2012-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/646589摘要：Dravet 症候群是小兒嚴重型嬰兒肌躍震顫癲癇，病患常在出生一歲內發病並隨年齡加劇，伴隨有認知、失智等神經發育問題，此疾病常因藥效不佳而預後極差，且台灣也有相關病例。相關研究顯示癲癇發作頻率深刻地影響神經發育，因此本計畫的目標在於尋找可降低癲癇發作頻率或可改善神經發育缺陷之抗癲癇藥物，期望達到可單獨或合併使用於治療Dravet 症候群病人。本計畫的獨特性是能利用本團隊最近成功產製、於第一型鈉離子通道 (NaV1.1; Scn1a) 引進亞洲病患點突變之 Dravet 小鼠模型 (Scn1aE1130X knock-in mouse) 來作為藥物篩選平台，此小鼠具有自發性癲癇及 50% 致死率，可模擬人類的 Dravet 症候群。本計畫要完成的目標如下:1. 藉由描繪及測量分析野生型及 Dravet 小鼠模式中的神經元形態差異來建立評估藥效參考資料。a. 描繪在出生後第 0、7、14 及 24 天的野生型及 Dravet 小鼠齒狀迴 (dentate gyrus) 中的顆粒狀細胞 (granule cells) 之樹突及軸突形態。b. 描繪在出生後第 0、7、14 及 24 天出生的野生型及 Dravet 小鼠齒狀迴中表現 Scn1a 之神經元的樹突及軸突形態。2. 利用 Dravet 小鼠來建立活體外完整組織培養 (ex vivo) 及活體內 (in vivo) 藥物測試平台。a. 投予司替戊醇 (stiripentol)、帝拔癇 (valporate)、以及氯巴占 (clobazam) 等三種藥物並觀察癲癇發作頻率是否減少及神經元形態是否有變化。b. 建立 Dravet 小鼠腦片培養系統用來藥物篩選並測試由其他合作實驗室提供的新藥對於神經元發育相關缺陷是否具療效。3. 藉由使用抑制神經元特異螢光標定 Dravet 小鼠來做臨床前藥物測試。a. 以結合小鼠遺傳學工具來更精確分析表現海馬迴內表現 Scn1a 的抑制性聯絡神經元及其主要神經元 (principal neuron) 之形態及電生理學變化。b. 用活體外完整組織培養 (ex vivo) 方式，測試表現 Scn1a 的神經元在藥物治療後其電生理是否有功能性恢復<br> Abstract: Dravet syndrome, also known as severe myoclonic epilepsy in infancy (SMEI), is a rare disease. The onset of the disease occurs within the first year after birth. The severity of the disease gradually reduces throughout the lives of patients with neurodevelopmental problems such as cognition and dementia. Most medications are disappointing for treatment and the prognosis is poor. There are Dravet syndrome patients in Taiwan. Studies indicate that the magnitude of mental deterioration seems to be highly correlated to the frequency of seizures. Therefore, the goal of this proposal is to screen antiepileptic drugs (AEDs) that can cause a positive impact on both the reduction of seizure frequency and the cognitive prognosis. We expect our candidate drugs can be used alone or as add-ons to treat Dravet syndrome. This proposal takes advantage of our recent success in generating a Dravet syndrome mouse model (Scn1aE1130X knock-in mouse) that will allow us to establish a drug testing platform. The mice show spontaneous seizure and a 50% lethality phenotype that mimic Dravet patients. Our specific aims are as the followings:Specific aim 1: Setting up a drug effect evaluation reference by neuronal morphometric description of a Dravet mouse modela. Establish the dendritic and axonal evaluation reference of the granule cell in dentate gyrus (DG) using wild type (WT) and Dravet mice at P0, P7, P14 and P24.b. Establish the dendritic and axonal evaluation reference of the Scn1a-expressing inhibitory interneurons in DG using WT and Dravet mice at P0, P7, P14 and P24.Specific aim 2: Establishing drug testing platforms ex vivo and in vivo using a Dravet mousea. Measure the reduction of seizure and the change of neuronal morphology in Dravet mice at P14 and P24 in vivo after stiripentol, valporate, and clobazam treatments.b. Establish a brain slice culture system for drug screening using a Dravet mouse and test new drug candidates provided by collaborative laboratories.Specific aim 3: Preclinical drug tests using a Scn1a-expressing interneuron-specific fluorescent protein labeled Dravet mousea. Morphometrical and electrophysiological analyses on Scn1a-expressing inhibitory interneurons as well as the principal excitatory neurons in the hippocampus.b. Evaluate the sodium channel functional recovery of Scnla-expressing neurons by ex vivo electrical recordings after candidate drug treatments.癲癇第一型鈉離子通道神經發育藥物EpilepsyScn1aneurodevelopmentdrug