Targeting type-2 metabotropic glutamate receptors to protect vulnerable hippocampal neurons against ischemic damage

Abstract

Background: To examine whether metabotropic glutamate (mGlu) receptors have any role in mechanisms that shape neuronal vulnerability to ischemic damage, we used the 4-vessel occlusion (4-VO) model of transient global ischemia in rats. 4-VO in rats causes a selective death of pyramidal neurons in the hippocampal CA1 region, leaving neurons of the CA3 region relatively spared. We wondered whether changes in the expression of individual mGlu receptor subtypes selectively occur in the vulnerable CA1 region during the development of ischemic damage, and whether post-ischemic treatment with drugs targeting the selected receptor(s) affords neuroprotection.

Results: We found that 4-VO caused significantly reduction in the transcript of mGlu2 receptors in the CA1 region at times that preceded the anatomical evidence of neuronal death. Down-regulation of mGlu2 receptors was associated with reduced H3 histone acetylation at the Grm2 promoter. The transcripts of other mGlu receptor subtypes were unchanged in the CA1 region of 4-VO rats. Ischemia did not cause changes in mGlu2 receptor mRNA levels in the resistant CA3 region, which, interestingly, were lower than in the CA1 region. Targeting the mGlu2 receptors with selective pharmacologic ligands had profound effects on ishemic neuronal damage. Post-ischemic oral treatment with the selective mGlu2 receptor NAM (negative allosteric modulator), ADX92639 (30 mg/kg), was highly protective against ischemic neuronal death. In contrast, s.c. administration of the mGlu2 receptor enhancer, LY487379 (30 mg/kg), amplified neuronal damage in the CA1 region and extended the damage to the CA3 region.

Conclusion: These findings suggest that the mGlu2 receptor is an important player in mechanisms regulating neuronal vulnerability to ischemic damage, and that mGlu2 receptor NAMs are potential candidates in the experimental treatments of disorders characterized by brain hypoperfusion, such as hypovolemic shock and cardiac arrest.

Fig. 1

Temporal profile of neuronal damage in CA1 and CA3 hippocampal regions. Representative Nissl staining of the CA1 and CA3 regions of rats subjected to 4-VO at different times after reperfusion (24 and 72 h)

Fig. 2

Expression profile of mGlu receptors in CA1 and CA3 regions at times preceding neuronal death. Quantitative PCR analysis of (a) mGlu2, (b) mGlu3, (c) mGlu1 and (d) mGlu5 receptors (mGluR1, -2, -3, and -5) at 12 and 24 h after reperfusion. mRNA values, expressed as copy number, were normalized to β-actin and are means ± S.E.M. of 3-5 rats per group; One-way ANOVA + Fisher's LSD: In (a) mGluR2 at 12 h post ischemia: F_(3,15) = 7.6, p < 0.05; mGluR2 at 24 h post ischemia: F_(3,15) = 7.1, p < 0.05; in (b) mGluR3 at 24 h: F_(3,14) = 2.6, p < 0.05; in (c) mGluR1 at 24 h post ischemia: F_(3,14) = 9.2, p < 0.05. Post-hoc analysis: p < 0.05 vs. the respective values obtained in sham operated rats (*); or vs. the respective values obtained in the CA1 region (#)

Fig. 3

Gene expression analysis of epigenetic factors following global ischemia at several times preceding neuronal death. Quantitative PCR analysis of (a) Dnmt1 and Dnmt3a, (b) Hdac1, Hdac2 and Hdac3, (c) Nr3c1 and (d) Gadd45β, at 6, 12 and 24 h after reperfusion. mRNA values, expressed as copy number, were normalized to β-actin and are means ± S.E.M. of 3-5 individual determinations; One-way ANOVA + Fisher's LSD. In (a) Dnmt3a at 6 h: F_(3,15) = 9.3, p < 0.05; In (b) Hdac1 at 6 h: F_(3,17) = 4.8, p < 0.05; at 24 h: F_(3,15) = 3.8, p < 0.05; Hdac2 at 12 h: F_(3,13) = 4.6, p < 0.05; Hdac3 at 6 h: F_(3,17) = 2.9, p < 0.05; in (c) Nr3c1 at 6 h: F_(3,17) = 4.3, p < 0.05; Nr3c1 at 12 h: F_(3,14) = 5.165 p < 0.05 and in (d) Gadd45β at 6 h: F_(3,17) = 48.4, p < 0.0001, at 12 h: F_(3,13) = 8.6 p < 0.005. Post-hoc analysis: p < 0.05 vs. the respective values obtained in sham operated rats (*); or vs. the respective values obtained in the CA1 region (#)

