RESUMO
Alternative splicing (AS) appears to be altered in Huntington's disease (HD), but its significance for early, pre-symptomatic disease stages has not been inspected. Here, taking advantage of Htt CAG knock-in mouse in vitro and in vivo models, we demonstrate a correlation between Htt CAG repeat length and increased aberrant linear AS, specifically affecting neural progenitors and, in vivo, the striatum prior to overt behavioral phenotypes stages. Remarkably, a significant proportion (36%) of the aberrantly spliced isoforms are not-functional and meant to non-sense mediated decay (NMD). The expanded Htt CAG repeats further reflect on a previously neglected, global impairment of back-splicing, leading to decreased circular RNAs production in neural progenitors. Integrative transcriptomic analyses unveil a network of transcriptionally altered micro-RNAs and RNA-binding proteins (Celf, hnRNPs, Ptbp, Srsf, Upf1, Ythd2) which might influence the AS machinery, primarily in neural cells. We suggest that this unbalanced expression of linear and circular RNAs might alter neural fitness, contributing to HD pathogenesis.
Assuntos
Doença de Huntington , Camundongos , Animais , Doença de Huntington/genética , Doença de Huntington/patologia , RNA Circular/genética , Splicing de RNA , Processamento Alternativo/genética , Perfilação da Expressão Gênica , Expansão das Repetições de Trinucleotídeos/genética , Proteína Huntingtina/genéticaRESUMO
Somatic expansion of the CAG repeat tract that causes Huntington's disease (HD) is thought to contribute to the rate of disease pathogenesis. Therefore, factors influencing repeat expansion are potential therapeutic targets. Genes in the DNA mismatch repair pathway are critical drivers of somatic expansion in HD mouse models. Here, we have tested, using genetic and pharmacological approaches, the role of the endonuclease domain of the mismatch repair protein MLH3 in somatic CAG expansion in HD mice and patient cells. A point mutation in the MLH3 endonuclease domain completely eliminated CAG expansion in the brain and peripheral tissues of a HD knock-in mouse model (HttQ111). To test whether the MLH3 endonuclease could be manipulated pharmacologically, we delivered splice switching oligonucleotides in mice to redirect Mlh3 splicing to exclude the endonuclease domain. Splice redirection to an isoform lacking the endonuclease domain was associated with reduced CAG expansion. Finally, CAG expansion in HD patient-derived primary fibroblasts was also significantly reduced by redirecting MLH3 splicing to the endogenous endonuclease domain-lacking isoform. These data indicate the potential of targeting the MLH3 endonuclease domain to slow somatic CAG repeat expansion in HD, a therapeutic strategy that may be applicable across multiple repeat expansion disorders.
Assuntos
Reparo do DNA , Endonucleases , Doença de Huntington/genética , Proteínas MutL , Processamento de Proteína , Expansão das Repetições de Trinucleotídeos , Animais , Células Cultivadas , Endonucleases/fisiologia , Feminino , Fibroblastos , Técnicas de Introdução de Genes , Instabilidade Genômica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas MutL/fisiologia , OligonucleotídeosRESUMO
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.
Assuntos
Proteínas de Ciclo Celular/genética , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Proteína Huntingtina/genética , Doença de Huntington/genética , Enzimas Multifuncionais/genética , Proteína 1 Homóloga a MutL/genética , Ribonucleotídeo Redutases/genética , Idade de Início , Animais , Modelos Animais de Doenças , Genes Modificadores/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Doença de Huntington/patologia , Camundongos , Camundongos Knockout , Fenótipo , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
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.
Assuntos
Corpo Estriado/metabolismo , Proteína Huntingtina/genética , Doença de Huntington/genética , Expansão das Repetições de Trinucleotídeos/genética , Animais , Corpo Estriado/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Introdução de Genes , Patrimônio Genético , Instabilidade Genômica/genética , Humanos , Proteína Huntingtina/biossíntese , Doença de Huntington/patologia , Camundongos , Mutação/genética , Neurônios/metabolismo , Neurônios/patologia , Fenótipo , Transcriptoma/genéticaRESUMO
Huntington's disease (HD) is a dominant neurodegenerative disorder that is due to expansion of an unstable HTT CAG repeat for which genome-wide genetic scans are now revealing chromosome regions that contain disease-modifying genes. We have explored a novel human-mouse cross-species functional prioritisation approach, by evaluating the HD modifier 6q23-24 linkage interval. This unbiased strategy employs C57BL/6J (B6J) Hdh(Q111) knock-in mice, replicates of the HD mutation, and the C57BL/6J-chr10(A/J)/NaJ chromosome substitution strain (CSS10), in which only chromosome 10 (chr10), in synteny with the human 6q23-24 region, is derived from the A/J (AJ) strain. Crosses were performed to assess the possibility of dominantly acting chr10 AJ-B6J variants of strong effect that may modulate CAG-dependent Hdh(Q111/+) phenotypes. Testing of F1 progeny confirmed that a single AJ chromosome had a significant effect on the rate of body weight gain and in Hdh(Q111) mice the AJ chromosome was associated subtle alterations in somatic CAG instability in the liver and the formation of intra-nuclear inclusions, as well as DARPP-32 levels, in the striatum. These findings in relatively small cohorts are suggestive of dominant chr10 AJ-B6 variants that may modify effects of the CAG expansion, and encourage a larger study with CSS10 and sub-strains. This cross-species approach may therefore be suited to functional in vivo prioritisation of genomic regions harbouring genes that can modify the early effects of the HD mutation.
