RESUMO
The neurobiological functions of a number of kinases expressed in the brain are unknown. Here, we report new findings on DCLK3 (doublecortin like kinase 3), which is preferentially expressed in neurons in the striatum and dentate gyrus. Its function has never been investigated. DCLK3 expression is markedly reduced in Huntington's disease. Recent data obtained in studies related to cancer suggest DCLK3 could have an anti-apoptotic effect. Thus, we hypothesized that early loss of DCLK3 in Huntington's disease may render striatal neurons more susceptible to mutant huntingtin (mHtt). We discovered that DCLK3 silencing in the striatum of mice exacerbated the toxicity of an N-terminal fragment of mHtt. Conversely, overexpression of DCLK3 reduced neurodegeneration produced by mHtt. DCLK3 also produced beneficial effects on motor symptoms in a knock-in mouse model of Huntington's disease. Using different mutants of DCLK3, we found that the kinase activity of the protein plays a key role in neuroprotection. To investigate the potential mechanisms underlying DCLK3 effects, we studied the transcriptional changes produced by the kinase domain in human striatal neurons in culture. Results show that DCLK3 regulates in a kinase-dependent manner the expression of many genes involved in transcription regulation and nucleosome/chromatin remodelling. Consistent with this, histological evaluation showed DCLK3 is present in the nucleus of striatal neurons and, protein-protein interaction experiments suggested that the kinase domain interacts with zinc finger proteins, including the transcriptional activator adaptor TADA3, a core component of the Spt-ada-Gcn5 acetyltransferase (SAGA) complex which links histone acetylation to the transcription machinery. Our novel findings suggest that the presence of DCLK3 in striatal neurons may play a key role in transcription regulation and chromatin remodelling in these brain cells, and show that reduced expression of the kinase in Huntington's disease could render the striatum highly vulnerable to neurodegeneration.
Assuntos
Corpo Estriado/enzimologia , Proteína Huntingtina/genética , Doença de Huntington/terapia , Mutação/genética , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Quinases Semelhantes a Duplacortina , Regulação para Baixo/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Força da Mão/fisiologia , Doença de Huntington/genética , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora , Neurônios/metabolismo , Fosfopiruvato Hidratase/metabolismo , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
A large number of gene products that are enriched in the striatum have ill-defined functions, although they may have key roles in age-dependent neurodegenerative diseases affecting the striatum, especially Huntington disease (HD). In the present study, we focused on Abhd11os, (called ABHD11-AS1 in human) which is a putative long noncoding RNA (lncRNA) whose expression is enriched in the mouse striatum. We confirm that despite the presence of 2 small open reading frames (ORFs) in its sequence, Abhd11os is not translated into a detectable peptide in living cells. We demonstrate that Abhd11os levels are markedly reduced in different mouse models of HD. We performed in vivo experiments in mice using lentiviral vectors encoding either Abhd11os or a small hairpin RNA targeting Abhd11os. Results show that Abhd11os overexpression produces neuroprotection against an N-terminal fragment of mutant huntingtin, whereas Abhd11os knockdown is protoxic. These novel results indicate that the loss lncRNA Abhd11os likely contribute to striatal vulnerability in HD. Our study emphasizes that lncRNA may play crucial roles in neurodegenerative diseases.
Assuntos
Corpo Estriado/metabolismo , Regulação para Baixo/genética , Regulação da Expressão Gênica/genética , Expressão Gênica/genética , Doença de Huntington/genética , Mutação , Proteínas do Tecido Nervoso/genética , Fármacos Neuroprotetores , Proteínas Nucleares/genética , RNA não Traduzido/genética , Serina Proteases/genética , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Proteína Huntingtina , Doença de Huntington/metabolismo , Masculino , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/genética , RNA não Traduzido/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina Proteases/metabolismoRESUMO
The mechanisms underlying preferential atrophy of the striatum in Huntington's disease (HD) are unknown. One hypothesis is that a set of gene products preferentially expressed in the striatum could determine the particular vulnerability of this brain region to mutant huntingtin (mHtt). Here, we studied the striatal protein µ-crystallin (Crym). Crym is the NADPH-dependent p38 cytosolic T3-binding protein (p38CTBP), a key regulator of thyroid hormone (TH) T3 (3,5,3'-triiodo-l-thyronine) transportation. It has been also recently identified as the enzyme that reduces the sulfur-containing cyclic ketimines, which are potential neurotransmitters. Here, we confirm the preferential expression of the Crym protein in the rodent and macaque striatum. Crym expression was found to be higher in the macaque caudate than in the putamen. Expression of Crym was reduced in the BACHD and Knock-in 140CAG mouse models of HD before onset of striatal atrophy. We show that overexpression of Crym in striatal medium-size spiny neurons using a lentiviral-based strategy in mice is neuroprotective against the neurotoxicity of an N-terminal fragment of mHtt in vivo. Thus, reduction of Crym expression in HD could render striatal neurons more susceptible to mHtt suggesting that Crym may be a key determinant of the vulnerability of the striatum. In addition our work points to Crym as a potential molecular link between striatal degeneration and the THs deregulation reported in HD patients.
