摘要:小兒重度肌痙攣癲癇症候群(severe myotonic epilepsy of infant, SMEI;又稱之為Dravet syndrome)是臨床上之罕見疾病,病患通常在新生兒時期已發病,並對藥物治療出現抗藥性以致癒後效果極差。文獻顯示,體內 Scn1a基因的突變導致其所轉錄之第一型鈉離子通道 (NaV1.1 channel) 功能失常與此疾病具重大關連性。研究證明,NaV1.1通道主要分布於海馬迴齒迴區 (dentate gyrus, DG) 內的抑制性聯絡神經元上,當其發生異常後會造成抑制性神經細胞活性降低,因而無法有效調控其下游的神經網絡,致使海馬迴整體興奮性升高並進一步引發癲癇,但其詳細致病機轉仍有待探討。此三年研究計畫中,我們實驗室將透過先前本團隊已成功建立之 Scn1a 基因替換小鼠動物模式來模擬人類 Dravet 症候群,藉由電生理之全細胞模式膜箝制 (whole-cell patch clamp) 方式,記錄小鼠海馬迴齒迴區腦薄片(brain slice) 中神經細胞的動作電位與神經傳遞物質變化,以得到此突變對海馬迴細胞神經生理功能之影響,並輔分子生物學和組織學技術,更深入闡明其病理機制。除此之外,我們更將以基因替換小鼠為動物模型探討神經幹細胞在Dravet syndrome 的角色與臨床治療之應用。最後,使用此動物模式作為抗癲癇藥物的篩選平台,以提供臨床上藥物治療的有效建議。其詳細研究目標及方法如下: 第一年目標: 探討 Scn1a缺失後對海馬迴 DG中的 granule cells興奮性之影響與機制。 本團隊先前已建立具備 Scn1a基因缺失之小鼠動物模式,並經由行為觀察、腦電波圖 (electroencephalography, EEG) 紀錄以及基本生理現象評估等方式,發現其與患有 Dravet症候群病人具有相似的臨床性徵,譬如:熱痙攣(febrile seizures)。因此,我們第一步將利用腦薄片電生理記錄搭配分子生物學與組織學技術等來探討 Scn1a基因缺陷後對海馬迴 DG區域內神經細胞活性的影響。 方法: (1) 電生理方面:我們將利用 Scn1a+/+、Scn1a+/-與 Scn1a-/-三種不同基因型之小鼠腦薄片來分析海馬迴 DG中的 granule cells之神經活性變化。如: spontaneous inhibitory postsynaptic currents (sIPSCs)、miniature IPSCs、spontaneous excitatory postsynaptic currents (sEPSCs) 與 minature EPSCs (mEPSCs);並使用 paired-pulse或者等方法來釐清突觸前或突觸後神經細胞所扮演的角色。 (2)分子生物學方面:利用 Western blot 與 qPCR 等技術分析不同 Na+ channel subtypes,如:NaV1.2, NaV1.3.....NaV1.6等蛋白於 Scn1a突變後是否發生補償現象。 (3)組織學方面:利用專一性抗體,如:NaV1.1, GAD (glutamic acid decarboxylase) 與 Prox1 (prospero homeobox 1) 標定 NaV1.1與神經細胞種類之相關性。以釐清 NaV1.1於 DG中的分布情況。 第二年目標: 探討 Scn1a突變後對海馬迴 DG中的神經新生之影響。 海馬迴中的 DG存在著持續性神經新生的現象,其過程中受到許多機制的影響;而 DG內的神經新生情況異常已知與許多疾病有關,如:癲癇症和阿茲海默氏症 (Alzheimer's disease)。證據顯示,罹患癲癇症的孩童往後容易出現學習或認知上的障礙;動物模式研究則發現,嬰幼兒期引發癲癇後會減少其海馬迴 DG內的神經新生情形,且對空間認知上的學習能力會明顯降低。Dravet症候群的病患通常一歲前即出現癲癇症狀且學習能力往往較同學齡兒童遲緩,但 Dravet 症候群與腦中的神經新生之間的關聯性尚不清楚。因此,本研究將使用 Scn1a基因缺失之小鼠來探討兩者的相關性。 方法: (1) 組織學方面:利用 BrdU (Bromodeoxyuridine) 標定新生細胞的方法,分析Scn1a+/+、Scn1a+/-與 Scn1a-/-三種不同基因型之小鼠海馬迴 DG腦區中的神經新生情況,以辨別 Scn1a 缺失後對神經幹細胞/前驅細胞之影響。後續並藉著專一性抗體如:nestin, GFAP (glial fibrillary acidic protein), DCX (Doublecortin), MAP2 (microtubule-associated protein 2) 與 NeuN (neuronal nuclei) 來識別不同階段之新生神經細胞,以釐清詳細的機制。 (2) 分子生物學方面:利用出生後 (postnatal day, P) 一至三天 (P1-P3) 之胎鼠,將其海馬迴中的神經幹細胞/前驅細胞分離出來,並使用離體培養方式得到神經球 (neurosphere),分別比較 Scn1a+/+、Scn1a+/-與 Scn1a-/-三種不同基因型小鼠海馬迴內分離出的神經球之生長情形;再者,藉由特定抗體如:nestin, GFAP, DCX, MAP2與 NeuN來觀察後續分化趨勢。 (3) 電生理學方面:已知離體培養之神經球可憑藉著特定誘導物分化成神經細胞、膠狀細胞 (glial cells) 與星狀細胞 (astrocyte)。因此,本實驗將利用whole-cell patch clamp方法來記錄 Scn1a缺失後對神經球分化而來的神經細胞功能之影響,如:靜止膜電位 (resting membrane potential)、動作電位 (action potential) 的產生與神經傳遞物質釋放現象。此外,我們亦將利用小鼠腦薄片作電刺激誘導長期增益效應 (long-term potentiation) 方法,比較 Scn1a+/+、Scn1a+/-與 Scn1a-/-不同基因型小鼠之間的差異。 第三年目標: 探討抗癲癇藥物 Stiripentol在 Dravet症候群的作用機制。 Dravet症候群的患童通常在一歲以內的嬰兒期即會有長時間的熱痙攣發作,一歲以後抽筋出現多樣化,尤其以肌躍震顫為特色,此時的發作愈趨頑固,幾乎所有抗痙攣藥物或治療方法都效果不彰。目前歐盟已正式核准 stiripentol 合併valproate與 clobazam使用來治療 Dravet症候群,其結果發現給予 stiripentol的組別能顯著降低抽筋情況發生。動物研究證實,stiripentol 可以增加腦中 GABA (gamma-aminobutyric acid) 的濃度,其作用可能是藉由抑制 GABA回收或降解來達成。