Assuntos
Cromossomos de Mamíferos , Cruzamentos Genéticos , Doença de Huntington/genética , Locos de Características Quantitativas , Alelos , Animais , Peso Corporal , Cromossomos Humanos , Modelos Animais de Doenças , Fosfoproteína 32 Regulada por cAMP e Dopamina/genética , Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Feminino , Técnicas de Introdução de Genes , Variação Genética , Instabilidade Genômica , Genótipo , Humanos , Proteína Huntingtina , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Fenótipo , Repetições de TrinucleotídeosRESUMO
Huntington's disease (HD) is a dominant neurological disorder caused by an expanded HTT exon 1 CAG repeat that lengthens huntingtin's polyglutamine tract. Lowering mutant huntingtin has been proposed for treating HD, but genetic modifiers implicate somatic CAG repeat expansion as the driver of onset. We find that branaplam and risdiplam, small molecule splice modulators that lower huntingtin by promoting HTT pseudoexon inclusion, also decrease expansion of an unstable HTT exon 1 CAG repeat in an engineered cell model. Targeted CRISPR-Cas9 editing shows this effect is not due to huntingtin lowering, pointing instead to pseudoexon inclusion in PMS1. Homozygous but not heterozygous inactivation of PMS1 also reduces CAG repeat expansion, supporting PMS1 as a genetic modifier of HD and a potential target for therapeutic intervention. Although splice modulation provides one strategy, genome-wide transcriptomics also emphasize consideration of cell-type specific effects and polymorphic variation at both target and off-target sites.
Assuntos
Doença de Huntington , Humanos , Doença de Huntington/genética , Éxons/genética , Perfilação da Expressão Gênica , Heterozigoto , Homozigoto , Proteínas MutL , Proteínas de NeoplasiasRESUMO
Circular RNA (circRNA) molecules have critical functions during brain development and in brain-related disorders. Here, we identified and validated a circRNA, circHTT(2,3,4,5,6), stemming from the Huntington's disease (HD) gene locus that is most abundant in the central nervous system (CNS). We uncovered its evolutionary conservation in diverse mammalian species, and a correlation between circHTT(2,3,4,5,6) levels and the length of the CAG-repeat tract in exon-1 of HTT in human and mouse HD model systems. The mouse orthologue, circHtt(2,3,4,5,6), is expressed during embryogenesis, increases during nervous system development, and is aberrantly upregulated in the presence of the expanded CAG tract. While an IRES-like motif was predicted in circH TT (2,3,4,5,6), the circRNA does not appear to be translated in adult mouse brain tissue. Nonetheless, a modest, but consistent fraction of circHtt(2,3,4,5,6) associates with the 40S ribosomal subunit, suggesting a possible role in the regulation of protein translation. Finally, circHtt(2,3,4,5,6) overexpression experiments in HD-relevant STHdh striatal cells revealed its ability to modulate CAG expansion-driven cellular defects in cell-to-substrate adhesion, thus uncovering an unconventional modifier of HD pathology.
RESUMO
Huntington's disease (HD), one of >50 inherited repeat expansion disorders (Depienne and Mandel, 2021), is a dominantly-inherited neurodegenerative disease caused by a CAG expansion in HTT (The Huntington's Disease Collaborative Research Group, 1993). Inherited CAG repeat length is the primary determinant of age of onset, with human genetic studies underscoring that the property driving disease is the CAG length-dependent propensity of the repeat to further expand in brain (Swami et al ., 2009; GeM-HD, 2015; Hensman Moss et al ., 2017; Ciosi et al ., 2019; GeM-HD, 2019; Hong et al ., 2021). Routes to slowing somatic CAG expansion therefore hold great promise for disease-modifying therapies. Several DNA repair genes, notably in the mismatch repair (MMR) pathway, modify somatic expansion in HD mouse models (Wheeler and Dion, 2021). To identify novel modifiers of somatic expansion, we have used CRISPR-Cas9 editing in HD knock-in mice to enable in vivo screening of expansion-modifier candidates at scale. This has included testing of HD onset modifier genes emerging from human genome-wide association studies (GWAS), as well as interactions between modifier genes, thereby providing new insight into pathways underlying CAG expansion and potential therapeutic targets.
