Dravet症候群小鼠的建立及病理分析與新藥開發之應用

Abstract

Dravet症候群主要是因為嬰兒時期發生肌陣攣癲癇伴隨有心智發展的延遲以及高死亡率，由於此嚴重型癲癇的病因主要為sodium channel type I α-subunit (SCN1A) 基因產生突變導致第一型鈉離子通道 (Nav1.1) 異常。病人對目前的抗癲癇藥物常出現抗藥性而預後極差，根據美國FDA的摘錄，本疾病屬於 “未滿足之醫療需求”的範疇，因此開發新藥物為臨床醫療重要的課題，除此之外，在癲癇產生的基礎研究上，已知“海馬迴腦區”為主要癲癇形成區域之一，其齒迴區負責控制興奮性訊息進入海馬迴，但由於目前尚未有相關文獻研究因SCN1A基因突變造成海馬迴齒迴區神經網路的本質及時間上變化，特別是尚未清楚了解癲癇形成的神經網路造成發育上缺陷是否與產生癲癇行為有時程上相關性，因此，為了探討齒迴區神經網路功能性及結構上的缺陷，我建立一個帶有人類SCN1A突變點的Dravet小鼠模式 (Scn1aE1099X/+)，此小鼠具有自發性癲癇以及對熱誘發癲癇有易感受度，表現型符合Dravet症候群病人的症狀，小鼠的自發性癲癇主要開始於出生後第三周，在第四周發作頻率大為提高，在這段時間，此小鼠齒迴區的表現Nav1.1的GABAergic神經元其Nav1.1表現量較其他海馬迴區域下降最多，除此之外，也發現齒迴區GABAergic神經元其動作電位之動力學改變、興奮程度降低、以及對齒迴區顆粒性細胞的抑制力下降，這些缺陷進而提高顆粒性細胞的自發性興奮訊息傳導及增加突觸前神經傳導物質釋放機率，除了電生理功能性相關的缺陷，也觀察到齒迴區顆粒性細胞的樹突型態發生異常，包括樹突結構複雜度的降低以及過多的樹突小刺，這些結構上的變化恰好與失去平衡神經網路興奮程度以及自發性癲癇高發作期於時間上重疊，因此齒迴區神經網路在結構上及功能上的發育缺陷與癲癇形成有重要的關係，同時也揭露此小鼠的神經發育過程中其神經網路連結有缺陷，更進一步推論這些結構異常可能在強化神經網路的不穩定性，使得更高層次如智力的發展受到影響，這些顯示Scn1a基因缺陷造成神經發育多方面的影響。 由於Dravet症候群在臨床上尚無良好的治療方式，目前Dravet症候群病患大多為SCN1A基因表現量不足所造成，若能以補足SCN1A表現量不足的方式進行源頭性的改善，應為較佳的治療策略，事實上在Dravet動物模式已證明提高原Scn1a表現量50%可部分改善動物的存活率由25%提高至50%，因此，在本論文的另一研究重點為開發新型藥物，目標為找到可以通讀無意義突變的異常等位基因的小分子藥物藉此提高SCN1A表現量，篩選藥物策略的獨特性為使用帶有病人突變點的小鼠cDNA後接冷光報導基因進行篩選，並由建立的小鼠平台驗證其功效，目前由Sigma LOPAC1280 library找到一化合物A具有通讀潛力，可在細胞及動物層次上再次表現蛋白，然而功能性驗證仍需更進一步驗證，若證明此化合物具通讀功能且具療效，將有機會治療各種無意義突變所導致的Dravet症候群病患，由於目前約有30%基因突變為無意義突變造成疾病，因此將有機會可推廣運用到所有由無意義突變引起的疾病。

Dravet syndrome (DS) is characterized by severe infant-onset myoclonic epilepsy along with delayed psychomotor development and heightened premature mortality. A primary monogenic cause is mutation of the SCN1A gene, which encodes the voltage-gated sodium channel subunit Nav1.1. Current treatment of DS patients has failed due to refractory of patients to clinical available drugs. According to FDA guideline, this disease is defined as “unmet medical need.” Therefore, to develop a novel drug for the disease remains an important issue. Besides, for the basic research of the disease, the nature and timing of changes caused by SCN1A mutation in the hippocampal dentate gyrus (DG) network, a core area for gating major excitatory input to hippocampus and a classic epileptogenic zone, are not well known. In particularly, it is still not clear whether the developmental deficit of this epileptogenic neural network temporally matches with the progress of seizure development. Here, we investigated the emerging functional and structural deficits of the DG network in a novel mouse model (Scn1aE1099X/+) that mimics the genetic deficit of human DS. Scn1aE1099X/+ (Het) mice, similarly to human DS patients, exhibited early spontaneous seizures and were more susceptible to hyperthermia-induced seizures starting at postnatal week (PW) 3, with seizures peaking at PW4. During the same period, the Het DG exhibited a greater reduction of Nav1.1-expressing GABAergic neurons compared to other hippocampal areas. Het DG GABAergic neurons showed altered action potential kinetics, reduced excitability, and generated fewer spontaneous inhibitory inputs into DG granule cells. The effect of reduced inhibitory input to DG granule cells was exacerbated by heightened spontaneous excitatory transmission and elevated excitatory release probability in these cells. In addition to electrophysiological deficit, we observed emerging morphological abnormalities of DG granule cells. Het granule cells exhibited progressively reduced dendritic arborization and excessive spines, which coincided with imbalanced network activity and the developmental onset of spontaneous seizures. Taken together, our results establish the existence of significant structural and functional developmental deficits of the DG network and the temporal correlation between emergence of these deficits and the onset of seizures in Het animals. Most importantly, our results uncover the developmental deficits of neural connectivity in Het mice. Such structural abnormalities likely further exacerbate network instability and compromise higher-order cognitive processing later in development, and thus highlight the multifaceted impacts of Scn1a deficiency on neural development. The current treatments for DS are unsatisfied. Most DS patients are haploinsufficiency of SCN1A. A novel therapeutic method is to increase the expression of SCN1A. According to the literature, heterozygous mice carrying Scn1a transgenes, which increase Scn1a expression levels by 50% in heterozygous mice, can partially improve the survival rate from 25% to 50%. Therefore, I propose a new therapeutic strategy: to discover and develop small molecule drugs that can read through premature terminal codons (PTC) in the mutant SCN1A allele chemically to produce sufficient Nav1.1 protein. The specificity of the strategy is using mouse Scn1a cDNA carrying human nonsense mutation to fuse with luciferase reporter gene. Compounds that can increase the luciferase will be further validated in our mouse model. By using this platform, we have identified a lead compound “compound A” from the Sigma LOPAC1280 library. Compound A can induce re-expression of the full-length Nav1.1 protein in cellular and animal models. However, the therapeutic effect still needs be determined. If the result is positive, the effective strategies will be a novel treatment for DS patients harboring nonsense mutations. Furthermore, given that ~30% of gene mutations that contribute to human diseases are nonsense mutations, our candidate compounds that can cause read-through of premature stop codons will have great potential for treating all nonsense-mutation-mediated diseases.