PROJECT SUMMARY
Neurodegenerative disease (ND) is a heterogeneous group of disorders characterized by the progressive loss
of specific neural clusters within the central nervous system (CNS). Mitochondrial dysfunction and branched-
chain amino acid (BCAA) metabolism have been linked to loss of CNS structure and function, hallmarks of ND
pathogenesis. However, the precise sequence of events leading to metabolic-induced neurodegeneration is
unknown. Growing evidence suggests chronic accumulation of BCAAs is pathogenic due to their pivotal role in
modulating the cerebral metabolism of glutamate. Recently, it was discovered that knockdown of BCAT1, a
crucial enzyme in the BCAA degradation pathway, is sufficient to induce widespread dopaminergic cell death
and motor impairment. Furthermore, increased mitochondrial respiration is required for BCAT1 neurotoxicity
through reactive oxygen species (ROS)-mediated damage, indicating an alternative mechanism between
disease etiology and metabolic dysfunction in disease. Our lab has recently identified pathogenic biallelic
missense variants in BCAT1 in a pediatric neurodegeneration patient. The mechanisms underlying abnormal
BCAA metabolism and BCAT1-associated neurotoxicity in ND pathogenesis have yet to be investigated. In this
study, human BCAT1 will be directly tested for its ability to induce neurodegeneration in vitro. I hypothesize that
loss of BCAT1 function disrupts BCAA catabolism and increases mitochondrial respiration to promote
neuronal degeneration through ROS-mediated oxidative damage. Using a combined model approach of
lentiviral RNAi-mediated and CRISPR/cas9 BCAT1 knockdown in differentiated neurons (B1N) and human-
induced pluripotent stem cell-derived neurons (B1hiPSC-dN), I will address this hypothesis and define the
mechanism of a novel pediatric neurodegenerative disorder in the following specific aims. In Aim 1A, I will test
if BCAT1 deficiency contributes to neuropathology by quantifying the presence of neurodegeneration in
mammalian neurons B1N and B1hiPSC-dN. To investigate the impact of BCAT1 downregulation on BCAA
catabolism, in Aim 1B, I will measure the accumulation of BCAAs and identify metabolic alterations in B1N and
B1hiPSC-dN using high-throughput metabolomic analyses. In Aim 2, I will assess whether BCAT1 deficiency is
sufficient to induce ROS-mediated oxidative damage. If so, this will further support a novel metabolic mechanism
underlying neuronal degeneration and suggest common therapeutic avenues. The findings from these aims will
provide the first in vitro investigation into the role of BCAT1 in ND pathogenesis and BCAA accumulation toxicity
in vitro, with significant implications for defining mitochondrial dysfunction disease mechanisms. As there is no
effective treatment available for neurodegeneration, understanding the metabolic contributions to disease
pathology will help to better inform future therapeutic interventions.
