Browsing by Author "Fischer, Kathryn"

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    Sucla2 Knock‐Out in Skeletal Muscle Yields Mouse Model of Mitochondrial Myopathy With Muscle Type–Specific Phenotypes
    (Wiley, 2024) Lancaster, Makayla S.; Hafen, Paul; Law, Andrew S.; Matias, Catalina; Meyer, Timothy; Fischer, Kathryn; Miller, Marcus; Hao, Chunhai; Gillespie, Patrick; McKinzie, David; Brault, Jeffrey J.; Graham, Brett H.; Medical and Molecular Genetics, School of Medicine
    Background: Pathogenic variants in subunits of succinyl-CoA synthetase (SCS) are associated with mitochondrial encephalomyopathy in humans. SCS catalyses the conversion of succinyl-CoA to succinate coupled with substrate-level phosphorylation of either ADP or GDP in the TCA cycle. This report presents a muscle-specific conditional knock-out (KO) mouse model of Sucla2, the ADP-specific beta subunit of SCS, generating a novel in vivo model of mitochondrial myopathy. Methods: The mouse model was generated using the Cre-Lox system, with the human skeletal actin (HSA) promoter driving Cre-recombination of a CRISPR-Cas9-generated Sucla2 floxed allele within skeletal muscle. Inactivation of Sucla2 was validated using RT-qPCR and western blot, and both enzyme activity and serum metabolites were quantified by mass spectrometry. To characterize the model in vivo, whole-body phenotyping was conducted, with mice undergoing a panel of strength and locomotor behavioural assays. Additionally, ex vivo contractility experiments were performed on the soleus (SOL) and extensor digitorum longus (EDL) muscles. SOL and EDL cryosections were also subject to imaging analyses to assess muscle fibre-specific phenotypes. Results: Molecular validation confirmed 68% reduction of Sucla2 transcript within the mutant skeletal muscle (p < 0.001) and 95% functionally reduced SUCLA2 protein (p < 0.0001). By 3 weeks of age, Sucla2 KO mice were 44% the size of controls by body weight (p < 0.0001). Mutant mice also exhibited 34%-40% reduced grip strength (p < 0.01) and reduced spontaneous exercise, spending about 88% less cumulative time on a running wheel (p < 0.0001). Contractile function was also perturbed in a muscle-specific manner; although no genotype-specific deficiencies were seen in EDL function, SUCLA2-deficient SOL muscles generated 40% less specific tetanic force (p < 0.0001), alongside slower contraction and relaxation rates (p < 0.001). Similarly, a SOL-specific threefold increase in mitochondria (p < 0.0001) was observed, with qualitatively increased staining for both COX and SDH, and the proportion of Type 1 myosin heavy chain expressing fibres within the SOL was nearly doubled (95% increase, p < 0.0001) in the Sucla2 KO mice compared with that in controls. Conclusions: SUCLA2 loss within murine skeletal muscle yields a model of SCS-deficient mitochondrial myopathy with reduced body weight, muscle weakness and exercise intolerance. Physiological and morphological analyses of hindlimb muscles showed remarkable differences in ex vivo function and cellular consequences between the EDL and SOL muscles, with SOL muscles significantly more impacted by Sucla2 inactivation. This novel model will provide an invaluable tool for investigations of muscle-specific and fibre type-specific pathogenic mechanisms to better understand SCS-deficient myopathy.
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    The Impact of Abstinence from Chronic Alcohol Consumption on the Mouse Striatal Proteome: Sex and Subregion-Specific Differences
    (2024-10) Duffus, Brittnie-lee Marie; Atwood, Brady; Oblak, Adrian; Mao, Yao-Ying; Baucum, AJ; Fischer, Kathryn
    Alcohol misuse is the third leading preventable cause of death in the world. The World Health Organization currently estimates that 1 in 20 deaths are directly alcohol related. One of the ways in which consuming excessive levels of alcohol can both directly and indirectly affect human mortality and morbidity, is through chronic inflammation. Recently, studies have suggested a link between increased alcohol use and the incidence of neuroinflammatory-related diseases. However, the mechanism in which alcohol potentially influences neuroinflammatory processes is still being uncovered. We implemented an unbiased proteomics exploration of alcohol-induced changes in the striatum, with a specific emphasis on proteins related to inflammation. The striatum is a brain region that is critically involved with the progression of alcohol use disorder. Using mass spectrometry following voluntary alcohol self-administration in mice, we show that distinct protein abundances and signaling pathways in different subregions of the striatum are disrupted by chronic exposure to alcohol compared to water drinking control mice. Further, in mice that were allowed to experience abstinence from alcohol compared to mice that were non-abstinent, the overall proteome and signaling pathways showed additional differences, suggesting that the responses evoked by chronic alcohol exposure are dependent on alcohol use history. To our surprise we did not find that chronic alcohol drinking or abstinence altered protein abundance or pathways associated with inflammation, but rather affected proteins and pathways associated with neurodegeneration and metabolic, cellular organization, protein translation, and molecular transport processes. These outcomes suggest that in this drinking model, alcohol-induced neuroinflammation in the striatum is not a primary outcome controlling altered neurobehavioral function, but these changes are rather mediated by altered striatal neuronal structure and cellular health.