Matrix metalloproteinase (MMP)-9 induced by Wnt signaling increases the proliferation and migration of embryonic neural stem cells at low O2 levels.

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-1α 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-1α. 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-1α and Wnt activation, responsible for increased rates of NSC proliferation and migration in 1% O(2).

Necdin and neurotrophin receptors: interactors of relevance for neuronal resistance to oxidant stress.

Necdin is a protein known to interact with the neurotrophin receptors, neurotrophic tyrosine kinase receptor type 1 (TrkA) and 75 kD low-affinity neurotrophin receptor (p75NTR). TrkA and p75NTR play roles in development and disease of the nervous system and chemoresistance of nervous system tumors. Necdin deletion is associated with Prader-Willi syndrome. The present studies demonstrate that the effects of necdin on the susceptibility of neuroblastoma cells to oxidant stress are dependent on the ratio of p75NTR to TrkA in the cell. In low p75NTR:TrkA ratio cells, necdin down-regulation decreases sensitivity to oxidant stress and expression of and signaling through TrkA. In high p75NTR:TrkA cells, necdin down-regulation is without effect. The effects of necdin deletion on the developing nervous system may depend on the relative expression of p75NTR and TrkA in the cells of particular regions of the nervous system.

Necdin and TrkA contribute to modulation by p75NTR of resistance to oxidant 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-deficient 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.

NDUFS4: creation of a mouse model mimicking a Complex I disorder.

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.

In vivo cardioprotection by S-nitroso-2-mercaptopropionyl glycine.

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 h 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 h 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 S-nitrosation and complex I inhibition in the cardioprotective mechanism of IPC and SNO-MPG in vivo.