RESUMO
Somatic expansion of the Huntington's disease (HD) CAG repeat drives the rate of a pathogenic process ultimately resulting in neuronal cell death. Although mechanisms of toxicity are poorly delineated, transcriptional dysregulation is a likely contributor. To identify modifiers that act at the level of CAG expansion and/or downstream pathogenic processes, we tested the impact of genetic knockout, in HttQ111 mice, of Hdac2 or Hdac3 in medium-spiny striatal neurons that exhibit extensive CAG expansion and exquisite disease vulnerability. Both knockouts moderately attenuated CAG expansion, with Hdac2 knockout decreasing nuclear huntingtin pathology. Hdac2 knockout resulted in a substantial transcriptional response that included modification of transcriptional dysregulation elicited by the HttQ111 allele, likely via mechanisms unrelated to instability suppression. Our results identify novel modifiers of different aspects of HD pathogenesis in medium-spiny neurons and highlight a complex relationship between the expanded Htt allele and Hdac2 with implications for targeting transcriptional dysregulation in HD.
Assuntos
Corpo Estriado/fisiopatologia , Histona Desacetilase 2/genética , Histona Desacetilases/genética , Doença de Huntington/genética , Neurônios/fisiologia , Animais , Núcleo Celular , Modelos Animais de Doenças , Histona Desacetilase 2/metabolismo , Histona Desacetilases/metabolismo , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/enzimologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Successful disease-modifying therapy for Huntington's disease (HD) will require therapeutic intervention early in the pathogenic process. Achieving this goal requires identifying phenotypes that are proximal to the HTT CAG repeat expansion. OBJECTIVE: To use Htt CAG knock-in mice, precise genetic replicas of the HTT mutation in patients, as models to study proximal disease events. METHODS: Using cohorts of B6J.HttQ111/+ mice from 2 to 18 months of age, we analyzed pathological markers, including immunohistochemistry, brain regional volumes and cortical thickness, CAG instability, electron microscopy of striatal synapses, and acute slice electrophysiology to record glutamatergic transmission at striatal synapses. We also incorporated a diet perturbation paradigm for some of these analyses. RESULTS: B6J.HttQ111/+ mice did not exhibit significant neurodegeneration or gliosis but revealed decreased striatal DARPP-32 as well as subtle but regional-specific changes in brain volumes and cortical thickness that parallel those in HD patients. Ultrastructural analyses of the striatum showed reduced synapse density, increased postsynaptic density thickness and increased synaptic cleft width. Acute slice electrophysiology showed alterations in spontaneous AMPA receptor-mediated postsynaptic currents, evoked NMDA receptor-mediated excitatory postsynaptic currents, and elevated extrasynaptic NMDA currents. Diet influenced cortical thickness, but did not impact somatic CAG expansion, nor did it show any significant interaction with genotype on immunohistochemical, brain volume or cortical thickness measures. CONCLUSIONS: These data show that a single HttQ111 allele is sufficient to elicit brain region-specific morphological changes and early neuronal dysfunction, highlighting an insidious disease process already apparent in the first few months of life.
Assuntos
Corpo Estriado/metabolismo , Proteína Huntingtina/genética , Doença de Huntington/genética , Sinapses/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Técnicas de Introdução de Genes/métodos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neostriado/metabolismo , Neurônios/metabolismo , Sinapses/genéticaRESUMO
Density-enhanced protein-tyrosine phosphatase-1 (DEP-1 also CD148) is a transmembrane molecule with a single intracellular PTP domain. It has recently been proposed to function as a tumor suppressor. We have previously shown that DEP-1 dephosphorylates the activated platelet-derived growth factor (PDGF) beta-receptor in a site-selective manner (Kovalenko et al. (2000). J. Biol. Chem. 275, 16219-16226). We analysed cell lines with inducible DEP-1 expression for cellular functions of DEP-1. Several aspects of PDGFbeta-receptor signaling were negatively affected by DEP-1 expression. These include PDGF-stimulated activation of inositol trisphosphate formation, Erk1/2, p21Ras, and Src. Activation of receptor-associated phosphoinositide-3 kinase activity and of Akt/PKB were weakly attenuated at early time points of stimulation. Inhibition of PDGF-stimulated signaling depended on DEP-1 catalytic activity. Importantly, DEP-1 inhibited PDGF-stimulated cell migration. The catalytically inactive DEP-1 C1239S variant enhanced cell migration and PDGF-stimulated Erk1/2 activation, suggesting a dominant negative interference with endogenous DEP-1. In contrast to cell migration, cell-substrate adhesion was promoted by active DEP-1 and delayed or suppressed by DEP-1 C1239S, correlating with positive effects of DEP-1 on adhesion-stimulated Src kinase. We propose that negative regulation of growth-factor stimulated cell migration and promotion of cell-matrix adhesion may be related to the function of DEP-1 as tumor suppressor.
