Pathogenesis and motor neuron degeneration of a novel disease associated with a P158A mutation in NAMPT

与 NAMPT P158A 突变相关的新型疾病的发病机制和运动神经元变性

• 批准号: 10444087
• 负责人: Shinghua Ding
• 金额: $ 49.92万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-15 至 2027-01-01
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10444087
• 关键词: 13 year old; Aging; Anabolism; Animals; Atrophic; Axon; Biochemical Reaction; Bioenergetics; Biological Assay; Biological Process; Brain; Cell Death; Cell division; Cells; Cessation of life; Citric Acid Cycle; Clinical; Code; DNA Repair; Data; Dendritic Spines; Development; Diagnosis; Disease; Disease Progression; Electron Transport; Electrophysiology (science); Energy Metabolism; Enzymes; Essential Genes; Fibroblasts; Foot Deformities; Functional disorder; Gene Expression Profiling; Genes; Genetic Diseases; Glycolysis; Goals; Hand deformities; Hereditary Motor and Sensory Neuropathies; Human; Image; In Vitro; Inborn Errors of Metabolism; Inherited; Ischemic Stroke; Lead; Link; Lower Extremity; Mammalian Cell; Mammals; Metabolic; Metabolic Pathway; Metabolic dysfunction; Metabolism; Mitochondria; Molecular; Motor; Motor Cortex; Motor Neurons; Mus; Muscle; Muscle Fibers; Muscle Weakness; Mutant Strains Mice; Mutation; Nerve Degeneration; Neuroglia; Neuromuscular Junction; Neurons; Neuropathy; Niacinamide; Nicotinamide Mononucleotide; Nicotinamide adenine dinucleotide; Oxidative Phosphorylation; Oxidative Stress; Paralysed; Pathogenesis; Pathologic; Pathway interactions; Patients; Pentosephosphate Pathway; Phenotype; Play; Preparation; Production; Proteins; Recombinants; Reporting; Respiration; Role; Sensory; Siblings; Signal Transduction; Single Nucleotide Polymorphism; Skin; Stable Isotope Labeling; Stress; Structure; Symptoms; Synapses; Technology; Testing; Time; Tissues; Transferase; Variant; Western Blotting; Wheelchairs; Wild Type Mouse; axonal degeneration; base; cofactor; fluorescence imaging; gene function; human disease; in vivo; induced pluripotent stem cell; metabolomics; mitochondrial dysfunction; motor impairment; motor neuron degeneration; mouse model; muscle degeneration; mutant; mutant mouse model; nervous system disorder; neuromuscular function; neuron loss; nicotinamide phosphoribosyltransferase; novel; patch clamp; protective effect; response; synaptic function; tool; two-photon

Project Summary Nicotinamide adenine dinucleotide (NAD+) is a cofactor required for glycolysis, the tricarboxylic acid cycle (TCA) and enzymatic reaction in electron transport chain (ETC). In mammalian cells, NAD+ salvage pathway, where nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme, is the predominant pathway for NAD+ biosynthesis. Although the dysregulation of NAD+ in aging and neurodegerative diseases has been reported, genetic diseases caused by NAMPT variants have not been clinically recognized and understood. Here we identified the first case of an inherited neurological disease caused by a homozygous single nucleotide polymorphism (SNP), i.e., a P158A mutation in the coding region of NAMPT gene. The major clinical features of patients include impaired motor coordination, muscle weakness, atrophy of lower extremities, positive Babiński sign. The patients were diagnosed as hereditary motor and sensory neuropathy involving axonal degeneration and neuromuscular junction (NMJ) dysfunction. Using skin-derived patient fibroblasts (p-FBs), our preliminary studies found that P158A mutation causes reduced bioenergetics, mitochondrial dysfunction, and decreased enzymatic activity of NAMPT for NAD+ biosynthesis compared with healthy control fibroblasts (c-FBs). The results indicate the pathological conditions related to the patients is initially resulted from bioenergetic stress and ultimately from neuronal and muscular degeneration. Thus, our project goal is to understand the pathogenesis and the mechanism of neuronal and muscular degeneration of this new disease. To achieve our goal, we generated many molecular tools including P158A-NAMPT mutant mice, c- & p-FBs-derived induced pluripotent stem cells (c- & p-iPSCs including isogenic and patient like p-iPSCs), and iPSC-induced motor neurons (c- & p- iMNs). We propose three Specific Aims. Aim 1 will test the hypothesis P158A mutation in NAMPT causes mitochondrial and synaptic dysfunction of p-iMNs. Using iMNs, we will study the effect of P158A mutation on cellular bioenergetics, glycolytic metabolism and mitochondrial respiration. We will also conduct combined metabolomic and transcriptional profiling to determine the molecular base of metabolic changes caused by P158A mutation. Aim 2 will test the hypothesis that P158A mutation in NAMPT causes MN degeneration. Using the mutant mice, we will study disease progression, upper and lower MN degeneration. We will use electrophysiological and two-photon (2-P) imaging to study the effect of P158A mutation on sensory response and cytosolic and mitochondrial Ca2+ signaling. Aim 3 will test the hypothesis that P158A mutation in NAMPT causes NMJ abnormalities and muscle degeneration. We will assess structural and functional abnormalities of NMJs and muscle contractile response of semitendinosus muscles isolated from the symptomatic mutant mice. A human disease caused by NAMPT mutation has not been reported so far. Our application represents a first in-depth study on the pathogenesis and mechanism of motor neuron and muscle degeneration of a new neurological disease caused by a mutation in NAMPT gene.

