RESUMEN
The muscleblind-like (Mbnl) family of RNA-binding proteins plays important roles in muscle and eye development and in myotonic dystrophy (DM), in which expanded CUG or CCUG repeats functionally deplete Mbnl proteins. We identified transcriptome-wide functional and biophysical targets of Mbnl proteins in brain, heart, muscle, and myoblasts by using RNA-seq and CLIP-seq approaches. This analysis identified several hundred splicing events whose regulation depended on Mbnl function in a pattern indicating functional interchangeability between Mbnl1 and Mbnl2. A nucleotide resolution RNA map associated repression or activation of exon splicing with Mbnl binding near either 3' splice site or near the downstream 5' splice site, respectively. Transcriptomic analysis of subcellular compartments uncovered a global role for Mbnls in regulating localization of mRNAs in both mouse and Drosophila cells, and Mbnl-dependent translation and protein secretion were observed for a subset of mRNAs with Mbnl-dependent localization. These findings hold several new implications for DM pathogenesis.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Distrofia Miotónica/metabolismo , Empalme del ARN , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Transcriptoma , Regiones no Traducidas 3' , Animales , Proteínas de Unión al ADN/genética , Proteínas de Drosophila , Drosophila melanogaster/metabolismo , Exones , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Mioblastos/metabolismo , Distrofia Miotónica/genética , Proteínas Nucleares , Especificidad de Órganos , Sitios de Empalme de ARN , Proteínas de Unión al ARN/genéticaRESUMEN
Understanding the normal function of the Huntingtin (HTT) protein is of significance in the design and implementation of therapeutic strategies for Huntington's disease (HD). Expansion of the CAG repeat in the HTT gene, encoding an expanded polyglutamine (polyQ) repeat within the HTT protein, causes HD and may compromise HTT's normal activity contributing to HD pathology. Here, we investigated the previously defined role of HTT in autophagy specifically through studying HTT's association with ubiquitin. We find that HTT interacts directly with ubiquitin in vitro. Tandem affinity purification was used to identify ubiquitinated and ubiquitin-associated proteins that copurify with a HTT N-terminal fragment under basal conditions. Copurification is enhanced by HTT polyQ expansion and reduced by mimicking HTT serine 421 phosphorylation. The identified HTT-interacting proteins include RNA-binding proteins (RBPs) involved in mRNA translation, proteins enriched in stress granules, the nuclear proteome, the defective ribosomal products (DRiPs) proteome and the brain-derived autophagosomal proteome. To determine whether the proteins interacting with HTT are autophagic targets, HTT knockout (KO) cells and immunoprecipitation of lysosomes were used to investigate autophagy in the absence of HTT. HTT KO was associated with reduced abundance of mitochondrial proteins in the lysosome, indicating a potential compromise in basal mitophagy, and increased lysosomal abundance of RBPs which may result from compensatory up-regulation of starvation-induced macroautophagy. We suggest HTT is critical for appropriate basal clearance of mitochondrial proteins and RBPs, hence reduced HTT proteostatic function with mutation may contribute to the neuropathology of HD.