Fig. 4

HDAC2 protein levels and Grm2 promoter H3-acetylation in the CA1 region following global ischemia. (a) Western blot analysis of HDAC2 in CA1 region at 12 and 24 h after reperfusion. Values are means ± S.E.M of 3-6 rats per group * p < 0.05 (Student's t test) vs. sham-operated rats; t₍₇₎ = 2.73 and t₍₇₎ = 2.61 at 12 and 24 h, respectively. (b) Chip analysis of H3 histone acetylation of Grm2 promoter in CA1 region at 12 and 24 h after reperfusion. Values (percent of input) are expressed as percent of controls (sham-operated), and are means ± S.E.M. of 3-8 rats per group. *p < 0.05 (Student's t-test) vs. sham-operated rats; t₍₅₎ = 1.43 and t₍₁₃₎ = 2.67 at 12 and 24 h, respectively

Fig. 5

In vitro pharmacological profile of the mGlu2 receptor NAM ADX92639. FLIPR data are shown in (a-c). Concentration-response curves of ADX92639 in HEK293 cells stably expressing human or rat mGlu2 receptors or human mGlu3 receptor and challenged with glutamate at the EC₈₀ value are shown in (a). Schild-plot analysis carried out in human mGlu2 cells challenged with glutamate in the absence or presence of increasing concentrations of ADX92639 is shown in (b). A reversibility experiment is shown in (c), where human mGlu2 receptor cells were treated with increasing concentrations of ADX92639 before being either washed or not prior to the addition of an EC₈₀ of glutamate. [³H]LY341495 (1 nM) binding in membrane prepared from human mGlu2 clones incubated in the absence or presence of increasing concentrations of ADX92639, glutamate or non-radioactive LY341495 is shown in (d). Values are means ± SD of two replicates per point and curves are representative of one from 2 to 6 separate experiments

Fig. 6

Neuronal damage analysis of post-ischemia treatment with an mGlu2 receptor NAM, ADX92639, or PAM, LY487379. Nissl staining and neuronal density in the CA1 and CA3 regions of sham-operated and 4-VO rats treated with selective mGlu2 receptor ligands. In (a) and (b), vehicle or the mGlu2 receptor NAM, ADX92639 (30 mg/kg), were orally administered three times at 12, 36, and 60 h after reperfusion; sham-operated rats were only treated with vehicle. Rats were killed at 72 h after reperfusion. Values are means ± S.E.M. of 3-8 rats per group. p < 0.05 vs. sham-operated rats (*) or vs. 4-VO rats treated with vehicle (#) (One-way ANOVA + Fisher LSD, F_(2,18) = 10). In (c) and (d), vehicle or the mGlu2 receptor PAM, LY487379 (30 mg/kg), were s.c. injected five times at 12, 24, 36, 48 and 60 h after reperfusion. Sham-operated rats were only treated with vehicle (data of two sham-operated rats treated with LY487379 are reported in the Results section). Rats were killed at 72 h after reperfusion. Values are means ± S.E.M. of 3-5 rats per group. p < 0.05 vs. sham-operated rats (*) or vs. 4-VO rats treated with vehicle (#) (One-way ANOVA + Fisher LSD; CA1: F_(2,12) = 29.7,p < 0.05; CA3 : F_(2,12) = 5.9, p < 0.05)