RESUMO
Cyclin-dependent kinase like-5 (CDKL5) disorder is a rare neurodevelopmental disease caused by mutations in the CDKL5 gene. The consequent misexpression of the CDKL5 protein in the nervous system leads to a severe phenotype characterized by intellectual disability, motor impairment, visual deficits and early-onset epilepsy. No therapy is available for CDKL5 disorder. It has been reported that a protein transduction domain (TAT) is able to deliver macromolecules into cells and even into the brain when fused to a given protein. We demonstrate that TAT-CDKL5 fusion protein is efficiently internalized by target cells and retains CDKL5 activity. Intracerebroventricular infusion of TAT-CDKL5 restored hippocampal development, hippocampus-dependent memory and breathing pattern in Cdkl5-null mice. Notably, systemically administered TAT-CDKL5 protein passed the blood-brain-barrier, reached the CNS, and rescued various neuroanatomical and behavioral defects, including breathing pattern and visual responses. Our results suggest that CDKL5 protein therapy may be an effective clinical tool for the treatment of CDKL5 disorder.
Assuntos
Síndromes Epilépticas/metabolismo , Síndromes Epilépticas/terapia , Proteínas Serina-Treonina Quinases/metabolismo , Espasmos Infantis/metabolismo , Espasmos Infantis/terapia , Animais , Encéfalo , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Neurônios/patologia , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase predominantly expressed in the brain. Mutations of the CDKL5 gene lead to CDKL5 disorder, a neurodevelopmental pathology that shares several features with Rett Syndrome and is characterized by severe intellectual disability. The phosphorylation targets of CDKL5 are largely unknown, which hampers the discovery of therapeutic strategies for improving the neurological phenotype due to CDKL5 mutations. Here, we show that the histone deacetylase 4 (HDAC4) is a direct phosphorylation target of CDKL5 and that CDKL5-dependent phosphorylation promotes HDAC4 cytoplasmic retention. Nuclear HDAC4 binds to chromatin as well as to MEF2A transcription factor, leading to histone deacetylation and altered neuronal gene expression. By using a Cdkl5 knockout (Cdkl5 -/Y) mouse model, we found that hypophosphorylated HDAC4 translocates to the nucleus of neural precursor cells, thereby reducing histone 3 acetylation. This effect was reverted by re-expression of CDKL5 or by inhibition of HDAC4 activity through the HDAC4 inhibitor LMK235. In Cdkl5 -/Y mice treated with LMK235, defective survival and maturation of neuronal precursor cells and hippocampus-dependent memory were fully normalized. These results demonstrate a critical role of HDAC4 in the neurodevelopmental alterations due to CDKL5 mutations and suggest the possibility of HDAC4-targeted pharmacological interventions.
Assuntos
Histona Desacetilases/biossíntese , Deficiência Intelectual/genética , Proteínas Serina-Treonina Quinases/genética , Síndrome de Rett/genética , Espasmos Infantis/genética , Animais , Modelos Animais de Doenças , Inibidores Enzimáticos/administração & dosagem , Síndromes Epilépticas , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/crescimento & desenvolvimento , Hipocampo/patologia , Histona Desacetilases/efeitos dos fármacos , Histona Desacetilases/genética , Humanos , Deficiência Intelectual/tratamento farmacológico , Deficiência Intelectual/fisiopatologia , Fatores de Transcrição MEF2/genética , Camundongos , Camundongos Knockout , Mutação , Células-Tronco Neurais/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fosforilação , Síndrome de Rett/tratamento farmacológico , Síndrome de Rett/patologia , Espasmos Infantis/tratamento farmacológico , Espasmos Infantis/patologiaRESUMO
A recently discovered neurodevelopmental disorder caused by the mutation of the cyclin-dependent kinase-like 5 gene (CDKL5) entails complex autistic-like behaviours similar to Rett syndrome, but its impact upon physiological functions remains largely unexplored. Sleep-disordered breathing is common and potentially life-threatening in patients with Rett syndrome; however, evidence is limited in children with CDKL5 disorder, and is lacking altogether in adults. The aim of this study was to test whether the breathing pattern during sleep differs between adult Cdkl5 knockout (Cdkl5-KO) and wild-type (WT) mice. Using whole-body plethysmography, sleep and breathing were recorded non-invasively for 8 h during the light period. Sleep apneas occurred more frequently in Cdkl5-KO than in WT mice. A receiver operating characteristic (ROC) analysis discriminated Cdkl5-KO significantly from WT mice based on sleep apnea occurrence. These data demonstrate that sleep apneas are a core feature of CDKL5 disorder and a respiratory biomarker of CDKL5 deficiency in mice, and suggest that sleep-disordered breathing should be evaluated routinely in CDKL5 patients.
