RESUMO
Reactive changes of glial cells during neuroinflammation impact brain disorders and disease progression. Elucidating the mechanisms that control reactive gliosis may help us to understand brain pathophysiology and improve outcomes. Here, we report that adult ablation of autism spectrum disorder (ASD)-associated CHD8 in astrocytes attenuates reactive gliosis via remodeling chromatin accessibility, changing gene expression. Conditional Chd8 deletion in astrocytes, but not microglia, suppresses reactive gliosis by impeding astrocyte proliferation and morphological elaboration. Astrocyte Chd8 ablation alleviates lipopolysaccharide-induced neuroinflammation and septic-associated hypothermia in mice. Astrocytic CHD8 plays an important role in neuroinflammation by altering the chromatin landscape, regulating metabolic and lipid-associated pathways, and astrocyte-microglia crosstalk. Moreover, we show that reactive gliosis can be directly mitigated in vivo using an adeno-associated virus (AAV)-mediated Chd8 gene editing strategy. These findings uncover a role of ASD-associated CHD8 in the adult brain, which may warrant future exploration of targeting chromatin remodelers in reactive gliosis and neuroinflammation in injury and neurological diseases.
RESUMO
Parkinson's disease (PD) is a debilitating neurodegenerative disorder. Its symptoms are typically treated with levodopa or dopamine receptor agonists, but its action lacks specificity due to the wide distribution of dopamine receptors in the central nervous system and periphery. Here, we report the development of a gene therapy strategy to selectively manipulate PD-affected circuitry. Targeting striatal D1 medium spiny neurons (MSNs), whose activity is chronically suppressed in PD, we engineered a therapeutic strategy comprised of a highly efficient retrograde adeno-associated virus (AAV), promoter elements with strong D1-MSN activity, and a chemogenetic effector to enable precise D1-MSN activation after systemic ligand administration. Application of this therapeutic approach rescues locomotion, tremor, and motor skill defects in both mouse and primate models of PD, supporting the feasibility of targeted circuit modulation tools for the treatment of PD in humans.
Assuntos
Terapia Genética , Doença de Parkinson , Animais , Humanos , Camundongos , Corpo Estriado/metabolismo , Levodopa/uso terapêutico , Levodopa/genética , Neurônios/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/terapia , Primatas , Receptores de Dopamina D1/metabolismo , Modelos Animais de DoençasRESUMO
Cornelia de Lange Syndrome (CdLS) is a rare, dominantly inherited multisystem developmental disorder characterized by highly variable manifestations of growth and developmental delays, upper limb involvement, hypertrichosis, cardiac, gastrointestinal, craniofacial, and other systemic features. Pathogenic variants in genes encoding cohesin complex structural subunits and regulatory proteins (NIPBL, SMC1A, SMC3, HDAC8, and RAD21) are the major pathogenic contributors to CdLS. Heterozygous or hemizygous variants in the genes encoding these five proteins have been found to be contributory to CdLS, with variants in NIPBL accounting for the majority (>60%) of cases, and the only gene identified to date that results in the severe or classic form of CdLS when mutated. Pathogenic variants in cohesin genes other than NIPBL tend to result in a less severe phenotype. Causative variants in additional genes, such as ANKRD11, EP300, AFF4, TAF1, and BRD4, can cause a CdLS-like phenotype. The common role that these genes, and others, play as critical regulators of developmental transcriptional control has led to the conditions they cause being referred to as disorders of transcriptional regulation (or "DTRs"). Here, we report the results of a comprehensive molecular analysis in a cohort of 716 probands with typical and atypical CdLS in order to delineate the genetic contribution of causative variants in cohesin complex genes as well as novel candidate genes, genotype-phenotype correlations, and the utility of genome sequencing in understanding the mutational landscape in this population.
