PROJECT 3 – ABSTRACT SUMMARY In alignment with the overall P01—Strategies for targeting Astrocyte Reactivity in AD and ADRD— Project 3 will target the astrocyte glutamate transporter EAAT2/Glt-1 (gene name SLC1A2) and assess the impact on cerebrovascular function, metabolism and neural network activity in Alzheimer’s disease (AD) and related dementias (ADRD). We and other groups have shown that SLC1A2 levels are reduced in AD and ADRD— a change that is recapitulated in many AD and ADRD mouse models. SLC1A2 is well known to guard against excitotoxic damage to synapses by minimizing synaptic glutamate spillover. This mechanism has been studied in many different disease models. However, much less is known about whether SLC1A2 similarly prevents glutamate from adversely affecting astrocyte endfeet and cerebrovessels, which are widely known to undergo dysfunction in AD and ADRD. SLC1A2 is also well known to drive the production of lactate— a key component of the astrocyte-neuron lactate shuttle. Nonetheless, few studies have investigated the possibility that loss of SLC1A2 in reactive astrocytes impairs lactate shuttling, leading to hypometabolism (another well-characterized pathology in AD and ADRD). We hypothesize that the loss of SLC1A2 is a key “loss of function phenotype” of reactive astrocytes that underlies impaired cerebro-vascular function/integrity, hypometabolism, and disrupted neural network activity in AD and ADRD. We will test this hypothesis across three Aims using two astrocyte- targeting approaches: a restoration approach where we use AAV-Gfa2 to overexpress SLC1A2 in astrocytes of a comorbid Aβ/vascular model (5xFAD mice with hyperhomocystenemia) and a knockdown approach where we will use AAV-GfaP-Cre to knock-down astrocytic SLC1A2 in FLOX-SLC1A2 mice. Aim 1 will determine the impact of SLC1A2 modulation on astrocyte Ca2+ signaling, endfeet and BBB integrity, cerebral blood flow, and neurovascular coupling using two photon (2P) imaging in awake mice, and MRI (in anesthetized mice). Aim 2 will determine the impact of SLC1A2 modulation on lactate and glucose signaling in awake mice using microelectrode arrays. And Aim 3 will test the effects of SLC1A2 modulation on neuronal network fidelity and synaptic function and plasticity using either 2P imaging of awake mice or electrophysiology on in situ brain slices. Points of interaction: All functional outcomes are assessed in our Animal Vascular-Metabolic-Neural Network core (Core B). Interrelationships between different astrocyte pathways (MMP9 modulation /Project 1, Insulin receptor modulation/Project 2, and KATP modulation/Project 4) will be assessed as a function of AD and ADRD comorbidities through the shared use of postmortem tissue across projects (cerebrovascular pathology/Project 1, amyloid pathology/Project 2, amyloid+vascular pathology/Project 3, LATE + hippocampal sclerosis pathology/Project 4). Similar interrelationships will be assessed across comorbidities in shared postmort...