Asunto(s)
Proteína Huntingtina , Lisosomas , Mitocondrias , Proteínas de Unión al ARN , Ubiquitina , Proteína Huntingtina/metabolismo , Proteína Huntingtina/genética , Lisosomas/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Humanos , Ubiquitina/metabolismo , Mitocondrias/metabolismo , Autofagia , Animales , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Ratones , Unión Proteica , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Péptidos/metabolismoRESUMEN
Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus that establishes life-long infection and increases the risk for the development of several cancers and autoimmune diseases. The mechanisms by which chronic EBV infection leads to subsequent disease remain incompletely understood. Lytic reactivation plays a central role in the development of EBV-driven cancers and may contribute to other EBV-associated diseases. Thus, the clinical use of antivirals as suppressive therapy for EBV lytic reactivation may aid efforts aimed at disease prevention. Current antivirals for EBV have shown limited clinical utility due to low potency or high toxicity, leaving open the need for potent antivirals suitable for long-term prophylaxis. In the present study, we show that tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide (TAF), drugs with excellent safety profiles used clinically for HIV prevention, inhibit EBV lytic DNA replication, with respective IC50 values of 0.30 µM and 84 nM. In a cell-based assay, TAF was 35- and 24-fold and TDF was 10- and 7-fold more potent than acyclovir and penciclovir, respectively, and TAF was also twice as potent as ganciclovir. The active metabolite of tenofovir prodrugs, tenofovir-diphosphate, inhibited the incorporation of dATP into a primed DNA template by the EBV DNA polymerase in vitro. In contrast to acyclovir, treatment of cells during latency for 24 h with TAF still inhibited EBV lytic DNA replication at 72 h after drug was removed. Our results suggest that tenofovir prodrugs may be particularly effective as inhibitors of EBV lytic reactivation, and that clinical studies to address critical questions about disease prevention are warranted.
Asunto(s)
Antivirales/farmacología , Replicación del ADN/efectos de los fármacos , Infecciones por Virus de Epstein-Barr/virología , Herpesvirus Humano 4/efectos de los fármacos , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Tenofovir/farmacología , Proteínas Virales/antagonistas & inhibidores , ADN Viral/genética , ADN Viral/metabolismo , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Herpesvirus Humano 4/enzimología , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiología , Humanos , Profármacos/farmacología , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
TDP-43 (encoded by the gene TARDBP) is an RNA binding protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS). However, how TARDBP mutations trigger pathogenesis remains unknown. Here, we use novel mouse mutants carrying point mutations in endogenous Tardbp to dissect TDP-43 function at physiological levels both in vitro and in vivo Interestingly, we find that mutations within the C-terminal domain of TDP-43 lead to a gain of splicing function. Using two different strains, we are able to separate TDP-43 loss- and gain-of-function effects. TDP-43 gain-of-function effects in these mice reveal a novel category of splicing events controlled by TDP-43, referred to as "skiptic" exons, in which skipping of constitutive exons causes changes in gene expression. In vivo, this gain-of-function mutation in endogenous Tardbp causes an adult-onset neuromuscular phenotype accompanied by motor neuron loss and neurodegenerative changes. Furthermore, we have validated the splicing gain-of-function and skiptic exons in ALS patient-derived cells. Our findings provide a novel pathogenic mechanism and highlight how TDP-43 gain of function and loss of function affect RNA processing differently, suggesting they may act at different disease stages.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/genética , Proteínas de Unión al ARN/genética , Empalme Alternativo/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Exones/genética , Humanos , Ratones , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Mutación , Empalme del ARN/genéticaRESUMEN
Huntington's disease (HD) is a chronic neurodegenerative disorder characterized by a late clinical onset despite ubiquitous expression of the mutant Huntingtin gene (HTT) from birth. Transcriptional dysregulation is a pivotal feature of HD. Yet, the genes that are altered in the prodromal period and their regulators, which present opportunities for therapeutic intervention, remain to be elucidated. Using transcriptional and chromatin profiling, we found aberrant transcription and changes in histone H3K27acetylation in the striatum of R6/1 mice during the presymptomatic disease stages. Integrating these data, we identified the Elk-1 transcription factor as a candidate regulator of prodromal changes in HD. Exogenous expression of Elk-1 exerted beneficial effects in a primary striatal cell culture model of HD, and adeno-associated virus-mediated Elk-1 overexpression alleviated transcriptional dysregulation in R6/1 mice. Collectively, our work demonstrates that aberrant gene expression precedes overt disease onset in HD, identifies the Elk-1 transcription factor as a key regulator linked to early epigenetic and transcriptional changes in HD, and presents evidence for Elk-1 as a target for alleviating molecular pathology in HD.
