RESUMEN
Friedreich ataxia (FRDA) is the most common inherited ataxia caused primarily by an intronic GAA.TTC triplet repeat expansion in the frataxin (FXN) gene. FXN RNA and protein levels are reduced in patients leading to progressive gait and limb ataxia, sensory loss, reduced tendon reflexes, dysarthria, absent lower limb reflexes, and loss of position and vibration sense. Neurological manifestations ensue from primary loss of dorsal root ganglia neurons and their associated axons ascending centrally in the spinal cord and peripherally in large myelinated nerves. Small noncoding RNAs such as microRNAs have been shown to be dysregulated in neurodegenerative diseases such as Alzheimer's and Huntington's disease. Here we report that hsa-miR-886-3p (miR-886-3p) was increased in patient cells as well as peripheral patient blood samples. Selective reduction in miR-886-3p by an anti-miR led to elevation of FXN message and protein levels without associated changes in histone marks at the FXN locus. Nevertheless, derepression of frataxin by a histone deacetylase inhibitor leads to a decrease in miR-886-3p. These results outline involvement of a small RNA, miR-886-3p in FRDA and a novel therapeutic approach to this disease using an anti-miR-886-3p.
Asunto(s)
Ataxia de Friedreich/genética , MicroARNs/metabolismo , Regulación hacia Arriba/genética , Línea Celular , Ataxia de Friedreich/metabolismo , Regulación de la Expresión Génica/fisiología , Histonas/metabolismo , Humanos , Proteínas de Unión a Hierro/genética , Proteínas de Unión a Hierro/metabolismo , MicroARNs/biosíntesis , Polimorfismo de Nucleótido Simple/genética , ARN Mensajero/biosíntesis , Receptor de Angiotensina Tipo 1/metabolismo , Repeticiones de Trinucleótidos/genética , FrataxinaRESUMEN
Oncogenic transformation of postmitotic neurons triggers cell death, but the identity of genes critical for degeneration remain unclear. The antitumor antibiotic mithramycin prolongs survival of mouse models of Huntington's disease in vivo and inhibits oxidative stress-induced death in cortical neurons in vitro. We had correlated protection by mithramycin with its ability to bind to GC-rich DNA and globally displace Sp1 family transcription factors. To understand how antitumor drugs prevent neurodegeneration, here we use structure-activity relationships of mithramycin analogs to discover that selective DNA-binding inhibition of the drug is necessary for its neuroprotective effect. We identify several genes (Myc, c-Src, Hif1α, and p21(waf1/cip1)) involved in neoplastic transformation, whose altered expression correlates with protective doses of mithramycin or its analogs. Most interestingly, inhibition of one these genes, Myc, is neuroprotective, whereas forced expression of Myc induces Rattus norvegicus neuronal cell death. These results support a model in which cancer cell transformation shares key genetic components with neurodegeneration.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Neuronas/efectos de los fármacos , Plicamicina/análogos & derivados , Plicamicina/farmacología , Factor de Transcripción Sp1/metabolismo , Análisis de Varianza , Animales , Animales Modificados Genéticamente , Western Blotting , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Inmunoprecipitación de Cromatina , Drosophila , Neuronas/citología , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Factor de Transcripción Sp1/genética , Relación Estructura-ActividadRESUMEN
An ideal therapeutic for stroke or spinal cord injury should promote survival and regeneration in the CNS. Arginase 1 (Arg1) has been shown to protect motor neurons from trophic factor deprivation and allow sensory neurons to overcome neurite outgrowth inhibition by myelin proteins. To identify small molecules that capture Arg1's protective and regenerative properties, we screened a hippocampal cell line stably expressing the proximal promoter region of the arginase 1 gene fused to a reporter gene against a library of compounds containing clinically approved drugs. This screen identified daidzein as a transcriptional inducer of Arg1. Both CNS and PNS neurons primed in vitro with daidzein overcame neurite outgrowth inhibition from myelin-associated glycoprotein, which was mirrored by acutely dissociated and cultured sensory neurons primed in vivo by intrathecal or subcutaneous daidzein infusion. Further, daidzein was effective in promoting axonal regeneration in vivo in an optic nerve crush model when given intraocularly without lens damage, or most importantly, when given subcutaneously after injury. Mechanistically, daidzein requires transcription and induction of Arg1 activity for its ability to overcome myelin inhibition. In contrast to canonical Arg1 activators, daidzein increases Arg1 without increasing CREB phosphorylation, suggesting its effects are cAMP-independent. Accordingly, it may circumvent known CNS side effects of some cAMP modulators. Indeed, daidzein appears to be safe as it has been widely consumed in soy products, crosses the blood-brain barrier, and is effective without pretreatment, making it an ideal candidate for development as a therapeutic for spinal cord injury or stroke.
