Drosophila and mouse models of PNPO deficiency

摘要 吡哆醇5 '-磷酸氧化酶（PNPO）是一种将维生素转化为非活性形式的限速酶。 维生素B6（VB 6）在饮食中，包括吡哆醇和吡哆胺，唯一的活性形式，吡哆醛5 '-磷酸（PLP）。 PLP是大脑中多巴胺、5-羟色胺和GABA合成所需的辅因子。 在人类中，已知PNPO缺乏会导致新生儿癫痫性脑病（NEE）。突变 NEE患者中PNPO的报告越来越多。最近的研究还确定PNPO是一个促进因素， 早发性癫痫和16个癫痫基因中的一个参与了常见的癫痫。但由于 由于缺乏动物模型，我们对PNPO对发育或成人功能的影响知之甚少 系统、回路或细胞水平的缺陷（例如涉及GABA、多巴胺或5-羟色胺合成） 在体内条件下。我们对轻度PNPO缺乏症如何与其他遗传缺陷相互作用知之甚少， 环境因素（例如饮食中的VB 6）导致癫痫发作或其他疾病。 我们已经鉴定了PNPO的果蝇同源物，并鉴定了果蝇突变体（sg 1195果蝇）， 部分PNPO缺乏。由于PNPO活性低，它们对膳食VB 6缺乏敏感。我们已经 产生了全局敲除以及敲入模型，其中内源性野生型（WT）飞PNPO 被患者体内发现的人类突变PNPO所取代。发育、寿命和癫痫发作期间的活力 这些果蝇的表型取决于特定的遗传操作以及饮食中VB 6的可用性。我们 他们还发现，PNPO缺乏加剧了果蝇中的其他癫痫突变等位基因， 协同作用。在目标1中，我们将定义果蝇模型的特定发育阶段，其中PNPO 缺乏会导致致命性和癫痫发作。在目标2中，我们将定义参与PNPO的脑特异性细胞类型。 在果蝇和小鼠模型中缺乏诱导的致死性和癫痫发作。我们将测试基因-基因相互作用（例如， PNPO和其他已知的癫痫基因）。在目标3中，我们将生成和描述苍蝇和老鼠模型 携带人类PNPO突变 1

Abstract Pyridoxine 5'-phosphate oxidase (PNPO) is a rate-limiting enzyme in converting inactive forms of Vitamin B6 (VB6) in diet, including pyridoxine and pyridoxamine, to the only active form, pyridoxal 5'-phosphate (PLP). PLP is a cofactor required for the syntheses of dopamine, serotonin and GABA in the brain. In humans, PNPO deficiency is known to cause neonatal epileptic encephalopathy (NEE). Mutations in PNPO have been increasingly reported in NEE patients. Recent studies also identify PNPO as a contributor to early-onset epilepsies and one of the16 epilepsy genes involved in the common epilepsies. However, due to the lack of animal models, we know little about the developmental or adult functional impact of PNPO deficiency at systems, circuit or cellular level (e.g. involvement of GABA, dopamine or serotonin synthesis) under in vivo conditions. We know little about how mild PNPO deficiency interacts with other genetic defects or environmental factors (e.g. VB6 in diet) to cause seizures or other conditions. We have identified the Drosophila homolog of PNPO and identified a Drosophila mutant (sgll95 flies) with partial PNPO deficiency. Due to low PNPO activity, they are sensitive to dietary VB6 deficiency. We have since generated global knock-down as well as knock-in models in which the endogenous wild-type (WT) fly PNPO was replaced by human mutant PNPO found in patients. Viability during development, lifespan and seizure phenotype of these flies depend on the specific genetic manipulation as well as availability of VB6 in diet. We have also found that PNPO deficiency exacerbated other epileptic mutant alleles in flies with significant synergistic interactions. In Aim 1, we will define specific developmental stages in fly models in which PNPO deficiency leads to lethality and seizures. In Aim 2, we will define brain specific cell types involved in PNPO- deficiency-induced lethality and seizures in fly and mouse models. We will test gene-gene interactions (e.g., PNPO and other known epilepsy genes). In Aim 3, we will generate and characterize fly and mouse models that carry human PNPO mutations. 1