Inhibition of Calcium-sensing Receptor Alleviates Chronic Intermittent Hypoxia-induced Cognitive Dysfunction via CaSR-PKC-ERK1/2 Pathway

doi:10.21203/rs.3.rs-2100396/v1

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Inhibition of Calcium-sensing Receptor Alleviates Chronic Intermittent Hypoxia-induced Cognitive Dysfunction via CaSR-PKC-ERK1/2 Pathway

https://doi.org/10.21203/rs.3.rs-2100396/v1

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published 05 Jan, 2023

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Obstructive sleep apnea hypopnea syndrome (OSAHS) is typically characterized by chronic intermittent hypoxia (CIH), associated with cognitive dysfunction in children. Calcium-sensing receptor (CaSR), a member of G-protein coupled receptors superfamily, has been demonstrated to mediate the apoptosis of hippocampal neurons in various diseases. However, the effect of CaSR on OSAHS remains elusive. In the present study, we investigated the role of CaSR in CIH-induced memory dysfunction and underlying mechanisms on regulation of PKC-ERK1/2 signaling pathway in vivo and in vitro. CIH exposures for 4 weeks in mice, modeling OSAHS, contributed to cognitive dysfunction, manifested as increased working memory errors, reference memory errors and total memory errors. CIH accelerated apoptosis of hippocampal neurons by increased TUNEL positive cells, up-regulated cleaved-Caspase3 and down-regulated Bcl-2 in mice, and resulted in the synaptic plasticity deficit via down-regulated synaptophysin (Syn) protein level. To further identify whether CaSR is involved in hippocampal neuronal apoptosis and synaptic plasticity induced by CIH, the mice were intraperitoneally injected with CaSR inhibitor (NPS2143) 30 min before CIH exposure everyday, and the results demonstrated CaSR inhibitor alleviated the apoptosis and synaptic plasticity deficit in the hippocampus of CIH mice, accompanied by decreased p-ERK1/2 and PKC. To clarify the mechanism of apoptosis and synaptic plasticity in CIH hippocampal neurons mediated by CaSR, we established intermittent hypoxia PC12 cell model and found that the activation of CaSR accelerated CIH-induced PC12 apoptosis and synaptic plasticity deficit by up-regulated p-ERK1/2 and PKC. Overall, our findings indicated that CaSR held a critical function on CIH-induced cognitive dysfunction in mice by accelerating hippocampal neuronal apoptosis and reducing synaptic plasticity via augmenting CaSR- PKC-ERK1/2 pathway; otherwise, inhibition of CaSR alleviated CIH-induced cognitive dysfunction.

Chronic intermittent hypoxia

Cognitive dysfunction

Calcium-sensing receptor

ERK1/2

Synaptophysin

Apoptosis

Obstructive sleep apnea-hypopnea syndrome (OSAHS) is mainly manifested by recurrent obstruction of the upper airway during sleep, contributing to hypopnea and sleep apnea. OSAHS can occur at any age, especially in preschool children [1]. Multiple systems were reported to be damaged by OSAHS in adults, including the cardiovascular system, respiratory system, and muscular system [2]. The others and our research have demonstrated the nervous system impairment, especially cognitive dysfunction, was observed more frequently in pediatric OSAHS [3, 4]. Chronic intermittent hypoxia (CIH) is recognized as the main feature of OSAHS. Large amounts of researches indicated that CIH exposure impaired cognitive function by inducing apoptosis and the change of synaptic plasticity in hippocampus [5, 6]. However, the underlying mechanisms remain unclear.

Calcium-sensing receptor (CaSR), a member of G-protein coupled receptors superfamily, senses extracellular levels of calcium ions and is essential for regulating systemic calcium homeostasis [7]. Apart from parathyroid glands, kidney, and bone, CaSR is also expressed in the hippocampus, and holds a critical function on neuronal proliferation and differentiation during early neural development [8]. Large amounts of researches demonstrated that CaSR expression was up-regulated in rat brains, specially in CA1 and CA3 of the hippocampus, after global or focal cerebral ischaemia/reperfusion [9, 10]. Moreover, CaSR, activated by extracellular calcium or CaSR agonists, increases the concentration of intracellular calcium ([Ca²⁺]i), which then induces the apoptosis of hippocampal neurons [11]. However, the mechanisms of CaSR-induced apoptosis are not well understood.

