGSz1-Gαz, represents a promising target for optimizing the analgesic actions of opioids without increasing the risk of dependence or addiction.
Asunto(s)
Analgésicos Opioides/farmacología , Proteínas RGS/antagonistas & inhibidores , Vía de Señalización Wnt , Analgesia , Animales , Condicionamiento Psicológico , Femenino , Proteínas de Unión al GTP/metabolismo , Inflamación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Morfina/farmacología , Neuronas/metabolismo , Sustancia Gris Periacueductal/metabolismo , Proteínas RGS/metabolismo , Análisis de Secuencia de ARN , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMEN
Huntington's disease is a dominantly inherited neurodegenerative disease caused by the expansion of a CAG repeat in the HTT gene. In addition to the length of the CAG expansion, factors such as genetic background have been shown to contribute to the age at onset of neurological symptoms. A central challenge in understanding the disease progression that leads from the HD mutation to massive cell death in the striatum is the ability to characterize the subtle and early functional consequences of the CAG expansion longitudinally. We used dense time course sampling between 4 and 20 postnatal weeks to characterize early transcriptomic, molecular and cellular phenotypes in the striatum of six distinct knock-in mouse models of the HD mutation. We studied the effects of the HttQ111 allele on the C57BL/6J, CD-1, FVB/NCr1, and 129S2/SvPasCrl genetic backgrounds, and of two additional alleles, HttQ92 and HttQ50, on the C57BL/6J background. We describe the emergence of a transcriptomic signature in HttQ111/+ mice involving hundreds of differentially expressed genes and changes in diverse molecular pathways. We also show that this time course spanned the onset of mutant huntingtin nuclear localization phenotypes and somatic CAG-length instability in the striatum. Genetic background strongly influenced the magnitude and age at onset of these effects. This work provides a foundation for understanding the earliest transcriptional and molecular changes contributing to HD pathogenesis.
Asunto(s)
Cuerpo Estriado/metabolismo , Proteína Huntingtina/genética , Enfermedad de Huntington/genética , Expansión de Repetición de Trinucleótido/genética , Animales , Cuerpo Estriado/patología , Modelos Animales de Enfermedad , Regulación del Desarrollo de la Expresión Génica , Técnicas de Sustitución del Gen , Antecedentes Genéticos , Inestabilidad Genómica/genética , Humanos , Proteína Huntingtina/biosíntesis , Enfermedad de Huntington/patología , Ratones , Mutación/genética , Neuronas/metabolismo , Neuronas/patología , Fenotipo , Transcriptoma/genéticaRESUMEN
Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expanded CAG repeat within the huntingtin (HTT) gene. The Q140 and HdhQ150 knock-in HD mouse models were generated such that HdhQ150 mice have an expanded CAG repeat inserted into the mouse Htt gene, whereas in the Q140s, mouse exon 1 Htt was replaced with a mutated version of human exon 1. By standardizing mouse strain background, breeding to homozygosity and employing sensitive behavioral tests, we demonstrate that the onset of behavioral phenotypes occurs earlier in the Q140 than the HdhQ150 knock-in mouse models and that huntingtin (HTT) aggregation appears earlier in the striata of Q140 mice. We have previously found that the incomplete splicing of mutant HTT from exon 1 to exon 2 results in the production of a small polyadenylated transcript that encodes the highly pathogenic mutant HTT exon 1 protein. In this report, we have identified a functional consequence of the sequence differences between these two models at the RNA level, in that the level of incomplete splicing, and of the mutant exon 1 HTT protein, are greater in the brains of Q140 mice. While differences in the human and mouse exon 1 HTT proteins (e.g., proline rich sequences) could also contribute to the phenotypic differences, our data indicate that the incomplete splicing of HTT and approaches to lower the levels of the exon 1 HTT transcript should be pursued as therapeutic targets.
