Sex dependent Mitochondrial Mechanisms of Neonatal Cerebral Hypoxic-ischemic Encephalopathy

Abstract

Many neurodevelopmental disorders are sex-biased, with males being particularly susceptible to central nervous system (CNS) abnormalities, but mechanisms underlying the sex-biased susceptibility are unclear. Neonatal hypoxic-ischemic encephalopathy (HIE) is one such disorder affecting 1.5-2/1000 live term births that contributes to lifelong cognitive and motor impairments, with males being at a greater risk for these adverse outcomes. Moreover, sex differences in neurobehavioral outcome are observed following the Rice-Vannucci (1981) rodent model of neonatal hypoxic-ischemia (HI). The unilateral carotid artery ligation in this model of HI results in an ipsilateral infarct, and a contralateral "hypoxia-only" hemisphere. Mitochondrial dysfunction is a common feature of CNS injury with increasing evidence suggesting marked sex differences in mitochondrial metabolism of humans and rodents. Following HI, mitochondrial bioenergetic dysfunction contributes to an extended secondary energy failure lasting days or weeks, making it a prime neuroprotective target. Acetyl-L-Carnitine (ALCAR) is neuroprotective following neurotrauma in juvenile and adult animal models; ALCAR is hypothesized to function as an alternative biofuel, antioxidant or by promoting mitochondrial biogenesis but the exact mechanism of neuroprotection is unclear. Emerging evidence suggests that mechanisms implicated in the pathophysiology of CNS injury are also sex dependent including oxidative phosphorylation, oxidative stress, antioxidant defense systems, mitochondrial biogenesis, autophagy and cell death signaling pathways. Therefore, these studies tested the hypotheses that following HIE: mitochondrial function, oxidative stress, antioxidant responses, mitochondrial quality control and cell death are sex dependent, and that ALCAR administration protects against these pathophysiological mechanisms. We observed that complex I mitochondrial respiration is impaired significantly more in males than females, which is associated with increased protein oxidation, impairment of mitochondrial glutathione peroxidase (GPx) activity, and decreased GPx4 immunoreactivity in male, but not female brain. Females have a higher level of reduced glutathione (GSH) than males in shams, decreased GSH, and increased non-mitochondrial GPx activity following HI in both cerebral hemispheres. There is no increase in protein oxidation in the female brain after HI. Furthermore, we find that ALCAR reduces protein oxidation in males following HI. Moreover, we determined mitochondrial fragmentation occurs, to different extents, in both sexes 24 hours after HI. Female mitochondria in the contralateral hemisphere are degraded by mitophagy while male mitochondrial proteins are tagged for removal but the mitophagy machinery is impaired, resulting in an accumulation of damaged mitochondria in the male brain following injury. Finally, we determined that there is significant neuronal cell death in both hemispheres in the male brain following HI, while neuronal death occurs exclusively in the ipsilateral hemisphere of the female brain. These sex-dependent mitochondrial mechanisms further the understanding of a sexually dimorphic neonatal brain injury and will aid in the advancement of sex-specific therapeutic development.