Role of MAST3 kinase in developmental and epileptic encephalopathy

MAST3 激酶在发育性脑病和癫痫性脑病中的作用

• 批准号: 10217382
• 负责人: ANGUS C. NAIRN
• 金额: $ 14.7万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-15 至 2023-04-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10217382
• 关键词: Acids; Address; Affect; Affinity Chromatography; Antibodies; Area; Biochemical; Biological; Biological Assay; Brain; Cell physiology; Cells; Cerebral Palsy; Cerebral cortex; Child; Childhood; Congenital cerebellar hypoplasia; Corpus Callosum; Corpus striatum structure; Cortical Malformation; Country; Data; Development; Developmental Delay Disorders; Developmental Therapeutics Program; Diagnosis; Diagnostic; Disease; Electrophysiology (science); Epilepsy; Family; Genes; Genetic Diseases; Genetic Research; Goals; Hippocampus (Brain); In Vitro; Income; Individual; Infection; Injury; Intellectual functioning disability; Intervention; Lead; Lentivirus Vector; Life; Link; Mass Spectrum Analysis; Microtubules; Missense Mutation; Modeling; Molecular; Morphology; Mutation; Neuraxis; Neurologic; Neurons; Pathogenesis; Patients; Pharmaceutical Preparations; Phenotype; Phosphorylation; Phosphotransferases; Physiological; Play; Prevalence; Promega; Property; Protein Phosphatase 2A Regulatory Subunit PR53; Protein Serine/Threonine Phosphatase; Protein phosphatase; Protein-Serine-Threonine Kinases; Proteins; Quality of life; Recombinant Proteins; Recombinants; Research; Rett Syndrome; Risk; Role; Signal Transduction; Syndrome; Techniques; Testing; United States; Variant; Vertebral column; childhood epilepsy; cyclic AMP-regulated phosphoprotein 16; epileptic encephalopathies; experimental study; gain of function; health care quality; improved; inhibitor/antagonist; knock-down; loss of function mutation; low and middle-income countries; mental development; mutant; nervous system disorder; neuron development; novel diagnostics; novel therapeutics; targeted treatment; tool

Epilepsy is one of the most common neurological disorders globally, estimated to affect 3.4 million people in the United States and 50 million people globally. Alarmingly, even in high-income countries, epilepsy in one- third of patients resists conventional drug intervention, highlighting the need to further understand the pathogenesis and molecular changes associated with this disease. Developmental and epileptic encephalopathy (DEE) is a relatively new concept in epilepsy research that describes genetic disorders in which a variation in a gene, such as a mutation, causes increased epileptiform activity and slowing of mental development. Recently, several developmental neurological conditions, such as Rett syndrome, cerebral palsy, and mega-corpus-callosum syndrome with cerebellar hypoplasia and cortical malformations have been associated with mutations in the microtubule-associated serine/threonine (MAST) protein kinase family. Specifically, our colleagues have discovered seven individuals with DEE with de novo missense mutations in the MAST3 gene: G510S, G515S, L516P, and V551L. Our group recently showed in brain that MAST3 phosphorylates ARPP-16, a small heat- and acid-stable protein enriched in striatum and cortex, at Ser46, converting it into a potent inhibitor of the serine/threonine phosphatase PP2A. Preliminary results suggest the DEE mutations in MAST3 cause a gain-of-function increase in MAST3 activity, promoting ARPP-16 phosphorylation at Ser46 which would lead to subsequent inhibition of specific forms of PP2A. Interestingly, other recent studies have identified loss of function mutations in the catalytic C, and substrate targeting B subunits, of PP2A in individuals with intellectual disability and developmental delay, the majority of which also have epilepsy. We hypothesize that increased MAST3 activity, and selective perturbation of PP2A signaling, may in part play a causative role in the development of childhood neurological conditions. The proposed research contains two specific aims to test the hypothesis that the DEE MAST3 mutations increase kinase activity and may change its interactome, causing detrimental changes in neuronal development. Aim 1 will determine which proteins and substrates interact with MAST3, how this interactome changes when the DEE MAST3 mutations are introduced, and how this affects kinase activity. This aim will be achieved using in vitro biochemical techniques, such as kinase assays with recombinant proteins, and pulldown assays in primary neuron cultures to identify interacting proteins and substrates. Aim 2 will investigate the role of the DEE MAST3 mutations in neuronal development and function using primary neuron cultures to visualize dendritic arborization and spines, morphological changes, and electrophysiological properties. Results gained from this study will further elucidate the potential for MAST3 to be a prominent force in the development of several neurological conditions early in life. Understanding this mechanism should lead to new diagnostic tools and interventions aimed at improving the quality of life of individuals affected by these disorders.

癫痫是全球最常见的神经系统疾病之一，估计会影响340万人 美国和全球5000万人。令人震惊的是，即使在高收入国家，癫痫 三分之一的患者抵制常规药物干预，强调了进一步了解 发病机理和与该疾病相关的分子变化。发育和癫痫病 脑病（DEE）是癫痫研究中相对较新的概念，描述了遗传疾病 基因的变异（例如突变）会导致癫痫样活动增加和心理减慢 发展。最近，几种发育神经系统疾病，例如RETT综合征，脑瘫， 和小脑发育不全和皮质畸形的大型毛 - 可甲状腺综合症已经存在 与微管相关的丝氨酸/苏氨酸（桅杆）蛋白激酶家族中的突变有关。 具体来说，我们 MAST3基因：G510S，G515S，L516P和V551L。我们的小组最近在大脑中表明mast3 磷酸化Arpp-16，一种富含纹状体和皮质中的小热和酸稳定的蛋白，在Ser46， 将其转换为丝氨酸/苏氨酸磷酸酶PP2A的有效抑制剂。初步结果表明 MAST3中的DEE突变会导致功能率增加MAST3活性，从而促进ARPP-16 Ser46处的磷酸化将导致随后抑制特定形式的PP2A。有趣的是， 其他最近的研究确定了催化C中功能突变的丧失，靶向b的底物 智力残疾和发育延迟的个体中PP2A的亚基，其中大多数也 有癫痫病。我们假设增加了MAST3活性，以及​​PP2A信号传导的选择性扰动， 可能有时在儿童神经系统状况的发展中起因作用。提议 研究包含两个特定的目的，以检验DEE MAST3突变增加激酶的假设。 活动并可能改变其相互作用组，从而导致神经元发育的不利变化。目标1意志 确定哪些蛋白质和底物与MAST3相互作用，这种相互作用的组在DEE时如何变化 引入MAST3突变，以及这如何影响激酶活性。使用体外将实现此目标 生化技术，例如重组蛋白的激酶测定法和原发性下拉分析 神经元培养物鉴定相互作用的蛋白质和底物。 AIM 2将调查DEE的作用 使用原代神经元培养物可视化树突状的神经元发育和功能中的MAST3突变 树博化和刺，形态变化和电生理特性。从中获得的结果 研究将进一步阐明MAST3成为发展的潜力 生命早期的神经条件。了解这种机制应导致新的诊断工具和 旨在改善受这些疾病影响的个体生活质量的干预措施。