The Complex I NADH dehydrogenase-ubiquinone-FeS 4 (NDUFS4) subunit gene is involved in proper Complex I function such that the loss of NDUFS4 decreases Complex I activity resulting in mitochondrial disease. Therefore, a mouse model harboring a point mutation in the NDUFS4 gene was created. An embryonic lethal phenotype was observed in homozygous (NDUFS4 -/-) mutant fetuses. Mitochondrial function was impaired in heterozygous animals based on oxygen consumption, and Complex I activity in NDUFS4 mouse mitochondria. Decreased Complex I activity with unaltered Complex II activity, along with an accumulation of lactate, were consistent with Complex I disorders in this mouse model.

The reversible S-nitrosation and inhibition of mitochondrial complex I is a potential mechanism of cardioprotection, recruited by ischemic preconditioning (IPC), S-nitrosothiols, and nitrite. Previously, to exploit this mechanism, the mitochondrial S-nitrosating agent S-nitroso-2-mercaptopropionyl glycine (SNO-MPG) was developed, and protected perfused hearts and isolated cardiomyocytes against ischemia-reperfusion (IR) injury. In the present study, the murine left anterior descending coronary artery (LAD) occlusion model of IR injury was employed, to determine the protective efficacy of SNO-MPG in vivo. Intraperitoneal administration of 1 mg/kg SNO-MPG, 30 min. prior to occlusion, significantly reduced myocardial infarction and improved EKG parameters, following 30 min. occlusion plus 2 or 24 hr. reperfusion. SNO-MPG protected to the same degree as IPC, and notably was also protective when administered at reperfusion. Cardioprotection was accompanied by increased mitochondrial protein S-nitrosothiol content, and inhibition of complex I, both of which were reversed after 2 hr. reperfusion. Finally, hearts from mice harboring a heterozygous mutation in the complex I NDUSF4 subunit were refractory to protection by either SNO-MPG or IPC, suggesting that a fully functional complex I, capable of reversible inhibition is critical for cardioprotection. Overall, these results are consistent with a role for mitochondrial Snitrosation and complex I inhibition in the cardioprotective mechanism of IPC and SNO-MPG in vivo.

Recent studies have shown that various neural and embryonic stem cells cultured in 1-8% oxygen (O 2 ), levels lower than those typically used in cell culture (20.9%), displayed increased rates of proliferation; however, the molecular mechanisms underlying these changes are largely undefined. In this study, using rigorously controlled O 2 levels, we found that neural stem cells (NSCs) from embryonic day 15 rat cortex increased their rate of proliferation and migration in 1% O 2 relative to 20% O 2 without changes in viability. We sought to identify molecular changes in NSCs grown in 1% O 2 that might account for these increases. In 1% O 2 , levels of the hypoxia-inducible transcription factor HIF-1␣ were transiently increased. Reduced adherence of NSCs in 1% O 2 to basement membrane-coated plates was observed, and quantitative RT-PCR analysis confirmed that the levels of mRNA for an assortment of cell adhesion and extracellular matrix molecules were altered. Most notable was a 5-fold increase in matrix metalloproteinase (MMP)-9 mRNA. Specific inhibition of MMP-9 activity, verified using a fluorescent substrate assay, prevented the increase in proliferation and migration in 1% O 2 . The canonical Wnt pathway was recently shown to be activated in stem cells in low O 2 via HIF-1␣. Inhibition of Wnt signaling by DKK-1 also prevented the increase in proliferation, migration, and MMP-9 expression. Thus, MMP-9 is a key molecular effector, downstream of HIF-1␣ and Wnt activation, responsible for increased rates of NSC proliferation and migration in 1% O 2 .A frequently overlooked variable for the in vitro expansion of stem/progenitor cells is the level of O 2 in cell culture (1-3). In fact, the level of O 2 (20.9% O 2 ) used for in vitro culture is hyperoxic for cells in vivo. The lung alveoli and bloodstream typically hold ϳ14% (4, 5) and 11.5% (6 -8) O 2 , respectively, and O 2 levels in various areas of the brain range from 0.1 to 9% (5, 7, 9 -12). Recent studies have shown that the use of lower O 2 levels in culture results in increased rates of proliferation of neural stem/progenitor cells (NSCs) 2 from various embryonic or adult brain regions (12-23), virally immortalized human NSCs (23), and embryonic stem (ES) cells (3,24,25) and that these O 2 levels can also influence the differentiation of NSCs (9 -16).A large body of work has described hypoxia-inducible transcription factors as key components of the O 2 -sensing machinery that responds to lowered O 2 in cells and organisms (5,10,26,27). It has been shown that levels of HIF-1␣ protein can vary significantly over physiological ranges of O 2 (28). Although HIF-1␣ is constitutively expressed, when O 2 levels are high, the protein is hydroxylated by a family of prolyl hydroxylase enzymes, catalyzing a modification that targets HIF-1␣ for proteasomal degradation. At lower O 2 levels, the HIF-1␣ protein undergoes less hydroxylation, is stabilized, and can then translocate to the nucleus where it activates several genes that modulate the cellular response to decreas...

Xenomitochondrial mice harboring trans-species mitochondria on a Mus musculus domesticus (MD) nuclear background were produced. We created xenomitochondrial ES cell cybrids by fusing Mus spretus (MS), Mus caroli (MC), Mus dunni (Mdu), or Mus pahari (MP) mitochondrial donor cytoplasts and rhodamine 6-G treated CC9.3.1 or PC4 ES cells. The selected donor backgrounds reflected increasing evolutionary divergence from MD mice and the resultant mitochondrial-nuclear mismatch targeted a graded respiratory chain defect. Homoplasmic (MS, MC, Mdu, and MP) and heteroplasmic (MC) cell lines were injected into MD ova, and liveborn chimeric mice were obtained (MS/MD 18 of 87, MC/MD 6 of 46, Mdu/MD 31 of 140, and MP/MD l of 9 founder chimeras, respectively). Seven MS/MD, 1 MC/MD, and 11 Mdu/MD chimeric founder females were mated with wild-type MD males, and 18 of 19 (95%) were fertile. Of fertile females, only one chimeric MS/MD (1% coat color chimerism) and four chimeric Mdu/MD females (80-90% coat color chimerism) produced homoplasmic offspring with low efficiency (7 of 135; 5%). Four male and three female offspring were homoplasmic for the introduced mitochondrial backgrounds. Three male and one female offspring proved viable. Generation of mouse lines using additional female ES cell lineages is underway. We hypothesize that these mice, when crossbred with neurodegenerative-disease mouse models, will show accelerated age-related neuronal loss, because of their suboptimal capacity for oxidative phosphorylation and putatively increased oxidative stress.

The neurotrophin receptor p75NTR provides protection from oxidant stress induced by 6-hydroxydopamine (6-OHDA) and resultant cell death. In the absence of p75NTR, TrkA is upregulated and its signaling pathway effectors are increasingly activated. Necdin, a MAGE protein and known interactor of p75NTR and TrkA, is a potential mediator of this phenomenon. Decreased expression of necdin protein in p75NTR-negative PC12 cells decreased TrkA expression and increased PC12 cell resistance to 6-OHDA. Inhibition of JNK phosphorylation by SP600125 also resulted in increased resistance to 6-OHDA, suggesting that TrkA signaling underlies the susceptibility of these cells to oxidant stress.