Neural progenitor cells are widespread throughout the adult central nervous system but only give rise to neurons in specific loci. Negative regulators of neurogenesis have therefore been postulated, but none have yet been identified as subserving a significant role in the adult brain. Here we report that nitric oxide (NO) acts as an important negative regulator of cell proliferation in the adult mammalian brain. We used two independent approaches to examine the function of NO in adult neurogenesis. In a pharmacological approach, we suppressed NO production in the rat brain by intraventricular infusion of an NO synthase inhibitor. In a genetic approach, we generated a null mutant neuronal NO synthase knockout mouse line by targeting the exon encoding active center of the enzyme. In both models, the number of new cells generated in neurogenic areas of the adult brain, the olfactory subependyma and the dentate gyrus, was strongly augmented, which indicates that division of neural stem cells in the adult brain is controlled by NO and suggests a strategy for enhancing neurogenesis in the adult central nervous system.

Mutations and deletions in mitochondrial DNA (mtDNA) lead to a number of human diseases characterized by neuromuscular degeneration. Accumulation of truncated mtDNA molecules (Δ‐mtDNA) lacking a specific 4977‐bp fragment, the common deletion, leads to three related mtDNA diseases : Pearson's syndrome; Kearns‐Sayre syndrome; and chronic progressive external ophthalmoplegia (CPEO). In addition, the proportion of Δ‐mtDNA present increases with age in a range of tissues. Consequently, there is considerable interest in the effects of the accumulation of Δ‐mtDNA on cell function. The 4977‐bp deletion affects genes encoding 7 polypeptide components of the mitochondrial respiratory chain, and 5 of the 22 tRNAs necessary for mitochondrial protein synthesis. To determine how the accumulation of Δ‐mtDNA affects oxidative phosphorylation we constructed a series of cybrids by fusing a human osteosarcoma cell line depleted of mtDNA (@gR⁰) with enucleated skin fibroblasts from a CPEO patient. The ensuing cybrids contained 0−86 %Δ‐mtDNA and all had volumes, protein contents, plasma‐membrane potentials and mitochondrial contents similar to those of the parental cell line. The bioenergetic consequences of accumulating Δ‐mtDNA were assessed by measuring the mitochondrial membrane potential, rate of ATP synthesis and ATP/ADP ratio. In cybrids containing less than 50−55 %Δ‐mtDNA, these bioenergetic functions were equivalent to those of cybrids with intact mtDNA. However, once the proportion of Δ‐mtDNA exceeded this threshold, the mitochondrial membrane potential, rate of ATP synthesis, and cellular ATP/ADP ratio decreased. These bioenergetic deficits will contribute to the cellular pathology associated with the accumulation of Δ‐mtDNA in the target tissues of patients with mtDNA diseases.

Nitric oxide reacts rapidly with superoxide to form the potent oxidant peroxynitrite. Mitochondria in vivo produce superoxide and in pathological situations the amount of superoxide produced increases; therefore, in the presence of nitric oxide, mitochondria will be a major site of peroxynitrite formation. Oxidative stress induces cyclosporin‐A‐sensitive mitochondrial calcium efflux and depolarisation which may contribute to tissue damage in pathological situations. To determine whether peroxynitrite could induce calcium efflux and depolarisation we exposed mitochondria to both nitric oxide and superoxide simultaneously, thus subjecting the mitochondria to a continual flux of peroxynitrite similar to that found in pathological situations. Our results show that: (a) exposure of mitochondria to nitric oxide plus super‐oxide induces cyclosporin‐A‐sensitive mitochondrial calcium efflux and depolarisation; (b) neither nitric oxide nor superoxide on their own induce calcium efflux or depolarisation under these conditions; (c) calcium efflux and depolarisation occur when peroxynitrite production (from nitric oxide and superoxide) exceeds about 0.99±0.03 nmol peroxynitrite min^‐1 mg mitochondrial protein^‐1. This rate of production of peroxynitrite is similar in magnitude to superoxide formation by mitochondria in pathological situations, suggesting that mitochondria can produce sufficient peroxynitrite in vivo to cause mitochondrial calcium efflux and depolarisation.Our experiments suggest a plausible model for tissue damage when mitochondrial superoxide production increases, for example in ischaemia‐reperfusion injury or following exposure to neurotoxins. In the presence of nitric oxide the mitochondrial superoxide production will lead to peroxynitrite formation which induces cyclosporin‐A‐sensitive mitochondrial calcium efflux and depolarisation. This disruption to mitochondrial function may in turn contribute to cell damage and death.