Asunto(s)
Conducta Animal/fisiología , Modelos Animales de Enfermedad , Proteína Huntingtina/genética , Enfermedad de Huntington/genética , Enfermedad de Huntington/psicología , Animales , Encéfalo/metabolismo , Encéfalo/patología , Femenino , Técnicas de Sustitución del Gen , Proteína Huntingtina/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , FenotipoRESUMEN
BACKGROUND: Huntington disease (HD) is a fatal neurodegenerative disorder involving reduced muscle coordination, mental and behavioral changes, and testicular degeneration. In order to further clarify the decreased fertility and penetration ability of the spermatozoa of transgenic HD minipig boars (TgHD), we applied a set of mitochondrial metabolism (MM) parameter measurements to this promising biological material, which can be collected noninvasively in longitudinal studies. OBJECTIVE: We aimed to optimize methods for MM measurements in spermatozoa and to establish possible biomarkers of HD in TgHD spermatozoa expressing the N-terminal part of mutated human huntingtin. METHODS: Semen samples from 12 TgHD and wild-type animals, aged 12-65 months, were obtained repeatedly during the study. Respiration was measured by polarography, MM was assessed by the detection of oxidation of radiolabeled substrates (mitochondrial energy-generating system; MEGS), and the content of the oxidative phosphorylation system subunits was detected by Western blot. Three possibly interfering factors were statistically analyzed: the effect of HD, generation and aging. RESULTS: We found 5 MM parameters which were significantly diminished in TgHD spermatozoa and propose 3 specific MEGS incubations and complex I-dependent respiration as potential biomarkers of HD in TgHD spermatozoa. CONCLUSIONS: Our results suggest a link between the gain of toxic function of mutated huntingtin in TgHD spermatozoa and the observed MM and/or glycolytic impairment. We determined 4 biomarkers useful for HD phenotyping and experimental therapy monitoring studies in TgHD minipigs.
Asunto(s)
Enfermedad de Huntington/complicaciones , Enfermedad de Huntington/patología , Mitocondrias/metabolismo , Espermatozoides/metabolismo , Espermatozoides/patología , Factores de Edad , Animales , Animales Modificados Genéticamente , Humanos , Proteína Huntingtina/genética , Enfermedad de Huntington/genética , Masculino , Proteínas Mitocondriales/metabolismo , Mutación/genética , Fosforilación Oxidativa , Complejo Piruvato Deshidrogenasa/metabolismo , Respiración , Semen/metabolismo , Porcinos , Porcinos Enanos , Ácidos Tricarboxílicos/metabolismo , Repeticiones de Trinucleótidos/genéticaRESUMEN
Huntington disease (HD) is a devastating, late-onset, inherited neurodegenerative disorder that manifests with personality changes, movement disorders, and cognitive decline. It is caused by a CAG repeat expansion in exon 1 of the HTT gene that translates to a polyglutamine tract in the huntingtin protein (HTT). The formation of HTT fragments has been implicated as an essential step in the molecular pathogenesis of HD and several proteases that cleave HTT have been identified. However, the importance of smaller N-terminal fragments has been highlighted by their presence in HD postmortem brains and by the fact that nuclear inclusions are only detected by antibodies to the N terminus of HTT. Despite an intense research effort, the precise length of these fragments and the mechanism by which they are generated remains unknown. Here we show that CAG repeat length-dependent aberrant splicing of exon 1 HTT results in a short polyadenylated mRNA that is translated into an exon 1 HTT protein. Given that mutant exon 1 HTT proteins have consistently been shown to be highly pathogenic in HD mouse models, the aberrant splicing of HTT mRNA provides a mechanistic basis for the molecular pathogenesis of HD. RNA-targeted therapeutic strategies designed to lower the levels of HTT are under development. Many of these approaches would not prevent the production of exon 1 HTT and should be reviewed in light of our findings.
