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. 2016 Oct 1;9(1):87.
doi: 10.1186/s13041-016-0268-5.

PERK Regulates G q Protein-Coupled Intracellular Ca 2+ Dynamics in Primary Cortical Neurons

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Free PMC article

PERK Regulates G q Protein-Coupled Intracellular Ca 2+ Dynamics in Primary Cortical Neurons

Siying Zhu et al. Mol Brain. .
Free PMC article

Abstract

PERK (EIF2AK3) is an ER-resident eIF2α kinase required for behavioral flexibility and metabotropic glutamate receptor-dependent long-term depression via its translational control. Motivated by the recent discoveries that PERK regulates Ca2+ dynamics in insulin-secreting β-cells underlying glucose-stimulated insulin secretion, and modulates Ca2+ signals-dependent working memory, we explored the role of PERK in regulating Gq protein-coupled Ca2+ dynamics in pyramidal neurons. We found that acute PERK inhibition by the use of a highly specific PERK inhibitor reduced the intracellular Ca2+ rise stimulated by the activation of acetylcholine, metabotropic glutamate and bradykinin-2 receptors in primary cortical neurons. More specifically, acute PERK inhibition increased IP3 receptor mediated ER Ca2+ release, but decreased receptor-operated extracellular Ca2+ influx. Impaired Gq protein-coupled intracellular Ca2+ rise was also observed in genetic Perk knockout neurons. Taken together, our findings reveal a novel role of PERK in neurons, which is eIF2α-independent, and suggest that the impaired working memory in forebrain-specific Perk knockout mice may stem from altered Gq protein-coupled intracellular Ca2+ dynamics in cortical pyramidal neurons.

Keywords: Ca2+; Gq protein-coupled receptor; PERK; Receptor-operated Ca2+ entry.

