RESUMO
Paroxysmal kinesigenic dyskinesia (PKD) is the most common paroxysmal dyskinesia, characterized by recurrent episodes of involuntary movements provoked by sudden changes in movement. Proline-rich transmembrane protein 2 (PRRT2) has been identified as the major causative gene for PKD. Here, we report that PRRT2 deficiency facilitates the induction of cerebellar spreading depolarization (SD) and inhibition of cerebellar SD prevents the occurrence of dyskinetic movements. Using Ca2+ imaging, we show that cerebellar SD depolarizes a large population of cerebellar granule cells and Purkinje cells in Prrt2-deficient mice. Electrophysiological recordings further reveal that cerebellar SD blocks Purkinje cell spiking and disturbs neuronal firing of the deep cerebellar nuclei (DCN). The resultant aberrant firing patterns in DCN are tightly, temporally coupled to dyskinetic episodes in Prrt2-deficient mice. Cumulatively, our findings uncover a pivotal role of cerebellar SD in paroxysmal dyskinesia, providing a potent target for treating PRRT2-related paroxysmal disorders.
Assuntos
Cerebelo/fisiologia , Distonia/patologia , Proteínas de Membrana/genética , Potenciais de Ação/efeitos dos fármacos , Animais , Cálcio/metabolismo , Distonia/metabolismo , Eletrocorticografia , Técnicas In Vitro , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/fisiologia , Técnicas de Patch-Clamp , Cloreto de Potássio/farmacologia , Células de Purkinje/fisiologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Canais de Sódio Disparados por Voltagem/química , Canais de Sódio Disparados por Voltagem/metabolismoRESUMO
OBJECTIVE: Mutations of the cyclin-dependent kinase-like 5 (CDKL5) gene cause severe neurodevelopmental disorders characterized by intractable epilepsy, intellectual disability, and autism. Multiple mouse models generated for mechanistic studies have exhibited phenotypes similar to some human pathological features, but none of the models has developed one of the major symptoms affecting CDKL5 deficiency disorder (CDD) patients: intractable recurrent seizures. As disrupted neuronal excitation/inhibition balance is closely associated with the activity of glutamatergic and γ-aminobutyric acidergic (GABAergic) neurons, our aim was to study the effect of the loss of CDKL5 in different types of neurons on epilepsy. METHODS: Using the Cre-LoxP system, we generated conditional knockout (cKO) mouse lines allowing CDKL5 deficiency in glutamatergic or GABAergic neurons. We employed noninvasive video recording and in vivo electrophysiological approaches to study seizure activity in these Cdkl5 cKO mice. Furthermore, we conducted Timm staining to confirm a morphological alteration, mossy fiber sprouting, which occurs with limbic epilepsy in both human and mouse brains. Finally, we performed whole-cell patch clamp in dentate granule cells to investigate cell-intrinsic properties and synaptic excitatory activity. RESULTS: We demonstrate that Emx1- or CamK2α-derived Cdkl5 cKO mice manifest high-frequency spontaneous seizure activities recapitulating the epilepsy of CDD patients, which ultimately led to sudden death in mice. However, Cdkl5 deficiency in GABAergic neurons does not generate such seizures. The seizures were accompanied by typical epileptic features including higher amplitude spikes for epileptiform discharges and abnormal hippocampal mossy fiber sprouting. We also found an increase in spontaneous and miniature excitatory postsynaptic current frequencies but no change in amplitudes in the dentate granule cells of Emx1-cKO mice, indicating enhanced excitatory synaptic activity. SIGNIFICANCE: Our study demonstrates that Cdkl5 cKO mice, serving as an animal model to study recurrent spontaneous seizures, have potential value for the pathological study of CDD-related seizures and for therapeutic innovation.
Assuntos
Síndromes Epilépticas/genética , Neurônios GABAérgicos/metabolismo , Ácido Glutâmico/metabolismo , Proteínas Serina-Treonina Quinases/genética , Convulsões/genética , Espasmos Infantis/genética , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Giro Denteado/citologia , Giro Denteado/metabolismo , Modelos Animais de Doenças , Eletroencefalografia , Síndromes Epilépticas/metabolismo , Síndromes Epilépticas/fisiopatologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Neurônios GABAérgicos/patologia , Proteínas de Homeodomínio , Camundongos , Camundongos Knockout , Fibras Musgosas Hipocampais/patologia , Neurônios/metabolismo , Neurônios/patologia , Técnicas de Patch-Clamp , Prosencéfalo , Convulsões/metabolismo , Convulsões/fisiopatologia , Espasmos Infantis/metabolismo , Espasmos Infantis/fisiopatologia , Fatores de TranscriçãoRESUMO
Deficiency in the E3 ubiquitin ligase UBE3A leads to the neurodevelopmental disorder Angelman syndrome (AS), while additional dosage of UBE3A is linked to autism spectrum disorder. The mechanisms underlying the downstream effects of UBE3A gain or loss of function in these neurodevelopmental disorders are still not well understood, and effective treatments are lacking. Here, using stable-isotope labeling of amino acids in mammals and ubiquitination assays, we identify PTPA, an activator of protein phosphatase 2A (PP2A), as a bona fide ubiquitin ligase substrate of UBE3A. Maternal loss of Ube3a (Ube3am-/p+) increased PTPA level, promoted PP2A holoenzyme assembly, and elevated PP2A activity, while maternal 15q11-13 duplication containing Ube3a down-regulated PTPA level and lowered PP2A activity. Reducing PTPA level in vivo restored the defects in dendritic spine maturation in Ube3am-/p+ mice. Moreover, pharmacological inhibition of PP2A activity with the small molecule LB-100 alleviated both reduction in excitatory synaptic transmission and motor impairment in Ube3am-/p+ mice. Together, our results implicate a critical role of UBE3A-PTPA-PP2A signaling in the pathogenesis of UBE3A-related disorders and suggest that PP2A-based drugs could be potential therapeutic candidates for treatment of UBE3A-related disorders.