RESUMEN
Huntington's disease (HD) is caused by a mutation in the Huntingtin (HTT) protein. We previously reported that the 23aa peptide of HTT protein, P42, is preventing HD pathological phenotypes, such as aggregation, reduction of motor performances and neurodegeneration. A systemic treatment with P42 during the pre-symptomatic phase of the disease showed therapeutic potential in R6/2 mice. We here tested P42 effects when administered during the post-symptomatic phase. The P42 treatment alleviated deficits in motor performances, even when symptoms have already started. Because changes in the level and activity of brain-derived neurotrophic factor (BDNF) have been shown to play a central role in HD, we analysed the influence of P42 on BDNF deficit and associated phenotypes. Our data suggest that P42 is involved in the spatio-temporal control of bdnf and trkB mRNA and their protein levels. Related to this enhancement of BDNF-TrkB signalling, R6/2 mice treated with P42, exhibit reduced anxiety, better learning and memory performances, and better long-term potentiation (LTP) response. Finally we identified a direct influence of P42 peptide on neuronal plasticity and activity. These results suggest that P42 offers an efficient therapeutic potential not only by preventing aggregation of mutant HTT at early stages of the disease, but also by favouring some physiological functions of normal HTT, as P42 is naturally part of it, at the different stages of the disease. This makes P42 peptide potentially suitable not only to prevent, but also to treat HD.
Asunto(s)
Ansiedad/tratamiento farmacológico , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/tratamiento farmacológico , Memoria/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Animales , Ansiedad/metabolismo , Ansiedad/patología , Factor Neurotrófico Derivado del Encéfalo/genética , Femenino , Humanos , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Transducción de SeñalRESUMEN
One way to better understand the molecular mechanisms involved in the construction of a nervous system is to identify the downstream effectors of major regulatory proteins. We previously showed that Engrailed (EN) and Gooseberry-Neuro (GsbN) transcription factors act in partnership to drive the formation of posterior commissures in the central nervous system of Drosophila. In this report, we identified genes regulated by both EN and GsbN through chromatin immunoprecipitation ("ChIP on chip") and transcriptome experiments, combined to a genetic screen relied to the gene dose titration method. The genomic-scale approaches allowed us to define 175 potential targets of EN-GsbN regulation. We chose a subset of these genes to examine ventral nerve cord (VNC) defects and found that half of the mutated targets show clear VNC phenotypes when doubly heterozygous with en or gsbn mutations, or when homozygous. This strategy revealed new groups of genes never described for their implication in the construction of the nerve cord. Their identification suggests that, to construct the nerve cord, EN-GsbN may act at three levels, in: (i) sequential control of the attractive-repulsive signaling that ensures contralateral projection of the commissural axons, (ii) temporal control of the translation of some mRNAs, (iii) regulation of the capability of glial cells to act as commissural guideposts for developing axons. These results illustrate how an early, coordinated transcriptional control may orchestrate the various mechanisms involved in the formation of stereotyped neuronal networks. They also validate the overall strategy to identify genes that play crucial role in axonal pathfinding.
