RESUMEN
Spinal and bulbar muscular atrophy (SBMA) is an inherited neuromuscular disorder caused by a polyglutamine (polyQ) repeat expansion in the androgen receptor (AR). PolyQ-AR neurotoxicity may involve generation of an N-terminal truncation fragment, as such peptides occur in SBMA patients and mouse models. To elucidate the basis of SBMA, we expressed N-terminal truncated AR in motor neuron-derived cells and primary cortical neurons. Accumulation of polyQ-AR truncation fragments in the cytosol resulted in neurodegeneration and apoptotic, caspase-dependent cell death. Using primary neurons from mice transgenic or deficient for apoptosis-related genes, we determined that polyQ-AR apoptotic activation is fully dependent on Bax. Jun N-terminal kinase (JNK) was required for apoptotic pathway activation through phosphorylation of c-Jun. Expression of polyQ-AR in DP5/Hrk null neurons yielded significant protection against apoptotic activation, but absence of Bim did not provide protection, apparently due to compensatory upregulation of DP5/Hrk or other BH3-only proteins. Misfolded AR protein in the cytosol thus initiates a cascade of events beginning with JNK and culminating in Bax-dependent, intrinsic pathway activation, mediated in part by DP5/Hrk. As apoptotic mediators are candidates for toxic fragment generation and other cellular processes linked to neuron dysfunction, delineation of the apoptotic activation pathway induced by polyQ-expanded AR may shed light on the pathogenic cascade in SBMA and other motor neuron diseases.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/genética , Neuropéptidos/metabolismo , Péptidos/metabolismo , Receptores Androgénicos/metabolismo , Expansión de Repetición de Trinucleótido/genética , Proteína X Asociada a bcl-2/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Línea Celular , Células Cultivadas , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Sistema Nervioso Central/fisiopatología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Ratones Transgénicos , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/fisiopatología , Neuronas/metabolismo , Neuronas/patología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Pliegue de Proteína , Receptores Androgénicos/química , Receptores Androgénicos/genética , Transducción de Señal/genéticaRESUMEN
Huntington's disease (HD) is a fatal, dominantly inherited disorder caused by polyglutamine repeat expansion in the huntingtin (htt) gene. Here, we observe that HD mice develop hypothermia associated with impaired activation of brown adipose tissue (BAT). Although sympathetic stimulation of PPARgamma coactivator 1alpha (PGC-1alpha) was intact in BAT of HD mice, uncoupling protein 1 (UCP-1) induction was blunted. In cultured cells, expression of mutant htt suppressed UCP-1 promoter activity; this was reversed by PGC-1alpha expression. HD mice showed reduced food intake and increased energy expenditure, with dysfunctional BAT mitochondria. PGC-1alpha is a known regulator of mitochondrial function; here, we document reduced expression of PGC-1alpha target genes in HD patient and mouse striatum. Mitochondria of HD mouse brain show reduced oxygen consumption rates. Finally, HD striatal neurons expressing exogenous PGC-1alpha were resistant to 3-nitropropionic acid treatment. Altered PGC-1alpha function may thus link transcription dysregulation and mitochondrial dysfunction in HD.
Asunto(s)
Tejido Adiposo Pardo/fisiopatología , Regulación de la Temperatura Corporal/genética , Proteínas de Choque Térmico/metabolismo , Enfermedad de Huntington/etiología , Factores de Transcripción/metabolismo , Animales , Temperatura Corporal/genética , Células Cultivadas , Modelos Animales de Enfermedad , Proteínas de Choque Térmico/genética , Enfermedad de Huntington/genética , Enfermedad de Huntington/metabolismo , Ratones , Ratones Transgénicos , Mitocondrias/fisiología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Transducción de Señal/genética , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Parkinson's disease (PD) is a neurodegenerative disorder characterized by fibrillar aggregates of alpha-synuclein in characteristic inclusions known as "Lewy bodies". As mutations altering alpha-synuclein structure or increasing alpha-synuclein expression level can cause familial forms of PD or related Lewy body disorders, alpha-synuclein is believed to play a central role in the process of neuron toxicity, degeneration and death in "synucleinopathies". beta-synuclein is closely related to alpha-synuclein and has been shown to inhibit alpha-synuclein aggregation and ameliorate alpha-synuclein neurotoxicity. We generated beta-synuclein transgenic mice and observed a marked reduction in alpha-synuclein protein expression in the cortex of mice over-expressing beta-synuclein. This reduction in alpha-synuclein protein expression was not accompanied by decreases in alpha-synuclein mRNA expression. Using the prion protein promoter alpha-synuclein A53T mouse model of PD, we demonstrated that over-expression of beta-synuclein could retard the progression of impaired motor performance, reduce alpha-synuclein aggregation and extend survival in doubly transgenic mice. We attributed the amelioration of alpha-synuclein neurotoxicity in such bigenic mice to the ability of beta-synuclein to reduce alpha-synuclein protein expression based upon I(125) autoradiography quantification. Our findings indicate that increased expression of beta-synuclein protein results in a reduction of alpha-synuclein protein expression. As increased expression of alpha-synuclein may cause or contribute to PD pathogenesis in sporadic and familial forms of disease, this observation has important implications for the development of therapies for PD.