Assuntos
Substâncias de Crescimento/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Fosfatases/fisiologia , Células 3T3 , Animais , Adesão Celular , Divisão Celular , Movimento Celular , Relação Dose-Resposta a Droga , Ativação Enzimática , Regulação da Expressão Gênica , Vetores Genéticos , Immunoblotting , Inositol 1,4,5-Trifosfato/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteína Fosfatase 1 , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores , Transdução de Sinais , Fatores de Tempo , Transfecção , Proteínas ras/metabolismoRESUMO
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat in the HTT gene encoding huntingtin. The disease has an insidious course, typically progressing over 10-15 years until death. Currently there is no effective disease-modifying therapy. To better understand the HD pathogenic process we have developed genetic HTT CAG knock-in mouse models that accurately recapitulate the HD mutation in man. Here, we describe results of a broad, standardized phenotypic screen in 10-46 week old heterozygous HdhQ111 knock-in mice, probing a wide range of physiological systems. The results of this screen revealed a number of behavioral abnormalities in HdhQ111/+ mice that include hypoactivity, decreased anxiety, motor learning and coordination deficits, and impaired olfactory discrimination. The screen also provided evidence supporting subtle cardiovascular, lung, and plasma metabolite alterations. Importantly, our results reveal that a single mutant HTT allele in the mouse is sufficient to elicit multiple phenotypic abnormalities, consistent with a dominant disease process in patients. These data provide a starting point for further investigation of several organ systems in HD, for the dissection of underlying pathogenic mechanisms and for the identification of reliable phenotypic endpoints for therapeutic testing.
Assuntos
Alelos , Doença de Huntington/genética , Doença de Huntington/patologia , Mutação/genética , Expansão das Repetições de Trinucleotídeos/genética , Animais , Ansiedade/genética , Ansiedade/patologia , Ansiedade/fisiopatologia , Sistema Nervoso Autônomo/patologia , Sistema Nervoso Autônomo/fisiopatologia , Comportamento Animal , Doenças Cardiovasculares/patologia , Doenças Cardiovasculares/fisiopatologia , Técnicas de Introdução de Genes , Glucose/metabolismo , Proteína Huntingtina , Doença de Huntington/fisiopatologia , Aprendizagem , Pulmão/anormalidades , Pulmão/patologia , Pulmão/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Atividade Motora , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Fenótipo , Teste de Desempenho do Rota-Rod , Olfato , Comportamento SocialRESUMO
The CAG trinucleotide repeat mutation in the Huntington's disease gene (HTT) exhibits age-dependent tissue-specific expansion that correlates with disease onset in patients, implicating somatic expansion as a disease modifier and potential therapeutic target. Somatic HTT CAG expansion is critically dependent on proteins in the mismatch repair (MMR) pathway. To gain further insight into mechanisms of somatic expansion and the relationship of somatic expansion to the disease process in selectively vulnerable MSNs we have crossed HTT CAG knock-in mice (HdhQ111) with mice carrying a conditional (floxed) Msh2 allele and D9-Cre transgenic mice, in which Cre recombinase is expressed specifically in MSNs within the striatum. Deletion of Msh2 in MSNs eliminated Msh2 protein in those neurons. We demonstrate that MSN-specific deletion of Msh2 was sufficient to eliminate the vast majority of striatal HTT CAG expansions in HdhQ111 mice. Furthermore, MSN-specific deletion of Msh2 modified two mutant huntingtin phenotypes: the early nuclear localization of diffusely immunostaining mutant huntingtin was slowed; and the later development of intranuclear huntingtin inclusions was dramatically inhibited. Therefore, Msh2 acts within MSNs as a genetic enhancer both of somatic HTT CAG expansions and of HTT CAG-dependent phenotypes in mice. These data suggest that the selective vulnerability of MSNs may be at least in part contributed by the propensity for somatic expansion in these neurons, and imply that intervening in the expansion process is likely to have therapeutic benefit.