{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,20]],"date-time":"2026-09-20T16:33:00Z","timestamp":1789921980383,"version":"4.0.1"},"posted":{"date-parts":[[2026,5,20]]},"group-title":"Cell Biology","reference-count":80,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,5,20]],"date-time":"2026-05-20T00:00:00Z","timestamp":1779235200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,14]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Multiple acyl-CoA dehydrogenase deficiency (MADD) is a rare lipid storage myopathy caused predominantly by pathogenic variants in ETFDH, which encodes ETF-QO in\n                  <jats:italic>Drosophila<\/jats:italic>\n                  , a mitochondrial electron-transfer protein required for fatty acid \u03b2-oxidation. Impaired ETF-QO function disrupts electron transfer from multiple acyl-CoA dehydrogenases to the ubiquinone pool, resulting in lipid accumulation, mitochondrial dysfunction, and progressive neuromuscular and cardiac abnormalities. Despite the availability of animal models of fatty acid oxidation disorders, genetically precise in vivo models carrying patient-relevant ETFDH missense variants and permitting longitudinal assessment of disease progression remain limited. Here, we generated CRISPR\/Cas9 knock-in\n                  <jats:italic>Drosophila melanogaster<\/jats:italic>\n                  models of late-onset MADD harboring three conserved Etf-QO missense substitutions (\n                  <jats:italic>L138R<\/jats:italic>\n                  ,\n                  <jats:italic>S307C<\/jats:italic>\n                  , and\n                  <jats:italic>L409F<\/jats:italic>\n                  ), corresponding to human ETFDH mutations\n                  <jats:italic>L127R<\/jats:italic>\n                  ,\n                  <jats:italic>S296C<\/jats:italic>\n                  , and\n                  <jats:italic>L399F<\/jats:italic>\n                  , respectively. These variants are located to the conserved FAD- and ubiquinone-binding regions of ETF-QO. Etf-QO mutant flies developed progressive deficits in locomotor activity and skeletal muscle performance accompanied by marked lipid droplet accumulation in indirect flight muscles, cardiac tissue, and fat bodies. Cardiac phenotyping further revealed impaired function, characterized by reduced fractional shortening, prolonged heart period, and increased arrhythmicity index. Consistent with mitochondrial bioenergetic dysfunction, Etf-QO mutants exhibited reduced oxygen consumption, increased oxidative stress, and decreased ATP levels. Molecular analyses indicated activation of cellular energy- and mitochondrial-stress responses, including increased AMPK and PGC-1\u03b1 signaling, together with elevated Pink1, Parkin, and SOD2 expression, suggesting engagement of mitochondrial quality-control and antioxidant defense pathways in response to ETF-QO dysfunction. Importantly, exercise paradigm consisting of 15 min of daily exercise for 2.5 weeks significantly improved locomotor, skeletal muscle, and cardiac performance while reducing lipid accumulation and reactive oxygen species burden. Together, these findings establish innovative CRISPR knock-in\n                  <jats:italic>Drosophila<\/jats:italic>\n                  models that recapitulate key neuromuscular, metabolic, mitochondrial, and cardiac features of ETFDH-associated MADD. The models reveal coordinated mitochondrial stress and energy-signaling responses to ETF-QO dysfunction and demonstrate that exercise can ameliorate multiple disease phenotypes. These findings provide a tractable\n                  <jats:italic>in vivo<\/jats:italic>\n                  platform for dissecting MADD pathogenesis and evaluating potential therapeutic strategies targeting mitochondrial dysfunction and lipid storage myopathies.