Date of Award

Fall 2021

Document Type

Open Access Dissertation

Department

Biomedical Engineering

First Advisor

Norma Frizzell

Abstract

Metabolic dysfunction burdens tissues with high energy demands, particularly the brain. Leigh syndrome is a mitochondrial encephalopathy stemming from genetic defects in the electron transport chain. Leigh syndrome patients develop lactic acidosis, ataxia, bilateral necrotizing lesions in the brainstem and basal ganglia, lesion microgliosis, and eventually death due to respiratory failure. The NADH dehydrogenase [ubiquinone] iron-sulfur protein 4 (NDUFS4) knockout mouse is an established model of Leigh syndrome due to impaired assembly of mitochondrial Complex I that develops motoric deficits and necrotizing lesions in the brainstem vestibular nuclei and olfactory bulb. In addition to the Complex I-derived bioenergetics defect, altered production of other metabolites, including fumarate, modulate protein function due to irreversible protein modification. Fumarate-induced protein succination (2SC) is increased in the NDUFS4 knockout (KO) brainstem and olfactory bulb. In this dissertation, we investigated altered metabolite production with an emphasis on itaconate-derived regulation of microglial and macrophage function in the NDUFS4 KO mouse.

We detected limited inflammatory markers in the NDUFS4 KO olfactory bulb, despite abundant microglia. We quantified impaired metabolism, reduced phagocytic capacity, and limited inflammatory activity in macrophages as a model of the microglial response during pro-inflammatory lipopolysaccharide stimulation. Knockout of NDUFS4 promoted a metabolic shift from mitochondrial to glycolytic metabolism, decreased the metabolic reserve, and impaired the accumulation of the immunometabolites itaconate and succinate during pro-inflammatory conditions. Itaconate-derived protein dicarboxypropylation provides a robust measure of the cellular capacity to increase immunometabolism, and we observe that this is recognized by an in-house antibody detecting succinate moieties. Using this antibody as a tool, we determined that isolated NDUFS4 KO microglia do not accumulate fumarate or itaconate-induced protein modifications in contrast to neurons, where fumarate-derived succination is significantly increased.

Given that the production of the anti-inflammatory metabolite itaconate was reduced, we hypothesized that exogenous itaconate, as the ester 4-octyl itaconate (4OI), might limit microglial accumulation and improve motor behavior in NDUFS4 KO mice. 4OI administration slowed the NDUFS4 knockout-induced decline in motor endurance in male mice with beneficial trends to improve other clinical symptoms, independent of microglial content in the olfactory bulb. The data suggest that itaconate supplementation may have therapeutic potential in the NDUFS4 KO, but the mechanisms underlying this benefit remain to be determined.

Finally, we improved methods to simultaneously detect enantiomers of lactate and 2-hydroxyglutarate. We hypothesized that fumarate-derived succination of a component of the α-ketoglutarate dehydrogenase complex would favor generation of L-2-hydroxyglutarate. Total levels of L and D enantiomers of 2- hydroxyglutarate were measured in parallel with lactate in tissues of the NDUFS4 knockout. The findings suggest additional metabolic flexibility in the NDUFS4 knockout, which may be harnessed for therapeutic benefits.

In summary, we find that the loss of NDUFS4 contributes to impaired metabolic flexibility and altered metabolic responses when challenged, elucidating novel alternative targets with therapeutic potential to treat this devastating disease.

Available for download on Friday, December 15, 2023

Included in

Biomedical Commons

Share

COinS