RESUMEN
Inherited alleles account for most of the genetic risk for schizophrenia. However, new (de novo) mutations, in the form of large chromosomal copy number changes, occur in a small fraction of cases and disproportionally disrupt genes encoding postsynaptic proteins. Here we show that small de novo mutations, affecting one or a few nucleotides, are overrepresented among glutamatergic postsynaptic proteins comprising activity-regulated cytoskeleton-associated protein (ARC) and N-methyl-d-aspartate receptor (NMDAR) complexes. Mutations are additionally enriched in proteins that interact with these complexes to modulate synaptic strength, namely proteins regulating actin filament dynamics and those whose messenger RNAs are targets of fragile X mental retardation protein (FMRP). Genes affected by mutations in schizophrenia overlap those mutated in autism and intellectual disability, as do mutation-enriched synaptic pathways. Aligning our findings with a parallel case-control study, we demonstrate reproducible insights into aetiological mechanisms for schizophrenia and reveal pathophysiology shared with other neurodevelopmental disorders.
Asunto(s)
Modelos Neurológicos , Mutación/genética , Red Nerviosa/metabolismo , Vías Nerviosas/metabolismo , Esquizofrenia/genética , Esquizofrenia/fisiopatología , Sinapsis/metabolismo , Trastornos Generalizados del Desarrollo Infantil/genética , Proteínas del Citoesqueleto/metabolismo , Exoma/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Humanos , Discapacidad Intelectual/genética , Tasa de Mutación , Red Nerviosa/fisiopatología , Proteínas del Tejido Nervioso/metabolismo , Vías Nerviosas/fisiopatología , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Esquizofrenia/metabolismo , Especificidad por SustratoRESUMEN
Integration of exogenous DNA into a host genome represents an important route to generate animal and cellular models for exploration into human disease and therapeutic development. In most models, little is known concerning structural integrity of the transgene, precise site of integration, or its impact on the host genome. We previously used whole-genome and targeted sequencing approaches to reconstruct transgene structure and integration sites in models of Huntington's disease, revealing complex structural rearrangements that can result from transgenesis. Here, we demonstrate in the R6/2 mouse, a widely used Huntington's disease model, that integration of a rearranged transgene with coincident deletion of 5,444 bp of host genome within the gene Gm12695 has striking molecular consequences. Gm12695, the function of which is unknown, is normally expressed at negligible levels in mouse brain, but transgene integration has resulted in cortical expression of a partial fragment (exons 8-11) 3' to the transgene integration site in R6/2. This transcript shows significant expression among the extensive network of differentially expressed genes associated with this model, including synaptic transmission, cell signalling and transcription. These data illustrate the value of sequence-level resolution of transgene insertions and transcription analysis to inform phenotypic characterization of transgenic models utilized in therapeutic research.
Asunto(s)
Modelos Animales de Enfermedad , Enfermedad de Huntington/patología , Ratones Transgénicos , Animales , Perfilación de la Expresión Génica , Reordenamiento Génico , Humanos , Ratones , Análisis de Secuencia de ADN , Eliminación de SecuenciaRESUMEN
Psychiatric diseases, including schizophrenia, bipolar disorder and major depression, are projected to lead global disease burden within the next decade. Pharmacotherapy, the primary--albeit often ineffective--treatment method, has remained largely unchanged over the past 50 years, highlighting the need for novel target discovery and improved mechanism-based treatments. Here, we examined in wild type mice the impact of chronic, systemic treatment with Compound 60 (Cpd-60), a slow-binding, benzamide-based inhibitor of the class I histone deacetylase (HDAC) family members, HDAC1 and HDAC2, in mood-related behavioral assays responsive to clinically effective drugs. Cpd-60 treatment for one week was associated with attenuated locomotor activity following acute amphetamine challenge. Further, treated mice demonstrated decreased immobility in the forced swim test. These changes are consistent with established effects of clinical mood stabilizers and antidepressants, respectively. Whole-genome expression profiling of specific brain regions (prefrontal cortex, nucleus accumbens, hippocampus) from mice treated with Cpd-60 identified gene expression changes, including a small subset of transcripts that significantly overlapped those previously reported in lithium-treated mice. HDAC inhibition in brain was confirmed by increased histone acetylation both globally and, using chromatin immunoprecipitation, at the promoter regions of upregulated transcripts, a finding consistent with in vivo engagement of HDAC targets. In contrast, treatment with suberoylanilide hydroxamic acid (SAHA), a non-selective fast-binding, hydroxamic acid HDAC 1/2/3/6 inhibitor, was sufficient to increase histone acetylation in brain, but did not alter mood-related behaviors and had dissimilar transcriptional regulatory effects compared to Cpd-60. These results provide evidence that selective inhibition of HDAC1 and HDAC2 in brain may provide an epigenetic-based target for developing improved treatments for mood disorders and other brain disorders with altered chromatin-mediated neuroplasticity.
