Ischemic insults promote epigenetic reprogramming of mu opioid receptor expression in hippocampal neurons

Abstract

Transient global ischemia is a neuronal insult that induces delayed, selective death of hippocampal CA1 pyramidal neurons. A mechanism underlying ischemia-induced cell death is activation of the gene silencing transcription factor REST (repressor element-1 silencing transcription factor)/NRSF (neuron-restrictive silencing factor) and REST-dependent suppression of the AMPA receptor subunit GluR2 in CA1 neurons destined to die. Here we show that REST regulates an additional gene target, OPRM1 (mu opioid receptor 1 or MOR-1). MORs are abundantly expressed by basket cells and other inhibitory interneurons of CA1. Global ischemia induces a marked decrease in MOR-1 mRNA and protein expression that is specific to the selectively vulnerable area CA1, as assessed by quantitative real-time RT-PCR, Western blotting, and ChIP. We further show that OPRM1 gene silencing is REST-dependent and occurs via epigenetic modifications. Ischemia promotes deacetylation of core histone proteins H3 and H4 and dimethylation of histone H3 at lysine-9 (H3-K9) over the MOR-1 promoter, an signature of epigenetic gene silencing. Acute knockdown of MOR-1 gene expression by administration of antisense oligodeoxynucleotides to hippocampal slices in vitro or injection of the MOR antagonist naloxone to rats in vivo affords protection against ischemia-induced death of CA1 pyramidal neurons. These findings implicate MORs in ischemia-induced death of CA1 pyramidal neurons and document epigenetic remodeling of expression of OPRM1 in CA1 inhibitory interneurons.

Fig. 1.

Global ischemia decreases MOR-1 mRNA and protein expression in CA1. (a) Real-time PCR of RNA samples from the CA1 and CA3 of control and experimental animals at 6, 24, and 48 h after ischemia. Bars represent relative mRNA quantity measured by the Ct values ± SEMs, where Ct is the number of cycles needed for detection of the fluorescence signal; significance was assessed by using the REST software with which transcript differences are tested for significance by a pairwise fixed reallocation–randomization test (55). ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001. (b and c) Representative Western blots probed with anti-MOR antibody (b) and relative MOR abundance in samples from the CA1 and CA3 at 48 h after sham operation (control) or at 12, 24, and 48 h after ischemia (c). Values are expressed as the ratio of band densities for experimental samples to band density of the corresponding control sample after correction for loading. Bars represent mean ± SEMs. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001 (Student's unpaired t test).

Fig. 2.

Ischemia enhances REST binding to the MOR-1 promoter in CA1. (a) ChIP analysis of cross-linked chromatin from the CA1 and CA3 of control (Ctl) and experimental animals at 12, 24, and 48 h after ischemia immunoprecipitated with an anti-REST antibody. Immunoprecipitation with nonimmune IgG showed negligible PCR product, indicating specificity of the ChIP technique. Left lanes show ODN length standards. (b) Band densities for PCR products were normalized to that of the corresponding control input. Bars represent mean ± SEMs. ∗, P < 0.05 (Student's unpaired t test).

Fig. 3.

Ischemia promotes deacetylation of H3 and H4 over the MOR-1 promoter in CA1. ChIP analysis (a and b) of CA1 and CA3 from control and experimental animals 12, 24, and 48 h after ischemia immunoprecipitated with antibody to acetyl-H3 (a and c) or acetyl-H4 (b and d). The IgG immunoprecipitate showed negligible PCR product, indicating little or no immunoprecipitation in the absence of primary antibody. Band densities for PCR products were normalized as in Fig. 2. Left lanes show ODN length standards in a and b. Bars represent mean ± SEMs. ∗, P < 0.05; ∗∗, P < 0.01 (Student's unpaired t test).

Fig. 4.

Ischemia enhances association of diMeK9-H3, but not diMeK4-H3, with the MOR-1 promoter in CA1. (a) ChlP analysis of cross-linked chromatin from the CA1 of control (Ctl) and experimental animals at 12, 24, and 48 h after ischemia immunoprecipitated with antibody to diMeK9-H3 (a Middle and b) or diMeK4-H3 (a Top and b) (Materials and Methods). (a) Representative ethidium bromide-stained agarose gel showing amplification of promoter fragments. Left lanes show ODN length standards. (b) Levels of diMeK9-H3 and diMeK4-H3 at MOR-1 promoter in CA1 were quantified by using real-time PCR. Ct values of immunoprecipitated samples were normalized to the corresponding value for input. Bars represent mean ± SEMs. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001 vs. control (Student's paired t test).

Fig. 5.

MOR-1 antisense suppresses MOR protein expression and rescues CA1 neurons. (a) MOR antisense suppressed MOR protein expression, assessed by Western blot analysis of four hippocampal slices from different animals. (b–m) PI labeling of cultured hippocampal slices. (b) Control slice. (f) Control slice treated with MOR antisense. (j) Control slice treated with MOR missense. (c–e) Slices at 24, 48, and 72 h after OGD (30 min). The pyramidal cell layer of CA1 exhibited intense PI labeling, indicative of cell death, evident at 24 and 72 h. (g–i) MOR antisense (10 μM, 72 h before OGD) reduces OGD-induced neuronal death. (k–m) MOR-1 missense (10 μM, 72 h before OGD) was without effect. (n) Quantitation of cell death, assessed as the percentage of PI fluorescence per unit area over CA1 normalized to the control. ∗, P < 0.05; ∗∗, P < 0.01; n.s. vs. OGD (one-way ANOVA, followed by Dunnett's multiple comparison test). Ctl, control; AS, antisense; MS, missense.

Fig. 6.

Naloxone protects CA1 neurons from postischemic neurodegeneration. (a–l) Representative toluidine blue-stained coronal brain sections at the level of the dorsal hippocampus from control and experimental animals subjected to saline (a and b), naloxone (1 mg/kg; c and d), ischemia and saline (e and f), ischemia and naloxone (0.1 mg/kg or 1 mg/kg, 1 h before ischemia; g–j), or ischemia and naloxone [1 mg/kg twice (1 h before and 1 h after ischemia); k and l). Animals were killed 7 days after reperfusion. (m) Summary data for a–l. Ischemia elicited selective, delayed death of CA1 pyramidal neurons. Naloxone at doses and times examined significantly protected CA1 neurons. ∗, P < 0.05; ∗∗, P < 0.01. Low- and high-magnification panel widths are 2.5 and 0.25 mm, respectively.