Cell Type-Specific Transcriptional Changes Underlying Memory Impairment in Temporal Lobe Epilepsy

项目摘要/摘要 摘要颞叶癫痫(TLE)是局灶性癫痫最常见的一种形式，对内科治疗往往难以奏效。 管理层。除癫痫发作外，患者还经常患有认知并发症，并伴有记忆障碍。 是在TLE中看到的特征缺陷。尽管严重影响了患者的生活质量，但其机制 人们对潜在的记忆力低下还不了解，直接解决它的疗法也是有限的。以前的研究 实验室已经在匹罗卡品的TLE小鼠模型中发现了局部细胞稳定性受损，这可能是 这些动物的空间记忆功能障碍。直到6周后才出现地方细胞缺陷。 致痫侮辱(即匹罗卡品诱导的癫痫持续状态，SE)，提示有病理上的 最初触发的过程需要几周的时间来降解海马体的编码。这一延迟提供了一个机会 对于治疗干预，防止海马区空间表征的退化，是一项重要的 检查全面了解记忆的细胞和分子基础的时间点 TLE功能障碍。这一提议的主要假设是，回路重组先于 导致定位细胞功能障碍和记忆损伤网络特性以及该调制电路 重组可以改善位置、细胞功能和记忆。鉴于细胞类型的巨大异质性， 海马区，我们对海马区组织进行了单核RNA测序，以确定细胞类型特异性 转录改变。我们在不同的时间点鉴定了差异表达基因(Deg)。 匹罗卡品诱导的SE和FOCUS对星形胶质细胞在认知中的作用的跟踪研究 正在处理。为了丰富与记忆损伤相关的变化，我们比较了3周时星形细胞的变化。 一种先前描述的阿尔茨海默病(AD)模型。和TLE一样，AD也是一种记忆障碍 损害，并有显著的海马区受累。在重叠的基因中，有一个功能上的 与蛋白降解相关的亚群：组织蛋白B、D和L(CTSB、CTSD、CTSL)和胱抑素C(Cst3)。这些 候选基因都与人类的AD病理有关，尽管它们的作用是否是保护性的 或者致病因素还没有完全确定。此外，虽然它们是通过对星形细胞的分析来鉴定的， 它们广泛表达于多种细胞类型。在这项提案中，我们将使用CRISPR介导的基因 有选择地从星形胶质细胞、小胶质细胞和中间神经元中剔除这些候选基因的缺失策略。 目标1将确定敲除对记忆任务性能的影响。在目标2中，不同 将根据基因敲除对海马电生理(尖波纹波)的影响进行评估 以及它们对Place细胞功能的影响。在目标3中，组织学研究将是 在细胞水平上评估基因敲除的影响，观察苔藓纤维发芽和 突触数量。这些实验将阐明候选基因在记忆过程和 确定每种细胞类型的相对贡献是什么。

PROJECT SUMMARY/ABSTRACT Temporal lobe epilepsy (TLE) is the most common form of focal epilepsy and is often refractory to medical management. In addition to seizures, patients often suffer from cognitive comorbidities, with memory impairment being the characteristic deficit seen in TLE. Despite the severe impact on patients’ quality of life, the mechanisms underlying poor memory are not understood and therapies that directly address it are limited. Previous studies in the lab have identified impaired place cell stability in the pilocarpine mouse model of TLE, which could underlie the spatial memory dysfunction in these animals. The place cell deficits do not emerge until 6-weeks after the epileptogenic insult (i.e. pilocarpine-induced status epilepticus, SE), suggesting that there are pathological processes triggered initially that take weeks to degrade hippocampal coding. This delay presents an opportunity for therapeutic intervention to prevent the degradation of hippocampal spatial representation, and is an important timepoint to examine for a comprehensive understanding of the cellular and molecular basis of memory dysfunction in TLE. The main hypothesis of this proposal is that circuit reorganization precedes the changes in network properties that cause place cell dysfunction and memory impairment, and that modulating circuit reorganization can improve place cell functioning and memory. Given the great cell type heterogeneity in the hippocampus, we performed single nuclei RNA sequencing on hippocampal tissue to determine cell type specific transcriptomic changes. We identified differentially expressed genes (DEGs) a different time points after pilocarpine-induced SE and focus follow-up studies on astrocytes given their emerging role in cognitive processing. To enrich for changes relevant to memory impairment, we compared the astrocytic DEGs at 3-weeks to a previously characterized Alzheimer’s disease (AD) model. AD, like TLE, is a disorder afflicted by memory impairment and has prominent hippocampal involvement. Within the overlapping genes, there is a functionally linked subset with roles in proteolysis: Cathepsins B, D, and L (Ctsb, Ctsd, Ctsl) and cystatin C (Cst3). These candidate genes have each been implicated in AD pathology in humans, although whether their role is protective or pathogenic is not fully determined. Additionally, while they were identified through analysis of astrocytic DEGs, they are broadly expressed across many cell types. In this proposal, we will use a CRISPR-mediated gene deletion strategy to selectively knockout each of these candidates from astrocytes, microglia, and interneurons. The effects of the knockouts on performance on a memory task will be determined in Aim 1. In Aim 2, the different knockouts will be evaluated in terms of their effects on hippocampal electrophysiology (sharp wave ripple occurrence, theta phase locking) and their effects on place cell function. In Aim 3, histological studies will be performed to evaluate the effects of the knockouts on a cellular level, looking at mossy fiber sprouting and synapse quantity. These experiments will elucidate the role of the candidate genes on memory processes and determine what the relative contributions are from each cell type.