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Calcium-sensing receptor (CaSR) as a novel target for ischemic neuroprotection.

Kim JY, Ho H, Kim N, Liu J, Tu CL, Yenari MA, Chang W - Ann Clin Transl Neurol (2014)

Bottom Line: Concurrent ablation of Gabbr1 gene blocked the neuroprotection caused by the Casr gene knockout.Coinjection of calcilytics with baclofen synergistically enhanced neuroprotection.Our study demonstrates a novel receptor interaction, which contributes to ischemic neuron death through CaSR upregulation and GABABR1 downregulation, and feasibility of neuroprotection by concurrently targeting these two receptors.

View Article: PubMed Central - PubMed

Affiliation: Endocrine Unit, University of California San Francisco and Veterans Affairs Medical Center San Francisco, California, 94121 ; Department of Neurology, University of California San Francisco and Veterans Affairs Medical Center San Francisco, California, 94121.

ABSTRACT

Object: Ischemic brain injury is the leading cause for death and long-term disability in patients who suffer cardiac arrest and embolic stroke. Excitotoxicity and subsequent Ca(2+)-overload lead to ischemic neuron death. We explore a novel mechanism concerning the role of the excitatory extracellular calcium-sensing receptor (CaSR) in the induction of ischemic brain injury.

Method: Mice were exposed to forebrain ischemia and the actions of CaSR were determined after its genes were ablated specifically in hippocampal neurons or its activities were inhibited pharmacologically. Since the CaSR forms a heteromeric complex with the inhibitory type B γ-aminobutyric acid receptor 1 (GABABR1), we compared neuronal responses to ischemia in mice deficient in CaSR, GABABR1, or both, and in mice injected locally or systemically with a specific CaSR antagonist (or calcilytic) in the presence or absence of a GABABR1 agonist (baclofen).

Results: Both global and focal brain ischemia led to CaSR overexpression and GABABR1 downregulation in injured neurons. Genetic ablation of Casr genes or blocking CaSR activities by calcilytics rendered robust neuroprotection and preserved learning and memory functions in ischemic mice, partly by restoring GABABR1 expression. Concurrent ablation of Gabbr1 gene blocked the neuroprotection caused by the Casr gene knockout. Coinjection of calcilytics with baclofen synergistically enhanced neuroprotection. This combined therapy remained robust when given 6 h after ischemia.

Interpretation: Our study demonstrates a novel receptor interaction, which contributes to ischemic neuron death through CaSR upregulation and GABABR1 downregulation, and feasibility of neuroprotection by concurrently targeting these two receptors.

No MeSH data available.


