Metabolic consequences of NDUFS4 gene deletion in immortalized mouse embryonic fibroblasts

Abstract

Human mitochondrial complex I (CI) deficiency is associated with progressive neurological disorders. To better understand the CI pathomechanism, we here studied how deletion of the CI gene NDUFS4 affects cell metabolism. To this end we compared immortalized mouse embryonic fibroblasts (MEFs) derived from wildtype (wt) and whole-body NDUFS4 knockout (KO) mice. Mitochondria from KO cells lacked the NDUFS4 protein and mitoplasts displayed virtually no CI activity, moderately reduced CII, CIII and CIV activities and normal citrate synthase and CV (F(o)F(1)-ATPase) activity. Native electrophoresis of KO cell mitochondrial fractions revealed two distinct CI subcomplexes of ~830kDa (enzymatically inactive) and ~200kDa (active). The level of fully-assembled CII-CV was not affected by NDUFS4 gene deletion. KO cells exhibited a moderately reduced maximal and routine O(2) consumption, which was fully inhibited by acute application of the CI inhibitor rotenone. The aberrant CI assembly and reduced O(2) consumption in KO cells were fully normalized by NDUFS4 gene complementation. Cellular [NAD(+)]/[NADH] ratio, lactate production and mitochondrial tetramethyl rhodamine methyl ester (TMRM) accumulation were slightly increased in KO cells. In contrast, NDUFS4 gene deletion did not detectably alter [NADP(+)]/[NADPH] ratio, cellular glucose consumption, the protein levels of hexokinases (I and II) and phosphorylated pyruvate dehydrogenase (P-PDH), total cellular adenosine triphosphate (ATP) level, free cytosolic [ATP], cell growth rate, and reactive oxygen species (ROS) levels. We conclude that the NDUFS4 subunit is of key importance in CI stabilization and that, due to the metabolic properties of the immortalized MEFs, NDUFS4 gene deletion has only modest effects at the live cell level. This article is part of a special issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

Succination is Increased on Select Proteins in the Brainstem of the NADH dehydrogenase (ubiquinone) Fe-S protein 4 (Ndufs4) Knockout Mouse, a Model of Leigh Syndrome

“…In summary, these results clearly demonstrate the localization of increased protein succination with specific regions of the nervous system where histopathological anomalies have been described in the Ndufs4 KO mouse, whereas the skeletal muscle and brain areas that are devoid of pathology do not show changes in protein succination. Interestingly, studies in fibroblasts from the Ndufs4 KO mouse, which are not affected by pathology despite the loss of Ndufs4, only showed minor deficits in mitochondrial respiration with unchanged proliferation or cell survival (57,58), further supporting the tissue specificity of our findings.…”

Acute stimulation of glucose influx upon mitoenergetic dysfunction requires LKB1, AMPK, Sirt2 and mTOR–RAPTOR

“…Similarly, genetic complex-I deficiency in mouse embryonic fibroblasts from NDUFS4 −/− mice is characterized by lower cellular levels of NAD + and increased levels of NADH (leading to a reduced NAD + :NADH ratio), without alterations in NADP + and NADPH levels (Valsecchi et al, 2012). Using a heart-specific NDUFS4 −/− mouse model, further evidence has been provided that complex-I deficiency is associated with a reduced NAD ratio and that this reduction is paralleled by a lower activity of the NAD + -dependent deacetylase sirtuin 3 (Sirt3) (Karamanlidis et al, 2013).…”

Cellular and animal models for mitochondrial complex I deficiency: A focus on the NDUFS4 subunit

“…Additionally, a reduction in NAD þ levels and increase in NADH levels were observed, paralleled by a decreased and increased extracellular glucose concentration and lactic acid, respectively. This suggests that Ndufs4 KO MEFs are slightly more glycolytic than wt MEFs, although no differences in key glycolytic proteins (i.e., hexokinases and phosphorylated pyruvate dehydrogenase) were observed between wt and Ndufs4 KO MEFs (38). Integrating the above results obtained from tissues and MEFs from the WBKO animal, we hypothesized that CIII stabilizes CI in the absence of the NDUFS4 subunit (37), and that due to the metabolic properties of the immortalized KO MEFs, Ndufs4 gene deletion has only modest effects in these cells (38).…”

“…Intact KO cells exhibited a moderate reduction in routine and maximal oxygen (O 2 ) consumption, which was fully inhibited by acute application of the CI inhibitor rotenone. The latter demonstrates that catalytically active CI is present in Ndufs4 KO MEFs (38). Additionally, a reduction in NAD þ levels and increase in NADH levels were observed, paralleled by a decreased and increased extracellular glucose concentration and lactic acid, respectively.…”

“…To further enhance our understanding of the cellular consequences of Ndufs4 gene deletion, we generated immortalized mouse embryonic fibroblasts (MEFs) from wt and WBKO mice (38). In contrast to NDUFS4 patient fibroblasts and WBKO tissue samples, Ndufs4 KO MEFs displayed virtually no CI activity.…”