Synaptic plasticity represents the information storage process of synapses and is considered to be related to learning and memory. Recent studies have shown that CaSR may exist widely in synapses, and CaSR plays a significant role in synaptic plasticity. Additionally, further research demonstrated that CaSR was likely to modify dendritic structure when synapses are involved in memory and learning [12]. The distribution of CaSR in nerve endings is related to the synthesis and release of neurotransmitters, participating in the regulation of synaptic response and synaptic plasticity. A recent study revealed that CaSR, which promotes dendritic development and axonal growth, is regulated via ERK signaling pathway [13]. Large amounts of evidence indicates that hippocampal synaptic plasticity is impaired after IH exposure [14, 15], which may be the basis of memory dysfunction in OSAHS. Nevertheless, it is unknown whether CaSR has an impact on cognitive function by regulating synaptic plasticity under intermittent hypoxic conditions.

CaSR can sense increased extracellular levels of calcium ions, lead to calcium influx, resulting in increased [Ca²⁺]i [16]. Intracellular calcium overload is a critical step for the apoptosis of hippocampal neurons undergoing H/R injury [17]. In addition, CaSR has been demonstrated to activate Ca²⁺-dependent protein kinase C (PKC) and induce the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) [18]. PKC, a family of serine/threonine protein kinases, participates in many cellular responses such as cell proliferation [19]. To date, conventional PKC isoforms have been identified require Ca²⁺ influx for their activation [20]. ERK1/2, as a subfamily of mitogen-activated protein kinases (MAPKs), is generally thought to be involved in cell apoptosis, proliferation and synaptic plasticity [21, 22]. Further studies indicated that CaSR activation aggravates the apoptosis and the change of synaptic plasticity in hippocampal neurons during H/R [11, 23] and the [Ca²⁺]i overload induced by the activation of CaSR contributes to the apoptosis of PC12 cells in oxygen and glucose deprivation/reoxygenation [24]. However, it remains unclear whether CaSR holds a function on CIH-induced apoptosis and synaptic plasticity in hippocampal neurons by PKC-ERK1/2 signaling pathway. Therefore, our present study aimed to investigate the role of CaSR in neurocognitive deficits of childhood OSAHS and the underlying mechanisms.

2.1. Experimental animals and grouping

All experimental protocols were approved by Animal Research Committee of Wenzhou Medical University (wydw 2021 − 0628). Forty healthy male C57BL/6 mice (18-22g; 4 weeks) were purchased from the Experimental Animal Center (License no. SCXK 2018-0017) of Wenzhou Medical University, Zhejiang Province, China. The mice were randomly divided into four groups: control group (C), chronic intermittent hypoxia model group (CIH), CIH with CaSR inhibitor (NPS2143) treatment group (CIH + NPS2143), and C with NPS2143 treatment group (C + NPS2143). The mice in C and CIH group were not subjected to NPS2143 injection. Mice in CIH + NPS2143 and C + NPS2143 groups were intraperitoneally injected with NPS2143 (3mg/kg, GLPBIO, Cat. No: GC169431) 30 min before CIH exposure everyday [25]. All animals used in the experiment were cared for in accordance with the ethical guidelines on animal experimentation of Laboratory Animals of China National Institutes of Health.

2.2. Animal models

The CIH model in mice is established by using a computer-controlled chamber with a nitrogen/oxygen delivery system to establish hypoxic-reoxygenation conditions that we previously designed [5]. In simple terms, in an intermittent hypoxic (IH) chamber, the oxygen concentration fluctuated from 9.0%±1.5–21%±0.5% in 90 s per cycle, 40 cycles per h, 7 h a day (from 8:00 to 15:00) during the light cycle, for 4 weeks. The CIH and CIH + NPS2143 groups were placed in the IH chamber, and the C and C + NPS2143 groups were placed in a cabin filled with compressed air.

2.3. 8-Arm radial maze test

An eight-arm radial maze was used to evaluate spatial memory of mice after CIH. As reported previously [26], two days before the CIH exposure, the mice were restricted to eat until their weight was reduced to 80–85 percent of their baseline body weight. After acclimation experiment, mice were trained following CIH at 19:00 every night, once a day for seven consecutive days. The 8-arm radial maze test was performed instantly after seven hours of IH exposure on the last day. During the test, mice covered with an opaque box were placed in the center of the maze for 15 seconds, and then released for free feeding when the box was removed. The hind paws of mice completely enter into one arm is considered that arm entry was complete. A trial was terminated immediately when all pellets were consumed or 5 minutes had elapsed. The performance of mice was evaluated according to the number of error choices as follows: working memory errors (WME: re-entry into arms where food pellet had been eaten), reference memory errors (RME: entry into a never-baited arm), and the total errors (TE: Mice enter a baited arm and eat the pellet as the right choice, otherwise it was counted as an error).