Assuntos
Síndrome de Cornélia de Lange , Proteínas Nucleares , Humanos , Proteínas Nucleares/genética , Síndrome de Cornélia de Lange/diagnóstico , Síndrome de Cornélia de Lange/genética , Síndrome de Cornélia de Lange/patologia , Fatores de Transcrição/genética , Proteínas de Ciclo Celular/genética , Fenótipo , Mutação , Genômica , Estudos de Associação Genética , Fatores de Elongação da Transcrição/genética , Histona Desacetilases/genética , Proteínas Repressoras/genéticaRESUMO
Searching for factors to improve knockin efficiency for therapeutic applications, biotechnology, and generation of non-human primate models of disease, we found that the strand exchange protein RAD51 can significantly increase Cas9-mediated homozygous knockin in mouse embryos through an interhomolog repair (IHR) mechanism. IHR is a hallmark of meiosis but only occurs at low frequencies in somatic cells, and its occurrence in zygotes is controversial. Using multiple approaches, we provide evidence for an endogenous IHR mechanism in the early embryo that can be enhanced by RAD51. This process can be harnessed to generate homozygotes from wild-type zygotes using exogenous donors and to convert heterozygous alleles into homozygous alleles without exogenous templates. Furthermore, we identify additional IHR-promoting factors and describe features of IHR events. Together, our findings show conclusive evidence for IHR in mouse embryos and describe an efficient method for enhanced gene conversion.
Assuntos
Reparo do DNA/genética , Conversão Gênica , Rad51 Recombinase/metabolismo , Alelos , Animais , Sequência de Bases , Proteína 9 Associada à CRISPR/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromossomos de Mamíferos/genética , Quebras de DNA de Cadeia Dupla , Embrião de Mamíferos , Feminino , Loci Gênicos , Recombinação Homóloga/genética , Homozigoto , Humanos , Mutação INDEL/genética , Camundongos Endogâmicos C57BL , Mosaicismo , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Ribonucleoproteínas/metabolismo , Zigoto/metabolismoRESUMO
Common polygenic diseases result from compounded risk contributed by multiple genetic variants, meaning that simultaneous correction or introduction of single nucleotide variants is required for disease modeling and gene therapy. Here, we show precise, efficient, and simultaneous multiplex base editing of up to three target sites across 11 genes/loci in cynomolgus monkey embryos using CRISPR-based cytidine- and adenine-base editors. Unbiased whole genome sequencing demonstrates high specificity of base editing in monkey embryos. Our data demonstrate feasibility of multiplex base editing for polygenic disease modeling in primate zygotes.
Assuntos
Edição de Genes/métodos , Animais , Sequência de Bases , Embrião de Mamíferos/metabolismo , Éxons/genética , Feto/metabolismo , Fígado/metabolismo , Macaca fascicularis/embriologia , Mutação/genéticaRESUMO
Diencephalic defects underlie an array of neurological diseases. Previous studies have suggested that retinoic acid (RA) signaling is involved in diencephalic development at late stages of embryonic development, but its roles and mechanisms of action during early neural development are still unclear. Here we demonstrate that mice lacking enzymatic activity of the acetyltransferase GCN5 ((Gcn5hat/hat )), which were previously characterized with respect to their exencephalic phenotype, exhibit significant diencephalic expansion, decreased diencephalic RA signaling, and increased diencephalic WNT and SHH signaling. Using a variety of molecular biology techniques in both cultured neuroepithelial cells treated with a GCN5 inhibitor and forebrain tissue from (Gcn5hat/hat ) embryos, we demonstrate that GCN5, RARα/γ, and the poorly characterized protein TACC1 form a complex in the nucleus that binds specific retinoic acid response elements in the absence of RA. Furthermore, RA triggers GCN5-mediated acetylation of TACC1, which results in dissociation of TACC1 from retinoic acid response elements and leads to transcriptional activation of RA target genes. Intriguingly, RA signaling defects caused by in vitro inhibition of GCN5 can be rescued through RA-dependent mechanisms that require RARß. Last, we demonstrate that the diencephalic expansion and transcriptional defects seen in (Gcn5hat/hat ) mutants can be rescued with gestational RA supplementation, supporting a direct link between GCN5, TACC1, and RA signaling in the developing diencephalon. Together, our studies identify a novel, nonhistone substrate for GCN5 whose modification regulates a previously undescribed, tissue-specific mechanism of RA signaling that is required to restrict diencephalic size during early forebrain development.SIGNIFICANCE STATEMENT Changes in diencephalic size and shape, as well as SNPs associated with retinoic acid (RA) signaling-associated genes, have been linked to neuropsychiatric disorders. However, the mechanisms that regulate diencephalic morphogenesis and the involvement of RA signaling in this process are poorly understood. Here we demonstrate a novel role of the acetyltransferase GCN5 in a previously undescribed mechanism of RA signaling in the developing forebrain that is required to maintain the appropriate size of the diencephalon. Together, our experiments identify a novel nonhistone substrate of GCN5, highlight an essential role for both GCN5 and RA signaling in early diencephalic development, and elucidate a novel molecular regulatory mechanism for RA signaling that is specific to the developing forebrain.