Assuntos
Corpo Estriado/patologia , Cristalinas/genética , Doença de Huntington/patologia , Proteínas do Tecido Nervoso/genética , Animais , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Expressão Gênica , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Macaca , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Ratos , Cristalinas muRESUMO
Genes selectively expressed in the striatum may be involved in the preferential vulnerability of striatal neurons to Huntington's disease (HD). Here, we investigated whether perturbations of Capucin expression, which is enriched in the striatum and downregulated in Huntington's disease models, could modify the neurotoxicity induced by the injection of a lentiviral vector encoding a short N-terminal fragment of mutant Huntingtin (mHtt) into the mouse striatum. Neither constitutive Capucin deficiency in knockout mice nor lentiviral vector-mediated Capucin overexpression in the striatum of adult wild type mice significantly modified vulnerability to the mHtt fragment in vivo, suggesting that Capucin has no impact on mHtt toxicity.
Assuntos
Corpo Estriado/metabolismo , Corpo Estriado/patologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Animais , Proteína Huntingtina , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , MutaçãoRESUMO
Using serial analysis of gene expression, we collected quantitative transcriptome data in 11 regions of the adult wild-type mouse brain: the orbital, prelimbic, cingulate, motor, somatosensory, and entorhinal cortices, the caudate-putamen, the nucleus accumbens, the thalamus, the substantia nigra, and the ventral tegmental area. With >1.2 million cDNA tags sequenced, this database is a powerful resource to explore brain functions and disorders. As an illustration, we performed interregional comparisons and found 315 differential transcripts. Most of them are poorly characterized and 20% lack functional annotation. For 78 differential transcripts, we provide independent expression level measurements in mouse brain regions by real-time quantitative RT-PCR. We also show examples where we used in situ hybridization to achieve infrastructural resolution. For 30 transcripts, we next demonstrated that regional enrichment is conserved in the human brain. We then quantified the expression levels of region-enriched transcripts in the R6/2 mouse model of Huntington disease and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson disease and observed significant alterations in the striatum, cerebral cortex, thalamus and substantia nigra of R6/2 mice and in the striatum of MPTP-treated mice. These results show that the gene expression data provided here for the mouse brain can be used to explore pathophysiological models and disclose transcripts differentially expressed in human brain regions.
Assuntos
Encéfalo/metabolismo , Sequência Conservada/genética , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Doenças Neurodegenerativas/genética , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Bases de Dados Genéticas , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Transcriptome analysis of mammalian brain structures is a potentially powerful approach in addressing the diversity of cerebral functions. Here, we used a microassay for serial analysis of gene expression (SAGE) to generate quantitative mRNA expression profiles of normal adult mouse striatum, nucleus accumbens, and somatosensory cortex. Comparison of these profiles revealed 135 transcripts heterogeneously distributed in the brain. Among them, a majority (78), although matching a registered sequence, are novel regional markers. To improve the anatomical resolution of our analysis, we performed in situ hybridization and observed unique expression patterns in discrete brain regions for a number of candidates. We assessed the distribution of the new markers in peripheral tissues using quantitative RT-PCR, Northern hybridization, and published SAGE data. In most cases, expression was higher in the brain than in peripheral tissues. Because the markers were selected according to their expression level, without reference to prior knowledge, our studies provide an unbiased, comprehensive molecular signature for various mammalian brain structures that can be used to investigate their plasticity under a variety of circumstances.