RESUMO
CDKL5 deficiency disorder (CDD) is a rare X-linked neurodevelopmental disorder that is characterised by early-onset seizures, intellectual disability, gross motor impairment, and autistic-like features. CDD is caused by mutations in the cyclin-dependent kinase-like 5 (CDKL5) gene that encodes a serine/threonine kinase with a predominant expression in the brain. Loss of CDKL5 causes neurodevelopmental alterations in vitro and in vivo, including defective dendritic arborisation and spine maturation, which most likely underlie the cognitive defects and autistic features present in humans and mice. Here, we show that treatment with epigallatocathechin-3-gallate (EGCG), the major polyphenol of green tea, can restore defects in dendritic and synaptic development of primary Cdkl5 knockout (KO) neurons. Furthermore, defective synaptic maturation in the hippocampi and cortices of adult Cdkl5-KO mice can be rescued through the intraperitoneal administration of EGCG, which is however not sufficient to normalise behavioural CDKL5-dependent deficits. EGCG is a pleiotropic compound with numerous cellular targets, including the dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) that is selectively inhibited by EGCG. DYRK1A controls dendritic development and spine formation and its deregulation has been implicated in neurodevelopmental and degenerative diseases. Treatment with another DYRK1A inhibitor, harmine, was capable of correcting neuronal CDKL5-dependent defects; moreover, DYRK1A levels were upregulated in primary Cdkl5-KO neurons in concomitance with increased phosphorylation of Tau, a well-accepted DYRK1A substrate. Altogether, our results indicate that DYRK1A deregulation may contribute, at least in part, to the neurodevelopmental alterations caused by CDKL5 deficiency.
Assuntos
Catequina/análogos & derivados , Síndromes Epilépticas/metabolismo , Polifenóis/metabolismo , Espasmos Infantis/metabolismo , Chá/metabolismo , Animais , Encéfalo/metabolismo , Catequina/metabolismo , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Quinases DyrkRESUMO
Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene are responsible for the onset of CDKL5 Deficiency Disorder (CDD), a neurological pathology characterised by severe infantile seizures, intellectual disability, impairment of gross motor skills, sleep and gastrointestinal disturbances. CDKL5 is a serine/threonine kinase the molecular network of which is not yet fully understood. Loss of CDKL5 both in vitro and in vivo leads to altered neuronal morphology including axon specification and outgrowth, dendritic arborisation and spine morphology suggesting a link between CDKL5 and the regulation of proper cytoskeleton functioning. Recently, we found that CDKL5 regulates the binding of CLIP170 to microtubules (MT). CLIP170 is a MT-plus end tracking protein (+TIP) that associates with MTs when present in its open, active conformation. Here we present evidence suggesting CLIP170 contributes to neuronal CDKL5-dependent defects and that it represents an important novel druggable target for CDD; indeed, CLIP170 is directly targeted by the neuroactive steroid pregnenolone (PREG), which induces the active conformation of the protein thus promoting MT-dynamics. We here show that PREG and a synthetic derivative pregnenolone-methyl-ether (PME) can restore the MT association of CLIP170 and revert morphological and molecular defects in Cdkl5-KO neurons at different stages of maturation. All together, these findings identify CLIP170 as possible novel druggable target for CDKL5 related disorders providing an intriguing prospective for future disease-modifying drug-based therapies.
Assuntos
Síndromes Epilépticas/tratamento farmacológico , Síndromes Epilépticas/genética , Pregnenolona/uso terapêutico , Espasmos Infantis/tratamento farmacológico , Espasmos Infantis/genética , Animais , Axônios/efeitos dos fármacos , Axônios/ultraestrutura , Células COS , Chlorocebus aethiops , Ensaio Cometa , Dendritos/efeitos dos fármacos , Dendritos/ultraestrutura , Proteína 4 Homóloga a Disks-Large/genética , Proteína 4 Homóloga a Disks-Large/metabolismo , Feminino , Cones de Crescimento/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Microtúbulos/efeitos dos fármacos , Microtúbulos/patologia , Neurônios/patologia , GravidezRESUMO
TALE (three amino acid loop extension) homeodomain proteins include the PBC and the MEINOX sub-families. MEINOX proteins form heterodimer complexes with PBC proteins. Heterodimerization is crucial to DNA binding and for nuclear localization. PBC-MEINOX heterodimers bind DNA also in combination with HOX proteins, thereby modulating their DNA-binding specificity. TALE proteins therefore play crucial roles in multiple developmental and differentiation pathways in vivo. We report the identification and characterization of a novel human gene homologous to PREP1, called PREP2. Sequence comparisons indicate that PREP1 and PREP2 define a novel sub-family of MEINOX proteins, distinct from the MEIS sub-family. PREP2 is expressed in a variety of human adult tissues and displays a more restricted expression pattern than PREP1. PREP2 is capable of heterodimerizing with PBC proteins. Heterodimerization with PBX1 appears to be essential for nuclear localization of both PREP2 and PBX1. A comparison between the functional properties of PREP1 and PREP2 reveals that PREP2-PBX display a faster DNA-dissociation rate than PREP1-PBX heterodimers, suggesting different roles in controlling gene expression. Like PREP1, PREP2-PBX heterodimers are capable of forming ternary complexes with HOXB1. The analysis of some PREP2 in vitro properties suggests a functional diversification among PREP and between PREP and MEIS MEINOX proteins.
Assuntos
Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Células 3T3 , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Clonagem Molecular , Citoplasma/química , Proteínas de Ligação a DNA/metabolismo , Dimerização , Humanos , Camundongos , Dados de Sequência Molecular , Fator de Transcrição 1 de Leucemia de Células Pré-B , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/biossíntese , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Transcrição GênicaRESUMO
Nuclear localization of the Extradenticle (EXD) and PBX1 proteins is regionally restricted during Drosophila and mammalian development. We studied the subcellular localization of EXD, PBX, and their partners Homothorax (HTH) and PREP1, in different cell contexts. HTH and PREP1 are cytoplasmic and require association with EXD/PBX for nuclear localization. EXD and PBX1 are nuclear in murine fibroblasts but not in Drosophila Schneider cells, in which they are actively exported to the cytoplasm. Coexpression of EXD/PBX with HTH/PREP1 causes nuclear localization of their heterodimers in both cell contexts. We propose that heterodimerization with HTH/PREP induces nuclear translocation of EXD and PBX1 in specific cell contexts by blocking their nuclear export.