Superoxide dismutase 2 ameliorates mitochondrial dysfunction in skin fibroblasts of Leber’s hereditary optic neuropathy patients

Abstract

BackgroundIn Leber’s hereditary optic neuropathy (LHON), mtDNA mutations mediate mitochondrial dysfunction and apoptosis of retinal ganglion cells. Mitochondrial superoxide dismutase 2 (SOD2) is a crucial antioxidase against reactive oxygen species (ROS). This study aims to investigate whether SOD2 could ameliorate mtDNA mutation mediated mitochondrial dysfunction in skin fibroblasts of LHON patients and explore the underlying mechanisms.MethodsThe skin of normal healthy subjects and severe LHON patients harboring m.11778G > A mutation was taken to prepare immortalized skin fibroblast cell lines (control-iFB and LHON-iFB). LHON-iFB cells were transfected with SOD2 plasmid or negative control plasmid, respectively. In addition, human neuroblastoma SH-SY5Y cells and human primary retinal pigmental epithelium (hRPE) cells were stimulated by H2O2 after gene transfection. The oxygen consumption rate (OCR) was measured with a Seahorse extracellular flux analyzer. The level of ATP production, mitochondrial membrane potential, ROS and malondialdehyde (MDA) were measured separately with the corresponding assay kits. The expression level of SOD2, inflammatory cytokines and p-IκBα/IκBα was evaluated by western-blot. Assessment of apoptosis was performed by TUNEL assay.ResultsLHON-iFB exhibited lower OCR, ATP production, mitochondrial membrane potential but higher level of ROS and MDA than control-iFB. Western-blot revealed a significantly increased expression of IL-6 and p-IκBα/IκBα in LHON-iFB. Compared with the negative control, SOD2 overexpression increased OCR, ATP production and elevated mitochondrial membrane potential, but impaired ROS and MDA production. Besides, western-blot demonstrated exogenous SOD2 reduced the protein level of IL-6 and p-IκBα/IκBα. TUNEL assays suggested SOD2 inhibited cells apoptosis. Analogously, in SH-SY5Y and hRPE cells, SOD2 overexpression increased ATP production and mitochondrial membrane potential, but decreased ROS, MDA levels and suppressed apoptosis.ConclusionSOD2 upregulation inhibited cells apoptosis through ameliorating mitochondrial dysfunction and reducing NF-κB associated inflammatory response. This study further support exogenous SOD2 may be a promising therapy for the treatment of LHON.

Case Report: Abnormalities of sperm motility and morphology in a patient with Leber hereditary optic neuropathy: Improvement after idebenone therapy

“…However, existence of an oxidative stress in the pathophysiology of LHON was proved by Rovcanin in a large series of patients ( 15 ). Efficacy of drugs reducing ROS is another argument in favor of the role of oxidative stress during this pathology ( 16 ).…”

Pathogenicity and Function Analysis of Two Novel SLC4A11 Variants in Patients With Congenital Hereditary Endothelial Dystrophy

“…Image fluorescence intensity was analyzed using ImageJ software (version 1.52a; National Institutes of Health [NIH]). 22 , 23 …”

“…The mitochondrial membrane potential assay was performed using the JC-1 assay kit (C2006; Beyotime, Shanghai, China). 22 , 23 Briefly, HEK293T cells were seeded into 12-well plates, and cultured them in complete DMEM medium for 48 hours after transfection with the plasmids. The cells were digested with EDTA-free trypsin, collected in centrifuge tubes, centrifuged, and incubated for 20 minutes at 37°C in a cell incubator protected from light.…”

“…The cells were washed twice with JC-1 staining buffer (1X) and detected by flow cytometry for mitochondrial membrane potential, which were expressed as the ratio of JC-1 aggregates to monomer. 22 , 23 …”

Compartmental Differences in the Retinal Ganglion Cell Mitochondrial Proteome

“…The abundance of these antioxidant proteins may suggest that the RGC somatodendritic mitochondria are subjected to especially high levels of oxidative stress and/or require particularly efficient antioxidant pathways to scavenge free radicals and avoid oxidative damage. It is notable that a number of mitochondrial optic neuropathies have been associated with RGC oxidative stress [68][69][70][71][72][73] .…”