Related in: MedlinePlus

Intraperitoneal (IP) injections of calcilytics protected neurons against ischemic injury and preserved hippocampal functions. (A) Representative TUNEL staining of hippocampi; (B) the % of neurons (normalized to the numbers of neurons in Sham-treated DMSO-injected mice) which were preserved; and (C) the percentage of TUNEL-(+) neurons which were shown in the hippocampi of 6-month-old wild-type C57/B6 mice subjected to a TGI (15 min) procedure, daily IP injections of NPS89636, NPS2143 (1 mg/kg), or vehicle (DMSO), and 3 days of reperfusion. *P < 0.05; **P < 0.01 versus DMSO group in (B); **P < 0.01 versus TGI-DMSO, #P < 0.05 NPS89636 versus NPS2143 in (C). N = 6–12 mice/group. Scale bar: 20 μm. (D) MWM tests were performed on 4-month-old wild-type C57/B6 mice subjected to Sham or TGI (10 min) procedure with or without daily injections of NPS2143 (1 mg/kg) for 2 weeks, as described in Materials and Methods. Left panel shows the effects of TGI and NPS2143 on learning efficiency as indicated by the pathlength in finding visible and hidden platforms during the 6-day 12-session training. All animal groups showed significant learning (two-way ANOVA: main effect of learning: ****P < 0.0001) as indicated by progressively shortened path lengths in seeking a hidden platform in sessions 6–12. However, the learning ability was significantly (*P < 0.05) impaired in TGI-DMSO mice when compared to Sham-DMSO mice. Interestingly, IP injections of NPS2143 significantly (*P < 0.05) restored the TGI-induced loss of learning ability (TGI-DMSO vs. TGI-NPS). Right panel shows effects of TGI and NPS on memory retention or recall as indicated by the length of time spent swimming in the previous platform in the final probe trial. *P < 0.05, **P < 0.01. N = 9–17 per group. (E) Micrographs show representative H&E staining of hippocampi (CA1) from mice subjected to Sham or TGI (10 min) procedures, followed by daily IP injection of vehicle (DMSO) or NPS2143 (NPS) for 14 days and 1-week MWM test. Brain samples were collected 1 day after the completion of MWM test. TUNEL, transferase dUTP nick end-labeling; TGI, transient global ischemia; MWM, Morris Water Maze; ANOVA, analysis of variance; CA, cornu ammonis.
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fig04: Intraperitoneal (IP) injections of calcilytics protected neurons against ischemic injury and preserved hippocampal functions. (A) Representative TUNEL staining of hippocampi; (B) the % of neurons (normalized to the numbers of neurons in Sham-treated DMSO-injected mice) which were preserved; and (C) the percentage of TUNEL-(+) neurons which were shown in the hippocampi of 6-month-old wild-type C57/B6 mice subjected to a TGI (15 min) procedure, daily IP injections of NPS89636, NPS2143 (1 mg/kg), or vehicle (DMSO), and 3 days of reperfusion. *P < 0.05; **P < 0.01 versus DMSO group in (B); **P < 0.01 versus TGI-DMSO, #P < 0.05 NPS89636 versus NPS2143 in (C). N = 6–12 mice/group. Scale bar: 20 μm. (D) MWM tests were performed on 4-month-old wild-type C57/B6 mice subjected to Sham or TGI (10 min) procedure with or without daily injections of NPS2143 (1 mg/kg) for 2 weeks, as described in Materials and Methods. Left panel shows the effects of TGI and NPS2143 on learning efficiency as indicated by the pathlength in finding visible and hidden platforms during the 6-day 12-session training. All animal groups showed significant learning (two-way ANOVA: main effect of learning: ****P < 0.0001) as indicated by progressively shortened path lengths in seeking a hidden platform in sessions 6–12. However, the learning ability was significantly (*P < 0.05) impaired in TGI-DMSO mice when compared to Sham-DMSO mice. Interestingly, IP injections of NPS2143 significantly (*P < 0.05) restored the TGI-induced loss of learning ability (TGI-DMSO vs. TGI-NPS). Right panel shows effects of TGI and NPS on memory retention or recall as indicated by the length of time spent swimming in the previous platform in the final probe trial. *P < 0.05, **P < 0.01. N = 9–17 per group. (E) Micrographs show representative H&E staining of hippocampi (CA1) from mice subjected to Sham or TGI (10 min) procedures, followed by daily IP injection of vehicle (DMSO) or NPS2143 (NPS) for 14 days and 1-week MWM test. Brain samples were collected 1 day after the completion of MWM test. TUNEL, transferase dUTP nick end-labeling; TGI, transient global ischemia; MWM, Morris Water Maze; ANOVA, analysis of variance; CA, cornu ammonis.

Mentions: We hypothesize that CaSR hyperactivity, as a result of receptor overexpression, stimulates intrinsic excitatory responses that contribute to neuronal death. We, therefore, tested whether two CaSR antagonists, namely calcilytics NPS89636 and NPS2413, which specifically inhibit CaSR-mediated signaling responses and intracellular Ca2+ mobilization in various cell types,51–53 protect against neuronal injury in wild-type (WT) C57/B6 mice. In WT mice, which were subjected to TGI, daily IP injections of vehicle (0.1% DMSO), and 72 h of reperfusion, the numbers of hippocampal neurons in the CA1, CA3, and DG were reduced by 53 ± 2%, 50 ± 2%, and 45 ± 3% (Fig. 4B, ▪) with 88 ± 3%, 67 ± 6%, and 43 ± 4% of them being TUNEL-(+) (Fig. 4A, DMSO; 4C, ▪), in the CA1, CA3, and DG, respectively. In contrast, daily IP injections of either NPS89636 or NPS2413 (1 mg/kg body wt), which began 1 h after the TGI procedure, significantly protected neurons from ischemic injury. In TGI mice injected with NPS89636, 59 ± 3%, 58 ± 2%, 64 ± 2% of hippocampal neurons was preserved in the CA1, CA3, and DG, respectively (Fig. 4B, ▪), with 57 ± 4%, 38 ± 2%, and 32 ± 1% of them being TUNEL-(+) (Fig. 4A, NPS89636; 4C, ▪). In TGI-treated NPS2143-injected mice, 68 ± 2%, 67 ± 2%, and 72 ± 1% of hippocampal neurons were preserved in the CA1, CA3, and DG, respectively (Fig. 4B, □), with only 39 ± 4%, 26 ± 2%, and 21 ± 1% of them being TUNEL-(+) (Fig. 4A, NPS-2143; 4C, □), indicating a greater potency of NPS2143 versus NPS89636 (P < 0.05) in neuroprotection. This is consistent with a lower half-maximal inhibitory concentration (IC50) for NPS2143 (43 nmol/L or 17 ng/mL) versus NPS89636 (271 nmol/L or 143 ng/mL) in blocking CaSR-mediated signaling responses.54–57 We did not observe significant changes in body temperatures of the mice with either calcilytics, excluding potential thermo effects on the neuronal responses.


