RESUMEN
Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) cause CDKL5 deficiency disorder (CDD), a neurodevelopmental disease characterized by severe infantile seizures and intellectual disability. The absence of CDKL5 in mice causes defective spine maturation that can at least partially explain the cognitive impairment in CDKL5 patients and CDD mouse models. The molecular basis for such defect may depend on the capacity of CDKL5 to regulate microtubule (MT) dynamics through its association with the MT-plus end tracking protein CLIP170 (cytoplasmic linker protein 170). Indeed, we here demonstrate that the absence of CDKL5 causes CLIP170 to be mainly in a closed inactive conformation that impedes its binding to MTs. Previously, the synthetic pregnenolone analogue, pregnenolone-methyl-ether (PME), was found to have a positive effect on CDKL5-related cellular and neuronal defects in vitro. Here, we show that PME induces the open active conformation of CLIP170 and promotes the entry of MTs into dendritic spines in vitro. Furthermore, the administration of PME to symptomatic Cdkl5-knock-out mice improved hippocampal-dependent behavior and restored spine maturation and the localization of MT-related proteins in the synaptic compartment. The positive effect on cognitive deficits persisted for 1 week after treatment withdrawal. Altogether, our results suggest that CDKL5 regulates spine maturation and cognitive processes through its control of CLIP170 and MT dynamics, which may represent a novel target for the development of disease-modifying therapies.
Asunto(s)
Síndromes Epilépticos , Proteínas Asociadas a Microtúbulos , Proteínas de Neoplasias , Pregnenolona , Animales , Síndromes Epilépticos/genética , Éteres/metabolismo , Hipocampo/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Proteínas de Neoplasias/genética , Pregnenolona/farmacología , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
CDKL5 deficiency disorder (CDD) is an X-linked neurodevelopmental disorder characterised by early-onset drug-resistant epilepsy and impaired cognitive and motor skills. CDD is caused by mutations in cyclin-dependent kinase-like 5 (CDKL5), which plays a well-known role in regulating excitatory neurotransmission, while its effect on neuronal inhibition has been poorly investigated. We explored the potential role of CDKL5 in the inhibitory compartment in Cdkl5-KO male mice and primary hippocampal neurons and found that CDKL5 interacts with gephyrin and collybistin, two crucial organisers of the inhibitory postsynaptic sites. Through molecular and electrophysiological approaches, we demonstrated that CDKL5 loss causes a reduced number of gephyrin puncta and surface exposed γ2 subunit-containing GABAA receptors, impacting the frequency of miniature inhibitory postsynaptic currents, which we ascribe to a postsynaptic function of CDKL5. In line with previous data showing that CDKL5 loss impacts microtubule (MT) dynamics, we showed that treatment with pregnenolone-methyl-ether (PME), which promotes MT dynamics, rescues the above defects. The impact of CDKL5 deficiency on inhibitory neurotransmission might explain the presence of drug-resistant epilepsy and cognitive defects in CDD patients. Moreover, our results may pave the way for drug-based therapies that could bypass the need for CDKL5 and provide effective therapeutic strategies for CDD patients.
Asunto(s)
Neuroesteroides , Espasmos Infantiles , Masculino , Ratones , Animales , Neuroesteroides/uso terapéutico , Pregnenolona/farmacología , Espasmos Infantiles/genética , Éteres , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Pregnenolone methyl-ether (PME) is a synthetic derivative of the endogenous neuroactive steroid pregnenolone (PREG), which is an important modulator of several brain functions. In addition to being the precursor of steroids, PREG acts directly on various targets including microtubules (MTs), the functioning of which is fundamental for the development and homeostasis of nervous system. The coordination of MT dynamics is supported by a plethora of MT-associated proteins (MAPs) and by a specific MT code that is defined by the post-translational modifications of tubulin. Defects associated with MAPs or tubulin post-translational modifications are linked to different neurological pathologies including mood and neurodevelopmental disorders. In this review, we describe the beneficial effect of PME in major depressive disorders (MDDs) and in CDKL5 deficiency disorder (CDD), two pathologies that are joint by defective MT dynamics. Growing evidence indeed suggests that PME, as well as PREG, is able to positively affect the MT-binding of MAP2 and the plus-end tracking protein CLIP170 that are both found to be deregulated in the above mentioned pathologies. Furthermore, PME influences the state of MT acetylation, the deregulation of which is often associated with neurological abnormalities including MDDs. By contrast to PREG, PME is not metabolised into other downstream molecules with specific biological properties, an aspect that makes this compound more suitable for therapeutic strategies. Thus, through the analysis of MDDs and CDD, this work focuses attention on the possible use of PME for neuronal pathologies associated with MT defects.
