RESUMO
BACKGROUND: Recent studies in Huntington's disease (HD) mouse models and patients suggest that hippocampal neurons and their cholinergic afferents are involved in the cognitive deficits seen in the disease. Nerve growth factor (NGF) is an essential regulator of cholinergic neuronal survival and neurotransmission. OBJECTIVE: We asked whether NGF might be involved in HD and if intra-cerebroventricular infusion of NGF can rescue hippocampal cholinergic neuronal markers, restore neurogenesis, and improve the spatial working memory in R6/1 mouse model of HD. METHODS: We quantified NGF protein level by enzyme-linked immunosorbent assay (ELISA), intracerebroventricularly infused NGF, assessed cholinergic neuronal markers by Western blotting and quantitative RT-PCR, evaluated neurogenesis by immunohistochemistry, and studied spatial working memory using radial maze. RESULTS: By quantifying NGF protein in the hippocampus of the R6/1 mice at different ages, we found progressive decreases in NGF protein levels. We then increased NGF levels in the R6/1 mice through intra-cerebroventricular infusion. We observed elevations of the cholinergic neurochemical markers vesicular acetylcholine transporter (VAChT) and choline acetyltransferase (ChAT) in the hippocampus and in the septal region, which contain the cell bodies of basal forebrain cholinergic neurons (BFCNs), but not in the striatum that harbors cholinergic interneurons. Finally, we found that NGF infusion also restored hippocampal neurogenesis and improved spatial working memory. CONCLUSIONS: Our results suggest that intracerebral injections of NGF might be a valuable therapy against cognitive symptoms in HD and should be further studied in HD animal models and patients.
Assuntos
Neurônios Colinérgicos/efeitos dos fármacos , Doença de Huntington , Memória de Curto Prazo/efeitos dos fármacos , Fator de Crescimento Neural/administração & dosagem , Neurogênese/efeitos dos fármacos , Animais , Western Blotting , Neurônios Colinérgicos/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Doença de Huntington/metabolismo , Imuno-Histoquímica , Infusões Intraventriculares , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In the neurons of Huntington's disease (HD) patients, gene regulatory networks are disrupted by aberrant nuclear localization of the master transcriptional repressor REST. Emerging evidence suggests that, in addition to protein-coding genes, noncoding RNAs (ncRNAs) may also contribute to neurodegenerative processes. To discover ncRNAs that are involved in HD, we screened genome-wide data for novel, noncoding targets of REST. This identified human accelerated region 1 (HAR1), a rapidly evolving cis-antisense locus that is specifically transcribed in the nervous system. We show that REST is targeted to the HAR1 locus by specific DNA regulatory motifs, resulting in potent transcriptional repression. Consistent with other REST target genes, HAR1 levels are significantly lower in the striatum of HD patients compared with unaffected individuals. These data represent further evidence that noncoding gene expression changes accompany neurodegeneration in Huntington's disease.
Assuntos
Doença de Huntington/genética , RNA não Traduzido/metabolismo , Proteínas Repressoras/metabolismo , Células 3T3 , Animais , Sequência de Bases , Células HEK293 , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA não Traduzido/genéticaRESUMO
Hundreds of microRNAs (miRNAs) are expressed in mammalian cells, where they aid in modulating gene expression by mediating mRNA transcript cleavage and/or regulation of translation rate. Functional studies to date have demonstrated that several of these miRNAs are important during development. However, the role of miRNAs in the regulation of stem cell growth and differentiation is not well understood. We show herein that microRNA (miR)-134 levels are maximally elevated at day 4 after retinoic acid-induced differentiation or day 2 after N2B27-induced differentiation of mouse embryonic stem cells (mESCs), but this change is not observed during embryoid body differentiation. The elevation of miR-134 levels alone in mESCs enhances differentiation toward ectodermal lineages, an effect that is blocked by a miR-134 antagonist. The promotion of mESC differentiation by miR-134 is due, in part, to its direct translational attenuation of Nanog and LRH1, both of which are known positive regulators of Oct4/POU5F1 and mESC growth. Together, the data demonstrate that miR-134 alone can enhance the differentiation of mESCs to ectodermal lineages and establish a functional role for miR-134 in modulating mESC differentiation through its potential to target and regulate multiple mRNAs.