Asunto(s)
Enfermedad de Huntington/genética , Proteínas Mutantes/genética , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Empalme del ARN , Animales , Secuencia de Bases , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Exones , Humanos , Proteína Huntingtina , Enfermedad de Huntington/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Datos de Secuencia Molecular , Proteínas Mutantes/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Repeticiones de TrinucleótidosRESUMEN
BACKGROUND: Huntington's disease is induced by CAG expansion in a single gene coding the huntingtin protein. The mutated huntingtin (mtHtt) primarily causes degeneration of neurons in the brain, but it also affects peripheral tissues, including testes. OBJECTIVE: We studied sperm and testes of transgenic boars expressing the N-terminal region of human mtHtt. METHODS: In this study, measures of reproductive parameters and electron microscopy (EM) images of spermatozoa and testes of transgenic (TgHD) and wild-type (WT) boars of F1 (24-48 months old) and F2 (12-36 months old) generations were compared. In addition, immunofluorescence, immunohistochemistry, Western blot, hormonal analysis and whole-genome sequencing were done in order to elucidate the effects of mtHtt. RESULTS: Evidence for fertility failure of both TgHD generations was observed at the age of 13 months. Reproductive parameters declined and progressively worsened with age. EM revealed numerous pathological features in sperm tails and in testicular epithelium from 24- and 36-month-old TgHD boars. Moreover, immunohistochemistry confirmed significantly lower proliferation activity of spermatogonia in transgenic testes. mtHtt was highly expressed in spermatozoa and testes of TgHD boars and localized in all cells of seminiferous tubules. Levels of fertility-related hormones did not differ in TgHD and WT siblings. Genome analysis confirmed that insertion of the lentiviral construct did not interrupt any coding sequence in the pig genome. CONCLUSIONS: The sperm and testicular degeneration of TgHD boars is caused by gain-of-function of the highly expressed mtHtt.
Asunto(s)
Proteína Huntingtina/metabolismo , Mutación , Espermatozoides/metabolismo , Espermatozoides/patología , Testículo/metabolismo , Testículo/patología , Envejecimiento/metabolismo , Envejecimiento/patología , Animales , Animales Modificados Genéticamente , Proliferación Celular/fisiología , Modelos Animales de Enfermedad , Vectores Genéticos , Humanos , Proteína Huntingtina/genética , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Lentivirus/genética , Masculino , Recuento de Espermatozoides , Porcinos , Porcinos EnanosRESUMEN
Apoptosis, or programmed cell death, is a cellular pathway involved in normal cell turnover, developmental tissue remodeling, embryonic development, cellular homeostasis maintenance and chemical-induced cell death. Caspases are a family of intracellular proteases that play a key role in apoptosis. Aberrant activation of caspases has been implicated in human diseases. In particular, numerous findings implicate Caspase-6 (Casp6) in neurodegenerative diseases, including Alzheimer disease (AD) and Huntington disease (HD), highlighting the need for a deeper understanding of Casp6 biology and its role in brain development. The use of targeted caspase-deficient mice has been instrumental for studying the involvement of caspases in apoptosis. The goal of this study was to perform an in-depth neuroanatomical and behavioral characterization of constitutive Casp6-deficient (Casp6-/-) mice in order to understand the physiological function of Casp6 in brain development, structure and function. We demonstrate that Casp6-/- neurons are protected against excitotoxicity, nerve growth factor deprivation and myelin-induced axonal degeneration. Furthermore, Casp6-deficient mice show an age-dependent increase in cortical and striatal volume. In addition, these mice show a hypoactive phenotype and display learning deficits. The age-dependent behavioral and region-specific neuroanatomical changes observed in the Casp6-/- mice suggest that Casp6 deficiency has a more pronounced effect in brain regions that are involved in neurodegenerative diseases, such as the striatum in HD and the cortex in AD.
Asunto(s)
Caspasa 6/fisiología , Degeneración Nerviosa/enzimología , Envejecimiento/patología , Envejecimiento/fisiología , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/patología , Animales , Apoptosis/fisiología , Secuencia de Bases , Conducta Animal/fisiología , Encéfalo/enzimología , Encéfalo/patología , Caspasa 6/deficiencia , Caspasa 6/genética , Humanos , Enfermedad de Huntington/enzimología , Enfermedad de Huntington/patología , Ratones , Ratones Noqueados , Actividad Motora/fisiología , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Neuronas/enzimología , Neuronas/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de N-Metil-D-Aspartato/fisiologíaRESUMEN
Unraveling the pathophysiology and testing candidate therapeutics in neurodegenerative disorders is, necessarily, highly dependent on model systems. Because Huntington's disease (HD) is caused by a single (expanded CAG tract) mutation in the huntingtin (HTT) gene, a richness of model systems, particularly mice, have been engineered to both dissect disease mechanisms and test potential therapeutics. Even so, as with other neurodegenerative diseases, very little success has been achieved in translating HD mouse model drug testing results to the clinic. Because of the considerable costs-human, opportunity, and financial-there is a pressing need to improve the use of existing HD models and also to develop models in higher species beyond rodent, such as sheep, minipig, and nonhuman primate, to bridge the translational gap from preclinical to clinical testing of candidate therapeutics.