Asunto(s)
Enfermedad de Parkinson/metabolismo , alfa-Sinucleína/metabolismo , Sinucleína beta/metabolismo , Animales , Humanos , Ratones , Ratones Transgénicos , Enfermedad de Parkinson/fisiopatología , Enfermedad de Parkinson/psicología , Priones/genética , Sinucleína beta/genéticaRESUMEN
In the mouse, Purkinje cell degeneration (pcd) is a recessive mutation characterized by degeneration of cerebellar Purkinje cells, retinal photoreceptors, olfactory bulb mitral neurons, and certain thalamic neurons, and is accompanied by defective spermatogenesis. Previous studies of pcd have led to the identification of Nna1 as the causal gene; however, how loss of Nna1 function results in neurodegeneration remains unresolved. One useful approach for establishing which functional domains of a protein underlie a recessive phenotype has been to determine the genetic basis of the various alleles at the locus of interest. Because none of the pcd alleles analyzed at the time of the identification of Nna1 provided insight into the molecular basis of Nna1 loss-of-function, we obtained a recent pcd remutation--pcd5J, and after determining that its phenotype is comparable to existing pcd severe alleles, we sought its genetic basis by sequencing Nna1. In this article we report that pcd5J results from the insertion of a single GAC triplet encoding an aspartic acid residue at position 775 of Nna1. Although this insertion does not affect Nna1 expression at the RNA level, Nna1pcd-5J protein expression is markedly decreased. Pulse-chase experiments reveal that the aspartic acid insertion dramatically destabilizes Nna1pcd-5J protein, accounting for the observation that pcd5J is a severe allele. The presence of a readily detectable genetic mutation in pcd5J confirms that Nna1 loss-of-function alone underlies the broad pcd phenotype and will facilitate further studies of how Nna1 loss-of-function produces neurodegeneration and defective spermatogenesis in pcd mice.
Asunto(s)
Ácido Aspártico/genética , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Degeneración Nerviosa/patología , Células de Purkinje/fisiología , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/genética , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/metabolismo , Alelos , Secuencia de Aminoácidos , Animales , Ácido Aspártico/metabolismo , Línea Celular , Cerebelo/metabolismo , Cerebelo/patología , Proteínas de Unión al GTP/biosíntesis , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Mutación , Degeneración Nerviosa/genética , Degeneración Nerviosa/metabolismo , ARN Mensajero/biosíntesis , Retina/metabolismo , Retina/patología , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/biosíntesisRESUMEN
In higher plants, the transcription of plastid genes is mediated by at least two types of RNA polymerase (RNAP); a plastid-encoded bacterial RNAP in which promoter specificity is conferred by nuclear-encoded sigma factors, and a nuclear-encoded phage-like RNAP. Green algae, however, appear to possess only the bacterial enzyme. Since transcription of much, if not most, of the chloroplast genome in Chlamydomonas reinhardtii is regulated by the circadian clock and the nucleus, we sought to identify sigma factor genes that might be responsible for this regulation. We describe a nuclear gene (RPOD) that is predicted to encode an 80 kDa protein that, in addition to a predicted chloroplast transit peptide at the N-terminus, has the conserved motifs (2.1- 4.2) diagnostic of bacterial sigma-70 factors. We also identified two motifs not previously recognized for sigma factors, adjacent PEST sequences and a leucine zipper, both suggested to be involved in protein-protein interactions. PEST sequences were also found in approximately 40% of sigma factors examined, indicating they may be of general significance. Southern blot hybridization and BLAST searches of the genome and EST databases suggest that RPODmay be the only sigma factor gene in C. reinhardtii. The levels of RPODmRNA increased 2- 3-fold in the mid-to-late dark period of light-dark cycling cells, just prior to, or coincident with, the peak in chloroplast transcription. Also, the dark-period peak in RPOD mRNA persisted in cells shifted to continuous light or continuous dark for at least one cycle, indicating that RPODis under circadian clock control. These results suggest that regulation of RPODexpression contributes to the circadian clock's control of chloroplast transcription.