Asunto(s)
Trastorno Depresivo Mayor , Éteres Metílicos , Síndromes Epilépticos , Humanos , Éteres Metílicos/metabolismo , Éteres Metílicos/farmacología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/farmacología , Microtúbulos/metabolismo , Pregnenolona/metabolismo , Pregnenolona/uso terapéutico , Proteínas Serina-Treonina Quinasas , Espasmos Infantiles , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/farmacologíaRESUMEN
CDKL5 deficiency disorder (CDD) is a severe neurodevelopmental encephalopathy caused by mutations in the X-linked CDKL5 gene that encodes a serine/threonine kinase. CDD is characterised by the early onset of seizures and impaired cognitive and motor skills. Loss of CDKL5 in vitro and in vivo affects neuronal morphology at early and late stages of maturation, suggesting a link between CDKL5 and the neuronal cytoskeleton. Recently, various microtubule (MT)-binding proteins have been identified as interactors of CDKL5, indicating that its roles converge on regulating MT functioning. MTs are dynamic structures that are important for neuronal morphology, migration and polarity. The delicate control of MT dynamics is fundamental for proper neuronal functions, as evidenced by the fact that aberrant MT dynamics are involved in various neurological disorders. In this review, we highlight the link between CDKL5 and MTs, discussing how CDKL5 deficiency may lead to deranged neuronal functions through aberrant MT dynamics. Finally, we discuss whether the regulation of MT dynamics through microtubule-targeting agents may represent a novel strategy for future pharmacological approaches in the CDD field.
Asunto(s)
Síndromes Epilépticos/metabolismo , Microtúbulos/metabolismo , Neuronas/metabolismo , Espasmos Infantiles/metabolismo , Animales , Humanos , Microtúbulos/efectos de los fármacos , Neuronas/efectos de los fármacos , Pregnenolona/farmacologíaRESUMEN
BACKGROUND: Recent evidence suggests that 2-week treatment with the non-psychotomimetic cannabinoid cannabidivarin (CBDV) could be beneficial towards neurological and social deficits in early symptomatic Mecp2 mutant mice, a model of Rett syndrome (RTT). AIM: The aim of this study was to provide further insights into the efficacy of CBDV in Mecp2-null mice using a lifelong treatment schedule (from 4 to 9 weeks of age) to evaluate its effect on recognition memory and neurological defects in both early and advanced stages of the phenotype progression. METHODS: CBDV 0.2, 2, 20 and 200 mg/kg/day was administered to Mecp2-null mice from 4 to 9 weeks of age. Cognitive and neurological defects were monitored during the whole treatment schedule. Biochemical analyses were carried out in brain lysates from 9-week-old wild-type and knockout mice to evaluate brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1) levels as well as components of the endocannabinoid system. RESULTS: CBDV rescues recognition memory deficits in Mecp2 mutant mice and delays the appearance of neurological defects. At the biochemical level, it normalizes BDNF/IGF1 levels and the defective PI3K/AKT/mTOR pathway in Mecp2 mutant mice at an advanced stage of the disease. Mecp2 deletion upregulates CB1 and CB2 receptor levels in the brain and these changes are restored after CBDV treatment. CONCLUSIONS: CBDV administration exerts an enduring rescue of memory deficits in Mecp2 mutant mice, an effect that is associated with the normalization of BDNF, IGF-1 and rpS6 phosphorylation levels as well as CB1 and CB2 receptor expression. CBDV delays neurological defects but this effect is only transient.
