Antisense oligonucleotide against GTPase Rab5b inhibits metabotropic agonist DHPG-induced neuroprotection

Abstract

(S)-3,5-dihydroxyphenylglycine (DHPG), a group I metabotropic glutamate receptor (mGluR1 and 5) agonist reduced NMDA-mediated membrane currents, NMDA-induced cell death and up-regulated Rab5b, a small GTPase involved in endocytosis [M. Blaabjerg, A. Baskys, J. Zimmer and M. P. Vawter, Changes in hippocampal gene expression after neuroprotective activation of group I metabotropic glutamate receptors, Molec. Brain Res. 117 (2003) 196-205; M. Blaabjerg, L. Fang, J. Zimmer and A. Baskys, Neuroprotection against NMDA excitotoxicity by group I metabotropic glutamate receptors is associated with reduction of NMDA stimulated currents, Experimental Neurol. 183 (2003) 573-580.]. To examine the role of Rab5b on DHPG-mediated neuroprotection in organotypic hippocampal cultures, we developed antisense oligonucleotide targeted to suppress Rab5b translation. Treatment of cultures with the antisense (24 h) but not scrambled sequence oligonucleotide suppressed DHPG-induced increase in Rab5b expression and significantly disrupted DHPG-induced protection against NMDA toxicity in a concentration-dependent manner (0.01-10 nM). Antisense but not scrambled oligonucleotide treatment reduced NMDA toxicity (to 74.4+/-5.9% of control) and this effect could be blocked by protein kinase C inhibitor staurosporine (0.2 microM) or with the protease inhibitor leupeptin (100 microM). Application of osmotic shock followed by K(+) depletion to disrupt endocytosis abolished the protective effect of DHPG. These data suggest that neuroprotection by DHPG against NMDA-mediated injury may involve facilitation of NMDA receptor endocytosis likely stimulated by DHPG-induced increase in Rab5b synthesis.