Effects of TRH and its analogues on primary cortical neuronal cell damage induced by various excitotoxic, necrotic and apoptotic agents

Abstract 

The tripeptide thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH2) has been shown to possess neuroprotective activity in in vitro and in vivo models. Since its potential utility is limited by relatively rapid metabolism, metabolically stabilized analogues have been constructed. In the present study we investigated the influence of TRH and its three stable analogues: Montirelin (MON, CG-3703), RGH-2202 (L-6-keto-piperidine-2carbonyl-l-leucyl-l-prolinamide) and Z-TRH (N-carbobenzyloxy-pGlutamyl-Histydyl-Proline) in various models of mouse cortical neuronal cell injury. Twenty four hour pre-treatment with TRH and its analogues in low micromolar concentrations attenuated the neuronal cell death evoked by excitatory amino acids (EAAs: glutamate, NMDA, kainate, quisqualate) and hydrogen peroxide. All the peptides showed neuroprotective action on staurosporine (St)-evoked apoptotic neuronal cell death, but this effect was caspase-3 independent. Interestingly, in mixed neuronal-glial cell preparations only MON decreased St- and glutamate-evoked neurotoxicity. None of the peptides inhibited the doxorubicin- and lactacystin-induced neuronal cortical cell death, agents acting via activation of death receptor (FAS) or inhibition of proteasome function, respectively. Furthermore, we found that neither inhibitors of PI3-K (wortmannin, LY 294002) nor MAPK/ERK1/2 (PD 098059, U 0126) were able to inhibit neuroprotective properties of TRH and MON in St model of apoptosis. The protection mediated by TRH and MON it that model was also not connected with influence of peptides on the pro-apoptotic GSK-3β and JNK protein kinase expression and activity. Further studies showed that calpains, calcium-activated proteases were induced by Glu, but not by St in cortical neurons. Moreover, the Glu-evoked increase in spectrin alpha II cleavage product induced by calpains was blocked by TRH. The obtained data showed that the potency of TRH and its analogues in inhibiting EAAs- and H₂O₂-induced neuronal cell death from the highest to lowest activity was: MON>TRH>Z-TRH>RHG. Interestingly, all peptides were active against St-induced apoptosis, however, on concentration basis MON was far more potent than the other peptides. None of the peptides inhibited Dox- and LC-evoked apoptotic cell death. Additionally, the data exclude potential role of pro-survival (PI3-K/Akt and MAPK/ERK1/2) and pro-apoptotic (GSK-3β and JNK) pathways in neuroprotective effects of TRH and its analogues on St-induced neuronal apoptosis. Moreover, the results point to involvement of the inhibition of calpains in the TRH neuroprotective effect in Glu model of neuronal cell death.

Abbreviations: Akt, serine/threonine protein kinase Akt, PKB, protein kinase B, AMPA, alpha-amino-3-hydroxy-5-methylisoxazole propionate, DIV, days in vitro, Dox, doxorubicin, adriamycin, ERK1/2, extracellularly regulated protein kinase 1/2, Glu, glutamate, GSK-3, β glycogen synthase kinase-3β, JNK, c-Jun NH(2)-terminal kinase, KA, kainate, LC, lactacystin, LDH, lactate dehydrogenase, MON, CG-3703, Montirelin, 6-Me-5-oxothiomorpholinyl-3-carbonyl-His-Pro-NH2, NMDA, N-methyl-d-aspartate, QA, quisqualate, PI3-K, phosphatidylinositol-3 kinase, RGH, RGH-2202, posatirelin L-6-keto-piperidine-2-carbonyl-l-leucyl-l-prolinamide, St, staurosporine, TRH, pGlu-His-Pro-NH2, Z-TRH, AWL-4102, N-(carbobenzyloxy)-pGlutamyl-Histydyl-Proline

Keywords: Montirelin, RGH-2202, Z-pGlutamyl-Histydyl-Proline, Glutamate, NMDA, Kainate, AMPA, Quisqualate, Staurosporine, Doxorubicin, Lactacystin, Calpain