Asunto(s)
Axones/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Animales , Sistema Nervioso Central/metabolismo , Inmunoprecipitación de Cromatina/métodos , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiología , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica/genética , Genoma , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Mutación , Neuroglía/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Transactivadores/genética , Transactivadores/fisiología , Factores de Transcripción/genética , Factores de Transcripción/fisiologíaRESUMEN
BACKGROUND: In Huntington's disease (HD), the ratio between normal and mutant Huntingtin (polyQ-hHtt) is crucial in the onset and progression of the disease. As a result, addition of normal Htt was shown to improve polyQ-hHtt-induced defects. Therefore, we recently identified, within human Htt, a 23aa peptide (P42) that prevents aggregation and polyQ-hHtt-induced phenotypes in HD Drosophila model. In this report, we evaluated the therapeutic potential of P42 in a mammalian model of the disease, R6/2 mice. RESULTS: To this end, we developed an original strategy for P42 delivery, combining the properties of the cell penetrating peptide TAT from HIV with a nanostructure-based drug delivery system (Aonys® technology), to form a water-in-oil microemulsion (referred to as NP42T) allowing non-invasive per mucosal buccal/rectal administration of P42. Using MALDI Imaging Mass Spectrometry, we verified the correct targeting of NP42T into the brain, after per mucosal administration. We then evaluated the effects of NP42T in R6/2 mice. We found that P42 (and/or derivatives) are delivered into the brain and target most of the cells, including the neurons of the striatum. Buccal/rectal daily administrations of NP42T microemulsion allowed a clear improvement of behavioural HD-associated defects (foot-clasping, rotarod and body weights), and of several histological markers (aggregation, astrogliosis or ventricular areas) recorded on brain sections. CONCLUSIONS: These data demonstrate that NP42T presents an unprecedented protective effect, and highlight a new therapeutic strategy for HD, associating an efficient peptide with a powerful delivery technology.
Asunto(s)
Cuerpo Estriado/efectos de los fármacos , Enfermedad de Huntington/tratamiento farmacológico , Actividad Motora/efectos de los fármacos , Proteínas del Tejido Nervioso/administración & dosificación , Proteínas del Tejido Nervioso/uso terapéutico , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/farmacocinética , Fragmentos de Péptidos/uso terapéutico , Administración Bucal , Administración Rectal , Animales , Cuerpo Estriado/metabolismo , Modelos Animales de Enfermedad , Femenino , Células HeLa , Humanos , Proteína Huntingtina , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/síntesis química , Proteínas del Tejido Nervioso/farmacocinética , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Prueba de Desempeño de Rotación con Aceleración ConstanteRESUMEN
BACKGROUND: Huntington's disease (HD) is caused by the abnormal expansion of the polyglutamine tract in the human Huntingtin protein (polyQ-hHtt). Although this mutation behaves dominantly, huntingtin loss of function also contributes to HD pathogenesis. Indeed, wild-type Huntingtin plays a protective role with respect to polyQ-hHtt induced defects. METHODOLOGY/PRINCIPAL FINDINGS: The question that we addressed here is what part of the wild-type Huntingtin is responsible for these protective properties. We first screened peptides from the Huntingtin protein in HeLa cells and identified a 23 aa peptide (P42) that inhibits polyQ-hHtt aggregation. P42 is part of the endogenous Huntingtin protein and lies within a region rich in proteolytic sites that plays a critical role in the pathogenesis process. Using a Drosophila model of HD, we tested the protective properties of this peptide on aggregation, as well as on different polyQ-hHtt induced neuronal phenotypes: eye degeneration (an indicator of cell death), impairment of vesicular axonal trafficking, and physiological behaviors such as larval locomotion and adult survival. Together, our results demonstrate high protective properties for P42 in vivo, in whole animals. These data also demonstrate a specific role of P42 on Huntington's disease model, since it has no effect on other models of polyQ-induced diseases, such as spinocerebellar ataxias. CONCLUSIONS/SIGNIFICANCE: Altogether our data show that P42, a 23 aa-long hHtt peptide, plays a protective role with respect to polyQ-hHtt aggregation as well as cellular and behavioral dysfunctions induced by polyQ-hHtt in vivo. Our study also confirms the correlation between polyQ-hHtt aggregation and neuronal defects. Finally, these results strongly suggest a therapeutic potential for P42, specific of Huntington's disease.