2.4. Animal sample collection

After the 8-arm radial maze test, five mice randomly selected from each group were anesthetized with 10% chloral hydrate via intraperitoneal injection. Their brain tissues were dissected into left and right hemispheres immediately on ice, and placed into 4% paraformaldehyde for Nissl staining, TUNEL assay and Immunohistochemistry. Subsequently, the remaining five mice in each group were dissected into left and right hippocampus, and were frozen at − 80°C for Western blotting analysis.

2.5. Nissl staining

Following dewaxing with dimethylbenzene, 4-µm-thick brain sections were treated with 100%, 95%, 85%, 75% gradient alcohol by turn. After rinsing with water, slices were immersed in Nissl staining solution (Beyotime Biotechnology, China) for 20 min at 37°C. Then, the tissue sections were dehydrated in 95% alcohol twice (5 min per time) and permeabilized with xylene twice (15 min each time). The images of hippocampal CA1 regions were captured by light microscope with 400× magnification (OLYMPUS 1X70-SIF2, Japan) and Image J software were applied to quantify the numbers of neurons. The size and number of Nissl body can reflect the degree of neuronal injury.

2.6. Terminal transferase-mediated dUTP nick end labeling (TUNEL) staining

TUNEL assay was used to detect apoptosis according to the manufacturer's recommended procedure. The tissue sections were dewaxed, sealed and treated with TUNEL reaction mixture for 30 min and POD for 60 min. The slices were then incubated with DAB, counter-stained with a hematoxylin dye and stained on Eukitt (sigma-aldrich). Apoptotic cells are those with brown granules in the nucleus, namely TUNEL positive staining cells. Under a 400x magnification light microscope, 5 sections in each group were randomly selected with 3 fields in each section. The positive staining cells in the hippocampus CA1 region were counted, and the apoptosis index was determined. Apoptosis index (AI) was calculated as number of TUNEL positive cells/total number of cells ×100 to evaluate apoptosis.

2.7. Immunohistochemistry

Brain tissue sections were dewaxed in xylene, rehydrated through a serial alcohol gradient, and washed in PBS. And then, the brain tissue sections were heated by microwave and cooled naturally to repair the antigen in 10 mmol/L citrate buffer with pH 6.0. The primary antibody rabbit anti-CaSR (1:200, Affinity), rabbit anti-p-ERK1/2 (1:200, Cell Signaling) and anti-synaptophysin (Syn) (1:100, Affinity) was used for immunostaining and brain tissue sections were incubated with above antibody at 4°C overnight. After incubation with the appropriate biotin-conjugated secondary antibody, stained with hematoxylin and eosin. Brain tissue sections were visualized through microscopy. Under a 400x magnification, 5 sections in each group were randomly selected with 3 fields in each section. The ingerated indensity in the hippocampus CA1 region were calculated via image J software (National Institutes of Health).

2.8. Tissue protein extraction

Quantitative expression level of CaSR, p-ERK1/2, ERK1/2, Syn, Bcl-2 and cleaved Caspase3 proteins in the hippocampus were measured by western blots. Hippocampal tissues of each group were homogenized in ice-cold Radio Immunoprecipitation Assay (RIPA) lysis buffer containing 1 mM Phenylmethanesulfonyl fluoride (PMSF) (Beyotime Biotechnology, China), followed by centrifugation at 12000 rpm and 4℃ for 15 minutes to obtain the supernatant. After supernatant was measured by BCA Protein Assay Reagent Kit (Beyotime Biotechnology, China) to concentrate the protein.