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Epigenómica , Enfermedad de Huntington/genética , Proteína Elk-1 con Dominio ets/genética , Proteína Elk-1 con Dominio ets/metabolismo , Animales , Cuerpo Estriado/metabolismo , Dependovirus , Modelos Animales de Enfermedad , Histonas/metabolismo , Proteína Huntingtina/genética , Enfermedad de Huntington/tratamiento farmacológico , Ratones , Ratones Transgénicos , Neuronas/metabolismo , Proteínas Nucleares/metabolismoRESUMEN
Neuroinflammation is an important contributor to neuronal pathology and death in neurodegenerative diseases and neuronal injury. Therapeutic interventions blocking the activity of the inflammatory kinase IKKß, a key regulator of neuroinflammatory pathways, is protective in several animal models of neurodegenerative disease and neuronal injury. In Huntington's disease (HD), however, significant questions exist as to the impact of blocking or diminishing the activity of IKKß on HD pathology given its potential role in Huntingtin (HTT) degradation. In cell culture, IKKß phosphorylates HTT serine (S) 13 and activates HTT degradation, a process that becomes impaired with polyQ expansion. To investigate the in vivo relationship of IKKß to HTT S13 phosphorylation and HD progression, we crossed conditional tamoxifen-inducible IKKß knockout mice with R6/1 HD mice. Behavioral assays in these mice showed a significant worsening of HD pathological phenotypes. The increased behavioral pathology correlated with reduced levels of endogenous mouse full-length phospho-S13 HTT, supporting the importance of IKKß in the phosphorylation of HTT S13 in vivo. Notably, many striatal autophagy genes were up-regulated in HD vs. control mice; however, IKKß knockout partially reduced this up-regulation in HD, increased striatal neurodegeneration, and enhanced an activated microglial response. We propose that IKKß is protective in striatal neurons early in HD progression via phosphorylation of HTT S13. As IKKß is also required for up-regulation of some autophagy genes and HTT is a scaffold for selective autophagy, IKKß may influence autophagy through multiple mechanisms to maintain healthy striatal function, thereby reducing neuronal degeneration to slow HD onset.
Asunto(s)
Enfermedad de Huntington , Quinasa I-kappa B , Animales , Autofagia/genética , Cuerpo Estriado/citología , Cuerpo Estriado/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Masculino , Ratones , Ratones Noqueados , Microglía/citología , Microglía/patología , Fosforilación/genéticaRESUMEN
CalDAG-GEFI (CDGI) is a protein highly enriched in the striatum, particularly in the principal spiny projection neurons (SPNs). CDGI is strongly down-regulated in two hyperkinetic conditions related to striatal dysfunction: Huntington's disease and levodopa-induced dyskinesia in Parkinson's disease. We demonstrate that genetic deletion of CDGI in mice disrupts dendritic, but not somatic, M1 muscarinic receptors (M1Rs) signaling in indirect pathway SPNs. Loss of CDGI reduced temporal integration of excitatory postsynaptic potentials at dendritic glutamatergic synapses and impaired the induction of activity-dependent long-term potentiation. CDGI deletion selectively increased psychostimulant-induced repetitive behaviors, disrupted sequence learning, and eliminated M1R blockade of cocaine self-administration. These findings place CDGI as a major, but previously unrecognized, mediator of cholinergic signaling in the striatum. The effects of CDGI deletion on the self-administration of drugs of abuse and its marked alterations in hyperkinetic extrapyramidal disorders highlight CDGI's therapeutic potential.