Asunto(s)
Drosophila melanogaster/efectos de los fármacos , Enfermedad de Huntington/metabolismo , Larva/efectos de los fármacos , Proteínas del Tejido Nervioso/química , Oligopéptidos/farmacología , Péptidos/química , Secuencia de Aminoácidos , Animales , Modelos Animales de Enfermedad , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Ojo/efectos de los fármacos , Ojo/metabolismo , Ojo/patología , Femenino , Regulación de la Expresión Génica , Células HeLa , Humanos , Proteína Huntingtina , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Masculino , Datos de Secuencia Molecular , Actividad Motora , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Oligopéptidos/química , Oligopéptidos/aislamiento & purificación , Péptidos/metabolismo , Unión Proteica , Multimerización de Proteína/efectos de los fármacos , Transporte de ProteínasRESUMEN
p53 is a key tumor suppressor that controls DNA damage response and genomic integrity. In response to genotoxic stress, p53 is stabilized and activated, resulting in controlled activation of genes involved in cell cycle arrest, DNA repair and/or apoptosis. ASAP is a centrosome- and spindle-associated protein, the deregulation of which induces severe mitotic defects. We show here that following double-strand break DNA formation, ASAP directly interacts with and stabilizes p53 by enhancing its p300-mediated acetylation and blocking its MDM2-mediated ubiquitination and degradation, leading to an increase of p53 transcriptional activity. Upon DNA damage, ASAP is transiently accumulated before being degraded upon persistent damage. This work links the p53 response with the cytoskeleton and confirms that the DNA-damaging signaling pathway is coordinated by centrosomal proteins. We reveal the existence of a new pathway through which ASAP signals the DNA damage response by regulating the p300-MDM2-p53 loop. These results point out ASAP as a possible target for the design of drugs to sensitize radio-resistant tumors.
Asunto(s)
Daño del ADN , Proteínas Asociadas a Microtúbulos/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Acetilación , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Proteína p300 Asociada a E1A/metabolismo , Células HEK293 , Humanos , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteína p53 Supresora de Tumor/genética , UbiquitinaciónRESUMEN
Homeodomain transcription factors classically exert their morphogenetic activities through the cell-autonomous regulation of developmental programs. In vertebrates, several homeoproteins have also been shown to have direct non-cell-autonomous activities in the developing nervous system. We present the first in vivo evidence for homeoprotein signaling in Drosophila. Focusing on wing development as a model, we first demonstrate that the homeoprotein Engrailed (En) is secreted. Using single-chain anti-En antibodies expressed under the control of a variety of promoters, we delineate the wing territories in which secreted En acts. We show that En is a short-range signaling molecule that participates in anterior crossvein development, interacting with the Dpp signaling pathway. This report thus suggests that direct signaling with homeoproteins is an evolutionarily conserved phenomenon that is not restricted to neural tissues and involves interactions with bona fide signal transduction pathways.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/embriología , Proteínas de Homeodominio/metabolismo , Factores de Transcripción/metabolismo , Alas de Animales/embriología , Animales , Animales Modificados Genéticamente , Anticuerpos , Drosophila/genética , Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Proteínas de Homeodominio/inmunología , Humanos , Transducción de Señal , Factores de Transcripción/inmunología , Alas de Animales/metabolismoRESUMEN
Huntington's disease (HD) is caused by the expansion of the polyglutamine (polyQ) tract in the human Huntingtin (hHtt) protein (polyQ-hHtt). Although this mutation behaves dominantly, htt loss of function may also contribute to HD pathogenesis. Using a Drosophila model of HD, we found that Engrailed (EN), a transcriptional activator of endogenous Drosophila htt (dhtt), is able to prevent aggregation of polyQ-hHtt. To interpret these findings, we tested and identified a protective role of N-terminal fragments of both Drosophila and Human wild-type Htt onto polyQ-hHtt-induced cellular defects. In addition, N-terminal parts of normal hHtt were also able to rescue eye degeneration due to the loss of Drosophila endogenous dhtt function. Thus, our data indicate that Drosophila and Human Htt share biological properties, and confirm a model whereby EN activates endogenous dhtt, which in turn prevents polyQ-hHtt-induced phenotypes. The protective role of wild-type hHtt N-terminal parts, specifically onto polyQ-hHtt-induced cellular toxicity suggests that the HD may be considered as a dominant negative disease rather than solely dominant.