Asunto(s)
Cannabinoides/farmacología , Disfunción Cognitiva/tratamiento farmacológico , Trastornos de la Memoria/tratamiento farmacológico , Proteína 2 de Unión a Metil-CpG/genética , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Cannabinoides/administración & dosificación , Disfunción Cognitiva/fisiopatología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Factor I del Crecimiento Similar a la Insulina/metabolismo , Masculino , Ratones , Ratones Noqueados , Síndrome de Rett/tratamiento farmacológico , Síndrome de Rett/fisiopatología , Proteína S6 Ribosómica/metabolismoRESUMEN
Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene cause a complex neurological disorder, characterized by infantile seizures, impairment of cognitive and motor skills and autistic features. Loss of Cdkl5 in mice affects dendritic spine maturation and dynamics but the underlying molecular mechanisms are still far from fully understood. Here we show that Cdkl5 deficiency in primary hippocampal neurons leads to deranged expression of the alpha-amino-3-hydroxy-5-methyl-4-iso-xazole propionic acid receptors (AMPA-R). In particular, a dramatic reduction of expression of the GluA2 subunit occurs concomitantly with its hyper-phosphorylation on Serine 880 and increased ubiquitination. Consequently, Cdkl5 silencing skews the composition of membrane-inserted AMPA-Rs towards the GluA2-lacking calcium-permeable form. Such derangement is likely to contribute, at least in part, to the altered synaptic functions and cognitive impairment linked to loss of Cdkl5. Importantly, we find that tianeptine, a cognitive enhancer and antidepressant drug, known to recruit and stabilise AMPA-Rs at the synaptic sites, can normalise the expression of membrane inserted AMPA-Rs as well as the number of PSD-95 clusters, suggesting its therapeutic potential for patients with mutations in CDKL5.
Asunto(s)
Síndromes Epilépticos/tratamiento farmacológico , Proteínas Serina-Treonina Quinasas/genética , Receptores AMPA/genética , Espasmos Infantiles/tratamiento farmacológico , Tiazepinas/administración & dosificación , Animales , Antidepresivos/administración & dosificación , Homólogo 4 de la Proteína Discs Large/genética , Síndromes Epilépticos/genética , Síndromes Epilépticos/patología , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/fisiopatología , Humanos , Ratones , Mutación , Neurogénesis/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/patología , Fosforilación , Cultivo Primario de Células , Proteínas Serina-Treonina Quinasas/deficiencia , Espasmos Infantiles/genética , Espasmos Infantiles/patología , Sinapsis/efectos de los fármacos , Sinapsis/genéticaRESUMEN
The cyclin-dependent kinase-like 5 (CDKL5) gene has been associated with rare neurodevelopmental disorders characterized by the early onset of seizures and intellectual disability. The CDKL5 protein is widely expressed in most tissues and cells with both nuclear and cytoplasmic localization. In post-mitotic neurons CDKL5 is mainly involved in dendritic arborization, axon outgrowth, and spine formation while in proliferating cells its function is still largely unknown. Here, we report that CDKL5 localizes at the centrosome and at the midbody in proliferating cells. Acute inactivation of CDKL5 by RNA interference (RNAi) leads to multipolar spindle formation, cytokinesis failure and centrosome accumulation. At the molecular level, we observed that, among the several midbody components we analyzed, midbodies of CDKL5-depleted cells were devoid of HIPK2 and its cytokinesis target, the extrachromosomal histone H2B phosphorylated at S14. Of relevance, expression of the phosphomimetic mutant H2B-S14D, which is capable of overcoming cytokinesis failure in HIPK2-defective cells, was sufficient to rescue spindle multipolarity in CDKL5-depleted cells. Taken together, these results highlight a hitherto unknown role of CDKL5 in regulating faithful cell division by guaranteeing proper HIPK2/H2B functions at the midbody.
