Our previous results showed that inhibition of protein tyrosine phosphatases (PTP) by orthovanadate is an appropriate strategy to mimic nerve growth factor (NGF) effects in neurons, including enhanced phosphorylation of TrkA, stimulation of downstream survival signaling pathways, and protection against apoptotic stress. In this study, we wanted to trigger such NGF-like survival signaling in primary hippocampal neurons with the more specific PTP inhibitors ethyl-3,4-dephostatin (DPN), 4-O-methyl-ethyl-3,4-dephostatin (Me-DPN), and methoxime-3,4-dephostatin. It was striking that only the nitric oxide (NO)-releasing dephostatin analogs DPN and Me-DPN, but not the nitrosamine-free methoxime derivative (which did not release NO), enhanced TrkA phosphorylation and protected the neurons against staurosporine (STS)-induced apoptosis. The established NO donor S-nitroso-N-acetylpenicillamine (SNAP) also enhanced TrkA phosphorylation and prevented apoptosis similarly to DPN and Me-DPN. Analysis of the major signaling pathways downstream of TrkA revealed that both SNAP and DPN enhanced phosphorylation of Akt and the mitogen-activated kinases (MAPK) Erk1/2. Blocking of these signaling pathways by the PI3-K inhibitor wortmannin or the MAPK kinase inhibitor U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene] equally abolished the neuroprotective effect of the NO donors. It was striking that inhibition of the soluble guanylyl cyclase (sGC) by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or protein kinase G (PKG) inhibition by (9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo-[1,2,3-fg: 3Ј,2Ј,1Ј-kl]pyrrolo [3,4-i] [1,6]benzodiazocine-10-carboxylic acid methyl ester (KT5823) also blocked the neuroprotective effect of the NO donors, and ODQ clearly attenuated SNAPinduced phosphorylation of TrkA, Akt, and MAPK. In conclusion, NO release by the dephostatin derivatives and subsequent stimulation of sGC and PKG is essential for their neuroprotective effects. In primary neurons, such NO-activated survival signaling involves NGF-like effects, including enhanced phosphorylation of TrkA and activation of PI3-K/Akt and MAPK pathways.Nitric oxide is an endogenous regulator of different cellular functions, including vascular tone (Gruetter et al., 1979;Furchgott and Zawadzki, 1980), neurotransmission (Vincent, 1994;Garthwaite and Boulton, 1995), inflammation (Nathan and Shiloh, 2000), and cellular signaling cascades (Dawson et al., 1992). In neurons, NO has been found to be a Janus-faced molecule that can mediate survival signaling (Troy et al., 2000;Contestabile and Ciani, 2004) but may on the other hand contribute to neuronal death and brain damage in neurological diseases [e.g., stroke (Zhang et al., 1996), Alzheimer's disease , amyotrophic lateral sclerosis (Urushitani and Shimohama, 2001), or Parkinson's disease (Liberatore et al., 1999)]. In the brain, NO can be Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
