Project Summary: Alzheimer’s Disease (AD) is characterized by a progressive loss of memories over time. Much like AD patients who lose the ability to recall former memories, rodent AD models show equivalent deficits in memory recall. Memory deterioration may result from a loss of synaptic long-term potentiation (LTP) and LTP-related molecular signaling mechanisms in neurons affected during AD pathogenesis, though this has not been directly examined. If we could protect LTP-like plasticity against destabilization that occurs during AD progression, we could prolong the persistence of adaptive memories, thus substantially reducing the symptomatic burden of AD patients, their families, and their caregivers. In our funded NIH Director’s Innovator Award (DP2AG067666), we proposed to develop a suite of molecular methods to modulate plasticity in a spatiotemporally-defined fashion. These approaches are based on the elucidation of the molecular mechanism of the small peptide ZIP, which we have found to work through macropinocytosis—an endocytotic process of internalizing extracellular fluid, solutes, and membrane in large endocytic vesicles known as the macropinosome. Furthermore, we find that ZIP’s memory erasing effects could be blocked by prior administration of amiloride. Importantly, we showed that amiloride not only blocks ZIP- induced destabilization of LTP, but also natural processes that remove AMPA glutamate receptors, such as those required for long-term depression (LTD)-induced behavioral extinction, and pathological processes in AD models that impair memory recall. These results demonstrate the potential of amiloride for preventing memory loss associated with AD. While we hypothesize that amiloride works by stabilizing surface AMPARs that are recruited to synapses during LTP, this has not been tested. In this administrative supplement application, we will elucidate the molecular mechanisms by which amiloride prevents the memory impairment in the genetic 5xFAD mouse model of AD. We will first examine the electrophysiological properties of neurons in the basolateral amygdala (BLA) that are critical for storage and remote recall of auditory fear conditioning memories. We will examine both the basal properties of these cells as well as the state of synaptic plasticity in both wild type control versus 5xFAD mice treated with saline or amiloride. Secondly, we will perform single nuclei RNA sequencing (snRNAseq) experiments to explore how amiloride administration may impact the molecular state of these cells. This mechanistic detail is absolutely critical towards understanding how amiloride protects memories from destabilization in rodent models of AD, and will serve as a key foundation of my future R01 application on AD.