Isolating hBCATm functions in human brain models

Project Summary Levels of the human branched-chain aminotransferase enzyme (BCAT) mitochondrial isoform (hBCATm) significantly increase in the brain microvascular endothelial cells of patients with Alzheimer’s disease (AD) as the disease progresses. The downstream effects of this are unknown and will be investigated in this study. Functionally, BCAT catalyzes the transamination of α-ketoglutarate and the branched-chain amino acids (BCAA) to respectively produce glutamate and branched-chain keto acids, the latter of which can be further metabolized to TCA cycle intermediates and ketone bodies. In the human brain, hBCATm is exclusively expressed in brain endothelial cells. We therefore hypothesize that BCAAs are metabolized by hBCATm in brain endothelial cells, and the products either shuttled to the astrocytes and neurons to support energy and glutamate demands, or utilized by the endothelial cell to support maintaining a strong blood-brain barrier (BBB). While astrocyte metabolism and metabolite shuttling have been studied in depth, how endothelial cells contribute towards brain metabolism is rarely investigated. Interestingly, hBCATm transamination can be inhibited by protein oxidation and it has a ‘moonlighting’ chaperone activity which may be enhanced by oxidation, although this has only been investigated using recombinant proteins outside of cells. It is known that increased amyloid beta and inflammation are prevalent in AD and drive increased reactive oxygen species and protein oxidation levels, but it is not known if hBCATm can be oxidized by this process. We hypothesize that an increase in BCAA transamination through hBCATm may support recovery from short term brain injuries, but in chronic, progressive diseases such as AD, hBCATm eventually becomes oxidized, causing a change in function. If not reversed, this will result in impairment to brain glutamate levels, energy turnover, and enhanced pathogenicity. The first aim of the study is to elucidate the impact of increased hBCATm expression on brain metabolism and BBB integrity. We will overexpress endothelial hBCATm in multicellular brain organoid models and assay using several biochemical strategies including mass spectrometry, immunochemistry analysis, and fluorescent redox probe labeling. The second aim of the study is to determine the oxidation state and enzyme activity of hBCATm during cellular stress. For this, brain organoids will be treated with stressors such as cytokines, and then hBCATm enriched and assayed for oxidation state, transamination activity, and chaperone activity. Understanding hBCATm activity is a major puzzle piece in brain physiology. This research will help us understand how the human brain adapts to insult. Understanding this process could lead to interventional and supportive treatments such as diet modification or drugs to support BCAA metabolism.