Asunto(s)
Modelos Animales de Enfermedad , Enfermedad de Huntington/terapia , Investigación Biomédica Traslacional/métodos , Animales , Humanos , Proteína Huntingtina , Enfermedad de Huntington/genética , Proteínas del Tejido Nervioso/genética , Especificidad de la Especie , Repeticiones de Trinucleótidos/genéticaRESUMEN
Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expanded CAG tract in the huntingtin (HTT) gene, leading to toxic gains of function. HTT-lowering treatments are in clinical trials, but the risks imposed are unclear. Recent studies have reported on the consequences of widespread HTT loss in mice, where one group described early HTT loss leading to fatal pancreatitis, but later loss as benign. Another group reported no pancreatitis but found widespread neurological phenotypes including subcortical calcification. To better understand the liabilities of widespread HTT loss, we knocked out Htt with two separate tamoxifen-inducible Cre lines. We find that loss of HTT at 2 mo of age leads to progressive tremors and severe subcortical calcification at examination at 14 mo of age but does not result in acute pancreatitis or histological changes in the pancreas. We, in addition, report that HTT loss is followed by sustained induction of circulating neurofilament light chain. These results confirm that global loss of HTT in mice is associated with pronounced risks, including progressive subcortical calcification and neurodegeneration.
Asunto(s)
Modelos Animales de Enfermedad , Proteína Huntingtina , Enfermedad de Huntington , Ratones Noqueados , Páncreas , Animales , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Ratones , Páncreas/patología , Páncreas/metabolismo , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Enfermedad de Huntington/metabolismo , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/etiología , Enfermedades Neurodegenerativas/patología , Masculino , Calcinosis/genética , Calcinosis/patología , Fenotipo , FemeninoRESUMEN
Huntington disease (HD) is an inherited progressive neurodegenerative disorder, characterized by motor, cognitive, and psychiatric deficits as well as neurodegeneration and brain atrophy beginning in the striatum and the cortex and extending to other subcortical brain regions. The genetic cause is an expansion of the CAG repeat stretch in the HTT gene encoding huntingtin protein (htt). Here, we generated an HD transgenic rat model using a human bacterial artificial chromosome (BAC), which contains the full-length HTT genomic sequence with 97 CAG/CAA repeats and all regulatory elements. BACHD transgenic rats display a robust, early onset and progressive HD-like phenotype including motor deficits and anxiety-related symptoms. In contrast to BAC and yeast artificial chromosome HD mouse models that express full-length mutant huntingtin, BACHD rats do not exhibit an increased body weight. Neuropathologically, the distribution of neuropil aggregates and nuclear accumulation of N-terminal mutant huntingtin in BACHD rats is similar to the observations in human HD brains. Aggregates occur more frequently in the cortex than in the striatum and neuropil aggregates appear earlier than mutant htt accumulation in the nucleus. Furthermore, we found an imbalance in the striatal striosome and matrix compartments in early stages of the disease. In addition, reduced dopamine receptor binding was detectable by in vivo imaging. Our data demonstrate that this transgenic BACHD rat line may be a valuable model for further understanding the disease mechanisms and for preclinical pharmacological studies.
