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

摘要 CACNA 1A基因突变，该基因编码P/Q型钙离子的成孔亚基 通道（Cav2.1），导致神经系统疾病，包括发作性共济失调2型（EA 2）和家族性 偏瘫偏头痛1型（FHM 1）。患者通常被归类为患有这些疾病之一，或 另一个，但症状往往是重叠的，区别已受到质疑。最近， CACNA 1A患者主要表现为认知缺陷，如智力残疾或发育障碍 延迟已经描述，这表明一个更突出的二分法可能在于突变，导致 严重的运动缺陷和主要特征为认知功能障碍的运动缺陷。运动功能障碍 例如共济失调，已被归因于小脑神经元兴奋性和起搏功能的破坏 浦肯野细胞，在那里这些通道表达最高。相反， 导致认知功能障碍仍然未知。然而，Cav2.1通道也在整个 神经系统突触前末梢，在那里它们调节突触囊泡的释放。变化的 不同的CACNA 1A突变的功能后果强调了描述其影响的重要性， 每个CACNA 1A突变对通道表达和功能的影响，以了解每个突变是如何导致 相关疾病表型。我们假设主要影响神经元兴奋性的突变导致 在经典的运动表型中，而那些影响突触特性的可能引起认知缺陷。到 开始来解决这个问题，我们提出了一系列CACNA 1A患者突变的特征，导致 主要是运动或主要是认知表现。我们已经验证，现在建议将联合收割机和 系统来表征这些突变的影响：异源表达系统（HEK 293 t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. elegans研究体内突触前 定位和突触功能。这一工作将为阐明其作用机制奠定基础， CACNA 1A突变影响神经元功能并导致多效性患者结局。

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.