2.9. Western blot

Equal amounts of lysates were electrophoretic isolated solution on 10% of sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), and then transferred to the PVFD membrane. The blots were sealed in blocking buffer at room temperature for 25 minutes and incubated with primary antibodies raised against rabbit anti-CaSR (1:1000, Affinity), rabbit anti-ERK1/2 (1:1000, Affinity), rabbit anti-p-ERK1/2 (1:2000, Cell Signaling), rabbit anti-cleaved Caspase-3 (1:1000, Cell Signaling), rabbit anti-Bcl-2 (1:1000, Affinity), rabbit anti-PKC (1:1000, Affinity), rabbit anti-Syn (1:1000, Affinity) or rabbit anti-β-tubulin (1:2000, Cell Signaling) at 4℃. After 16-18h, the blots were cleaned with Tris-buffered saline and Tween 20 (TBST) for 3 times and incubated with secondary antibody for 1 h at room temperature. After adequate flushing of the blots, the Immuno-reactive blots were observed with the enhanced chemiluminescence (ECL) reagent. Different band intensities corresponding to immunoblot detection of protein samples were quantified using ImageJ software. All experiments were performed at least 3 times and all data presented in the graphs are the mean of 5 independent samples.

2.10. Cell culture, CIH model and chemical treatment

PC12 cells purchased from iCell Biosciece Inc were planted at a density of 2×10⁵ cells/ml on plates and was grown in culture medium containing serum, glucose and sodium pyruvate at 37°C in a humidified atmosphere (95% air and 5% CO₂). To establish the CIH model, the cells were treated with hypoxia (94% N₂ / 5% CO₂ / 1% O₂) for 1h / reoxygenation (74% N₂ / 5% CO₂ / 21% O₂) for 0.5h for 6 cycles [27]. The groups are as follows: (i) control group (C): the cells were maintained in a standard incubator; (ii) C + DMSO group: the cells in a standard incubator were treated with DMSO (1µM). (iii) C + NPS2143 group: the cells in C group were treated with NPS2143 (CaSR inhibitor, 5µM; GLPBIO, Cat.No: GC169431); (iv) C + PD98059 group: cells in the standard incubator were treated with PD98059 (MEK inhibitor, 15µM; GLPBIO, Cat.No:GC12819); (v) C + CHE group: PC12 cells in C group were treated with Chelerythrine chloride (CHE, PKC inhibitor, 10µM; Sigma, CAS: 3895-92-9) (vi) IH group: the cells were exposed to intermittent hypoxia; (vii) IH + NPS2143 group: the cells were pretreated with NPS2143 (5µM) before IH exposure; (viii) IH + PD98059 group: the cells were pretreated with PD98059 (15µM) before IH exposure. (IX) IH + CHE group: the cells were pretreated with CHE (10µM) before IH treatment. The reagents were pretreated half an hour before IH exposure.

2.11. Cell Counting Kit 8 (CCK-8)

Cell viabilities in every group were detected via a CCK-8 kit (DojinDo, Japan) after IH exposure for 9 h. In addition, cytotoxicity of CaSR inhibitors (NPS2143) and MEK inhibitors (PD98059) was determined by CCK8. The cells treated with 2.0×10⁵ cells/ml were planted on 96-well plates and cultured at 37°C in a humidified atmosphere (95% air and 5% CO₂) for 24–48 hours depended on cell growth rate. After treatment with reagents of different concentrations, the cells rinsed with PBS were added CCK8 reaction mixture (10 µl CCK-8 stock solution, 90 µl DMEM medium) to each well and incubated at 37℃ for 1h. Cell viability was determined by measuring absorbance at 450nm with a fluorometer.

2.12. Cellular immunofluorescence

The cells grown on the coverslips were exposed IH for 9 h and then immediately were immobilized with 4% paraformaldehyde (PFA) for 30 min, permeated with 0.02% Triton X-100 and blocked with 10% goat serum at room temperature for 30 min. Cells were incubated at 4℃ overnight with following primary antibodies: rabbit anti-CaSR (1:200, Affinity), rabbit anti-p-ERK1/2 (1:200, Cell Signaling), rabbit anti-PKC (1:200, Affinity) and rabbit anti-Syn (1:200, Affinity). Next day, the DAPI-treated cells incubated with the secondary antibody conjugated to Alexa fluor 488 donkey anti-rabbit IgG (1:200, Affinity) or Alexa fluor 594 donkey anti-rabbit IgG (1:200, Affinity) were visualized through fluorescence microscopy (400x magnification). To measure fluorescence intensity of indicated protein, three random images were selected from each slide, 5 slides every group, and were quantified with Image J software.

