GABA訊息調控Dravet syndrome癲癇症之致病機轉研究

Abstract

Dravet症候群（DS）是一種因先天基因突變引起的兒童難治型癲癇，常於出生後5-8個月確診。約有80%的患者皆在Scn1a基因突變導致電壓型鈉離子通道(NaV1.1)功能缺陷。已知DS的致病機制是由於GABA抑制性訊號減少，導致海馬體中神經迴路的興奮性及抑制性調節失衡，進而引發癲癇發作。儘管許多研究檢視DS的病理生理學，但其癲癇發作的確切機制仍不清楚。已知GABA訊號在調節神經新生中亦有極重要的作用，因此，GABA訊號的失衡與多種疾病中的神經新生受損有關。在顳葉型癲癇模式中，異常的海馬迴神經新生為癲癇反覆發作的重要病態生理特徵之一。然而，癲癇形成的原因至今仍成謎。為了釐清神經新生的變化是否在癲癇發作之前已發生異常，因而降低癲癇閥值並增加DS中癲癇的敏感性，我們建立了一個DS的動物模型，並使用神經幹細胞培養進行體外研究。本篇論文結果顯示，在Dravet症候群的海馬齒狀回及出生後八天的體外神經幹細胞中，GABA表現量皆顯著下降，神經幹細胞過度增生及分化成神經元數量增加。另外，體外神經幹細胞分化出的神經元之分支顯著變多，其神經束長度也顯著的增長。於神經幹細胞培養中補充GABA、活化GABABR或抑制細胞內鈣離子後，細胞增生數量及神經細胞皆顯著減少，神經元之分支及長度也近似於野生型。在小鼠癲癇未形成前，海馬齒狀回中異位幹細胞數量也顯著增加。在出生後八天之DS小鼠長期活化GABABR，可有效改善癲癇發作的閥值。推論其可能的調控機轉是因DS小鼠缺少GABA活化GABAB R，未能抑制細胞內鈣離子增加及停止細胞週期進程，導致神經幹細胞過度增生。這些早期存在的異常變化，隨著發育過程逐漸累積，成為Dravet症候群癲癇形成的原因。

Dravet syndrome (DS) is a severe form of epilepsy in children caused by genetic mutation and is typically diagnosed between 5 and 8 months of age. Approximately 80% of patients mutations in the Scn1a gene, which result in loss-of-function of the voltage-gated sodium channel NaV1.1. The currently accepted pathogenic mechanism of DS involves impaired GABAergic inhibitory signaling, leading to an imbalance between excitation and inhibition within hippocampal neural circuits and consequently triggering seizures. Although numerous studies have investigated the pathophysiology of DS, the mechanisms underlying seizure formation remain unclear. GABAergic signaling is also critically involved in the regulation of adult neurogenesis, and dysregulation of GABA signaling has been associated with impaired neurogenesis in various neurological disorders. In temporal lobe epilepsy models, aberrant hippocampal neurogenesis is recognized as one of the main symptoms associated with recurrent seizures; however, whether abnormal neurogenesis is a cause or a consequence of epilepsy remains unclear. To determine whether alterations in neurogenesis before seizure onset and thereby lower seizure threshold and increase seizure susceptibility in DS, we established a DS mouse model and performed in vitro neural stem cell (NSC) culture. Our results demonstrated that expression of GABA was significantly reduced in both the dentate gyrus of DS mice and cultured NSC/NPCs isolated at postnatal day 8. This reduction was accompanied by excessive proliferation of NSCs and enhanced neuronal differentiation. Furthermore, neurons differentiated from DS-derived NSCs exhibited a significant increase in neurite branching and length. Supplementation with GABA, activation of GABAB receptors, or inhibition of intracellular calcium signaling in cultured NSCs markedly reduced cell proliferation and neural differentiation, while neuronal branching and neurite length were restored comparable to wild-type controls. In addition, the number of ectopically migrated neural stem cells in the dentate gyrus was significantly increased before the onset of seizures in DS mice. Chronic activation of GABAB receptors beginning at postnatal day 8 effectively improved seizure threshold in DS mice. These findings suggest that insufficient GABA-mediated activation of GABAB receptors in DS mice fails to suppress intracellular calcium elevation and cell-cycle progression, leading to excessive proliferation of neural stem cells. The accumulation of these early developmental abnormalities may ultimately contribute to epileptogenesis in Dravet syndrome.