RESUMEN
Spinocerebellar ataxia type 7 (SCA7) is an inherited neurodegenerative disorder caused by expansion of a polyglutamine tract in the ataxin-7 protein. A unique feature of SCA7 is degeneration of photoreceptor cells in the retina, resulting in cone-rod dystrophy. In an SCA7 transgenic mouse model that we developed, it was found that the cone-rod dystrophy involves altered photoreceptor gene expression due to interference with Crx, a homeodomain transcription factor containing a glutamine-rich region. To determine the basis of the Crx-ataxin-7 interaction, Crx and ataxin-7 truncation and point mutants were generated, and the ability of mutant versions of either protein to co-immunoprecipitate the normal version of the other protein was tested. Thus Crx's ataxin-7 interaction domain was localized to its glutamine-rich region and ataxin-7's Crx binding domain was mapped to its glutamine tract. The importance of each protein's respective glutamine region for a productive interaction was confirmed by performing Crx transactivation assays in HEK293 cells and correlating the extent of Crx transcription interference with the intactness of each protein's glutamine region. It was also established that ataxin-7 must localize to the nucleus to repress Crx transactivation, and the likely nuclear localization signals were mapped to ataxin-7's carboxy-terminal region. Finally, using chromatin immunoprecipitation, it was demonstrated that Crx and ataxin-7 engage in a functionally significant interaction by co-occupying the promoter and enhancer regions of Crx-regulated retinal genes in vivo. The results suggest that one mechanism of SCA7 disease pathogenesis is transcription dysregulation, and that Crx transcription interference is a predominant factor in SCA7 cone-rod dystrophy retinal degeneration.
Asunto(s)
Regulación de la Expresión Génica , Glutamina/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas del Tejido Nervioso/genética , Degeneración Retiniana/metabolismo , Ataxias Espinocerebelosas/genética , Transactivadores/metabolismo , Animales , Ataxina-7 , Western Blotting , Núcleo Celular/metabolismo , Células Cultivadas , Cartilla de ADN , Modelos Animales de Enfermedad , Vectores Genéticos , Inmunohistoquímica , Luciferasas , Ratones , Mutagénesis , Proteínas del Tejido Nervioso/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Degeneración Retiniana/complicaciones , Degeneración Retiniana/genética , Ataxias Espinocerebelosas/complicaciones , Transcripción Genética/genética , Técnicas del Sistema de Dos Híbridos , beta-GalactosidasaRESUMEN
Spinocerebellar ataxia (SCA) type 7 is an inherited neurodegenerative disorder caused by expansion of a polyglutamine tract within the ataxin-7 protein. To determine the molecular basis of polyglutamine neurotoxicity in this and other related disorders, we produced SCA7 transgenic mice that express ataxin-7 with 24 or 92 glutamines in all neurons of the CNS, except for Purkinje cells. Transgenic mice expressing ataxin-7 with 92 glutamines (92Q) developed a dramatic neurological phenotype presenting as a gait ataxia and culminating in premature death. Despite the absence of expression of polyglutamine-expanded ataxin-7 in Purkinje cells, we documented severe Purkinje cell degeneration in 92Q SCA7 transgenic mice. We also detected an N-terminal truncation fragment of ataxin-7 in transgenic mice and in SCA7 patient material with both anti-ataxin-7 and anti-polyglutamine specific antibodies. The appearance of truncated ataxin-7 in nuclear aggregates correlates with the onset of a disease phenotype in the SCA7 mice, suggesting that nuclear localization and proteolytic cleavage may be important features of SCA7 pathogenesis. The non-cell-autonomous nature of the Purkinje cell degeneration in our SCA7 mouse model indicates that polyglutamine-induced dysfunction in adjacent or connecting cell types contributes to the neurodegeneration.