Excess adrenal steroid production is a prevalent but underdiagnosed etiology of hypertension and
cardiovascular disease and poorly managed with current medical therapies. Diverse lines of complementary
evidence point to dysfunction of adrenal mitochondrial redox pathways as an unexplored pathogenic driver for
these conditions. For example, although physiologic stimuli and genetic mutations that increase production of
the adrenal steroid aldosterone act through a shared pathway of elevated cytosolic Ca2+, Ca2+ elevations are
buffered by mitochondria. Adrenal mitochondria have a unique electron transport chain that uses the redox
cofactor NADPH to support several steps in steroidogenesis, and mitochondrial Ca2+ uptake can stimulate
production of the steroids aldosterone and cortisol in a manner correlating with increased mitochondrial
NADPH. Importantly, human loss of function mutations in nicotinamide nucleotide transhydrogenase (NNT)
produce a mitochondrial NADPH deficiency that, in turn, can cause severe cortisol and aldosterone deficiency.
While together this suggests a key role for mitochondrial redox biology in adrenal steroid physiology, to date
exploration has been limited in this area due to limited knowledge of the basic biology of adrenal mitochondria.
Preliminary data presented here highlight a novel purification methodology to produce the first proteomic
characterization of adrenal mitochondria, which show notable enrichment for the redox enzymes NNT and
NQO1 (NAD(P)H:quinone acceptor oxidoreductase 1). Adrenal mitochondrial preparations are validated with
bioenergetic interrogation in a custom-built fluorimeter, and NNT and NQO1-deficient human adrenal cells are
generated with CRISPR. With these unique resources, studies are proposed to address the central hypothesis
that novel redox pathways of adrenal mitochondria regulate steroidogenesis. Mechanism will be elucidated
with bioenergetic studies of isolated mitochondria and permeabilized human adrenal cells, stable isotope
tracing of permeabilized cells, targeted LC-MS measurements of steroids and central redox
cofactors/metabolites, and activity-based metabolite profiling with recombinant enzymes on fresh adrenal
extracts. The studies will launch a new direction in the study of adrenal steroidogenesis. The applicant, Dr.
Patrick Ward, Instructor at Massachusetts General Hospital, has delineated a 5 year career plan building upon
his Ph.D. in cancer metabolism and his clinical training in internal medicine and endocrinology. Dr. Ward will
be mentored by Dr. Vamsi Mootha, a world leader in mitochondrial biology with an exceptional training record,
and advised by an outstanding committee with significant expertise in adrenal biology, hypertension, and
cardiovascular disease. Dr. Ward’s outstanding mentorship team and institutional environment, coupled with
the novel resources he has already generated, uniquely position him to execute his proposed studies and
subsequently transition to independence while rapidly ascending as a leader in adrenal metabolism.