项目摘要 烟酰胺腺嘌呤二核苷酸（NAD+）是糖酵解、三羧酸循环（TCA）所需的辅因子 以及电子传递链（ETC）中的酶促反应。在哺乳动物细胞中，NAD+补救途径，其中 烟酰胺磷酸核糖基转移酶（NAMPT）是限速酶，是主要的途径， NAD+生物合成。尽管NAD+在衰老和神经退行性疾病中的失调已经被证实， 据报道，由NAMPT变异体引起的遗传疾病尚未在临床上被认识和理解。这里 我们发现了第一例由纯合单核苷酸引起的遗传性神经疾病， 多态性（SNP），即，NAMPT基因编码区P158 A突变。主要临床特征 患者包括运动协调受损、肌无力、下肢萎缩、巴氏阳性 标志.患者被诊断为遗传性运动和感觉神经病，涉及轴突变性 和神经肌肉接头（NMJ）功能障碍。使用皮肤来源的患者成纤维细胞（p-FB），我们的初步 研究发现，P158 A突变导致生物能量学降低，线粒体功能障碍， 与健康对照成纤维细胞（c-FB）相比，NAMPT对NAD+生物合成的酶活性。的 结果表明，与患者相关的病理状况最初是由生物能量应激引起的， 最终导致神经元和肌肉退化因此，我们的项目目标是了解发病机制 并对这种新疾病的神经和肌肉变性机制进行了探讨。为了实现我们的目标，我们 产生了许多分子工具，包括P158 A-NAMPT突变小鼠，c-和p-FB衍生的诱导多能 干细胞（c-iPSC和p-iPSC，包括同基因的和患者样的p-iPSC）和iPSC诱导的运动神经元（c-iPSC和p-iPSC）。 iMN）。我们提出三个具体目标。目的1将检验P158 A突变导致NAMPT的假设， p-iMN的线粒体和突触功能障碍。利用iMNs，我们将研究P158 A突变对 对细胞生物能量学、糖酵解代谢和线粒体呼吸的影响。我们还将联合 代谢组学和转录谱分析，以确定代谢变化引起的分子基础， P158 A突变。目的2验证NAMPT中P158 A突变导致MN变性的假设。 使用突变小鼠，我们将研究疾病进展，上下MN变性。我们将使用 电生理和双光子（2-P）成像，以研究P158 A突变对感觉反应的影响 以及胞质和线粒体Ca 2+信号传导。目的3验证NAMPT中P158 A突变可能与NAMPT的发生有关的假设。 导致NMJ异常和肌肉退化。我们将评估结构和功能异常 从症状突变体中分离的NMJ和半腱肌的肌肉收缩反应 小鼠NAMPT突变引起的人类疾病至今未见报道。我们的应用程序代表了 首次深入研究了运动神经元和肌肉变性的发病机制和机制， 由NAMPT基因突变引起的神经系统疾病。