Asunto(s)
Proteínas de Homeodominio/fisiología , Enfermedad de Huntington/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Factores de Transcripción/fisiología , Animales , Animales Modificados Genéticamente , Inmunoprecipitación de Cromatina , Mapeo Cromosómico , Proteínas de Drosophila , Ojo/patología , Proteínas de Homeodominio/genética , Humanos , Modelos Animales , Péptidos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Factores de Transcripción/genéticaRESUMEN
One challenging question in neurogenesis concerns the identification of cues that trigger axonal growth and pathfinding to form stereotypic neuronal networks during the construction of a nervous system. Here, we show that in Drosophila, Engrailed (EN) and Gooseberry-Neuro (GsbN) act together as cofactors to build the posterior commissures (PCs), which shapes the ventral nerve cord. Indeed, we show that these two proteins are acting together in axon growth and midline crossing, and that this concerted action occurs at early development, in neuroblasts. More precisely, we identified that their expressions in NB 6-4 are necessary and sufficient to trigger the formation of the PCs, demonstrating that segmentation genes such as EN and GsbN play a crucial role in the determination of NB 6-4 in a way that will later influence growth and guidance of all the axons that form the PCs. We also demonstrate a more specific function of GsbN in differentiated neurons, leading to fasciculations between axons, which might be required to obtain PC mature axon bundles.
Asunto(s)
Tipificación del Cuerpo , Proteínas de Drosophila/fisiología , Drosophila melanogaster/embriología , Proteínas de Homeodominio/fisiología , Neuronas/metabolismo , Proteínas Nucleares/fisiología , Transactivadores/fisiología , Factores de Transcripción/fisiología , Animales , Técnicas del Sistema de Dos HíbridosRESUMEN
In all metazoans, the expression of group B HMG domain Sox transcription factors is associated with the earliest stages of CNS development. In Drosophila, SoxNeuro (SoxN) is involved in dorso-ventral patterning of the neuroectoderm, and in the formation and segregation of neuroblasts. In this report, we show that SoxN expression persists in a subset of neurons and glial cells of the ventral nerve cord at embryonic stages 15/16. In an attempt to address SoxN function in late stages of CNS development, we have used a chromatin immunoprecipitation approach to isolate genomic regions bound in vivo by SoxN. We identified several genes involved in the regulation of axon scaffolding as potential direct target genes of SoxN, including beat1a, semaphorin2a, fasciclin2, longitudinal lacking and tailup/islet. We present genetic evidence for a direct involvement of SoxN in axonal patterning. Indeed, overexpressing a transcriptionally hyperactive mutated SoxN protein in neurons results in specific defects in axon scaffolding, which are also observed in transheterozygous combinations of SoxN null mutation and mutations in its target genes.
Asunto(s)
Axones/fisiología , Proteínas de Drosophila/fisiología , Drosophila/embriología , Proteínas del Grupo de Alta Movilidad/fisiología , Factores de Transcripción/fisiología , Animales , Tipificación del Cuerpo , Sistema Nervioso Central/embriología , Sistema Nervioso Central/metabolismo , Inmunoprecipitación de Cromatina , Drosophila/metabolismo , Proteínas de Drosophila/genética , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas del Grupo de Alta Movilidad/genética , Mutación , Neuroglía/fisiología , Neuronas/fisiología , Factores de Transcripción SOX , Factores de Transcripción/genéticaRESUMEN
Sry high mobility group (HMG) box (Sox) transcription factors are involved in the development of central nervous system (CNS) in all metazoans. Little is known on the molecular mechanisms that regulate their transcriptional activity. Covalent posttranslational modification by small ubiquitin-like modifier (SUMO) regulates several nuclear events, including the transcriptional activity of transcription factors. Here, we demonstrate that SoxNeuro, an HMG box-containing transcription factor involved in neuroblast formation in Drosophila, is a substrate for SUMO modification. SUMOylation assays in HeLa cells and Drosophila S2 cells reveal that lysine 439 is the major SUMO acceptor site. The sequence in SoxNeuro targeted for SUMOylation, IKSE, is part of a small inhibitory domain, able to repress in cis the activity of two adjacent transcriptional activation domains. Our data show that SUMO modification represses SoxNeuro transcriptional activity in transfected cells. Overexpression in Drosophila embryos of a SoxN form that cannot be targeted for SUMOylation strongly impairs the development of the CNS, suggesting that SUMO modification of SoxN is crucial for regulating its activity in vivo. Finally, we present evidence that SUMO modification of group B1 Sox factors was conserved during evolution, because Sox3, the human counterpart of SoxN, is also negatively regulated through SUMO modification.