Assuntos
Diencéfalo/anatomia & histologia , Diencéfalo/metabolismo , Transdução de Sinais/fisiologia , Tretinoína/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Animais , Diencéfalo/embriologia , Ativação Enzimática , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tamanho do Órgão/fisiologiaRESUMO
Myopathies decrease muscle functionality. Mutations in ryanodine receptor 1 (RyR1) are often associated with myopathies with microscopic core-like structures in the muscle fiber. In this study, we identify a mouse RyR1 model in which heterozygous animals display clinical and pathological hallmarks of myopathy with core-like structures. The RyR1 mutation decreases sensitivity to activated calcium release and myoplasmic calcium levels, subsequently affecting mitochondrial calcium and ATP production. Mutant muscle shows a persistent potassium leak and disrupted expression of regulators of potassium homeostasis. Inhibition of KATP channels or increasing interstitial potassium by diet or FDA-approved drugs can reverse the muscle weakness, fatigue-like physiology and pathology. We identify regulators of potassium homeostasis as biomarkers of disease that may reveal therapeutic targets in human patients with myopathy of central core disease (CCD). Altogether, our results suggest that amelioration of potassium leaks through potassium homeostasis mechanisms may minimize muscle damage of myopathies due to certain RyR1 mutations.
Assuntos
Doenças Musculares/patologia , Potássio/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Biomarcadores/metabolismo , Biópsia , Cálcio/metabolismo , Dieta , Etilnitrosoureia , Regulação da Expressão Gênica/efeitos dos fármacos , Glibureto/farmacologia , Heterozigoto , Homeostase/efeitos dos fármacos , Humanos , Canais KATP/metabolismo , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Músculo Esquelético/ultraestrutura , Doenças Musculares/genética , Mutação/genética , Miopatia da Parte Central/genética , Miopatia da Parte Central/patologia , NAD/metabolismo , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
The formation of the embryonic brain and spinal cord begins as the neural plate bends to form the neural folds, which meet and adhere to close the neural tube. The neural ectoderm and surrounding tissues also coordinate proliferation, differentiation, and patterning. This highly orchestrated process is susceptible to disruption, leading to neural tube defects (NTDs), a common birth defect. Here, we highlight genetic and epigenetic contributions to neural tube closure. We describe an online database we created as a resource for researchers, geneticists, and clinicians. Neural tube closure is sensitive to environmental influences, and we discuss disruptive causes, preventative measures, and possible mechanisms. New technologies will move beyond candidate genes in small cohort studies toward unbiased discoveries in sporadic NTD cases. This will uncover the genetic complexity of NTDs and critical gene-gene interactions. Animal models can reveal the causative nature of genetic variants, the genetic interrelationships, and the mechanisms underlying environmental influences.
Assuntos
Encéfalo/crescimento & desenvolvimento , Epigênese Genética , Tubo Neural/crescimento & desenvolvimento , Medula Espinal/crescimento & desenvolvimento , Animais , Encéfalo/embriologia , Feminino , Crista Neural/embriologia , Crista Neural/crescimento & desenvolvimento , Placa Neural/embriologia , Placa Neural/crescimento & desenvolvimento , Tubo Neural/embriologia , Medula Espinal/embriologiaRESUMO
Cornelia de Lange syndrome (CdLS) is a multisystem genetic disorder with distinct facies, growth failure, intellectual disability, distal limb anomalies, gastrointestinal and neurological disease. Mutations in NIPBL, encoding a cohesin regulatory protein, account for >80% of cases with typical facies. Mutations in the core cohesin complex proteins, encoded by the SMC1A, SMC3 and RAD21 genes, together account for â¼5% of subjects, often with atypical CdLS features. Recently, we identified mutations in the X-linked gene HDAC8 as the cause of a small number of CdLS cases. Here, we report a cohort of 38 individuals with an emerging spectrum of features caused by HDAC8 mutations. For several individuals, the diagnosis of CdLS was not considered prior to genomic testing. Most mutations identified are missense and de novo. Many cases are heterozygous females, each with marked skewing of X-inactivation in peripheral blood DNA. We also identified eight hemizygous males who are more severely affected. The craniofacial appearance caused by HDAC8 mutations overlaps that of typical CdLS but often displays delayed anterior fontanelle closure, ocular hypertelorism, hooding of the eyelids, a broader nose and dental anomalies, which may be useful discriminating features. HDAC8 encodes the lysine deacetylase for the cohesin subunit SMC3 and analysis of the functional consequences of the missense mutations indicates that all cause a loss of enzymatic function. These data demonstrate that loss-of-function mutations in HDAC8 cause a range of overlapping human developmental phenotypes, including a phenotypically distinct subgroup of CdLS.