Asunto(s)
Proteínas Portadoras/metabolismo , División Celular , Centrosoma/metabolismo , Citocinesis/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Proteínas Portadoras/genética , Ciclo Celular , Células HeLa , Humanos , Ratones , Fosforilación , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
CDKL5 is a protein kinase that plays a key role for neuronal functions as testified by the onset of complex neuronal dysfunctions in patients with genetic lesions in CDKL5. Here we identify a novel interactor of CDKL5, IQGAP1, a fundamental regulator of cell migration and polarity. In accordance with a functional role of this interaction, depletion of CDKL5 impairs cell migration and impedes the localization of IQGAP1 at the leading edge. Moreover, we demonstrate that CDKL5 is required for IQGAP1 to form a functional complex with its effectors, Rac1 and the microtubule plus end tracking protein CLIP170. These defects eventually impact on the microtubule association of CLIP170, thus deranging their dynamics. CLIP170 is a cellular target of the neurosteroid pregnenolone; by blocking CLIP170 in its active conformation, pregnenolone is capable of restoring the microtubule association of CLIP170 in CDKL5 deficient cells and rescuing morphological defects in neurons devoid of CDKL5. These findings provide novel insights into CDKL5 functions and pave the way for target-specific therapeutic strategies for individuals affected with CDKL5-disorder.
Asunto(s)
Pregnenolona/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Actinas/metabolismo , Animales , Células COS , Movimiento Celular/fisiología , Chlorocebus aethiops , Células HeLa , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/patología , Proteínas de Neoplasias/metabolismo , Neuronas/metabolismo , Neurotransmisores/metabolismo , Unión Proteica , Proteínas Activadoras de ras GTPasa/genética , Proteínas Activadoras de ras GTPasa/metabolismoRESUMEN
MeCP2 is a transcriptional regulator whose functional alterations are responsible for several autism spectrum and mental disorders. Post-translational modifications (PTMs), and particularly differential phosphorylation, modulate MeCP2 function in response to diverse stimuli. Understanding the detailed role of MeCP2 phosphorylation is thus instrumental to ascertain how MeCP2 integrates the environmental signals and directs its adaptive transcriptional responses. The evolutionarily conserved serine 164 (S164) was found phosphorylated in rodent brain but its functional role has remained uncharacterized. We show here that phosphorylation of S164 in brain is dynamically regulated during neuronal maturation. S164 phosphorylation highly impairs MeCP2 binding to DNA in vitro and largely affects its nucleosome binding and chromatin affinity in vivo. Strikingly, the chromatin-binding properties of the global MeCP2 appear also extensively altered during the course of brain maturation. Functional assays reveal that proper temporal regulation of S164 phosphorylation controls the ability of MeCP2 to regulate neuronal morphology. Altogether, our results support the hypothesis of a complex PTM-mediated functional regulation of MeCP2 potentially involving a still poorly characterized epigenetic code. Furthermore, they demonstrate the relevance of the Intervening Domain of MeCP2 for binding to DNA.
Asunto(s)
Encéfalo/metabolismo , Cromatina/metabolismo , Proteína 2 de Unión a Metil-CpG/metabolismo , Animales , Metilación de ADN , Dendritas/metabolismo , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Células 3T3 NIH , Nucleosomas/metabolismo , Fosforilación , Unión Proteica , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Conejos , Serina/metabolismoRESUMEN
Mutations in the X-linked CDKL5 (cyclin-dependent kinase-like 5) gene have been associated with several forms of neurodevelopmental disorders, including atypical Rett syndrome, autism spectrum disorders, and early infantile epileptic encephalopathy. Accordingly, loss of CDKL5 in mice results in autistic-like features and impaired neuronal communication. Although the biological functions of CDKL5 remain largely unknown, recent pieces of evidence suggest that CDKL5 is involved in neuronal plasticity. Herein, we show that, at all stages of development, neuronal depolarization induces a rapid increase in CDKL5 levels, mostly mediated by extrasomatic synthesis. In young neurons, this induction is prolonged, whereas in more mature neurons, NMDA receptor stimulation induces a protein phosphatase 1-dependent dephosphorylation of CDKL5 that is mandatory for its proteasome-dependent degradation. As a corollary, neuronal activity leads to a prolonged induction of CDKL5 levels in immature neurons but to a short lasting increase of the kinase in mature neurons. Recent results demonstrate that many genes associated with autism spectrum disorders are crucial components of the activity-dependent signaling networks regulating the composition, shape, and strength of the synapse. Thus, we speculate that CDKL5 deficiency disrupts activity-dependent signaling and the consequent synapse development, maturation, and refinement.
