Author ORCID Identifier

https://orcid.org/0000-0001-6123-3308

Date Available

4-25-2024

Year of Publication

2024

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department/School/Program

Neuroscience

First Advisor

Dr. Patrick Sullivan, PhD

Abstract

A large percent of both clinical and pre-clinical traumatic brain injury (TBI) studies report better outcomes females after severe injury compared to males. Mitochondrial dysfunction is a well-characterized driver of secondary injury and an attractive neurotherapeutic target; though, there is limited research available on mitochondrial dysfunction in females. However, a few studies have shown total mitochondria from the cortex of females appear protected from TBI-induced bioenergetic dysfunction. These results were replicated in these studies so further characterization was performed. Additionally, protein expression of some oxidative phosphorylation (OXPHOS) complexes were elevated in the uninjured cortex of females relative to uninjured males, which may contribute to spared bioenergetic function. The total brain mitochondrial population can be separated into two fractions with distinct functional profiles after TBI: synaptic and non-synaptic mitochondria. These outcomes were established in male models of TBI so it is unclear whether females have similar patterns of injury. A time course of bioenergetic dysfunction was created to characterize sex differences in synaptic and non-synaptic mitochondrial function. While synaptic mitochondria from both sexes were impaired after injury, the starkest sex difference was detected in non-synaptic mitochondria 24h post-controlled cortical impact (CCI) TBI. This fraction in males showed bioenergetic impairment and increased Ca2+ loading 24h post-CCI, while mitochondria from females had no functional deficits. Further, non-synaptic mitochondria from sham females had increased OXPHOS protein expression compared to sham males, which may be a result of estrogenic upregulation of mitochondrial proteins. The results suggest spared non-synaptic function in females masks synaptic dysfunction in total mitochondria after TBI. Female sex hormones have been suggested to be neuroprotective in neurological disorders like stroke and dementia. Indirect estrogen actions have been shown to converge on mitochondria to influence function. Direct administration of the most potent estrogen during the pre-menopausal period, 17β-estradiol (E2), was shown to reduce peroxide production and uncouple ATP synthase in isolated liver mitochondria. No studies have examined the direct role of E2 on mitochondrial dysfunction after TBI, though I hypothesized it would alleviate this impairment. Total mitochondria from the ipsilateral (injured) and contralateral (unimpacted control) cortices of male and female mice were isolated 24h after CCI and then treated with E2 immediately before functional assessment. E2 appeared to uncouple mitochondria from uninjured tissue in both sexes and, interestingly, these uncoupling effects were also observed in ipsilateral mitochondria from females, but not males. Mild mitochondrial uncoupling has been shown to be therapeutically relevant to treat TBI and may be another way in which E2 protects mitochondria from injury. Mitochondrial uncouplers have been shown to improve outcomes after TBI, but are limited by a narrow safety window. The uncoupling prodrug, MP201, improves mitochondrial bioenergetics, cognitive performance, and cortical tissue sparing after CCI in male mice. The mechanisms in which MP201 and E2 promote mitochondrial protection suggests there would be an additive effect in recovery after TBI. Ovariectomized mice were implanted with physiological concentrations of E2 or blank hormone 3d before CCI or sham injury. These mice were then treated with one dose of MP201 or vehicle 6h after brain surgery. Neither E2 nor MP201 improved synaptic mitochondrial bioenergetics 24h post-CCI relative to sham. To explore the long-term effects of these treatments, behavioral testing was performed after daily dosing of MP201 for one week following CCI. E2 replacement improved cognitive performance in sham mice compared to blank sham. MP201 improved cognitive performance in blank mice, but worsened performance in E2 mice. This suggests uncoupling compounds are not suitable for TBI patients undergoing hormone replacement therapy. The results from these studies highlight the importance of measuring mitochondrial dysfunction in both sexes and accounting for hormonal status when developing mitochondrial-targeted therapeutics to treat TBI.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2024.209

Funding Information

This study was supported by the "Neurobiology of Central Nervous System Injury and Repair" Training grant (no.: 5T32 NS077889) from 2021-2023, the Lexington Veterans Affairs Merit Award (no.: 2I01BX003405) from 2020-2024, the Kentucky Spinal Cord and Head Injury Research Trust (no.: 20-7A) from 2020-2024, the National Institutes of Health (no.: R01 NS112693-01A1) from 2020-2024, and the National Institutes of Health (no.: P20 GM148326-01) in 2024.

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