PROJECT ABSTRACT Alzheimer’s disease (AD) is a devastating and growing epidemic without means of definitive diagnosis, prevention, or disease-modifying treatment. AD features a decades-long prodromal period in which amyloid-β (Aβ) accumulation precedes neurodegeneration and cognitive impairment. This time period represents a critical window of therapeutic intervention to intercept before irreversible neurodegeneration. Synaptic loss is the strongest pathological correlate to cognitive decline, although neuron specific and microglial dependent mechanisms contributing to synaptic loss are unknown. To this end, there are critical gaps in our understanding of (1) early molecular changes occurring in synapses driven by Aβ pathology, (2) the impact of age on Aβ- induced molecular changes in the synapse, and (3) the role of microglia in these early synaptic changes. The overall goal of this proposal is to identify age-dependent and synapse-specific effects of Aβ deposition in mouse models of AD, and to determine the role microglia play in Aβ-driven synaptic proteomic changes. My central hypothesis is that microglia are key mediators of early synaptic proteomic changes that occur as a result of progressive Aβ pathology. I will rigorously test my hypothesis through two specific aims. First (Aim 1), I will characterize early and age-dependent effects of Aβ pathology on protein-level changes in neuronal synapses. I will study neuronal as well as synapse-specific changes in a mouse model of Aβ pathology (5xFAD) by expressing a promiscuous biotin ligase (TurboID) to specifically label proteins expressed in neurons followed by synaptic fractionation. This will allow us to purify neuronal proteins from synapse-fractionated preparations without requiring cell type isolation. By applying mass spectrometry (MS) to these samples, I will define protein changes occurring specifically in neurons as a result of Aβ pathology and aging (1.5, 3 and 6 months) using a combination of differential expression and network approaches (Aim 1). These studies will reveal protein signatures altered specifically by amyloid pathology which emerge before synaptic loss and cognitive impairment. Next (Aim 2), I will mechanistically determine how microglia contribute to early Aβ-driven pathological changes in neurons using an in-vivo microglia depletion strategy (CSF1R inhibition). Neuron-labeled 5xFAD and WT mice will undergo microglia depletion and we will apply MS to identify early neuronal protein changes that are microglia-dependent and microglia-independent. By integrating mouse proteomic data from both aims with existing human brain proteomic generated through the accelerating medicines partnership (AMP-AD), I will be able to identify molecular changes occurring in neurons in early pre-symptomatic stages of AD progression. Successful completion of these aims will address critical gaps in the fields of neuroproteomics and AD, particularly the need to resolve early molecular changes...