Project Summary/Abstract Although small-molecule BACE1 inhibitors have been successfully developed to lower amyloid- (A ) levels in brain, the phase II/III clinical trials continue to fail in patients with mild-to-moderate and prodromal Alzheimer's disease (AD). Currently, the safety and efficacy of these agents are unknown. In fact, multiple BACE1 inhibitors showed, in common, dose-dependent cognitive worsening (rather than improvement), most likely reflecting side effects associated with BACE1 over-inhibition (i.e., `on-target' class problems) rather than compound-specific effects. We hypothesize that combining 2 therapeutic agents that target separate pathways may offer synergistic benefits and allow for the lower dose of individual drugs to reduce the side effect. However, due to lack of preclinical evidence, the combination treatment is scarcely undergoing clinical trials to improve the efficacy and safety of BACE1 inhibitors. In this project, we will explore a novel combination intervention with BACE1 inhibitors and symptomatic drugs in AD model, which may be able to increase the effectiveness of BACE1 inhibitors within the safe dose range. Specifically, we will test whether combined chronic treatment with the BACE1 inhibitor (GRL-8234 or verubecestat) and memantine can provide synergistic benefits in 5XFAD mice at 12 months of age (the endpoint following 2-month treatment), which develop profound A pathology and are not responsive to each drug monotherapy. In Aim 1, we will apply a broad battery of hippocampus-dependent cognitive assays (e.g., the contextual fear conditioning, water maze, spontaneous alternation Y-maze, and nesting behavior) to evaluate the synergy between BACE1 inhibitor and memantine treatments in reversing behavioral impairments in 5XFAD mice. Hippocampal CA1 slice electrophysiology will also be performed to address synaptic benefits (e.g., LTP) the combination (but not individual) treatment can produce. In Aim 2, we will conduct biochemistry and immunostaining experiments to clarify the underlying mechanisms. Memantine cannot block low-molecular-weight A oligomer toxicity or mechanistically access the intracellular buildup of C99 (a direct precursor to A ) as an NMDA receptor antagonist, whereas BACE1 inhibitors reduce both pathogenic -products of APP irrespective of disease stages. Meanwhile, memantine but not BACE1 inhibitors can reverse the multiple proximate causes of cognitive symptoms that are triggered by the over-activation of extrasynaptic NMDA receptors (e.g., dysregulated downstream signaling and inflammatory immune responses). In addition to these biological measures for efficacy, changes in the BACE1 cleavage of physiological substrates other than APP (e.g., synaptic proteins SEZ6 and CHL1) and hippocampal neurogenesis will also be assessed to address the mechanisms of potential adverse effects. Collectively, if successful, our mouse model study will provide proof of concept for new combination AD therapy ...