The critical neuropathology underlying the cognitive decline in Alzheimer's disease is the loss of synapses. A leading view of the pathogenesis of Alzheimer's disease is that synaptic abnormalities are produced that lead to enhanced synapse elimination. The synaptopathy in AD is thought to be due largely to the production of toxic soluble oligomers of the Aβ1-42 peptide (oAβ). Soluble Aβ oligomers, but not monomers, have been shown to cause synaptic dysfunction, manifest by inhibition of LTP, enhancement of LTD, loss of dendritic spines, biochemical abnormalities, and hyperactivity. The enhanced LTD and hyperactivity appear to be due to elevation of extracellular glutamate as a consequence of impaired glutamate reuptake. Although substantial evidence has accumulated to support this hypothesis, there are significant gaps in our understanding of how oAβ perturbs glutamate homeostasis. Specifically, the identity of the glutamate transporter or transporters targeted by oAβ to produce the defect in glutamate homeostasis is unknown, as are the molecular mechanisms by which glutamate transport function is compromised by oAβ. These gaps loom greater in light of recent evidence that monoclonal antibodies (aducanumab; BAN2401) targeting soluble Aβ oligomers in AD patients may slow cognitive decline. The major glutamate transporter in the forebrain is GLT- 1 (human homolog EAAT2), which represents 1% of brain protein. GLT-1 is expressed in both astrocytes and glutamatergic axon terminals. Recent work by the applicant using a conditional GLT-1 knockout (KO) has shown that GLT-1 expressed in axon terminals is the dominant transporter mediating glutamate uptake into crude synaptosome preparations, also known as plasma membrane vesicles (PMVs). GLT-1 expressed in presynaptic terminals has also been shown to play an important role in synaptic mitochondrial metabolism. Several studies suggest that in the human and in mouse models deficits in glutamate transporter expression and/or function occur in AD. In critical experiments, glutamate uptake into PMVs derived from brain slices was decreased when the slices were treated with oAβ, implicating neuronal GLT-1. The central hypothesis motivating this project is that GLT-1 is the primary mechanistic target of oAβ causing glutamate dyshomeostasis. Given these findings it is important to ascertain whether GLT-1 is the specific glutamate transporter targeted by oAβ, whether oAβ affects GLT-1 function in astrocytes or neurons, or both, and the molecular basis for the interaction of oAβ with GLT-1. The specific goals of this project are to: Aim 1: Identify the glutamate transporter whose function is impaired by oAβ. Aim 2: Determine the cellular localization of effects of oAβ on GLT-1 using a conditional GLT-1 KO. The pursuit of these goals will lead to a molecular understanding of how oAβ, the salient AD cytotoxins, perturb glutamate homeostasis in AD and ultimately lead to novel approaches to prevent and treat AD.