Asunto(s)
Dendritas , Factores de Intercambio de Guanina Nucleótido/genética , Neostriado/fisiopatología , Plasticidad Neuronal , Sistema Nervioso Parasimpático/fisiopatología , Sinapsis , Animales , Enfermedades de los Ganglios Basales/genética , Enfermedades de los Ganglios Basales/fisiopatología , Enfermedades de los Ganglios Basales/psicología , Estimulantes del Sistema Nervioso Central/farmacología , Potenciales Postsinápticos Excitadores/genética , Hipercinesia/genética , Hipercinesia/psicología , Potenciación a Largo Plazo , Masculino , Ratones , Ratones Noqueados , Actividad Motora , Polimorfismo de Nucleótido Simple , Receptor Muscarínico M1/genética , Receptor Muscarínico M1/fisiología , Trastornos Relacionados con Sustancias/genética , Trastornos Relacionados con Sustancias/fisiopatología , Trastornos Relacionados con Sustancias/psicologíaRESUMEN
Myotonic dystrophy (dystrophia myotonica, DM) is a multi-systemic disease caused by expanded CTG or CCTG microsatellite repeats. Characterized by symptoms in muscle, heart and central nervous system, among others, it is one of the most variable diseases known. A major pathogenic event in DM is the sequestration of muscleblind-like proteins by CUG or CCUG repeat-containing RNAs transcribed from expanded repeats, and differences in the extent of MBNL sequestration dependent on repeat length and expression level may account for some portion of the variability. However, many other cellular pathways are reported to be perturbed in DM, and the severity of specific disease symptoms varies among individuals. To help understand this variability and facilitate research into DM, we generated 120 RNASeq transcriptomes from skeletal and heart muscle derived from healthy and DM1 biopsies and autopsies. A limited number of DM2 and Duchenne muscular dystrophy samples were also sequenced. We analyzed splicing and gene expression, identified tissue-specific changes in RNA processing and uncovered transcriptome changes strongly correlating with muscle strength. We created a web resource at http://DMseq.org that hosts raw and processed transcriptome data and provides a lightweight, responsive interface that enables browsing of processed data across the genome.
Asunto(s)
Músculo Esquelético/metabolismo , Miocardio/metabolismo , Distrofia Miotónica/genética , Adulto , Empalme Alternativo/genética , Secuencia de Bases , Femenino , Perfilación de la Expresión Génica/métodos , Corazón/fisiología , Humanos , Masculino , Repeticiones de Microsatélite/genética , Músculo Esquelético/fisiología , Distrofia Miotónica/metabolismo , Análisis de Componente Principal , ARN/genética , Empalme del ARN/genética , Proteínas de Unión al ARN/metabolismo , Transcriptoma/genéticaRESUMEN
Disruptive or excessive repetitive motor patterns (stereotypies) are cardinal symptoms in numerous neuropsychiatric disorders. Stereotypies are also evoked by psychomotor stimulants such as amphetamine. The acquisition of motor sequences is paralleled by changes in activity patterns in the striatum, and stereotypies have been linked to abnormal plasticity in these reinforcement-related circuits. Here, we designed experiments in mice to identify transcriptomic changes that underlie striatal plasticity occurring alongside the development of drug-induced stereotypic behavior. We identified three schedules of amphetamine treatment inducing different degrees of stereotypy and used bulk RNAseq to compare striatal gene expression changes among groups of mice treated with the different drug-dose schedules and vehicle-treated, cage-mate controls. Mice were identified as naïve, sensitized, or tolerant to drug-induced stereotypy. All drug-treated groups exhibited expression changes in genes that encode members of the extracellular signal-regulated kinase (ERK) cascades known to regulate psychomotor stimulant responses. In the sensitized group with the most prolonged stereotypy, we found dysregulation of 20 genes that were not changed in other groups. Gene set enrichment analysis indicated highly significant overlap with genes regulated by neuregulin 1 (Nrg1). Nrg1 is known to be a schizophrenia and autism susceptibility gene that encodes a ligand for Erb-B receptors, which are involved in neuronal migration, myelination, and cell survival, including that of dopamine-containing neurons. Stimulant abuse is a risk factor for schizophrenia onset, and these two disorders share behavioral stereotypy phenotypes. Our results raise the possibility that drug-induced sensitization of the Nrg1 signaling pathway might underlie these links.