2.13. Western blot analysis

Quantitative expression of CaSR, p-ERK1/2, ERK1/2, PKC, Syn and cleaved Caspase-3 proteins were measured by western blot analysis after IH exposure for 9 h. Rinsed three times with PBS, cells were homogenized in RIPA lysis buffer (Beyotime, China) added with 1% PMSF (Beyotime, China) as well as 1% phosphatase inhibitor (Beyotime, China). After centrifugation, total proteins were extracted in superior solution and BCA Protein Assay Kit (Beyotime, China) were applied to determine the concentration of the above protein. The following steps were the same as the tissue protein western blot experiment above.

2.14. Statistical analysis

All data are normally distributed, expressed as means ± SD. The RME, WME and TE were compared via repeated measures two-way analysis of variance (ANOVA), whereas all other comparisons between groups with different treatments were analyzed by analysis of variance (ANOVA), followed by LSD or Dunnett’s T3 post-hoc analysis. All statistical analyses were conducted using SPSS software (IBM Corp., Armonk, NY, USA). The statistical significance level was set at 0.05 for all comparisons.

3.1. Inhibition of CaSR alleviated CIH-induced cognitive dysfunction of mice.

Eight-arm radial maze test is a widely recognized method to detect spatial memory and learning function. In this study, we used eight-arm maze to evaluate the effect of CIH on learning and memory function. Repeated measures ANOVA showed that WME, RME and TE were gradually reduced in each group with seven-day training (Fig. 1a-c). Following the learning and memory processes, these data quantify the formation of spatial memory throughout training. In the testing day, the number of WME, RME and TE increased significantly after intermittent hypoxia exposure(all p < 0.001), indicating that CIH induced the memory impairment, while inhibition of CaSR decreased significantly the above errors(WME, p = 0.007; RME, p = 0.017, TE, p = 0.008; CIH + NPS2143 vs CIH group, Fig. 1d). We found from the data in behavioral test that inhibition of CaSR significantly alleviated CIH-induced cognitive dysfunction.

3.2. Inhibition of CaSR alleviated neuronal injury and apoptosis in CA1 region of the hippocampus with CIH exposure

Neural apoptosis in hippocampus is one of the important mechanisms associated with cognitive dysfunction. In our study, Nissl staining was analyzed to assess neuronal viability in the hippocampus. A decreasing neuronal viability was showed in the hippocampal CA1 region in CIH group (p < 0.001; Fig. 2a-b). In addition, TUNEL staining was conducted to detect the apoptosis of neurons. Compared with control mice, CIH resulted in an increase of TUNEL-positive neurons in the hippocampal CA1 region with nuclear concentration (p < 0.001; Fig. 2c-d). On the contrary, after treatment with CaSR inhibitor, the neuronal injury and apoptosis rate in hippocampal CA1 region were significantly decreased, indicating that inhibition of CaSR alleviated CIH-induced neuronal apoptosis.

In order to further clarify the severity degree of CIH-induced neuronal apoptosis, we examined the expression level of cleaved Caspase3 and Bcl-2 via Western Blot assay. Cleaved Caspase3, the active form of Caspase3, is known as an indicator of apoptosis because it is increased during cell death [28]. The expression level of cleaved Caspase3 reflected the severity of apoptosis. Bcl-2 exists in mitochondria as heterodimer or homodimer, which can prevent cell apoptosis. We detected Bcl-2 and cleaved Caspase3 expression after 4 weeks of CIH exposure. As shown in Fig. 2e-g, compared with C group, the level of cleaved Caspase3 was up-regulated (p < 0.001) and Bcl-2 was decreased in CIH group (p < 0.001). In contrast, the levels of cleaved Caspase-3 were down-regulated (p = 0.018), while Bcl-2 was increased significantly in CIH + NPS2143 group compared to CIH group (p = 0.003). These data suggested that inhibition of CaSR could significantly decrease CIH-induced Caspase3-dependent cell apoptosis.