另外,更多證據指出 stiripentol會抑制 cytochrome 450酵素進而增強許多抗癲癇藥物的效果;最新的實驗則證明 stiripentol亦會延長 GABAA接受器開啟時間,上述情況都可能與臨床上 stiripentol能改善 Dravet症候群抽筋發作相關,但 stiripentol的治療機制仍有待確認。本實驗預計使用 Dravet症候群動物模式來探討 stiripentol的作用機轉,以提供臨床藥物治療上或未來新藥的研究和開發更多參考。 方法: (1) 電生理學方面:分析 stiripentol對 Scn1a+/+、Scn1a+/-與 Scn1a-/-三種不同基因型之小鼠海馬迴腦薄片的神經活性影響,如: sIPSCs、mIPSCs、sEPSCs與 mEPSCs;並探討不同 stiripentol濃度的效力。並釐清 stiripentol於突觸前或突觸後神經細胞所扮演的角色。 (2) 分子生物學方面:利用western blot與 qPCR來分析 stiripentol處理後之Dravet症候群小鼠海馬迴 DG中 nestin, GFAP, DCX, MAP2與 NeuN.等蛋白質和 mRNA表現量。 (3) 組織學方面:利用 nestin, GFAP, DCX, MAP2與 NeuN等專一性抗體來探討stiripentol對 Dravet症候群小鼠海馬迴 DG中的神經細胞新生影響。 (4) 行為學方面:利用 Morris 水迷宮 (Morris water maze) 來評估 stiripentol 對Dravet症候群小鼠之學習能力改善情況。 待本計畫完成後,我們除了對 Dravet 症候群的致病機轉有更深入的了解外,其實驗結果將可提供給新抗癲癇藥物的研發或臨床用藥參考。
Abstract: Dravet syndrome (severe myotonic epilepsy of infant, SMEI) is a kind of rare disease in clinic. The patients usually have early onset seizures during neonatal period and poor outcome due to refractory to classical antiepileptic medical therapy. Previous studies demonstrated that mutation on Scn1a gene, which encodeds NaV1.1 channels in central nervous system, is related to Dravet syndrome. According to the animal model studies, NaV1.1 channels has been found to express predominant on soma and axons of hippocampal parvalbumin interneurons. The Scn1a-mutant mice showed hyperpolarized resting membrane potential and reduced depolarization-evoked action potential number when compared with wild-type, therefore, caused general hyperexcitability that may lead to epilepsy but the pathological mechanisms still unclear. In order to find out effective clinical medical therapy for the Dravet syndrome patients. An animal model for Dravet syndrome has been established in our previous studied. In this project, we will investigate the pathomechanism of Dravet syndrome by using the electrophysiology, molecular biology, stem cell biology and histology in Scn1a-mutant mice. These results will be helpful for screen and determine the effective treatments from conventional antiepileptic drugs (AEDs) or develop new strategies against Dravet syndrome. First year: Study the effects of Scn1a-mutation in neuronal excitation in hippocampal DG granule cells. We have previously generated that mice homozygous/heterozygous for a missense mutation in Scn1a developed early onset epileptic seizures or febrile seizures characteristic in Dravet syndrome patients. First, we examined whether reduced NaV1.1 expression altered the firing properties of neurons by electrophysiological, molecular biological and histological techniques. (1) Elucidation of neural activity in Scn1a-deficit mice in hippocampus DG. (2) Elucidation of compensatory effects in others Na+ channel subtypes. (3) Identity of neuronal subtypes which express NaV1.1 by specific marker such as GAD or Prox1. Second