Asunto(s)
Preparaciones Farmacéuticas , Transcriptoma , Anfetamina , Animales , Cuerpo Estriado , Ratones , Conducta EstereotipadaRESUMEN
Identifying large expansions of short tandem repeats (STRs), such as those that cause amyotrophic lateral sclerosis (ALS) and fragile X syndrome, is challenging for short-read whole-genome sequencing (WGS) data. A solution to this problem is an important step toward integrating WGS into precision medicine. We developed a software tool called ExpansionHunter that, using PCR-free WGS short-read data, can genotype repeats at the locus of interest, even if the expanded repeat is larger than the read length. We applied our algorithm to WGS data from 3001 ALS patients who have been tested for the presence of the C9orf72 repeat expansion with repeat-primed PCR (RP-PCR). Compared against this truth data, ExpansionHunter correctly classified all (212/212, 95% CI [0.98, 1.00]) of the expanded samples as either expansions (208) or potential expansions (4). Additionally, 99.9% (2786/2789, 95% CI [0.997, 1.00]) of the wild-type samples were correctly classified as wild type by this method with the remaining three samples identified as possible expansions. We further applied our algorithm to a set of 152 samples in which every sample had one of eight different pathogenic repeat expansions, including those associated with fragile X syndrome, Friedreich's ataxia, and Huntington's disease, and correctly flagged all but one of the known repeat expansions. Thus, ExpansionHunter can be used to accurately detect known pathogenic repeat expansions and provides researchers with a tool that can be used to identify new pathogenic repeat expansions.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Expansión de las Repeticiones de ADN , Secuenciación Completa del Genoma/métodos , Algoritmos , Proteína C9orf72/genética , Bases de Datos Genéticas , Humanos , Medicina de Precisión , Sensibilidad y Especificidad , Programas InformáticosRESUMEN
MicroRNAs (miRNAs) regulate physiological and pathological processes by inducing posttranscriptional repression of target messenger RNAs (mRNAs) via incompletely understood mechanisms. To discover factors required for human miRNA activity, we performed an RNAi screen using a reporter cell line of miRNA-mediated repression of translation initiation. We report that reduced expression of ribosomal protein genes (RPGs) dissociated miRNA complexes from target mRNAs, leading to increased polysome association, translation, and stability of miRNA-targeted mRNAs relative to untargeted mRNAs. RNA sequencing of polysomes indicated substantial overlap in sets of genes exhibiting increased or decreased polysomal association after Argonaute or RPG knockdowns, suggesting similarity in affected pathways. miRNA profiling of monosomes and polysomes demonstrated that miRNAs cosediment with ribosomes. RPG knockdowns decreased miRNAs in monosomes and increased their target mRNAs in polysomes. Our data show that most miRNAs repress translation and that the levels of RPGs modulate miRNA-mediated repression of translation initiation.
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MicroARNs/fisiología , Iniciación de la Cadena Peptídica Traduccional/genética , Proteínas Ribosómicas/genética , Células HeLa , Humanos , MicroARNs/genética , Interferencia de ARN , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/fisiología , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The dopamine systems of the brain powerfully influence movement and motivation. We demonstrate that striatonigral fibers originating in striosomes form highly unusual bouquet-like arborizations that target bundles of ventrally extending dopamine-containing dendrites and clusters of their parent nigral cell bodies. Retrograde tracing showed that these clustered cell bodies in turn project to the striatum as part of the classic nigrostriatal pathway. Thus, these striosome-dendron formations, here termed "striosome-dendron bouquets," likely represent subsystems with the nigro-striato-nigral loop that are affected in human disorders including Parkinson's disease. Within the bouquets, expansion microscopy resolved many individual striosomal fibers tightly intertwined with the dopamine-containing dendrites and also with afferents labeled by glutamatergic, GABAergic, and cholinergic markers and markers for astrocytic cells and fibers and connexin 43 puncta. We suggest that the striosome-dendron bouquets form specialized integrative units within the dopamine-containing nigral system. Given evidence that striosomes receive input from cortical regions related to the control of mood and motivation and that they link functionally to reinforcement and decision-making, the striosome-dendron bouquets could be critical to dopamine-related function in health and disease.
