Assessing Synaptic and Intrinsic Effects of Patient-Derived ID-Associated CACNA1A Mutations Using Multiple Models

使用多种模型评估患者源性 ID 相关 CACNA1A 突变的突触和内在影响

• 批准号: 10657084
• 负责人: PERI T KURSHAN
• 金额: $ 25.2万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10657084
• 关键词: Address; Affect; Ataxia; Awareness; Behavioral; Biological Models; Biophysics; Brain; Caenorhabditis elegans; Calcium Channel; Cell surface; Cells; Cerebellum; Characteristics; Classification; Clustered Regularly Interspaced Short Palindromic Repeats; Cognitive; Cognitive deficits; Complementary DNA; Data; Defect; Developmental Delay Disorders; Disease; Dominant-Negative Mutation; Electrophysiology (science); Embryo; Episodic ataxia; Familial Hemiplegic Migraine; Foundations; Genes; Goals; Homologous Gene; Human; Image; Impaired cognition; Intellectual functioning disability; Kidney; Lead; Mediating; Mediator; Methods; Modeling; Molecular; Motor; Movement Disorders; Mutate; Mutation; Nematoda; Nervous System; Neurobehavioral Manifestations; Neuromuscular Junction; Neurons; P-Q type voltage-dependent calcium channel; Patient-Focused Outcomes; Patients; Phenotype; Play; Presynaptic Terminals; Property; Publishing; Purkinje Cells; Q-Type Calcium Channels; Role; Structure; Surface; Symptoms; Synapses; Synaptic Vesicles; System; Techniques; Testing; Therapeutic Intervention; Transfection; Work; biophysical properties; cell fixing; confocal imaging; disease phenotype; immunocytochemistry; in vivo; in vivo Model; motor deficit; motor disorder; mouse model; mutant mouse model; nervous system disorder; neuronal excitability; novel; pharmacologic; presynaptic; synaptic function; vesicular release; voltage

Abstract Mutations in the CACNA1A gene, which encodes the pore-forming subunit of the P/Q type calcium channel (Cav2.1), lead to neurological disorders including Episodic Ataxia type 2 (EA2) and Familial Hemiplegic Migraine type 1 (FHM1). Patients have typically been classified as having one of these disorders or the other, but symptoms are often overlapping and the distinction has been called into question. More recently, CACNA1A patients presenting primarily with cognitive defects such as intellectual disability or developmental delay have been described, suggesting that a more salient dichotomy may lie between mutations that lead to severe motor deficits and those that are characterized primarily by cognitive dysfunction. Motor dysfunction such as ataxia has been attributed to disruption of neuronal excitability and pacemaking function of cerebellar Purkinje cells, where these channels are most highly expressed. In contrast, t he underlying mechanisms leading to cognitive dysfunction remain unknown. However, Cav2.1 channels are also expressed throughout the nervous system at presynaptic terminals where they mediate synaptic vesicle release. The varying functional consequences of different CACNA1A mutations underscore the importance of delineating the impact of each CACNA1A mutation on channel expression and function to understand how each causes the associated disease phenotypes. We hypothesize that mutations that effect primarily neuronal excitability result in classical motor phenotypes, while those that effect synaptic properties may give rise to cognitive deficits. To begin to address this, we propose to characterize an array of CACNA1A patient mutations resulting in either primarily motor or primarily cognitive presentations. We have validated and now propose to combine two model systems to characterize the effect of these mutations: a heterologous expression system (HEK293t cells) to assess cell-surface expression and biophysical properties using molecular, imaging, and whole cell electrophysiology techniques, as well as the nematode C. elegans to investigate in vivo presynaptic localization and synaptic function. This work will lay the foundation for elucidating the mechanism by which CACNA1A mutations affect neuronal function and lead to pleiotropic patient outcomes.

抽象的 Cacna1a基因中的突变，该基因编码P/Q型钙的孔形成亚基 通道（Cav2.1），导致神经系统疾病，包括情节性共济失调（EA2）和家族性疾病 偏瘫偏头痛1型（FHM1）。患者通常被归类为其中一种疾病或 另一种但症状通常是重叠的，区别已受到质疑。最近， CACNA1A患者主要出现认知缺陷，例如智力障碍或发育 已经描述了延迟，表明更明显的二分法可能位于导致的突变之间 严重的运动缺陷以及主要由认知功能障碍的特征。电动机功能障碍 诸如共济失调归因于小脑神经元兴奋性和起搏功能的破坏 这些通道最高度表达的Purkinje细胞。相反，他的基本机制 导致认知功能障碍仍然未知。但是，Cav2.1频道也在整个过程中表达 突触前末端的神经系统介导突触囊泡释放。变化 不同CACNA1A突变的功能后果强调了描述影响的重要性 在通道表达和功能上的每个cacna1a突变的理解如何引起 相关疾病表型。我们假设主要影响神经元兴奋性结果的突变 在经典运动表型中，而影响突触特性的表型可能会导致认知缺陷。到 开始解决这个问题，我们建议表征一系列CACNA1A患者突变，导致任何一种 主要是运动或主要是认知表现。我们已经验证并提议结合两个模型 表征这些突变效果的系统：异源表达系统（HEK293T细胞） 使用分子，成像和全细胞评估细胞表面表达和生物物理特性 电生理技术以及线虫C.秀丽隐杆线虫在体内研究 定位和突触功能。这项工作将为阐明该机制的基础 CACNA1A突变会影响神经元功能并导致多效性患者结局。