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 p...