Asunto(s)
Dopamina/metabolismo , Neuronas Dopaminérgicas/ultraestructura , Enfermedad de Parkinson/fisiopatología , Sustancia Negra/ultraestructura , Animales , Ganglios Basales/fisiología , Ganglios Basales/ultraestructura , Mapeo Encefálico , Cuerpo Estriado/metabolismo , Cuerpo Estriado/fisiología , Cuerpo Estriado/ultraestructura , Dendrímeros/química , Dendritas/fisiología , Dendritas/ultraestructura , Neuronas Dopaminérgicas/metabolismo , Humanos , Ratones , Neostriado/metabolismo , Neostriado/fisiología , Neostriado/ultraestructura , Enfermedad de Parkinson/metabolismo , Sustancia Negra/metabolismo , Sustancia Negra/fisiologíaRESUMEN
Mutations that cause neurological phenotypes are highly informative with regard to mechanisms governing human brain function and disease. We report autosomal recessive mutations in the enzyme glutamate pyruvate transaminase 2 (GPT2) in large kindreds initially ascertained for intellectual and developmental disability (IDD). GPT2 [also known as alanine transaminase 2 (ALT2)] is one of two related transaminases that catalyze the reversible addition of an amino group from glutamate to pyruvate, yielding alanine and α-ketoglutarate. In addition to IDD, all affected individuals show postnatal microcephaly and â¼80% of those followed over time show progressive motor symptoms, a spastic paraplegia. Homozygous nonsense p.Arg404* and missense p.Pro272Leu mutations are shown biochemically to be loss of function. The GPT2 gene demonstrates increasing expression in brain in the early postnatal period, and GPT2 protein localizes to mitochondria. Akin to the human phenotype, Gpt2-null mice exhibit reduced brain growth. Through metabolomics and direct isotope tracing experiments, we find a number of metabolic abnormalities associated with loss of Gpt2. These include defects in amino acid metabolism such as low alanine levels and elevated essential amino acids. Also, we find defects in anaplerosis, the metabolic process involved in replenishing TCA cycle intermediates. Finally, mutant brains demonstrate misregulated metabolites in pathways implicated in neuroprotective mechanisms previously associated with neurodegenerative disorders. Overall, our data reveal an important role for the GPT2 enzyme in mitochondrial metabolism with relevance to developmental as well as potentially to neurodegenerative mechanisms.
Asunto(s)
Encéfalo/crecimiento & desarrollo , Mitocondrias/enzimología , Enfermedades del Sistema Nervioso/genética , Transaminasas/genética , Secuencia de Aminoácidos/genética , Animales , Encéfalo/metabolismo , Encéfalo/patología , Ciclo del Ácido Cítrico/genética , Homocigoto , Humanos , Ácidos Cetoglutáricos/metabolismo , Ratones , Mitocondrias/patología , Mutación Missense , Enfermedades del Sistema Nervioso/patología , Fenotipo , Ácido Pirúvico/metabolismo , Transaminasas/metabolismoRESUMEN
BACKGROUND: Advances in DNA sequencing technology have resulted in an abundance of personalized data with challenging clinical utility and meaning for clinicians. This wealth of data has potential to dramatically impact the quality of healthcare. Nurses are at the focal point in educating patients regarding relevant healthcare needs; therefore, an understanding of sequencing technology and utilizing these data are critical. AIM: The objective of this study was to explicate the role of nurses and nurse scientists as integral members of healthcare teams in improving understanding of DNA sequencing data and translational genomics for patients. APPROACH: A history of the nurse role in newborn screening is used as an exemplar. DISCUSSION: This study serves as an exemplar on how genome sequencing has been utilized in nursing science and incorporates linkages of other omics approaches used by nurses that are included in this special issue. This special issue showcased nurse scientists conducting multi-omic research from various methods, including targeted candidate genes, pharmacogenomics, proteomics, epigenomics, and the microbiome. From this vantage point, we provide an overview of the roles of nurse scientists in genome sequencing research and provide recommendations for the best utilization of nurses and nurse scientists related to genome sequencing.