\n                <\/jats:p>","DOI":"10.64898\/2026.05.18.726022","type":"posted-content","created":{"date-parts":[[2026,5,21]],"date-time":"2026-05-21T07:50:15Z","timestamp":1779349815000},"source":"Crossref","is-referenced-by-count":1,"title":["Innovative CRISPR Etf-QO Mutant Knock-in Model Mimics MADD-associated Lipid Storage Myopathy and Reveal Therapeutic Potential of Exercise Intervention"],"prefix":"10.64898","author":[{"given":"Sachin","family":"Budhathoki","sequence":"first","affiliation":[{"name":"Department of Pathology, Division of Molecular and Cellular Pathology, Heersink School of Medicine, Heersink School of Medicine, The University of Alabama at Birmingham, AL 35294, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yiming","family":"Guo","sequence":"additional","affiliation":[{"name":"Department of Pathology, Division of Molecular and Cellular Pathology, Heersink School of Medicine, Heersink School of Medicine, The University of Alabama at Birmingham, AL 35294, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mary","family":"Doamekpor","sequence":"additional","affiliation":[{"name":"Department of Pathology, Division of Molecular and Cellular Pathology, Heersink School of Medicine, Heersink School of Medicine, The University of Alabama at Birmingham, AL 35294, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0353-4633","authenticated-orcid":false,"given":"Girish","family":"Melkani","sequence":"additional","affiliation":[{"name":"Department of Pathology, Division of Molecular and Cellular Pathology, Heersink School of Medicine, Heersink School of Medicine, The University of Alabama at Birmingham, AL 35294, USA"},{"name":"UAB Nathan Shock Center, The University of Alabama at Birmingham, AL 35294, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026091921351910000_2026.05.18.726022v2.1","doi-asserted-by":"crossref","first-page":"1756286419843359","DOI":"10.1177\/1756286419843359","article-title":"Metabolic lipid muscle disorders: biomarkers and treatment","volume":"12","year":"2019","journal-title":"Therapeutic advances in neurological disorders"},{"key":"2026091921351910000_2026.05.18.726022v2.2","doi-asserted-by":"publisher","DOI":"10.1007\/s11910-010-0154-y"},{"key":"2026091921351910000_2026.05.18.726022v2.3","doi-asserted-by":"publisher","DOI":"10.64898\/2026.05.18.726022"},{"key":"2026091921351910000_2026.05.18.726022v2.4","doi-asserted-by":"publisher","DOI":"10.1159\/000443556"},{"key":"2026091921351910000_2026.05.18.726022v2.5","doi-asserted-by":"publisher","DOI":"10.1242\/dev.044230"},{"key":"2026091921351910000_2026.05.18.726022v2.6","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-physiol-021115-105045"},{"key":"2026091921351910000_2026.05.18.726022v2.7","doi-asserted-by":"publisher","DOI":"10.3390\/jcm7120472"},{"key":"2026091921351910000_2026.05.18.726022v2.8","doi-asserted-by":"publisher","DOI":"10.1016\/j.biocel.2020.105899"},{"key":"2026091921351910000_2026.05.18.726022v2.9","doi-asserted-by":"publisher","DOI":"10.1097\/MD.0000000000021944"},{"key":"2026091921351910000_2026.05.18.726022v2.10","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0604567103"},{"key":"2026091921351910000_2026.05.18.726022v2.11","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbadis.2014.06.031"},{"key":"2026091921351910000_2026.05.18.726022v2.12","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymgme.2006.01.009"},{"key":"2026091921351910000_2026.05.18.726022v2.13","doi-asserted-by":"publisher","DOI":"10.1016\/j.neurol.2015.11.008"},{"key":"2026091921351910000_2026.05.18.726022v2.14","doi-asserted-by":"publisher","DOI":"10.1002\/jmd2.70035"},{"key":"2026091921351910000_2026.05.18.726022v2.15","doi-asserted-by":"crossref","first-page":"747360","DOI":"10.3389\/fneur.2021.747360","article-title":"Clinical presentations and genetic characteristics of late-onset MADD due to ETFDH mutations in five patients: A case series","volume":"12","year":"2021","journal-title":"Frontiers in Neurology"},{"key":"2026091921351910000_2026.05.18.726022v2.16","unstructured":"Prasun P . 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