Nitric Oxide Stimulates the Proliferation of Neural Stem Cells Bypassing the Epidermal Growth Factor Receptor

Abstract

Nitric oxide (NO) was described to inhibit the proliferation of neural stem cells. Some evidence suggests that NO, under certain conditions, can also promote cell proliferation, although the mechanisms responsible for a potential proliferative effect of NO in neural stem cells have remained unaddressed. In this work, we investigated and characterized the proliferative effect of NO in cell cultures obtained from the mouse subventricular zone. We found that the NO donor NOC-18 (10 lM) increased cell proliferation, whereas higher concentrations (100 lM) inhibited cell proliferation. Increased cell proliferation was detected rapidly following exposure to NO and was prevented by blocking the mitogen-activated kinase (MAPK) pathway, independently of the epidermal growth factor (EGF) receptor. Downstream of the EGF receptor, NO activated p21Ras and the MAPK pathway, resulting in a decrease in the nuclear presence of the cyclin-dependent kinase inhibitor 1, p27 KIP1 , allowing for cell cycle progression. Furthermore, in a mouse model that shows increased proliferation of neural stem cells in the hippocampus following seizure injury, we observed that the absence of inducible nitric oxide synthase (iNOS 2/2 mice) prevented the increase in cell proliferation observed following seizures in wild-type mice, showing that NO from iNOS origin is important for increased cell proliferation following a brain insult. Overall, we show that NO is able to stimulate the proliferation of neural stem cells bypassing the EGF receptor and promoting cell division. Moreover, under pathophysiological conditions in vivo, NO from iNOS origin also promotes proliferation in the hippocampus. STEM CELLS

Biochemistry of Nitric Oxide

“…It is well known that most of the exogenous carcinogens act via production of reactive oxygen or reactive nitrogen species. The reactive oxygen species are generated both physiologically and pathologically in mammals and induce many kind of cellular damage [88] including DNA damage [89]. Since DNA plays a central role in information transfer, attention has focussed on oxidative damage as significant source of mutations that lead to cancer and other human pathologies [90].…”

Assessing the Influence of Neuroinflammation on Neurogenesis: In Vitro Models Using Neural Stem Cells and Microglia as Valuable Research Tools

“…The isolation method described by Saura and collaborators is, therefore, one of the methods that seems to offer the best value yield/purity (Saura et al, 2003). We favor the isolation of microglial cells by shaking from mixed glial cultures treated with low levels of M-CSF as an alternative to the method of Saura (Saura et al, 2003), with a high purity of the microglia obtained (>90%) and, unlike previous methods, with a high yield (Carreira et al, 2010).…”

“…On the other hand, neurogenesis seems to be induced by microglial cells activated by IL-4 or low level of IFN-gamma, which has been associated with increased neuroprotection (Wong et al, 2004;Song et al, 2005;Baron et al, 2008). Moreover, some inflammatory mediators like NO seem to have opposite roles in regulating neurogenesis in inflammatory conditions (Carreira et al, 2010). Apparently, microglial cells and the factors they release play a dual role in neurogenesis acting as antiproliferative or proliferative agents.…”

“…Decreased (Contestabile et al, 2003;MorenoLopez et al, 2004;Matarredona et al, 2005;Ciani et al, 2006;Covacu et al, 2006;Fritzen et al, 2007;Luo et al, 2007;Carreira et al, 2010) TNF-alpha…”

“…On the other hand, the isolation of adult microglial cells and subsequent culture with low concentrations of macrophage colony-stimulating factor (M-CSF) leads to increased proliferation and survival of cells that persists for several weeks (Suzumura et al, 1990;Ponomarev et al, 2005). M-CSF seems to be a key factor for the maintenance and survival of microglial cells in vitro, and has been used in several works (Wegiel et al, 1998;Ponomarev et al, 2005;Carreira et al, 2010). Other methods are also described for the isolation of microglial cells, which include isolation from CNS tissue by Percoll gradient (Dick et al, 1995;Ford et al, 1995), isolation from primary cultures by nutritional deprivation (Hao et al, 1991) or by collecting floating cells in mixed glial cultures (Ganter et al, 1992), but the yield is generally very low.…”