Asunto(s)
Arginasa/genética , AMP Cíclico/metabolismo , Isoflavonas/farmacología , Regeneración Nerviosa/efectos de los fármacos , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Regiones Promotoras Genéticas/fisiología , Análisis de Varianza , Animales , Animales Recién Nacidos , Arginasa/metabolismo , Células CHO , Células Cultivadas , Cerebelo/citología , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Proteína GAP-43/metabolismo , Ganglios Espinales/citología , Ensayos Analíticos de Alto Rendimiento/métodos , Hipocampo/citología , Masculino , Glicoproteína Asociada a Mielina/farmacología , Regeneración Nerviosa/fisiología , Neuronas/citología , Enfermedades del Nervio Óptico/tratamiento farmacológico , Enfermedades del Nervio Óptico/patología , Estrés Oxidativo/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores de Estrógenos/metabolismo , Bibliotecas de Moléculas PequeñasRESUMEN
Friedreich ataxia (FRDA), the most common hereditary ataxia, is caused by mutations in the frataxin (FXN) gene. The vast majority of FRDA mutations involve expansion of a GAA*TTC-repeat tract in intron 1, which leads to an FXN mRNA deficit. Bisulfite mapping demonstrates that the region adjacent to the repeat was methylated in both unaffected and affected individuals. However, methylation was more extensive in patients. Additionally, three residues were almost completely methylation-free in unaffected individuals but almost always methylated in those with FRDA. One of these residues is located within an E-box whose deletion caused a significant drop in promoter activity in reporter assays. Elevated levels of histone H3 dimethylated on lysine 9 were seen in FRDA cells consistent with a more repressive chromatin organization. Such chromatin is known to reduce transcription elongation. This may be one way in which the expanded repeats contribute to the frataxin deficit in FRDA. Our data also suggest that repeat-mediated chromatin changes may also affect transcription initiation by blocking binding of factors that increase frataxin promoter activity. Our results also raise the possibility that the repeat-mediated increases in DNA methylation in the FXN gene in FRDA patients are secondary to the chromatin changes.
Asunto(s)
Expansión de las Repeticiones de ADN , Epigénesis Genética , Ataxia de Friedreich/genética , Intrones , Proteínas de Unión a Hierro/genética , Secuencia de Aminoácidos , Línea Celular , Cromatina/química , Metilación de ADN , Elementos E-Box , Histonas/metabolismo , Humanos , Modelos Genéticos , Datos de Secuencia Molecular , Células Musculares/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , FrataxinaRESUMEN
Fragile X syndrome, the most common heritable form of mental retardation, is caused by silencing of the FMR1 (fragile X mental retardation-1 gene). The protein product of this gene, FMRP (fragile X mental retardation protein), is thought to be involved in the translational regulation of mRNAs important for learning and memory. In mammals, there are two homologues of FMRP, namely FXR1P (fragile X-related protein 1) and FXR2P. Disruption of Fxr2 in mice produces learning and memory deficits, and Fmr1 and Fxr2 double-knockout mice have exaggerated impairments in certain neurobehavioral phenotypes relative to the single gene knockouts. This has led to the suggestion that FMR1 and FXR2 functionally overlap and that increasing the expression of FXR2P may ameliorate the symptoms of an FMRP deficiency. Interestingly, the region upstream of the FXR2 translation start site acts as a bidirectional promoter in rodents, driving transcription of an alternative transcript encoding the ABP (androgen-binding protein) [aABP (alternative ABP promoter)]. To understand the regulation of the human FXR2 gene, we cloned the evolutionarily conserved region upstream of the FXR2 translation start site and showed that it also has bidirectional promoter activity in both neuronal and muscle cells as evidenced by luciferase reporter assay studies. Alignment of the human, mouse, rat, rabbit and dog promoters reveals several highly conserved transcription factor-binding sites. Gel electrophoretic mobility-shift assays, chromatin immunoprecipitation studies and co-transfection experiments with plasmids expressing these transcription factors or dominant-negative versions of these factors showed that NF-YA (nuclear transcription factor Yalpha), AP2 (activator protein 2), Nrf1 (nuclear respiratory factor/alpha-Pal) and Sp1 (specificity protein 1) all bind to the FXR2 promoter both in vitro and in vivo and positively regulate the FXR2 promoter.
