Activation of the galanin receptor 2 (GalR2) protects the hippocampus from neuronal damage

Abstract

Expression of the neuropeptide galanin is up-regulated in many brain regions following nerve injury and in the basal forebrain of patients with Alzheimer's disease. We have previously demonstrated that galanin modulates hippocampal neuronal survival, although it was unclear which receptor subtype(s) mediates this effect. Here we report that the protective role played by galanin in hippocampal cultures is abolished in animals carrying a loss-of-function mutation in the second galanin receptor subtype (GalR2-MUT). Exogenous galanin stimulates the phosphorylation of the serine/threonine kinase Akt and extracellular signal-regulated kinase (ERK) in wild-type (WT) cultures by 435 +/- 5% and 278 +/- 2%, respectively. The glutamate-induced activation of Akt was abolished in cultures from galanin knockout animals, and was markedly attenuated in GalR2-MUT animals, compared with WT controls. In contrast, similar levels of glutamate-induced ERK activation were observed in both loss-of-function mutants, but were further increased in galanin over-expressing animals. Using specific inhibitors of either ERK or Akt confirms that a GalR2-dependent modulation in the activation of the Akt and ERK signalling pathways contributes to the protective effects of galanin. These findings imply that the rise in endogenous galanin observed either after brain injury or in various disease states is an adaptive response that reduces apoptosis by the activation of GalR2, and hence Akt and ERK.

Fig. 1

Effect of treatment with 2 mM glutamate on GalR2-MUT (loss-of-function mutation in the galanin receptor 2) and wild-type (WT) hippocampal organotypic cultures, in either the presence or the absence of 1 μM of either galanin or Gal2-11. Cultures were then placed in serum-free media for a 24-h recovery period and imaged to measure the percentage of cell death. Results and representative images demonstrate significantly greater cell death in the CA1 and CA3 regions of the hippocampus in GalR2-MUT than WT cultures. The protective effects of either galanin or Gal2-11 are abolished in GalR2-MUT cultures. Bars represent mean ± SEM, n = 3. *Significant difference (p < 0.05) from WT glutamate-treated cultures, **significant difference (p < 0.01) from WT glutamate-treated cultures.

Fig. 2

Effect of acute treatment with 1 μM galanin on the percentage of phosphorylated Akt and extracellular signal-regulated kinase (ERK) in wild-type (WT) hippocampal organotypic cultures for the indicated time points. Histograms represent quantitative western blot analysis of phosphorylated Akt (top) and ERK1 (bottom). Data are expressed as a ratio of the normalized percentages of ether phospho- and total-Akt or ERK. Representative blots for each phospho-protein are shown above each histogram. Results demonstrate a robust stimulation of pAkt and pERK1 within 2 min of the addition of 1 μM galanin. Bars represent the mean ± SEM, n = 3. *Significantly different (p < 0.05) from control cultures; **significantly different (p < 0.01) from control cultures.

Fig. 3

Modulation of Akt activation in response to 2 mM glutamate toxicity in galanin knockout (GalKO) and GalR2-MUT (mutation in the galanin receptor 2) hippocampal organotypic cultures, and 6 mM glutamate toxicity in galanin over-expressing (GalOE) hippocampal organotypic cultures, for the indicated time points, each compared with their respective strain-matched wild-type (WT) controls. Histograms represent quantitative western blot analysis of phosphorylated Akt in GalKO and WT cultures (top left), GalR2-MUT and WT cultures (middle left), and GalOE and WT cultures (bottom left). Data are expressed as a ratio of the normalized percentages of phospho- and total-Akt. Representative blots for each genotype are shown on the right. Results demonstrate a significant stimulation of pAkt within 5 min of the addition of glutamate in all three WT strains. The increase in pAkt levels is abolished in the GalKO cultures, greatly attenuated in the GalR2-MUT cultures, and is unchanged in the GalOE cultures (compared with the WT cultures treated with glutamate). Bars represent the mean ± SEM, n = 3. *Significantly different (p < 0.05) from WT control cultures; **Significantly different (p < 0.01) from WT control cultures.

Fig. 4

Modulation of extracellular signal-regulated kinase 1 (ERK1) activation in response to 2 mM glutamate toxicity in galanin knockout (GalKO) and GalR2-MUT (mutation in the galanin receptor 2) hippocampal organotypic cultures, and 6 mM glutamate toxicity in galanin over-expressing (GalOE) hippocampal organotypic cultures, for the indicated time points, each compared with their respective strain-matched wild-type (WT) controls. Histograms represent quantitative western blot analysis of phosphorylated ERK1 in GalKO and WT cultures (top left), GalR2-MUT and WT cultures (middle left), and GalOE and WT cultures (bottom left). Data are expressed as a ratio of the normalized percentages of phospho- and total-ERK1. Representative blots for each genotype are shown on the right. Results demonstrate a significant stimulation of pERK1 within 10 min of the addition of glutamate in all three WT strains. The increase in pERK1 levels was not statistically different in the GalKO and GalR2-MUT cultures, compared with the WT cultures treated with glutamate. A significantly greater increase was observed in the GalOE cultures, 10 and 15 min after the addition of glutamate, compared with the WT cultures. Bars represent the mean ± SEM, n = 3. *Significantly different (p < 0.05) from WT control cultures, **significantly different (p < 0.01) from WT control cultures.

Fig. 5

Effect of treatment with 2 mM glutamate on wild-type (WT) hippocampal organotypic cultures in either the presence or the absence of 1 μM or 10 nM galanin, and/or 5 μM LY294009, and/or 300 nM PD98059. Cultures were then placed in serum-free media for a 24-h recovery period and imaged to measure the percentage of cell death in the CA1 region (solid bars) of the hippocampus. Results and representative images demonstrate an increase in cell death with either inhibitor after glutamate treatment. The protective effects of 1 μM galanin are abrogated with inhibitors of either the ERK or the Akt signalling pathways, whereas the protective effects of 10 nM galanin is abrogated by the Akt, but not the ERK, inhibitor. Bars represent mean ± SEM, n = 3. *Significantly different (p < 0.05) from glutamate-treated cultures; **significantly different (p < 0.01) from glutamate-treated cultures, ⁺significantly different (p < 0.05) from galanin- and glutamate-treated cultures, ⁺⁺significantly different (p < 0.01) from galanin- and glutamate-treated cultures.