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

颞叶癫痫记忆障碍背后的细胞类型特异性转录变化

• 批准号: 10662500
• 负责人: Victoria Ho
• 金额: $ 24.05万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-15 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10662500
• 关键词: Acute; Address; Adult; Affect; Alzheimer' s Disease; Alzheimer' s disease model; Alzheimer' s disease pathology; Animals; Astrocytes; Automobile Driving; Autophagocytosis; Behavior; Behavioral; Calcium; Candidate Disease Gene; Cathepsins; Cathepsins B; Cell physiology; Cells; Characteristics; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Cognitive; Cysteine; Development; Disease; Electrophysiology (science); Environment; Epilepsy; Episodic memory; Exhibits; Family; Fire - disasters; Follow-Up Studies; Functional disorder; Gene Deletion; Genes; Genetic Transcription; Heterogeneity; Hippocampus; Homeostasis; Human; Image; Impaired cognition; Impairment; Interneurons; Knock-out; Link; Location; Maps; Mediating; Medical; Memory; Memory impairment; Microglia; Microscope; Molecular; Mus; Neurons; Organism; Overlapping Genes; Partial Epilepsies; Pathogenicity; Pathologic; Pathologic Processes; Patients; Peptide Hydrolases; Performance; Pharmaceutical Preparations; Phase; Pilocarpine; Property; Proteolysis; Quality of life; Refractory; Role; Seizures; Status Epilepticus; Synapses; Temporal Lobe Epilepsy; Therapeutic Intervention; Time; Tissues; Up-Regulation; axon growth; axonal sprouting; carboxypeptidase C; cell type; comorbidity; differential expression; experimental study; extracellular; histological studies; improved; inhibitor; knockout gene; memory process; miniaturize; mossy fiber; mouse model; novel; post gamma-globulins; prevent; side effect; single nucleus RNA-sequencing; spatial memory; targeted treatment; transcriptomics; water maze

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.

项目总结/摘要 颞叶癫痫（TLE）是局灶性癫痫的最常见形式，并且通常是药物难治性的。 管理除了癫痫发作，患者还经常患有认知合并症，伴有记忆障碍 是颞叶癫痫的特征性缺陷。尽管对患者的生活质量有严重影响， 潜在的记忆力差还不清楚，直接解决它的疗法也很有限。以前的研究 在实验室中，已经确定了TLE的毛果芸香碱小鼠模型中位置细胞稳定性受损，这可能是TLE的基础。 这些动物的空间记忆障碍。位置细胞缺陷直到6周后才出现。 致痫性损伤（即匹鲁卡品诱导的癫痫持续状态，SE），表明存在病理性 最初触发的过程需要数周时间来降解海马编码。这一拖延提供了一个机会 用于治疗干预以防止海马空间表征的退化，并且是重要的 时间点检查，以全面了解记忆的细胞和分子基础 TLE中的功能障碍。这一建议的主要假设是，电路重组之前， 网络特性导致位置细胞功能障碍和记忆障碍， 重组可以改善位置细胞功能和记忆。考虑到细胞类型的巨大异质性， 海马，我们对海马组织进行单核RNA测序，以确定细胞类型特异性 转录组学变化。我们发现差异表达的基因（DEG）在不同的时间点后， 毛果芸香碱诱导的SE和对星形胶质细胞的重点随访研究，考虑到其在认知功能中的新作用 处理.为了丰富与记忆障碍相关的变化，我们比较了3周时星形胶质细胞的DEG， 先前表征的阿尔茨海默病（AD）模型。AD和TLE一样， 有明显的海马受累。在重叠的基因中，有一个功能性的 在蛋白水解中起作用的连锁亚群：组织蛋白酶B、D和L（Cts B、Cts d、Cts 1）和胱抑素C（Cst 3）。这些 每个候选基因都与人类AD病理学有关，尽管它们的作用是否是保护性的， 或者致病性不完全确定。此外，虽然它们是通过分析星形胶质细胞DEG鉴定的， 它们在许多细胞类型中广泛表达。在本提案中，我们将使用CRISPR介导的基因 删除策略以从星形胶质细胞、小胶质细胞和中间神经元中选择性地敲除这些候选物中的每一个。 目标1中将确定敲除对记忆任务表现的影响。在目标2中， 将根据它们对海马电生理学（尖波波纹涟漪的影响来评价敲除 发生，θ相位锁定）及其对位置细胞功能的影响。在目标3中，组织学研究将 进行了评估敲除对细胞水平的影响，观察苔藓纤维发芽， 突触数量这些实验将阐明候选基因在记忆过程中的作用， 确定每种细胞类型的相对贡献。