Asunto(s)
Cromosomas Artificiales Bacterianos/genética , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Proteínas del Tejido Nervioso/genética , Empalme Alternativo , Animales , Ansiedad/genética , Ansiedad/psicología , Conducta Animal/fisiología , Western Blotting , Peso Corporal/fisiología , Ingestión de Alimentos/fisiología , Trastornos Neurológicos de la Marcha/psicología , Dosificación de Gen , Humanos , Proteína Huntingtina , Enfermedad de Huntington/psicología , Inmunohistoquímica , Actividad Motora/fisiología , Tomografía de Emisión de Positrones , Equilibrio Postural/fisiología , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Huntington's disease (HD) is caused by a mutation in the huntingtin (htt) gene encoding an expansion of glutamine repeats at the N terminus of the Htt protein. Proteolysis of Htt has been identified as a critical pathological event in HD models. In particular, it has been postulated that proteolysis of Htt at the putative caspase-6 cleavage site (at amino acid Asp-586) plays a critical role in disease progression and pathogenesis. However, whether caspase-6 is indeed the essential enzyme that cleaves Htt at this site in vivo has not been determined. To evaluate, we crossed the BACHD mouse model with a caspase-6 knock-out mouse (Casp6(-/-)). Western blot and immunocytochemistry confirmed the lack of caspase-6 protein in Casp6(-/-) mice, regardless of HD genotype. We predicted the Casp6(-/-) mouse would have reduced levels of caspase-6 Htt fragments and increased levels of full-length Htt protein. In contrast, we found a significant reduction of full-length mutant Htt (mHtt) and fragments in the striatum of BACHD Casp6(-/-) mice. Importantly, we detected the presence of Htt fragments consistent with cleavage at amino acid Asp-586 of Htt in the BACHD Casp6(-/-) mouse, indicating that caspase-6 activity cannot fully account for the generation of the Htt 586 fragment in vivo. Our data are not consistent with the hypothesis that caspase-6 activity is critical in generating a potentially toxic 586 aa Htt fragment in vivo. However, our studies do suggest a role for caspase-6 activity in clearance pathways for mHtt protein.
Asunto(s)
Ácido Aspártico/metabolismo , Caspasa 6/metabolismo , Regulación de la Expresión Génica/genética , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/fisiopatología , Proteínas del Tejido Nervioso/metabolismo , Factores de Edad , Aminoácidos/genética , Aminoácidos/metabolismo , Animales , Ácido Aspártico/genética , Peso Corporal/genética , Encéfalo/metabolismo , Encéfalo/patología , Caspasa 6/deficiencia , Células Cultivadas , Cuerpo Estriado/citología , Modelos Animales de Enfermedad , Embrión de Mamíferos , Conducta Exploratoria/fisiología , Femenino , Proteína Huntingtina , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Imagen por Resonancia Magnética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Actividad Motora/genética , Proteínas del Tejido Nervioso/genética , Neuronas , Proteolisis , ARN Interferente Pequeño/metabolismo , Prueba de Desempeño de Rotación con Aceleración Constante , Expansión de Repetición de Trinucleótido/genética , Ubiquitinación/genéticaRESUMEN
BACKGROUND: One of the means of easing increased pressure on emergency care worldwide has been the development of advanced musculoskeletal physiotherapy practice in the emergency department setting. This model of care is in its infancy in Ireland. AIMS: To evaluate the effectiveness of an advanced practice physiotherapist working as a primary contact clinician in the emergency department at St. James's Hospital, Dublin. METHODS: A three-month retrospective chart review was undertaken for patients assigned the advanced practice physiotherapist as their primary clinician during their emergency department attendance. Three widely accepted measures of quality in emergency medicine were used to evaluate effectiveness, namely, time from attendance to discharge, time from triage to assessment, and unplanned reattendance within seven days. RESULTS: A total of 129 patients were included in this study. Time from attendance to discharge was significantly less in the APP group (mean 208.5 min, standard deviation 122.4 min) than in the ED group (mean 377.1 min, standard deviation 314.7 min) (mean difference - 168.61 (95% C.I - 191.24- - 145.98)) (p < 0.001). Time from triage to assessment was significantly less in the APP group (mean 72.1 min, standard deviation 51.9 min) than in the ED group (mean 94.1 min, standard deviation 96.5 min) (mean difference - 22.08 (95% C.I - 31.28- - 12.89)) (p < 0.001). The unplanned reattendance rate was 3.9%. No adverse events were identified. CONCLUSIONS: The findings of this study indicate that an advanced practice physiotherapist can provide a timely, effective, and safe service for patients attending the emergency department with musculoskeletal complaints in Ireland.
