Project Summary: Chronic inflammation and glutamate dysregulation are pathological processes that precede and hasten brain aging and degeneration in major depression (MD). Herein, we propose the kynurenine (KYN) pathway as a mediator of the association between inflammation and glutamate pathologies. Kynurenines are neuroactive molecules that readily access the brain from the periphery via dedicated transporters known as large neutral amino acid transporters (LAT-1) located in the blood-brain barrier. Once inside the brain, KYN is converted by activated microglial cells into metabolites such as quinolinic acid (QA) and 3-hydroxykynurenine (3HK). These downstream metabolites of KYN exert robust excitotoxicity (QA) and oxidative stress (3HK) and ultimately can lead to neuronal dysfunction and death. Extensive preclinical data and our preliminary data indicate that the production and transfer of kynurenines into the brain from peripheral tissues is substantially increased by biological factors such as immune activation and aging. Preclinical data also indicate that blockade of KYN influx into the brain via competitive blockade of LAT-1 by leucine (a higher affinity ligand) abrogates depressive-like behavior induced by immune activation in rodents. Our preliminary data in humans indicate that peripheral KYN drives KYN metabolites in the brain, leading to increased basal ganglia glutamate, reduced coherence of local neuronal activity, decreased functional connectivity among brain regions, and depressive behaviors, including anhedonia and psychomotor slowing. Based on these data, we propose a 6- week challenge paradigm in which middle-aged MD patients with increased inflammation will be randomized to either leucine or lysine (n= 75 total). In contrast to leucine, lysine does not inhibit the influx of KYN as it uses a different set of transporters (cationic transporters) to access the brain. We will estimate glutamate and inositol (astroglial marker) using magnetic resonance spectroscopy (MRS); and study the coherence of local brain activity and functional connectivity in basal ganglia and dorsomedial prefrontal cortical (dmPFC) regions using resting-state functional magnetic resonance imaging (fMRI) at baseline, 1 and 6-weeks of treatment. Using standardized instruments, we will measure anhedonia and psychomotor slowing while sampling blood and cerebrospinal fluid (6 weeks only) for inflammatory mediators and KYN metabolites. This study design will test the hypotheses that a 6-week leucine challenge will decrease glutamate and inositol (Aim 1), improve local activity coherence and functional connectivity in the basal ganglia and dmPFC (Aim 2), and reduce anhedonia and psychomotor slowing (Aim 3). If supported, these aims will implicate KYN dysregulation as a key mediator of the brain and behavioral changes induced by inflammation and aging in MD. Moreover, this proof-of- mechanism proposal will identify KYN and its transporters in the BBB as key targets...