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

摘要 编码P/Q型钙离子成孔亚单位的CACNA1A基因突变 通道(Cav2.1)，导致神经系统疾病，包括发作性共济失调2型(EA2)和家族性 偏瘫偏头痛1型(FHM1)。患者通常被归类为患有这些疾病之一或 另一种，但症状往往是重叠的，这种区别受到了质疑。最近， CACNA1a患者主要表现为认知缺陷，如智力残疾或发育不良 已经描述了延迟，这表明更显著的二分法可能存在于导致 严重的运动障碍和以认知功能障碍为主要特征的疾病。运动功能障碍 例如共济失调被归因于神经元的兴奋性和小脑的起搏功能的破坏 浦肯野细胞，在那里这些通道表达最高。相比之下，潜在的机制 导致认知功能障碍的原因尚不清楚。然而，Cav2.1通道也在整个过程中表达 突触前终末的神经系统，在那里它们调节突触小泡的释放。变化无常 不同CACNA1A突变的功能后果强调了描述影响的重要性 了解每个CACNA1A突变对通道表达和功能的影响，以了解每个突变如何导致 相关疾病表型。我们假设，主要影响神经元兴奋性的突变会导致 在经典的运动表型中，而那些影响突触属性的因素可能会导致认知障碍。至 为了开始解决这个问题，我们建议表征一组CACNA1A患者突变导致的 主要是运动性或认知性表现。我们已经过验证，现在建议将两种模式结合起来 表征这些突变影响的系统：异源表达系统(HEK293t细胞) 使用分子、成像和全细胞技术评估细胞表面表达和生物物理特性 电生理学技术，以及线虫在体内研究突触前 定位和突触功能。这项工作将为阐明其致病机制奠定基础。 CACNA1A突变会影响神经元功能，并导致多效性患者的预后。

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.