Calcium-sensing receptor (CaSR) as a novel target for ischemic neuroprotection.

Kim JY, Ho H, Kim N, Liu J, Tu CL, Yenari MA, Chang W - Ann Clin Transl Neurol (2014)

Intraperitoneal (IP) injections of calcilytics protected neurons against ischemic injury and preserved hippocampal functions. (A) Representative TUNEL staining of hippocampi; (B) the % of neurons (normalized to the numbers of neurons in Sham-treated DMSO-injected mice) which were preserved; and (C) the percentage of TUNEL-(+) neurons which were shown in the hippocampi of 6-month-old wild-type C57/B6 mice subjected to a TGI (15 min) procedure, daily IP injections of NPS89636, NPS2143 (1 mg/kg), or vehicle (DMSO), and 3 days of reperfusion. *P < 0.05; **P < 0.01 versus DMSO group in (B); **P < 0.01 versus TGI-DMSO, #P < 0.05 NPS89636 versus NPS2143 in (C). N = 6–12 mice/group. Scale bar: 20 μm. (D) MWM tests were performed on 4-month-old wild-type C57/B6 mice subjected to Sham or TGI (10 min) procedure with or without daily injections of NPS2143 (1 mg/kg) for 2 weeks, as described in Materials and Methods. Left panel shows the effects of TGI and NPS2143 on learning efficiency as indicated by the pathlength in finding visible and hidden platforms during the 6-day 12-session training. All animal groups showed significant learning (two-way ANOVA: main effect of learning: ****P < 0.0001) as indicated by progressively shortened path lengths in seeking a hidden platform in sessions 6–12. However, the learning ability was significantly (*P < 0.05) impaired in TGI-DMSO mice when compared to Sham-DMSO mice. Interestingly, IP injections of NPS2143 significantly (*P < 0.05) restored the TGI-induced loss of learning ability (TGI-DMSO vs. TGI-NPS). Right panel shows effects of TGI and NPS on memory retention or recall as indicated by the length of time spent swimming in the previous platform in the final probe trial. *P < 0.05, **P < 0.01. N = 9–17 per group. (E) Micrographs show representative H&E staining of hippocampi (CA1) from mice subjected to Sham or TGI (10 min) procedures, followed by daily IP injection of vehicle (DMSO) or NPS2143 (NPS) for 14 days and 1-week MWM test. Brain samples were collected 1 day after the completion of MWM test. TUNEL, transferase dUTP nick end-labeling; TGI, transient global ischemia; MWM, Morris Water Maze; ANOVA, analysis of variance; CA, cornu ammonis.
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fig04: Intraperitoneal (IP) injections of calcilytics protected neurons against ischemic injury and preserved hippocampal functions. (A) Representative TUNEL staining of hippocampi; (B) the % of neurons (normalized to the numbers of neurons in Sham-treated DMSO-injected mice) which were preserved; and (C) the percentage of TUNEL-(+) neurons which were shown in the hippocampi of 6-month-old wild-type C57/B6 mice subjected to a TGI (15 min) procedure, daily IP injections of NPS89636, NPS2143 (1 mg/kg), or vehicle (DMSO), and 3 days of reperfusion. *P < 0.05; **P < 0.01 versus DMSO group in (B); **P < 0.01 versus TGI-DMSO, #P < 0.05 NPS89636 versus NPS2143 in (C). N = 6–12 mice/group. Scale bar: 20 μm. (D) MWM tests were performed on 4-month-old wild-type C57/B6 mice subjected to Sham or TGI (10 min) procedure with or without daily injections of NPS2143 (1 mg/kg) for 2 weeks, as described in Materials and Methods. Left panel shows the effects of TGI and NPS2143 on learning efficiency as indicated by the pathlength in finding visible and hidden platforms during the 6-day 12-session training. All animal groups showed significant learning (two-way ANOVA: main effect of learning: ****P < 0.0001) as indicated by progressively shortened path lengths in seeking a hidden platform in sessions 6–12. However, the learning ability was significantly (*P < 0.05) impaired in TGI-DMSO mice when compared to Sham-DMSO mice. Interestingly, IP injections of NPS2143 significantly (*P < 0.05) restored the TGI-induced loss of learning ability (TGI-DMSO vs. TGI-NPS). Right panel shows effects of TGI and NPS on memory retention or recall as indicated by the length of time spent swimming in the previous platform in the final probe trial. *P < 0.05, **P < 0.01. N = 9–17 per group. (E) Micrographs show representative H&E staining of hippocampi (CA1) from mice subjected to Sham or TGI (10 min) procedures, followed by daily IP injection of vehicle (DMSO) or NPS2143 (NPS) for 14 days and 1-week MWM test. Brain samples were collected 1 day after the completion of MWM test. TUNEL, transferase dUTP nick end-labeling; TGI, transient global ischemia; MWM, Morris Water Maze; ANOVA, analysis of variance; CA, cornu ammonis.
Mentions: We hypothesize that CaSR hyperactivity, as a result of receptor overexpression, stimulates intrinsic excitatory responses that contribute to neuronal death. We, therefore, tested whether two CaSR antagonists, namely calcilytics NPS89636 and NPS2413, which specifically inhibit CaSR-mediated signaling responses and intracellular Ca2+ mobilization in various cell types,51–53 protect against neuronal injury in wild-type (WT) C57/B6 mice. In WT mice, which were subjected to TGI, daily IP injections of vehicle (0.1% DMSO), and 72 h of reperfusion, the numbers of hippocampal neurons in the CA1, CA3, and DG were reduced by 53 ± 2%, 50 ± 2%, and 45 ± 3% (Fig. 4B, ▪) with 88 ± 3%, 67 ± 6%, and 43 ± 4% of them being TUNEL-(+) (Fig. 4A, DMSO; 4C, ▪), in the CA1, CA3, and DG, respectively. In contrast, daily IP injections of either NPS89636 or NPS2413 (1 mg/kg body wt), which began 1 h after the TGI procedure, significantly protected neurons from ischemic injury. In TGI mice injected with NPS89636, 59 ± 3%, 58 ± 2%, 64 ± 2% of hippocampal neurons was preserved in the CA1, CA3, and DG, respectively (Fig. 4B, ▪), with 57 ± 4%, 38 ± 2%, and 32 ± 1% of them being TUNEL-(+) (Fig. 4A, NPS89636; 4C, ▪). In TGI-treated NPS2143-injected mice, 68 ± 2%, 67 ± 2%, and 72 ± 1% of hippocampal neurons were preserved in the CA1, CA3, and DG, respectively (Fig. 4B, □), with only 39 ± 4%, 26 ± 2%, and 21 ± 1% of them being TUNEL-(+) (Fig. 4A, NPS-2143; 4C, □), indicating a greater potency of NPS2143 versus NPS89636 (P < 0.05) in neuroprotection. This is consistent with a lower half-maximal inhibitory concentration (IC50) for NPS2143 (43 nmol/L or 17 ng/mL) versus NPS89636 (271 nmol/L or 143 ng/mL) in blocking CaSR-mediated signaling responses.54–57 We did not observe significant changes in body temperatures of the mice with either calcilytics, excluding potential thermo effects on the neuronal responses.