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Enfermería Neonatal/métodos , Tamizaje Neonatal/enfermería , Rol de la Enfermera , Análisis de Secuencia de ADN , Pruebas Genéticas , Genoma Humano , Humanos , Recién Nacido , Investigación Metodológica en EnfermeríaRESUMEN
Although dominant gain-of-function triplet repeat expansions in the Huntingtin (HTT) gene are the underlying cause of Huntington disease (HD), understanding the normal functions of nonmutant HTT protein has remained a challenge. We report here findings that suggest that HTT plays a significant role in selective autophagy. Loss of HTT function in Drosophila disrupts starvation-induced autophagy in larvae and conditional knockout of HTT in the mouse CNS causes characteristic cellular hallmarks of disrupted autophagy, including an accumulation of striatal p62/SQSTM1 over time. We observe that specific domains of HTT have structural similarities to yeast Atg proteins that function in selective autophagy, and in particular that the C-terminal domain of HTT shares structural similarity to yeast Atg11, an autophagic scaffold protein. To explore possible functional similarity between HTT and Atg11, we investigated whether the C-terminal domain of HTT interacts with mammalian counterparts of yeast Atg11-interacting proteins. Strikingly, this domain of HTT coimmunoprecipitates with several key Atg11 interactors, including the Atg1/Unc-51-like autophagy activating kinase 1 kinase complex, autophagic receptor proteins, and mammalian Atg8 homologs. Mutation of a phylogenetically conserved WXXL domain in a C-terminal HTT fragment reduces coprecipitation with mammalian Atg8 homolog GABARAPL1, suggesting a direct interaction. Collectively, these data support a possible central role for HTT as an Atg11-like scaffold protein. These findings have relevance to both mechanisms of disease pathogenesis and to therapeutic intervention strategies that reduce levels of both mutant and normal HTT.
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Autofagia , Proteínas Asociadas a Microtúbulos/fisiología , Animales , Animales Modificados Genéticamente , Drosophila , Proteínas de Drosophila , Proteína Huntingtina , Ratones , Proteínas Asociadas a Microtúbulos/genéticaRESUMEN
The earliest stages of Huntington disease are marked by changes in gene expression that are caused in an indirect and poorly understood manner by polyglutamine expansions in the huntingtin (HTT) protein. To explore the hypothesis that DNA methylation may be altered in cells expressing mutated HTT, we use reduced representation bisulfite sequencing (RRBS) to map sites of DNA methylation in cells carrying either wild-type or mutant HTT. We find that a large fraction of the genes that change in expression in the presence of mutant huntingtin demonstrate significant changes in DNA methylation. Regions with low CpG content, which have previously been shown to undergo methylation changes in response to neuronal activity, are disproportionately affected. On the basis of the sequence of regions that change in methylation, we identify AP-1 and SOX2 as transcriptional regulators associated with DNA methylation changes, and we confirm these hypotheses using genome-wide chromatin immunoprecipitation sequencing (ChIP-Seq). Our findings suggest new mechanisms for the effects of polyglutamine-expanded HTT. These results also raise important questions about the potential effects of changes in DNA methylation on neurogenesis and cognitive decline in patients with Huntington disease.