year: Study the mechanism of neurogensis in hippocampal DG in the Scn1a-mutation mice. Hippocampus DG is one of the brain region to keep generation of newly neurons throughout the lifespan of individuals. It has been konwn that abnormal pattern of neurogenesis in hippocampus DG may contribute to some neurological disorders such as epilepsy and Alzheimer's disease. Clinical evidences have suggested that seizures in newborns are more harmful than seizures occurring in older children and characteristic in learning or cognitive deficits. Moreover, previous studies indicates that after recurrent seizures in the neonatal animal, there is a reduction in newly born granule cells in hippocampus and impaired spatial learning. However, the mechanisms of neurogenesis in the brain of Dravet syndrome is still unclear. In this project, we will investigate the process of newborn granule cells in DG in Scn1a-deficit mice. (1) Investigate the neurogenesis in DG in Scn1a+/+、Scn1a+/-, and Scn1a-/- - mice by BrdU-labeling or specific marker such as nestin, GFAP, DCX, MAP2 and NeuN. (2) Elucidate the mechanisms of Nav1.1 deficit on neural stem/progenitor cells development in vitro by neurosphere culture, which isolated from Scn1a-deficit mice. (3) Record the neuronal activity which differentiate from neurosphere by whole-cell patch clamp technique. Third year: Study the mechanism of Stiripentol on Dravet syndrome. The patient with Dravet syndrome is usually refractory to classical antiepileptic medical therapy. Stiripentol is registered as an orphan drug in Europe. Recently, clinical studies have suggested that stiripentol combinated with clobazam and valproate exhibits higher anticonvulsant properties in children with Dravet syndrome. In animal model, it has been known that stiripentol could increased GABA concentration in the brain and prolonged other anticonvulsant drugs efficacy. However, the mechanism of stiripentol on anti-seizures remain to be elucidated. In this project, we will use the electrophysiological, molecular biological, histological and behavioral methods to investigate the role of stiripentol in the mouse model of Dravet syndrome. (1) Study the effects of stiripentol on neurotransmission in Scn1a-mutation mice by electrophysiological techniques. (2) Analyzed the expression level of nestin, GFAP, DCX, MAP2 and NeuN in Scn1a-mutation mice after stiripentol treatment. (3) Study the nestin, GFAP, DCX, MAP2 and NeuN locolization by specific marker in hippocampal DG in Scn1a-mutation mice after stiripentol application. (4) Compare the Morris water maze performance in Scn1a-deficit mice before or after stiripentol injection. After complete this project, we may understand the molecular regulation mechanism of Scn1a-mutation disorders and provide useful information for clinical physicians to management Dravet syndrome patients.