Asunto(s)
Neuronas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteína Fosfatasa 1/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/fisiología , Animales , Apoptosis , Western Blotting , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Electrofisiología , Técnica del Anticuerpo Fluorescente , Hipocampo/citología , Hipocampo/metabolismo , Ratones , Neuronas/citología , Fosforilación , Biosíntesis de Proteínas , Proteína Fosfatasa 1/genética , Proteínas Serina-Treonina Quinasas/genética , Proteolisis , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de N-Metil-D-Aspartato/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de SeñalRESUMEN
Mutations in MECP2 cause a broad spectrum of neuropsychiatric disorders of which Rett syndrome represents the best defined condition. Both neuronal and non-neuronal functions of the methyl-binding protein underlie the related pathologies. Nowadays MeCP2 is recognized as a multifunctional protein that modulates its activity depending on its protein partners and posttranslational modifications. However, we are still missing a comprehensive understanding of all MeCP2 functions and their involvement in the related pathologies. The study of human mutations often offers the possibility of clarifying the functions of a protein. Therefore, we decided to characterize a novel MeCP2 phospho-isoform (Tyr-120) whose relevance was suggested by a Rett syndrome patient carrying a Y120D substitution possibly mimicking a constitutively phosphorylated state. Unexpectedly, we found MeCP2 and its Tyr-120 phospho-isoform enriched at the centrosome both in dividing and postmitotic cells. The molecular and functional connection of MeCP2 to the centrosome was further reinforced through cellular and biochemical approaches. We show that, similar to many centrosomal proteins, MeCP2 deficiency causes aberrant spindle geometry, prolonged mitosis, and defects in microtubule nucleation. Collectively, our data indicate a novel function of MeCP2 that might reconcile previous data regarding the role of MeCP2 in cell growth and cytoskeleton stability and that might be relevant to understand some aspects of MeCP2-related conditions. Furthermore, they link the Tyr-120 residue and its phosphorylation to cell division, prompting future studies on the relevance of Tyr-120 for cortical development.
Asunto(s)
Centrosoma/metabolismo , Proteína 2 de Unión a Metil-CpG/metabolismo , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Proteína 2 de Unión a Metil-CpG/genética , Ratones , Microtúbulos/metabolismo , Mitosis , Mutación Missense , Fosforilación , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de Proteínas , Síndrome de Rett/genéticaRESUMEN
Although Rett syndrome (RTT) represents one of the most frequent forms of severe intellectual disability in females worldwide, we still have an inadequate knowledge of the many roles played by MeCP2 (whose mutations are responsible for most cases of RTT) and their relevance for RTT pathobiology. Several studies support a role of MeCP2 in the regulation of synaptic plasticity and homeostasis. At the molecular level, MeCP2 is described as a repressor capable of inhibiting gene transcription through chromatin compaction. Indeed, it interacts with several chromatin remodeling factors, such as HDAC-containing complexes and ATRX. Other studies have inferred that MeCP2 functions also as an activator; a role in regulating mRNA splicing and in modulating protein synthesis has also been proposed. Further, MeCP2 avidly binds both 5-methyl- and 5-hydroxymethyl-cytosine. Recent evidence suggests that it is the highly disorganized structure of MeCP2, together with its post-translational modifications (PTMs) that generate and regulate this functional versatility. Indeed, several reports have demonstrated that differential phosphorylation of MeCP2 is a key mechanism by which the methyl binding protein modulates its affinity for its partners, gene expression and cellular adaptations to stimuli and neuronal plasticity. As logic consequence, generation of phospho-defective Mecp2 knock-in mice has permitted associating alterations in neuronal morphology, circuit formation, and mouse behavioral phenotypes with specific phosphorylation events. MeCP2 undergoes various other PTMs, including acetylation, ubiquitination and sumoylation, whose functional roles remain largely unexplored. These results, together with the genome-wide distribution of MeCP2 and its capability to substitute histone H1, recall the complex regulation of histones and suggest the relevance of quickly gaining a deeper comprehension of MeCP2 PTMs, the respective writers and readers and the consequent functional outcomes.