Drosophila and mouse models of PNPO deficiency

摘要 5‘-磷酸吡哆醇氧化酶(PNPO)是一种转化非活性维生素的限速酶 饮食中的B6(VB6)，包括吡哆醇和吡哆胺，到唯一的活性形式，吡哆醛5‘-磷酸(PLP)。 PLP是大脑中合成多巴胺、5-羟色胺和GABA所必需的辅因子。 在人类中，已知PNPO缺乏会导致新生儿癫痫脑病(NEE)。基因突变 PNPO在NEE患者中的报道越来越多。最近的研究也发现PNPO是导致 早发性癫痫和常见癫痫涉及的16个癫痫基因之一。然而，由于 由于缺乏动物模型，我们对PNPO的发育或成人功能影响知之甚少 系统、电路或细胞水平的缺陷(例如，涉及GABA、多巴胺或5-羟色胺的合成) 在活体条件下。我们对轻微的PNPO缺乏如何与其他遗传缺陷或 环境因素(如饮食中的维生素B6)会导致癫痫发作或其他情况。 我们已经鉴定了PNPO的果蝇同源物，并鉴定了一种果蝇突变体(Sgll95 Fars)。 部分PNPO缺乏症。由于PNPO活性低，它们对饲料中VB6的缺乏很敏感。从那以后我们就 生成了全球击倒和敲入模型，在该模型中，内源野生型(WT)飞行PNPO 被在患者中发现的人类突变PNPO所取代。发育、寿命和癫痫发作期间的生存能力 这些果蝇的表型取决于特定的基因操作以及饲料中VB6的可获得性。我们 还发现，PNPO缺乏会加剧果蝇其他癫痫突变等位基因，并具有显著的 协同互动。在目标1中，我们将定义PNPO在果蝇模型中的特定发育阶段 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的大脑特定细胞类型- 苍蝇和小鼠模型中的缺乏性致死和癫痫发作。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，我们将生成并描述苍蝇和老鼠模型 携带人类PNPO突变的基因。 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