275 - Mitochondrial bioenergetic deficits in hypoxic ischemic brain injury are reversed by Allopregnanolone.

Publication Number: 4271.275

Nahla Zaghloul, University of Florida, Gainesville, FL, FL, United States; Michael D.. Weiss, University of Florida, Gainesville, FL, United States; Mohamed Ahmed, University of Florida College of Medicine, GAINESVILLE, FL, United States

Background: Hypoxic-ischemic (HI) brain injury in premature infants causes major cognitive deficits and cerebral palsy (CP), with no therapy currently available for premature infants. HI causes mitochondrial dysfunction, energy failure, and neuronal death. Allopregnanolone (ALLO) is an endogenous neuro-steroid essential for brain growth and neuronal and glial cell survival. Premature infants are devoid of placental ALLO supply.

Objective: To evaluate the effect of Allopregnanolone on mitochondrial ultrastructure and function in hypoxic-ischemic brain injury.

Design/Methods: A neonatal mouse model of HI, where carotid artery ligation was performed at P5 and then treated with three doses of ALLO (10 mg/kg/dose IP) on P5, P8, and P11 or saline. At P12, transmission electron microscopy, mitochondrial respiration, and biogenesis were analyzed. Key mitochondrial enzyme protein expression, markers of oxidative stress, and targeted mitochondrial transcriptomic analysis were analyzed. At P60, the mice underwent neurobehavioral evaluation.

Results: Transmission electron microscopy revealed small, fragmented mitochondria with loss of mitochondrial membrane and cristae in hippocampal and cortical neurons of the HI group, whereas ALLO preserved the mitochondrial ultrastructure. ALLO significantly mitigated deficits in mitochondrial respiration (measured by Seahorse XF-96 metabolic analyzer) and biogenesis (mitochondrial DNA copy number and Peroxisome proliferator-activated receptor gamma coactivator 1-alpha {PGC-1α} western blot), restored key mitochondrial enzyme levels (oxoglutarate dehydrogenase {OGDH} and pyruvate dehydrogenase subunit E1α {PDHE1α} of the Krebs cycle), and reduced HI-induced reactive oxygen species H2O2 and lipid peroxidation. To explore the mechanisms by which ALLO regulates the brain metabolism, we conducted targeted transcriptome analysis of mitochondria isolated from cortical and hippocampal tissue. ALLO upregulated genes involved in glucose metabolism, mitochondrial bioenergetics and neural repair, while downregulated genes involved in mitochondrial uncoupling and dynamics, glutamate and inflammatory responsive genes. ALLO-treated HI mice exhibited significantly improved motor function measured by grip strength meter, coordination measured by rotarod, learning, and memory measured by novel object recognition test.

Conclusion(s): ALLO emerges as a unique natural neurotherapeutic steroid for HI brain injury, reducing neuroinflammation and oxidative stress, preserving mitochondrial structure, enzyme function, bioenergetics, and improving long-term neurodevelopmental outcomes.