Mitochondrial energy metabolism and reactive oxygen species level disruption in ACAD9- deficient fibroblasts

Guilhian Leipnitz, PhD  (1,2), Al-Walid Mohsen, PhD , (1) Anuradha Karunanidhi, MS (1), Bianca Seminotti, PhD (1) Vera Y. Roginskaya, MS  (2) , Bennett Van Houten, PhD  (2) , Jerry Vockley, MD, PhD (2) .

1Division Medical Genetics, Department Pediatrics, University of Pittsburgh, Pittsburgh, PA, USA

2 Department of Biochemistry, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil

3Department Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA, USA

*e-mail:; phone: +1 412 692-91XX

Background: Acyl-CoA dehydrogenase 9 (ACAD9) is a homodimeric flavoprotein reported to carry out a dual role catalyzing the first step in long-chain fatty acid    -oxidation and acting as an assembly factor for mitochondrial respiratory chain complex I. Individuals with complex I deficiency having mutations in ACAD9 gene present with progressive encephalomyopathy, recurrent Reye syndrome, and cardiomyopathy that can be fatal.

Objective: Evaluating the effect of ACAD9 deficiency on mitochondrial bioenergetics and reactive oxygen species production in fibroblasts of an ACAD9-deficient patient.

Methods: Fibroblasts were cultured in medium with or without glucose to assess the ability  of ACAD9 deficient cells to accommodate the shift of energy source from glucose and the effect of such shift on oxidative phosphorylation and ATP synthesis. We measured ACAD9 protein, oxygen consumption, mitochondrial membrane potential and mass, ATP production, and superoxide generation  using  western  blotting, Seahorse flux analyzer, immunostaining, fluorimetry, and luminometry.

Results: Western blots demonstrated no significant alterations on ACAD9 protein content in patient cells cultured in the absence or presence of glucose. Seahorse analyzer showed decreased basal respiration and reserve capacity in both media. Fluorescence staining and luminometry demonstrated increased superoxide production, decreased mitochondrial membrane potential and ATP production  in  the  presence  or  absence of glucose. However, mitochondrial mass was increased only in medium without glucose.

Conclusion: Our findings show that fibroblasts with ACAD9 deficiency have mitochondrial dysfunction characterized by bioenergetics disruption and increased superoxide generation, which may contribute to  the  pathophysiology underlying  the  symptoms observed in ACAD9-related complex I deficiency.

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