RESUMO
In investigating the role of metal ions in the pathogenesis of Huntington's disease, we examined the effects of clioquinol, a metal-binding compound currently in clinical trials for Alzheimer's disease treatment, on mutant huntingtin-expressing cells. We found that PC12 cells expressing polyglutamine-expanded huntingtin exon 1 accumulated less mutant protein and showed decreased cell death when treated with clioquinol. This effect was polyglutamine-length-specific and did not alter mRNA levels or protein degradation rates. Clioquinol treatment of transgenic Huntington's mice (R6/2) improved behavioral and pathologic phenotypes, including decreased huntingtin aggregate accumulation, decreased striatal atrophy, improved rotarod performance, reduction of weight loss, normalization of blood glucose and insulin levels, and extension of lifespan. Our results suggest that clioquinol is a candidate therapy for Huntington's disease and other polyglutamine-expansion diseases.
Assuntos
Clioquinol/farmacologia , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Glicemia/metabolismo , Peso Corporal/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular , Feminino , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Doença de Huntington/fisiopatologia , Insulina/sangue , Masculino , Camundongos , Camundongos Transgênicos , Mutação/genética , Proteínas do Tecido Nervoso/química , Proteínas Nucleares/química , Pâncreas/metabolismo , Pâncreas/patologia , Peptídeos/genética , Peptídeos/metabolismo , RNA Mensageiro/genética , Ratos , Taxa de SobrevidaRESUMO
The multi-C2H2 zinc-finger domain containing transcriptional regulators of the spalt (SAL) family plays important developmental regulatory roles. In a competitive subtractive hybridization screen of genes expressed in Xenopus laevis hindlimb regeneration blastemas, we identified a SAL family member that, by phylogenetic analysis, falls in the same clade as human SALL4 and have designated it as XlSALL4. Mutations of human SALL4 have been linked to Okihiro syndrome, which includes preaxial (anterior) limb defects. The expression pattern of XlSALL4 transcripts during normal forelimb and hindlimb development and during hindlimb regeneration at the regeneration-competent and regeneration-incompetent stages is temporally and regionally dynamic. We show for the first time that a SAL family member (XlSALL4) is expressed at the right place and time to play a role regulating both digit identity along the anterior/posterior axis and epimorphic limb regeneration.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Membro Posterior/embriologia , Membro Posterior/crescimento & desenvolvimento , Regeneração/fisiologia , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/genética , Sequência de Aminoácidos , Animais , Biblioteca Gênica , Membro Posterior/química , Membro Posterior/metabolismo , Humanos , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Fatores de Tempo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Xenopus laevis/embriologiaRESUMO
Suppression polymerase chain reaction-based subtractive hybridization was used to identify genes that are expressed during Xenopus laevis hindlimb regeneration. Subtractions were done by using RNAs extracted from the regeneration-competent stage (stage 53) and regeneration-incompetent stage (stage 59) of limb development. Forward and reverse subtractions were done between stage 53 7-day blastema and stage 53 contralateral limb (competent stage), stage 59 7-day pseudoblastema and stage 59 contralateral limb (incompetent stage), and stage 53 7-day blastema and stage 59 7-day pseudoblastema. Several thousand clones were analyzed from the various subtracted libraries, either by random selection and sequencing (1,920) or by screening subtracted cDNA clones (6,150), arrayed on nylon membranes, with tissue-specific probes. Several hundred clones were identified from the array screens whose expression levels were at least twofold higher in experimental tissue vs. control tissue (e.g., blastema vs. limb) and selected for sequencing. In addition, primers were designed to assay several of the randomly selected clones and used to assess the level of expression of these genes during regeneration and normal limb development. Approximately half of the selected clones were differentially expressed, as expected, including several that demonstrate blastema-specific enhancement of expression. Three distinct categories of expression were identified in our screens: (1) clones that are expressed in both regeneration-competent blastemas and -incompetent pseudoblastemas, (2) clones that are expressed at highest levels in regeneration-competent blastemas, and (3) clones that are expressed at highest levels in regeneration-incompetent pseudoblastemas. Characterizing the role of each of these three categories of genes will be important in furthering our understanding of the process of tissue regeneration.
Assuntos
Extremidades/fisiologia , Expressão Gênica , Regeneração/genética , Xenopus laevis/fisiologia , Animais , Biologia Computacional , Biblioteca Gênica , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Xenopus laevis/genéticaRESUMO
Members of the myocyte enhancer factor 2 (MEF2) family of transcription factors contain highly conserved sequences within their MADS box and MEF2 domain. These motifs are required for DNA binding and dimerization properties, as well as for MEF2 association with various transcriptional activator or repressor proteins. The D-mef2 gene encodes the MEF2 protein of Drosophila and genetic studies have shown that normal D-MEF2 function is needed for muscle cell differentiation during embryogenesis and indirect flight muscle formation during pupal development. We have characterized three additional lethal alleles of D-mef2 and identified the specific mutation in each that alters a conserved amino acid present within the MADS box of all known MEF2 proteins. Mutation of these invariant residues results in the inability of mutant D-MEF2 proteins to bind DNA in vitro, muscle defects within the embryo, and adverse effects on the structure of indirect flight muscles within the adult. Since the crystal structure of a MEF2 core protein bound to DNA has been previously solved, our results correlate the mutation of specific MADS box amino acids utilized for target DNA recognition with severe myogenic phenotypes manifested during Drosophila development.