Culmsee Carsten, Gerling Norbert, Landshamer Stefan, Rickerts Bianca, Duchstein Hans-Jürgen, Umezawa Kazuo, Klumpp Susanne, Krieglstein Josef
Institute of Pharmacology and Toxicology, Department of Pharmacy, Phillips-University, Marburg, Germany.
Mol Pharmacol. 2005 Oct;68(4):1006-17. doi: 10.1124/mol.105.013086. Epub 2005 Jul 18.
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:3',2',1'-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 SNAP-induced 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.
我们之前的研究结果表明，原钒酸盐抑制蛋白酪氨酸磷酸酶（PTP）是模拟神经生长因子（NGF）对神经元作用的一种合适策略，包括增强TrkA的磷酸化、刺激下游存活信号通路以及保护细胞免受凋亡应激。在本研究中，我们想用更具特异性的PTP抑制剂乙基-3,4-去磷抑素（DPN）、4-O-甲基-乙基-3,4-去磷抑素（Me-DPN）和甲氧肟-3,4-去磷抑素在原代海马神经元中触发这种类似NGF的存活信号。令人惊讶的是，只有释放一氧化氮（NO）的去磷抑素类似物DPN和Me-DPN，而不是不释放NO的无亚硝胺甲肟衍生物，增强了TrkA磷酸化并保护神经元免受星形孢菌素（STS）诱导的凋亡。已确立的NO供体S-亚硝基-N-乙酰青霉胺（SNAP）同样增强了TrkA磷酸化并预防了凋亡，其作用与DPN和Me-DPN相似。对TrkA下游主要信号通路的分析表明，SNAP和DPN均增强了Akt和丝裂原活化激酶（MAPK）Erk1/2的磷酸化。PI3-K抑制剂渥曼青霉素或MAPK激酶抑制剂U0126 [1,4-二氨基-2,3-二氰基-1,4-双（2-氨基苯基硫）丁二烯]阻断这些信号通路同样消除了NO供体的神经保护作用。令人惊讶的是，1H-[1,2,4]恶二唑并[4,3-a]喹喔啉-1-酮（ODQ）抑制可溶性鸟苷酸环化酶（sGC）或（9S,10R,12R）-2,3,9,10,11,12-六氢-10-甲氧基-2,9-二甲基-1-氧代-9,12-环氧-1H-二吲哚并-[1,2,3-fg:3',2',1'-kl]吡咯并[3,4-i][1,6]苯并二氮杂卓-10-羧酸甲酯（KT5823）抑制蛋白激酶G（PKG）也阻断了NO供体的神经保护作用，并且ODQ明显减弱了SNAP诱导的TrkA、Akt和MAPK的磷酸化。总之，去磷抑素衍生物释放NO以及随后对sGC和PKG的刺激对其神经保护作用至关重要。在原代神经元中这种由NO激活的存活信号涉及类似NGF的作用，包括增强TrkA磷酸化以及激活PI3-K/Akt和MAPK通路。