Asunto(s)
Metilación de ADN , Proteínas Mutantes/genética , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Animales , Línea Celular , Inmunoprecipitación de Cromatina , Islas de CpG , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Proteína Huntingtina , Enfermedad de Huntington/genética , Enfermedad de Huntington/metabolismo , Ratones , Factores de Transcripción SOXB1/metabolismo , Factor de Transcripción AP-1/metabolismoRESUMEN
Aggregation of misfolded proteins is characteristic of a number of neurodegenerative diseases, including Huntington disease (HD). The CCT/TRiC (chaperonin containing TCP-1/TCP-1 ring) chaperonin complex can inhibit aggregation and cellular toxicity induced by expanded repeat Huntingtin (mHtt) fragments. The substrate-binding apical domain of CCT/TRiC subunit CCT1, ApiCCT1, is sufficient to inhibit aggregation of expanded repeat mHtt fragments in vitro, providing therapeutic promise for HD. However, a key hurdle in considering ApiCCT1 as a potential treatment is in delivery. Because ApiCCT1 has a region of similarity to the HIV Tat protein cell-transduction domain, we tested whether recombinant ApiCCT1 (ApiCCT1(r)) protein could enter cells following exogenous delivery and modulate an established panel of mHtt-mediated cell-based phenotypes. Cell fractionation studies demonstrate that exogenous ApiCCT1(r) can penetrate cell membranes and can localize to the nucleus, consistent with a strategy that can target both cytosolic and nuclear pathogenic events in HD. ApiCCT1(r) application does indeed modulate HD cellular phenotypes by decreasing formation of visible inclusions, fibrillar oligomers, and insoluble mHtt derived from expression of a truncated mHtt exon 1 fragment. ApiCCT1(r) also delays the onset of inclusion body formation as visualized via live imaging. ApiCCT1(r) reduces mHtt-mediated toxicity in immortalized striatal cells derived from full-length knock-in HD mice, suggesting that therapeutic benefit may extend beyond effects on aggregation. These studies provide the basis for a potentially robust and unique therapeutic strategy to target mHtt-mediated protein pathogenesis.
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Chaperoninas/administración & dosificación , Mutación , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Supervivencia Celular , Chaperoninas/química , Electroforesis en Gel de Poliacrilamida , Proteína Huntingtina , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Células PC12 , Fenotipo , RatasRESUMEN
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
Transcriptional dysregulation is an early feature of Huntington disease (HD). We observed gene-specific changes in histone H3 lysine 4 trimethylation (H3K4me3) at transcriptionally repressed promoters in R6/2 mouse and human HD brain. Genome-wide analysis showed a chromatin signature for this mark. Reducing the levels of the H3K4 demethylase SMCX/Jarid1c in primary neurons reversed down-regulation of key neuronal genes caused by mutant Huntingtin expression. Finally, reduction of SMCX/Jarid1c in primary neurons from BACHD mice or the single Jarid1 in a Drosophila HD model was protective. Therefore, targeting this epigenetic signature may be an effective strategy to ameliorate the consequences of HD.
Asunto(s)
Encéfalo/metabolismo , Histonas/metabolismo , Enfermedad de Huntington/metabolismo , Lisina/metabolismo , Animales , Animales Modificados Genéticamente , Western Blotting , Encéfalo/patología , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Células Cultivadas , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Femenino , Perfilación de la Expresión Génica , Histona Demetilasas , Humanos , Proteína Huntingtina , Enfermedad de Huntington/genética , Masculino , Metilación , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Neuronas/metabolismo , Oxidorreductasas N-Desmetilantes/genética , Oxidorreductasas N-Desmetilantes/metabolismo , Regiones Promotoras Genéticas/genética , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The ability to quantitatively evaluate the impact of a potential therapeutic intervention for Huntington disease (HD) in animal models for the disease is a critical step in the pathway to development of an effective therapy for this devastating neurodegenerative disorder. We report here an approach that combines a cell-based assay's quantitative accuracy and direct relationship to molecular processes with the ability to directly monitor effects in HD model mouse neurons. To accomplish this goal, we have developed an accurate quantitative reporter assay for a transcript known to be down-regulated as an early consequence of mutant huntingtin expression. This system uses mouse strains carrying a GFP reporter for the expression of the dopamine receptor D2, expressed in the medium spiny neurons of the basal ganglion. This receptor consistently demonstrates reduced expression in patients and murine models, and the FACS-based assay gives a highly accurate and quantitative readout of this pathology in mouse neurons expressing mutant huntingtin. For four genetic models and one viral model, a highly reproducible time course of loss of reporter expression is observed. This quantitative measure of HD pathology can be used to measure the effects of HD therapeutics in small cohorts with high confidence. We further demonstrate that the introduction of an shRNA against the huntingtin transgene by virus can improve this pathological status in medium spiny neurons transduced with the construct. We believe this system can be of great utility in the validation of effective therapeutic interventions for HD.