Asunto(s)
Afecto/efectos de los fármacos , Conducta Animal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Cromatina/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/antagonistas & inhibidores , Acetilación/efectos de los fármacos , Animales , Benzamidas/química , Benzamidas/farmacología , Encéfalo/citología , Encéfalo/metabolismo , Cromatina/genética , Histona Desacetilasa 1/antagonistas & inhibidores , Histona Desacetilasa 2/antagonistas & inhibidores , Inhibidores de Histona Desacetilasas/química , Histonas/metabolismo , Litio/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/citología , Neuronas/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Reproducibilidad de los Resultados , Factores de Tiempo , Transcripción Genética/efectos de los fármacosRESUMEN
Long-term memory formation is known to be critically dependent upon de novo gene expression in the brain. As a consequence, pharmacological enhancement of the transcriptional processes mediating long-term memory formation provides a potential therapeutic strategy for cognitive disorders involving aberrant neuroplasticity. Here we focus on the identification and characterization of small molecule inhibitors of histone deacetylases (HDACs) as enhancers of CREB (cAMP response element-binding protein)-regulated transcription and modulators of chromatin-mediated neuroplasticity. Using a CREB reporter gene cell line, we screened a library of small molecules structurally related to known HDAC inhibitors leading to the identification of a probe we termed crebinostat that produced robust activation of CREB-mediated transcription. Further characterization of crebinostat revealed its potent inhibition of the deacetylase activity of recombinant class I HDACs 1, 2, 3, and class IIb HDAC6, with weaker inhibition of the class I HDAC8 and no significant inhibition of the class IIa HDACs 4, 5, 7, and 9. In cultured mouse primary neurons, crebinostat potently induced acetylation of both histone H3 and histone H4 as well as enhanced the expression of the CREB target gene Egr1 (early growth response 1). Using a hippocampus-dependent, contextual fear conditioning paradigm, mice systemically administered crebinostat for a ten day time period exhibited enhanced memory. To gain insight into the molecular mechanisms of memory enhancement by HDAC inhibitors, whole genome transcriptome profiling of cultured mouse primary neurons treated with crebinostat, combined with bioinformatic analyses of CREB-target genes, was performed revealing a highly connected protein-protein interaction network reflecting modules of genes important to synaptic structure and plasticity. Consistent with these findings, crebinostat treatment increased the density of synapsin-1 punctae along dendrites in cultured neurons. Finally, crebinostat treatment of cultured mouse primary neurons was found to upregulate Bdnf (brain-derived neurotrophic factor) and Grn (granulin) and downregulate Mapt (tau) gene expression-genes implicated in aging-related cognitive decline and cognitive disorders. Taken together, these results demonstrate that crebinostat provides a novel probe to modulate chromatin-mediated neuroplasticity and further suggests that pharmacological optimization of selective of HDAC inhibitors may provide an effective therapeutic approach for human cognitive disorders. This article is part of a Special Issue entitled 'Cognitive Enhancers'.