Bottom Line: Concurrent ablation of Gabbr1 gene blocked the neuroprotection caused by the Casr gene knockout.Coinjection of calcilytics with baclofen synergistically enhanced neuroprotection.Our study demonstrates a novel receptor interaction, which contributes to ischemic neuron death through CaSR upregulation and GABABR1 downregulation, and feasibility of neuroprotection by concurrently targeting these two receptors.

View Article: PubMed Central - PubMed

Affiliation: Endocrine Unit, University of California San Francisco and Veterans Affairs Medical Center San Francisco, California, 94121 ; Department of Neurology, University of California San Francisco and Veterans Affairs Medical Center San Francisco, California, 94121.

ABSTRACT

Object: Ischemic brain injury is the leading cause for death and long-term disability in patients who suffer cardiac arrest and embolic stroke. Excitotoxicity and subsequent Ca(2+)-overload lead to ischemic neuron death. We explore a novel mechanism concerning the role of the excitatory extracellular calcium-sensing receptor (CaSR) in the induction of ischemic brain injury.

Method: Mice were exposed to forebrain ischemia and the actions of CaSR were determined after its genes were ablated specifically in hippocampal neurons or its activities were inhibited pharmacologically. Since the CaSR forms a heteromeric complex with the inhibitory type B γ-aminobutyric acid receptor 1 (GABABR1), we compared neuronal responses to ischemia in mice deficient in CaSR, GABABR1, or both, and in mice injected locally or systemically with a specific CaSR antagonist (or calcilytic) in the presence or absence of a GABABR1 agonist (baclofen).

Results: Both global and focal brain ischemia led to CaSR overexpression and GABABR1 downregulation in injured neurons. Genetic ablation of Casr genes or blocking CaSR activities by calcilytics rendered robust neuroprotection and preserved learning and memory functions in ischemic mice, partly by restoring GABABR1 expression. Concurrent ablation of Gabbr1 gene blocked the neuroprotection caused by the Casr gene knockout. Coinjection of calcilytics with baclofen synergistically enhanced neuroprotection. This combined therapy remained robust when given 6 h after ischemia.

Interpretation: Our study demonstrates a novel receptor interaction, which contributes to ischemic neuron death through CaSR upregulation and GABABR1 downregulation, and feasibility of neuroprotection by concurrently targeting these two receptors.

No MeSH data available.


Related in: MedlinePlus