3.3. Inhibition of CaSR attenuated synaptic plasticity deficit in the hippocampus of mice following CIH.

Synaptophysin (Syn), as an important synaptic protein, hold a critical function on the regulation of synapse plasticity. To determine the the effect of CIH on synaptic plasticity, Syn expression levels in hippocampus were measured via immunohistochemistry (IHC) and Western Blot (WB) assay. The IHC results revealed that CIH exposure decreased Syn staining in hippocampal CA1 region(p < 0.001, Fig. 3A-B). Meanwhile, WB analysis showed that the levels of the Syn protein in CIH group was significantly down-regulated compared with that in the control group (p < 0.001, Fig. 3C-D). However, Syn staining was increased and Syn level was up-regulated significantly in mice treated with NPS2143 following CIH exposure (p = 0.049, p < 0.001; CIH + NPS2143 vs CIH group, Fig. 3b and 3d). These findings indicated that inhibition of CaSR might attenuated the synaptic plasticity deficit in the hippocampus after CIH exposure.

3.4. CaSR was involved in hippocampal neuronal apoptosis and synaptic plasticity deficit induced by CIH, accompanied by decreased p-ERK1/2.

Large amounts of evidence demonstrated CaSR was associated with apoptosis [29]. In our present study, CaSR inhibitor was used to explore the role of CaSR in CIH-induced hippocampal neuronal apoptosis. In Fig. 1d, inhibition of CaSR improved spatial memory function with reduced RME, WME and TE (p = 0.007, 0.017, 0.008; CIH + NPS2143 vs CIH group). Nissl staining was performed to determine the neuronal viability and showed that hippocampal neurons in the CIH + NPS2143 group maintained a better functional status than those in the CIH group (Fig. 2a-b). In addition, it was demonstrated that cell apoptosis was significantly alleviated in CIH + NPS2143 group with decreased TUNEL-positive neurons (p < 0.001), down-regulated cleaved Caspase3 (p = 0.018) and up-regulated Bcl-2 (p = 0.003), comparing with the CIH group (Fig. 2c-g). At the same time, we also found that treatment with NPS2143 significantly alleviated synaptic plasticity deficit in the mice following CIH exposure, manifested by up-regulated Syn protein level.

ERK1/2 was involved in cell proliferation and synaptic plasticity [30, 31]. In our study, CaSR and p-ERK1/2 expression in hippocampal CA1 region was determined by immunohistochemistry. As shown in Fig. 4a-c, compared with C group, the level of CaSR and p-ERK1/2 was elevated in hippocampal CA1 region (all p < 0.001), while in the CIH + NPS2143 group, CaSR and p-ERK1/2 were down-regulated (p = 0.002, p < 0.001, CIH + NPS2143 vs CIH group). To further investigate the changes of CaSR and ERK1/2 in hippocampus, CaSR and p-ERK1/2 protein expression via western blots was determined. These data in Fig. 4d-f revealed CIH increased CaSR and p-ERK1/2 expression in hippocampus (all p < 0.001). Meanwhile, we found NPS2143 pretreatment decreased the above protein levels (p = 0.004, 0.013), while ERK1/2 was no difference in different groups (p = 0.072). These data indicated CaSR was involved in hippocampal neuronal apoptosis and synaptic plasticity deficit induced by CIH, accompanied by increased p-ERK1/2, while inhibition of CaSR alleviated CIH-induced neuronal apoptosis and synaptic plasticity deficit, with decreased p-ERK1/2.

3.5. IH induced Caspase3-dependent apoptosis in PC12 cells.

To further elucidate the underlying mechanism of CIH-induced apoptosis in hippocampus, we established the IH (hypoxia for 1 h / reoxygenation for 0.5 h reoxygenation) model in PC12 cells according to our previous methods [27]. In the experiment, cell viability was determined by CCK8 assay and the level of cleaved-Caspase3 was detected via western blots. As shown in Fig. 5, IH induced cell injury in an IH cycle-dependent manner. Compared to the control group, cell viability was gradually decreased from 2 to 6 cycles (IH2-6; all p < 0.001; Fig. 3a) and the level of cleaved-Caspase3 was increased for up to 4 cycles (IH4; all p < 0.001; Fig. 3b). IH for 6 cycles provided maximal cell injury compared to less cycles of IH. Therefore, hypoxia / reoxygenation for 6 cycles (a total of 9 hours) was selected for subsequent experiments. In addition, the data suggested IH induced Caspase3-dependent apoptosis in PC12 cells.