Assuntos
Fontanelas Cranianas/anormalidades , Síndrome de Cornélia de Lange/enzimologia , Anormalidades do Olho/enzimologia , Genes Ligados ao Cromossomo X , Histona Desacetilases/genética , Hipertelorismo/enzimologia , Proteínas Repressoras/genética , Sequência de Aminoácidos , Criança , Pré-Escolar , Estudos de Coortes , Fontanelas Cranianas/enzimologia , Síndrome de Cornélia de Lange/genética , Anormalidades do Olho/genética , Feminino , Histona Desacetilases/química , Histona Desacetilases/metabolismo , Humanos , Hipertelorismo/genética , Lactente , Masculino , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Fenótipo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Alinhamento de SequênciaRESUMO
Cornelia de Lange syndrome (CdLS) is a dominantly inherited congenital malformation disorder, caused by mutations in the cohesin-loading protein NIPBL for nearly 60% of individuals with classical CdLS, and by mutations in the core cohesin components SMC1A (~5%) and SMC3 (<1%) for a smaller fraction of probands. In humans, the multisubunit complex cohesin is made up of SMC1, SMC3, RAD21 and a STAG protein. These form a ring structure that is proposed to encircle sister chromatids to mediate sister chromatid cohesion and also has key roles in gene regulation. SMC3 is acetylated during S-phase to establish cohesiveness of chromatin-loaded cohesin, and in yeast, the class I histone deacetylase Hos1 deacetylates SMC3 during anaphase. Here we identify HDAC8 as the vertebrate SMC3 deacetylase, as well as loss-of-function HDAC8 mutations in six CdLS probands. Loss of HDAC8 activity results in increased SMC3 acetylation and inefficient dissolution of the 'used' cohesin complex released from chromatin in both prophase and anaphase. SMC3 with retained acetylation is loaded onto chromatin, and chromatin immunoprecipitation sequencing analysis demonstrates decreased occupancy of cohesin localization sites that results in a consistent pattern of altered transcription seen in CdLS cell lines with either NIPBL or HDAC8 mutations.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Síndrome de Cornélia de Lange/genética , Síndrome de Cornélia de Lange/metabolismo , Histona Desacetilases/genética , Mutação/genética , Proteínas Repressoras/genética , Acetilação , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Anáfase , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteoglicanas de Sulfatos de Condroitina/química , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Cromatina/genética , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Proteínas Cromossômicas não Histona/química , Cristalografia por Raios X , Proteínas de Ligação a DNA , Feminino , Fibroblastos , Células HeLa , Histona Desacetilases/química , Histona Desacetilases/deficiência , Histona Desacetilases/metabolismo , Humanos , Masculino , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Prófase , Conformação Proteica , Proteínas/genética , Proteínas Repressoras/química , Proteínas Repressoras/deficiência , Proteínas Repressoras/metabolismo , Transcrição Gênica , CoesinasRESUMO
The evolutionarily conserved cohesin complex was originally described for its role in regulating sister-chromatid cohesion during mitosis and meiosis. Cohesin and its regulatory proteins have been implicated in several human developmental disorders, including Cornelia de Lange (CdLS) and Roberts syndromes. Here we show that human mutations in the integral cohesin structural protein RAD21 result in a congenital phenotype consistent with a "cohesinopathy." Children with RAD21 mutations display growth retardation, minor skeletal anomalies, and facial features that overlap findings in individuals with CdLS. Notably, unlike children with mutations in NIPBL, SMC1A, or SMC3, these individuals have much milder cognitive impairment than those with classical CdLS. Mechanistically, these mutations act at the RAD21 interface with the other cohesin proteins STAG2 and SMC1A, impair cellular DNA damage response, and disrupt transcription in a zebrafish model. Our data suggest that, compared to loss-of-function mutations, dominant missense mutations result in more severe functional defects and cause worse structural and cognitive clinical findings. These results underscore the essential role of RAD21 in eukaryotes and emphasize the need for further understanding of the role of cohesin in human development.