RESUMEN
Huntington's disease arises from a toxic gain of function in the huntingtin (HTT) gene. As a result, many HTT-lowering therapies are being pursued in clinical studies, including those that reduce HTT RNA and protein expression in the liver. To investigate potential impacts, we characterized molecular, cellular, and metabolic impacts of chronic HTT lowering in mouse hepatocytes. Lifelong hepatocyte HTT loss is associated with multiple physiological changes, including increased circulating bile acids, cholesterol and urea, hypoglycemia, and impaired adhesion. HTT loss causes a clear shift in the normal zonal patterns of liver gene expression, such that pericentral gene expression is reduced. These alterations in liver zonation in livers lacking HTT are observed at the transcriptional, histological, and plasma metabolite levels. We have extended these phenotypes physiologically with a metabolic challenge of acetaminophen, for which the HTT loss results in toxicity resistance. Our data reveal an unexpected role for HTT in regulating hepatic zonation, and we find that loss of HTT in hepatocytes mimics the phenotypes caused by impaired hepatic ß-catenin function.
Asunto(s)
Hepatocitos , Hígado , Animales , Ratones , Acetaminofén , FenotipoRESUMEN
BACKGROUND: Synaptic changes occur early in patients with Huntington's disease (HD) and in mouse models of HD. An analysis of synaptic changes in HD transgenic sheep (OVT73) is fitting since they have been shown to have some phenotypes. They also have larger brains, longer lifespan, and greater motor and cognitive capacities more aligned with humans, and can provide abundant biofluids for in vivo monitoring of therapeutic interventions. OBJECTIVE: The objective of this study was to determine if there were differences between 5- and 10-year-old OVT73 and wild-type (WT) sheep in levels of synaptic proteins in brain and in neurofilament light chain (NfL) in cerebrospinal fluid (CSF) and plasma. METHODS: Mutant huntingtin (mHTT) and other proteins were measured by western blot assay in synaptosomes prepared from caudate, motor, and piriform cortex in 5-year-old and caudate, putamen, motor; and piriform cortex in 10-year-old WT and OVT73 sheep. Levels of NfL, a biomarker for neuronal damage increased in many neurological disorders including HD, were examined in CSF and plasma samples from 10-year-old WT and OVT73 sheep using the Simoa NfL Advantage kit. RESULTS: Western blot analysis showed mHTT protein expression in synaptosomes from OVT73 sheep was 23% of endogenous sheep HTT levels at both ages. Significant changes were detected in brain levels of PDE10A, SCN4B, DARPP32, calmodulin, SNAP25, PSD95, VGLUT 1, VAMP1, and Na+/K+-ATPase, which depended on age and brain region. There was no difference in NfL levels in CSF and plasma in OVT73 sheep compared to age-matched WT sheep. CONCLUSIONS: These results show that synaptic changes occur in brain of 5- and 10-year-old OVT73 sheep, but levels of NfL in biofluids are unaffected. Altogether, the data support a prodromal disease state in OVT73 sheep that involves the caudate, putamen and cortex.
RESUMEN
Huntington's disease arises from a toxic gain of function in the huntingtin ( HTT ) gene. As a result, many HTT-lowering therapies are being pursued in clinical studies, including those that reduce HTT RNA and protein expression in the liver. To investigate potential impacts, we characterized molecular, cellular, and metabolic impacts of chronic HTT lowering in mouse hepatocytes. Lifelong hepatocyte HTT loss is associated with multiple physiological changes, including increased circulating bile acids, cholesterol and urea, hypoglycemia, and impaired adhesion. HTT loss causes a clear shift in the normal zonal patterns of liver gene expression, such that pericentral gene expression is reduced. These alterations in liver zonation in livers lacking HTT are observed at the transcriptional, histological and plasma metabolite level. We have extended these phenotypes physiologically with a metabolic challenge of acetaminophen, for which the HTT loss results in toxicity resistance. Our data reveal an unexpected role for HTT in regulating hepatic zonation, and we find that loss of HTT in hepatocytes mimics the phenotypes caused by impaired hepatic ß-catenin function.