Asunto(s)
Sistema Nervioso Central/embriología , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila/embriología , Regulación del Desarrollo de la Expresión Génica , Proteínas del Grupo de Alta Movilidad/genética , Proteína SUMO-1/metabolismo , Factores de Transcripción/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Animales Modificados Genéticamente , Arginina/metabolismo , Secuencia Conservada , Proteínas de Unión al ADN/química , Drosophila/citología , Drosophila/genética , Proteínas de Drosophila/química , Embrión no Mamífero , Evolución Molecular , Genes Reporteros , Células HeLa , Proteínas del Grupo de Alta Movilidad/química , Humanos , Inmunohistoquímica , Luciferasas/metabolismo , Lisina/química , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Factores de Transcripción SOX , Factores de Transcripción SOXB1 , Proteína SUMO-1/genética , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Factores de Transcripción/química , Transcripción GenéticaRESUMEN
SRY, a Y chromosome-encoded DNA-binding protein, is required for testis organogenesis in mammals. Expression of the SRY gene in the genital ridge is followed by diverse early cell events leading to Sertoli cell determination/differentiation and subsequent sex cord formation. Little is known about SRY regulation and its mode of action during testis development, and direct gene targets for SRY are still lacking. In this study, we demonstrate that interaction of the human SRY with histone acetyltransferase p300 induces the acetylation of SRY both in vitro and in vivo at a single conserved lysine residue. We show that acetylation participates in the nuclear localisation of SRY by increasing SRY interaction with importin beta, while specific deacetylation by HDAC3 induces a cytoplasmic delocalisation of SRY. Finally, by analysing p300 and HDAC3 expression profiles during both human or mouse gonadal development, we suggest that acetylation and deacetylation of SRY may be important mechanisms for regulating SRY activity during mammalian sex determination.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Acetilación , Acetiltransferasas/metabolismo , Transporte Activo de Núcleo Celular , Animales , Proteínas de Ciclo Celular/metabolismo , Línea Celular , ADN/metabolismo , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Gónadas/embriología , Gónadas/metabolismo , Histona Acetiltransferasas , Histona Desacetilasas/metabolismo , Humanos , Lisina/genética , Lisina/metabolismo , Masculino , Ratones , Proteínas Nucleares/genética , Unión Proteica , Proteína de la Región Y Determinante del Sexo , Factores de Transcripción/genética , Factores de Transcripción p300-CBPRESUMEN
SOX9 transcription factor is involved in chondrocyte differentiation and male sex determination. Heterozygous defects in the human SOX9 gene cause campomelic dysplasia. The mechanisms behind SOX9 function are not understood despite the description of different target genes. This study therefore sets out to identify SOX9-associated proteins to unravel how SOX9 interacts with the cellular transcription machinery. We report the ability of SOX9 to interact with TRAP230, a component of the thyroid hormone receptor-associated protein (TRAP) complex. Both in vitro and in vivo assays have confirmed that the detected interaction is specific and occurs endogenously in cells. Using co-transfection experiments, we have also shown that the TRAP230 interacting domain can act in a dominant-negative manner regarding SOX9 activity. Our results add SOX9 to the list of activators that communicate with the general transcription machinery through the TRAP complex and suggest a basis for the collaboration of SOX9 with different coactivators that could contact the same coactivator/integrator complex.