Investigating Neuroprotective Roles of BCAT1 During Hypoxia

About This Grant

PROJECT SUMMARY: Hypoxic ischemic encephalopathy (HIE) is a developmental brain injury caused by transient reductions in oxygen supply to the term fetus. HIE is a significant cause of neurodevelopmental disorders like autism and epilepsy, suggesting that prenatal hypoxia exposure can cause lifelong neurological dysfunction. The underlying molecular mechanism(s) are not well understood, but an emerging hypothesis suggests that disruptions to epigenetics during prenatal hypoxia may be causal to neurodevelopmental phenotypes. Notably, hypoxia’s effect on cellular metabolism influences pools of metabolite precursors for epigenetic marks, highlighting the importance of investigating the metabolic-epigenetic axis of perinatal hypoxic brain injury. A paradigm of mild prenatal hypoxia demonstrated the upregulation of all three branched chain amino acids (BCAAs), leucine, isoleucine, and valine, in fetal mouse brain. Additionally, our analysis revealed that expression of branched chain amino acid transaminase 1 (Bcat1), the enzyme that catalyzes the catabolism of the BCAAs, is also significantly upregulated in almost all neuronal types by prenatal hypoxia. BCAT1 transaminates the BCAAs, in that it catalyzes the reversible transfer of the amino group from the BCAAs to alpha-ketoglutarate (a-KG) to form glutamate and the branched chain ketoacids (BCKAs). BCAA supplementation has recently emerged as a therapeutic for traumatic brain injury, and thus understanding hypoxia’s influence on BCAA metabolism could reveal a critical role for BCAT1 in neonatal hypoxic brain injury. Additionally, BCAT1 regulates the concentrations of glutamate, the overproduction of which can induce neuronal injury during prolonged periods of hypoxia, and a-KG, which is an essential cofactor for chromatin modifying enzymes. Moreover, accumulating evidence suggests BCAT1 may play a role in reducing oxidative stress in multiple cell types, making it an appealing potential neuroprotective target for studies of neonatal hypoxic brain injury. I hypothesize that BCAT1 plays a neuroprotective role during hypoxia by modulating histone methylation and reactive oxygen species (ROS) in neurons. In this proposed set of aims, I will address this hypothesis utilizing neurons differentiated from human induced pluripotent stem cells (hiPSCs) and primary mouse neurons to assess BCAT1’s impact on BCAA metabolism, epigenetics and oxidative stress during hypoxia. In Aim 1, I will use stable isotope tracing and steady state amino acid measurement by liquid chromatography-mass spectrometry (LC-MS) to define hypoxia’s effect on neuronal BCAA metabolism. Additionally, I will use Cleavage Under Targets and Tagmentation (CUT&Tag) and bulk mRNA-sequencing (RNA-seq) to profile BCAT1’s impact on histone methylation and gene expression during hypoxia. In Aim 2, I will test potential mechanisms of neuroprotection by measuring ROS, cell viability, and neuronal morphology in BCAT1-/- neurons exposed to hypoxia. Characterizing hypoxia’s influence on BCAT1 regulation of BCAA metabolism, epigenetics, and oxidative stress is crucial to evaluating the potential for BCAA supplementation or restriction as a novel therapeutic.