Asunto(s)
Factor de Unión a CCAAT/metabolismo , Factor Nuclear 1 de Respiración/metabolismo , Proteínas de Unión al ARN/genética , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción AP-2/metabolismo , Animales , Secuencia de Bases , Factor de Unión a CCAAT/genética , Línea Celular , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Clonación Molecular , Secuencia Conservada , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Humanos , Datos de Secuencia Molecular , Células Musculares/citología , Factor Nuclear 1 de Respiración/genética , Plásmidos/genética , Regiones Promotoras Genéticas , Conejos , Alineación de Secuencia , Factor de Transcripción Sp1/genética , Factor de Transcripción AP-2/genética , TransfecciónRESUMEN
Japanese encephalitis (JE) remains a major public health threat with vaccination as the only measure for its prevention. Epitope-based vaccination is a promising approach for achieving protective immunity and avoid immunopathology in Japanese encephalitis virus (JEV) infection due to flavivirus cross-reactivity. We have mapped B-cell epitopes from JEV envelope protein, responsible for elicitation of neutralizing antibodies. Incorporation of T helper (T(H)) epitopes, along with these, imparted protective immunity to the host. In the present study, based on in silico epitope selection we optimized and proposed a polytope DNA construct (P-JEV) consisting B-cell and T(H) epitopes from the JEV envelope (E) protein as well as non-structural protein-1 (NS1). The immunogenicity and protective efficacy of P-JEV was assessed by in vitro and in vivo experiments. The expressed P-JEV showed reactivity in in vitro assays with JEV monoclonal antibodies. Protective efficacy of P-JEV was assessed in BALB/c mice. Our findings indicate that P-JEV may be a candidate vaccine for the prevention of JEV infection.
Asunto(s)
Virus de la Encefalitis Japonesa (Especie)/inmunología , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito T/inmunología , Vacunas contra la Encefalitis Japonesa/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Virus de la Encefalitis Japonesa (Especie)/genética , Epítopos de Linfocito B/genética , Epítopos de Linfocito T/genética , Vacunas contra la Encefalitis Japonesa/administración & dosificación , Vacunas contra la Encefalitis Japonesa/genética , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Proteínas del Envoltorio Viral/genéticaRESUMEN
Caused by a polyglutamine expansion in the huntingtin protein, Huntington's disease leads to striatal degeneration via the transcriptional dysregulation of a number of genes, including those involved in mitochondrial biogenesis. Here we show that transglutaminase 2, which is upregulated in HD, exacerbates transcriptional dysregulation by acting as a selective corepressor of nuclear genes; transglutaminase 2 interacts directly with histone H3 in the nucleus. In a cellular model of HD, transglutaminase inhibition de-repressed two established regulators of mitochondrial function, PGC-1alpha and cytochrome c and reversed susceptibility of human HD cells to the mitochondrial toxin, 3-nitroproprionic acid; however, protection mediated by transglutaminase inhibition was not associated with improved mitochondrial bioenergetics. A gene microarray analysis indicated that transglutaminase inhibition normalized expression of not only mitochondrial genes but also 40% of genes that are dysregulated in HD striatal neurons, including chaperone and histone genes. Moreover, transglutaminase inhibition attenuated degeneration in a Drosophila model of HD and protected mouse HD striatal neurons from excitotoxicity. Altogether these findings demonstrate that selective TG inhibition broadly corrects transcriptional dysregulation in HD and defines a novel HDAC-independent epigenetic strategy for treating neurodegeneration.
Asunto(s)
Proteínas de Unión al GTP/antagonistas & inhibidores , Enfermedad de Huntington/enzimología , Enfermedad de Huntington/genética , Transcripción Genética , Transglutaminasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Citocromos c/genética , Citocromos c/metabolismo , Modelos Animales de Enfermedad , Drosophila , Metabolismo Energético , Inhibidores Enzimáticos/farmacología , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Histonas/metabolismo , Humanos , Enfermedad de Huntington/metabolismo , Ratones , Mitocondrias/metabolismo , Nitrocompuestos/toxicidad , Péptidos/farmacología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Regiones Promotoras Genéticas , Propionatos/toxicidad , Proteína Glutamina Gamma Glutamiltransferasa 2 , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transglutaminasas/genética , Transglutaminasas/metabolismoRESUMEN
Decreased histone acetyltransferase activity and transcriptional dysfunction have been implicated in almost all neurodegenerative conditions. Increasing net histone acetyltransferase activity through inhibition of the histone deacetylases (HDACs) has been shown to be an effective strategy to delay or halt progression of neurological disease in cellular and rodent models. These findings have provided firm rationale for Phase I and Phase II clinical trials of HDAC inhibitors in Huntington's disease, spinal muscular atrophy, and Freidreich's ataxia. In this review, we discuss the current findings and promise of HDAC inhibition as a strategy for treating neurological disorders. Despite the fact that HDAC inhibitors are in an advanced stage of development, we suggest other approaches to modulating HDAC function that may be less toxic and more efficacious than the canonical agents developed so far.