Figures

Fig. 1
Fig. 1
Gq protein-coupled intracellular Ca2+ ([Ca2+]i) rise is impaired by acute PERK inhibition. a [Ca2+]i of primary cortical neurons in response to 250 μM carbachol treatment (DMSO n = 21, PI n = 17; ***p < 0.001, two-tailed student’s t-Test). b [Ca2+]i of primary cortical neurons in response to 50 μM DHPG treatment (DMSO n = 36, PI n = 57; ***p < 0.001, two-tailed student’s t-Test). c [Ca2+]i of primary cortical neurons in response to 1 μM bradykinin treatment (DMSO n = 25, PI n = 37; ***p < 0.001, two-tailed student’s t-Test). In all of the experiments above, cells were pretreated with 500 nM PERK inhibitor (PI) or DMSO for 15 min before recording. In the representative graph on the left, each Ca2+ trace represents the average of 6–11 neurons that were imaged from the same coverslip. Basal Ca2+ oscillation over 100 sec before treatment and drug-stimulated [Ca2+]i rise over 200 sec were quantified by calculating the area under the curve (AUC). Final analysis is presented as AUC/100 sec and shown in the bar graph on the right
Fig. 2
Fig. 2
IP3-AM induced intracellular Ca2+ ([Ca2+]i) rise is impaired by acute PERK inhibition. [Ca2+]i. of primary cortical neurons in response to 1 μM IP3-AM treatment (DMSO n = 48, PI n = 48; *** p < 0.001, two-tailed student’s t-Test). Cells were pretreated with 500 nM PERK inhibitor (PI) or DMSO for 15 min before recording. In the representative graph on the left, each Ca2+ trace represents the average of 12–14 neurons that were imaged from the same coverslip. Basal Ca2+ oscillation over 100 sec before treatment and IP3-AM-stimulated [Ca2+]i rise over 600 sec were quantified by calculating the area under the curve (AUC). Final analysis is presented as AUC/100 sec and shown in the bar graph on the right
Fig. 3
Fig. 3
Acute PERK inhibition increases IP3 receptor mediated ER Ca2+ release. a [Ca2+]i. of primary cortical neurons in response to 250 μM carbachol treatment in Ca2+ free bath (DMSO n = 29, PI = 26; * p < 0.05, two-tailed student’s t-Test). b [Ca2+]i. of primary cortical neurons in response to 50 μM DHPG treatment in Ca2+- free bath (DMSO n = 33, PI = 39; * p < 0.05, two-tailed student’s t-Test). In both experiments, cells were pretreated with 500 nM PERK inhibitor (PI) or DMSO for 15 min before recording. Drug treatment started 100 sec after Ca2+- free bath perfusion. In the representative graph on the left, each Ca2+ trace represents the average of 8–12 neurons that were imaged from the same coverslip. Basal Ca2+ oscillation over 100 sec before treatment and drug-stimulated [Ca2+]i rise over 20–30 sec were quantified by calculating the area under the curve (AUC), and shown in the middle and right bar graphs respectively
Fig. 4
Fig. 4
Acute PERK inhibition impairs receptor-operated Ca2+ entry, but not store-operated Ca2+ entry. a [Ca2+]i. of thapsigargin (TG) pretreated primary cortical neurons in response to 50 μM DHPG treatment. Cells were pretreated with 500 nM PERK inhibitor (PI) or DMSO for 15 min before recording, and perfused with 1 μM TG for 300 sec before 50 μM DHPG treatment. In the representative graph on the left, each Ca2+ trace represents the average of 8–9 neurons that were imaged from the same coverslip. Basal Ca2+ oscillation over 100 sec before treatment and DHPG-stimulated [Ca2+]i rise over 500 sec were quantified by calculating the area under the curve (AUC). Final analysis is presented as AUC/100 sec and shown in the bar graph on the right (DMSO n = 37, PI n = 35; *** p < 0.001, two-tailed student’s t-Test). b Store-operated Ca2+ entry in primary cortical neurons. Cells were pretreated with 500 nM PI or DMSO for 15 min before recording, and perfused with 1 μM TG in Ca2+- free bath for 300 sec before reintroduction of 2 mM Ca2+. In the representative graph on the left, each Ca2+ trace represents the average of 9–12 neurons that were imaged from the same coverslip. Store-operated Ca2+ entry over 500 sec was quantified by calculating the area under the curve (AUC). Final analysis is presented as AUC/100 sec and shown in the bar graph on the right (DMSO n = 45, PI n = 36; n.s. not significant, two-tailed student’s t-Test)
Fig. 5
Fig. 5
Gq protein-coupled intracellular Ca2+ ([Ca2+]i) mobilization is impaired in genetic Perk knockout primary cortical neurons. a Western blot analysis confirmed almost complete knockdown of PERK in the cerebral cortex of BrPKO mice at postnatal day 0 (BrPKO: Nestin-Cre Perk-floxed; *** p < 0.001, two-tailed student’s t-Test). b No difference in synapse density was observed between WT and BrPKO primary cortical neurons. Representative image on the left shows the immunofluorescent staining of Synapsin 1(red) and MAP2 (green) in primary cortical neurons. Synapse density quantification in the bar graph on the right represents pooled data from 3 mice per genotype (5 neurons were randomly picked for synapse density quantification per animal, n = 15 for each genotype; WT and BrPKO neurons were cultured from the pups in the same litter; n.s. not significant, two-tailed student’s t-Test). c DHPG stimulated [Ca2+]i rise is impaired in genetic Perk KO primary cortical neurons. In the representative graph on the left, each Ca2+ trace represents the average of 8–10 neurons that were imaged from the same coverslip. Basal Ca2+ oscillation over 100 sec before treatment and DHPG-stimulated [Ca2+]i rise over 200 sec were quantified by calculating the area under the curve (AUC). Final analysis is presented as AUC/100 sec and shown in the bar graph on the right (WT n = 44, BrPKO n = 34; *** p < 0.001, two-tailed student’s t-Test)
Fig. 6
Fig. 6
Proposed model for PERK’s regulation of Gq protein-coupled Ca2+ dynamics in pyramidal neurons. Upon extracellular ligand binding, Gq protein-coupled receptor is activated, which subsequently activates Gq/PLC. Activated PLC hydrolyzes PIP2 into IP3 and DAG. Increased cytosol IP3 induces ER Ca2+ depletion by binding with ER-resident IP3R, which may activate PERK due to Ca2+ dissociation from its regulatory domain in the ER. Activated PERK may then restore ER Ca2+ level by inhibiting IP3R mediated ER Ca2+ release and activating receptor-operated Ca2+ entry

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