Assuntos
Mutação , Proteínas Serina-Treonina Quinases/deficiência , Síndromes da Apneia do Sono/genética , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Serina-Treonina Quinases/genética , Curva ROC , Respiração , Síndrome de Rett/complicações , Síndrome de Rett/genética , Síndromes da Apneia do Sono/complicaçõesRESUMO
Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been identified in a rare neurodevelopmental disorder characterized by early-onset seizures, severe developmental delay, intellectual disability and Rett syndrome-like features. CDKL5 is highly expressed in the brain during early postnatal stages, suggesting its importance for brain maturation. Using a newly-generated Cdkl5 knockout (Cdkl5 -/Y) mouse, we recently found that loss of Cdkl5 impairs postnatal hippocampal development with a reduction in neuronal precursor survival and maturation. These defects were accompanied by increased activity of the glycogen synthase kinase 3ß (GSK3ß) a crucial inhibitory regulator of many neurodevelopmental processes. The goal of the current study was to establish whether inhibition of GSK3ß corrects hippocampal developmental defects due to Cdkl5 loss. We found that treatment with the GSK3ß inhibitor SB216763 restored neuronal precursor survival, dendritic maturation, connectivity and hippocampus-dependent learning and memory in the Cdkl5 -/Y mouse. Importantly, these effects were retained one month after treatment cessation. At present, there are no therapeutic strategies to improve the neurological defects of subjects with CDKL5 disorder. Current results point at GSK3ß inhibitors as potential therapeutic tools for the improvement of abnormal brain development in CDKL5 disorder.
Assuntos
Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Hipocampo/efeitos dos fármacos , Indóis/farmacologia , Deficiências da Aprendizagem/tratamento farmacológico , Maleimidas/farmacologia , Fármacos Neuroprotetores/farmacologia , Proteínas Serina-Treonina Quinases/deficiência , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Modelos Animais de Doenças , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Hipocampo/enzimologia , Hipocampo/crescimento & desenvolvimento , Hipocampo/patologia , Deficiências da Aprendizagem/enzimologia , Deficiências da Aprendizagem/patologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Camundongos Knockout , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/enzimologia , Células-Tronco Neurais/patologia , Neurogênese/efeitos dos fármacos , Neurogênese/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Neurônios/patologia , Nootrópicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/genética , Memória EspacialRESUMO
Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been identified in a neurodevelopmental disorder characterized by early-onset intractable seizures, severe developmental delay, intellectual disability, and Rett's syndrome-like features. Since the physiological functions of CDKL5 still need to be elucidated, in the current study we took advantage of a new Cdkl5 knockout (KO) mouse model in order to shed light on the role of this gene in brain development. We mainly focused on the hippocampal dentate gyrus, a region that largely develops postnatally and plays a key role in learning and memory. Looking at the process of neurogenesis, we found a higher proliferation rate of neural precursors in Cdkl5 KO mice in comparison with wild type mice. However, there was an increase in apoptotic cell death of postmitotic granule neuron precursors, with a reduction in total number of granule cells. Looking at dendritic development, we found that in Cdkl5 KO mice the newly-generated granule cells exhibited a severe dendritic hypotrophy. In parallel, these neurodevelopmental defects were associated with impairment of hippocampus-dependent memory. Looking at the mechanisms whereby CDKL5 exerts its functions, we identified a central role of the AKT/GSK-3ß signaling pathway. Overall our findings highlight a critical role of CDKL5 in the fundamental processes of brain development, namely neuronal precursor proliferation, survival and maturation. This evidence lays the basis for a better understanding of the neurological phenotype in patients carrying mutations in the CDKL5 gene.