Bianca Seminotti1*, PhD, Al-Walid Mohsen1, PhD, Guilhian Leipnitz 1,2, PhD, Anuradha Karunanidhi 1, MS, PhD, Peter Wipf 3, PhD, Jerry Vockley 1,4, MD, PhD.
1Division Medical Genetics, Department Pediatrics, University of Pittsburgh, Pittsburgh, PA, USA
2PPG Ciências Biológicas: Bioquímica, Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
3Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, USA
4Department of Human Genetics, University of Pittsburgh, Pittsburgh, PA, USA
Very long-chain acyl-CoA dehydrogenase (VLCAD) enzyme deficiency is the most common mitochondrial β-oxidation (FAO) defect of long-chain fatty acids. The clinical phenotype is heterogeneous, including hepatomegaly, ardiomyopathy and hypoketotic hypoglycemia, frequently induced by prolonged fasting and infectious illnesses. Skeletal myopathy associated with rhabdomyolysis may also be occasioned. Nevertheless, the cellular mechanisms related to the pathophysiology remain unclear.
VLCAD deficient fibroblasts were cultured in medium without glucose for 48 h
to shift cellular metabolism to fatty acids as energy source. We evaluated reactive oxygen species production, the immunocontent of proteins involved in endoplasmic reticulum-mitochondria crosstalk and function (DDIT3, IP3, Grp75, Grp78 and VDAC1) and the transcription factors Nrf2 and NF-kB.
Superoxide production and DCFH oxidation were higher in VLCAD deficient fibroblasts.JP4-039 and XJB-5-131, mitochondrial targeted free radical scavengers, were able to decrease the levels of reactive oxygen species.
We also observed increased Nrf2 and NF-kB antigens in VLCAD deficient cells, which were slightly decreased by JP4-039, as compared to control cells. Finally, we observed increased DDIT3 antigen, and decreased IP3 receptor, Grp75
and VDAC1 antigens, but no changes in Grp78 and Mfn2 antigen signal.
Our results showed increased reactive oxygen species levels that were improved by the treatment with the antioxidants JP4-039 and XJB-5-131. The expression
of transcription factors related to oxidative stress and inflammatory signaling pathways in VLCAD deficient fibroblasts was altered, and partially restored
by JP4-039. Finally, alterations in the proteins involved in the endoplasmic reticulum-mitochondria crosstalk were observed, indicating a disturbance
in this process and its function