RESUMEN
We have developed an inducible Huntington's disease (HD) mouse model that allows temporal control of whole-body allele-specific mutant huntingtin (mHtt) expression. We asked whether moderate global lowering of mHtt (~50%) was sufficient for long-term amelioration of HD-related deficits and, if so, whether early mHtt lowering (before measurable deficits) was required. Both early and late mHtt lowering delayed behavioral dysfunction and mHTT protein aggregation, as measured biochemically. However, long-term follow-up revealed that the benefits, in all mHtt-lowering groups, attenuated by 12 months of age. While early mHtt lowering attenuated cortical and striatal transcriptional dysregulation evaluated at 6 months of age, the benefits diminished by 12 months of age, and late mHtt lowering did not ameliorate striatal transcriptional dysregulation at 12 months of age. Only early mHtt lowering delayed the elevation in cerebrospinal fluid neurofilament light chain that we observed in our model starting at 9 months of age. As small-molecule HTT-lowering therapeutics progress to the clinic, our findings suggest that moderate mHtt lowering allows disease progression to continue, albeit at a slower rate, and could be relevant to the degree of mHTT lowering required to sustain long-term benefits in humans.
Asunto(s)
Enfermedad de Huntington , Ratones , Humanos , Animales , Lactante , Enfermedad de Huntington/tratamiento farmacológico , Enfermedad de Huntington/genética , Agregado de Proteínas , Proteína Huntingtina/genética , Proteína Huntingtina/líquido cefalorraquídeo , Modelos Animales de Enfermedad , Cuerpo Estriado/metabolismo , Progresión de la EnfermedadRESUMEN
Huntington's disease is caused by a CAG / polyglutamine repeat expansion. Mutated CAG repeats undergo somatic instability, resulting in tracts of several hundred CAGs in the brain; and genetic modifiers of Huntington's disease have indicated that somatic instability is a major driver of age of onset and disease progression. As the CAG repeat expands, the likelihood that exon 1 does not splice to exon 2 increases, resulting in two transcripts that encode full-length huntingtin protein, as well as the highly pathogenic and aggregation-prone exon 1 huntingtin protein. Strategies that target the huntingtin gene or transcripts are a major focus of therapeutic development. It is essential that the levels of all isoforms of huntingtin protein can be tracked, to better understand the molecular pathogenesis, and to assess the impact of huntingtin protein-lowering approaches in preclinical studies and clinical trials. Huntingtin protein bioassays for soluble and aggregated forms of huntingtin protein are in widespread use on the homogeneous time-resolved fluorescence and Meso Scale Discovery platforms, but these do not distinguish between exon 1 huntingtin protein and full-length huntingtin protein. In addition, they are frequently used to quantify huntingtin protein levels in the context of highly expanded polyglutamine tracts, for which appropriate protein standards do not currently exist. Here, we set out to develop novel huntingtin protein bioassays to ensure that all soluble huntingtin protein isoforms could be distinguished. We utilized the zQ175 Huntington's disease mouse model that has â¼190 CAGs, a CAG repeat size for which protein standards are not available. Initially, 30 combinations of six antibodies were tested on three technology platforms: homogeneous time-resolved fluorescence, amplified luminescent proximity homogeneous assay and Meso Scale Discovery, and a triage strategy was employed to select the best assays. We found that, without a polyglutamine-length-matched standard, the vast majority of soluble mutant huntingtin protein assays cannot be used for quantitative purposes, as the highly expanded polyglutamine tract decreased assay performance. The combination of our novel assays, with those already in existence, provides a tool-kit to track: total soluble mutant huntingtin protein, soluble exon 1 huntingtin protein, soluble mutant huntingtin protein (excluding the exon 1 huntingtin protein) and total soluble full-length huntingtin protein (mutant and wild type). Several novel aggregation assays were also developed that track with disease progression. These selected assays can be used to compare the levels of huntingtin protein isoforms in a wide variety of mouse models of Huntington's disease and to determine how these change in response to genetic or therapeutic manipulations.