癲癇為一種神經疾病，然而造成癲癇的原因仍有許多未知，目前也沒有完全根除的治療方式。癲癇病患除了必須長期服用抗癲癇藥物，醫師也常建議搭配維生素B6(吡哆醇)來強化神經功能，然而臨床上發現許多病患在長期服用高劑量的吡哆醇後會有神經受損的副作用。在本篇研究中，我們使用藥物誘導斑馬魚胚胎產生癲癇反應。在戊烯四唑和銀杏毒素誘發的癲癇中能看到斑馬魚快速游動及繞圈；而在咖啡因誘導的癲癇中，斑馬魚會有靜止以及顫抖的行為。在發育早期將斑馬魚浸泡於誘發癲癇的藥物中，會引起斑馬魚發育異常。根據目前醫學治療，我們同時在斑馬魚癲癇發作時給予吡哆醇，但是我們發現給予誘發癲癇後的斑馬魚胚胎吡哆醇會導致其大量且快速的壞死。同時，瘦體素的基因表現在癲癇發生後明顯上升，而在大量誘發瘦體素蛋白表達後給予吡哆醇，同樣導致斑馬魚胚胎出現快速的壞死並死亡。這樣的結果證實瘦體素參與在癲癇斑馬魚補充吡哆醇後致死的機制中。為了進一步探究癲癇斑馬魚補充吡哆醇後致死的機制，我們在癲癇斑馬魚補充吡哆醇的同時，加入多種已知參與在細胞死亡機制的分子抑制劑，並觀察其存活率。結果發現抑制細胞程序性壞死可以提高癲癇斑馬魚補充吡哆醇後的存活率，證實補充吡哆醇導致癲癇斑馬魚的死亡是透過細胞程序性壞死機制。除了藥物誘發的癲癇，我們亦透過基因調控方式引發癲癇反應。我們利用嗎啉基注射降低吡哆醇5'-磷酸氧化酶蛋白表達，使斑馬魚胚胎的活性維生素B6降低，進而發現產生癲癇反應的斑馬魚胚胎，出現發育及行為異常。而這樣的異常發育和行為能夠透過表達人類及斑馬魚的吡哆醇5'-磷酸氧化酶蛋白而回復，證實人類和斑馬魚的吡哆醇5'-磷酸氧化酶是有發育上的保留性的。同時我們也給予癲癇反應的斑馬魚胚胎不同的維生素B6。讓我們驚訝的是，我們發現除了有生物活性的維生素B6之外，吡哆胺也能夠回復斑馬魚在吡哆醇5'-磷酸氧化酶缺少時產生的異常。透過使用吡哆胺減少斑馬魚胚胎中缺乏吡哆醇5'-磷酸氧化酶造成的發育及行為異常，可能提供臨床上使用維生素B6 的癲癇病患另一個選擇性。而透過給予不同的維生素B6 也能夠降低單一維生素B6 的攝取劑量，達到減少生物毒性、增加安全性的治療。

Generalized seizures are neural symptoms of epilepsy that cannot be seized completely to date. Several nutritional supplements and medications, such as benzodiazepines, phenytoin and vitamin B6 (pyridoxine), have been recommended to relieving patients’ symptoms. However, some clinical reports showed neurological side effects in patients with pyridoxine supplementation. In this study, we first established zebrafish seizure models by exposing zebrafish to chemical compounds, including pentylenetetrazol (PTZ), ginkgotoxin, and caffeine. Epileptic-like behavior, including burst swimming and cycling, were induced by PTZ and ginkgotoxin. Freezing and trembling phenotypes were observed after caffeine treatment. Besides, seizure inducers also resulted in multiple developmental defects in brain, heart, eyes and muscle. Because of the controversial effects of pyridoxine on epileptic patients, we next exposed epileptic larvae to pyridoxine. Epileptic larvae treated with pyridoxine showed necrotic death and increased leptin-b transcripts. Overexpression of leptin-b in pyridoxine-exposed zebrafish embryos displayed similar toxicity, suggesting that leptin-b participated in the pyridoxine-induced necroptotic death in the epileptic model. Necroptosis inhibitors effectively increased the survival in pyridoxine-exposed epileptic zebrafish, indicating that pyridoxine toxicity in epileptic embryos was mediated by necroptosis. In addition to chemical-induced epilepsy, we established a zebrafish genetic model of epilepsy through the knockdown of Pyridoxine 5’-phosphate oxidase (Pnpo). PNPO is the critical enzyme catalyzing the synthesis of pyridoxal 5’-phosphate (PLP), the bioactive form of vitamin B6. Knockdown of Pnpo resulted in similar epileptic-like behavior and developmental defects observed in the chemical-induced epilepsy model. Both zebrafish and human Pnpo transcripts effectively rescued the defects in Pnpo morphants, indicating the conserved function between human and zebrafish PNPO. Various B6 vitamers, including pyridoxamine, pyridoxine, pyridoxal, and PLP, were applied to Pnpo morphants for rescue. To our surprise, in addition to PLP, an inactive form of vitamin B6, the pyridoxamine, also rescued the defects in Pnpo morphants. In conclusion, our findings showed that pyridoxine treatment triggered the necroptosis in seizure larvae. The success of using pyridoxamine to relieve the behavior and developmental defects in seizure larvae indicates that pyridoxamine, possessing both advantages of safety and biostability, may be an alternative choice of vitamin B6 supplementation for the clinical use on seizure patients.

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