3.6. CaSR mediated PC12 cell apoptosis induced by IH.

To investigate the role of CaSR in IH-induced cell apoptosis, NPS2143, an inhibitor of CaSR, was selected to pretreat PC12 cells. Firstly, according to previous studies [32], several different concentrations of inhibitors in our experiment were selected. Cell toxicity of the inhibitors was detected via CCK8 assay, and then the expression level of CaSR was determined by western blots. In Fig. 6a, CCK8 assay showed no apparent cytotoxicity on PC12 cells for up to 10µM of NPS2143 (NPS2143(10)), but cell viability in C + NPS2143(15) group was significantly decreased (p = 0.006), compared the other groups, which suggested that 15 µM of NPS2143 was injury to PC12 cells. In Fig. 6b-c, western blot assay showed that various concentrations of NPS2143 decreased the level of CaSR in PC12 cells induced by IH exposure, and pretreatment with 5µM NPS2143 (IH + NPS2143(5)) provided maximal inhibition compared to lower concentrations (2.5µM; IH + NPS2143(5) vs IH + NPS2143(2.5) group, p = 0.011). Moreover, there was no statistically significant difference in inhibitory effect compared with higher concentration of NPS2143 (10µM; IH + N5 vs IH + N10 group, p = 0.257). Therefore, pretreatment of NPS2143 at 5µM was selected for subsequent experiments in IH.

We investigated whether CaSR held a critical function on PC12 cells apoptosis with IH exposure. Immunofluorescence assay and western blot (Fig. 6d,e,g and h-k) demonstrated that CaSR and p-ERK1/2 expression was increased in PC12 cells after IH (all p < 0.001), followed increasing cleaved Caspase3 protein level (p < 0.001), which was consistent with the results in vitro. The above makers of PC12 cells pretreated with NPS2143 were significantly decreased (IH + NPS2143 vs IH group, all p < 0.001). Moreover, the changes of cleaved Caspase3 was consistent with the above trend. As shown in Fig. 6g and l, the level of PKC was up-regulated (p < 0.001) in CIH-exposed PC12 cells, while NPS2143 pretreatment down-regulated the level of PKC (p = 0.003). Collectively, these data suggested that CaSR played an important role in IH-induced cell apoptosis by increasing p-ERK1/2 and PKC, while inhibition of CaSR alleviated the IH-induced cell apoptosis via decreased p-ERK1/2 and PKC.

3.7. CaSR modulated the synaptic plasticity in PC12 cells with IH exposure.

Synapse-associated proteins Syn and GAP-43 play an important part in the regulation of synapse plasticity. Syn and GAP-43 expression levels in PC12 cells were measured via immunofluorescence (IF) and WB assay. The IF results revealed that IH exposure decreased Syn level in hippocampal CA1 region(p < 0.001, Fig. 7A-B). Meanwhile, WB analysis showed that the levels of the Syn and GAP-43 protein in IH group was significantly down-regulated compared with that in the control group (p = 0.007; p = 0.003, Fig. 7C-E). However, Syn and GAP-43 levels were up-regulated significantly in mice treated with NPS2143 following IH exposure (p = 0.033, p = 0.019; CIH + NPS2143 vs CIH group). These findings indicated that inhibition of CaSR might attenuate synaptic plasticity deficit in the hippocampus after IH exposure.

3.8. Phosphorylated ERK1/2 and PKC were involved in the IH-induced cell apoptosis and synaptic plasticity deficit.

To identify whether p-ERK1/2 held a critical function on IH-induced cell apoptosis and synaptic plasticity deficit, PD98059, a potent and selective MEK inhibitor, which can inhibit ERK1/2 signaling, was selected to pretreat PC12 cells. Firstly, according to previous research [24]. several different concentrations of inhibitors in our experiment were selected. The toxicity of inhibitors to cells was detected via CCK8 assay, and then the protein level of p-ERK1/2 was determined by western blots. In Fig. 8a, CCK8 assay showed no apparent cytotoxicity on PC12 cells with the concentrations from 15µM to 30µM of PD98059 under normal cell viability (all p > 0.05). In Fig. 8b, western blot assay showed that various concentrations of PD98059 decreased the level of p-ERK1/2 in PC12 cells induced by IH exposure, and pretreatment with 15µM PD98059 (IH + PD98059(15)) provided maximal inhibition compared to higher concentrations (IH + PD98059(15) vs IH + PD98059(20) group, p = 0.001; IH + PD98059(15) vs IH + PD98059(25) group, p < 0.001). Therefore, pretreatment of PD98059 at 15µM was selected for subsequent experiments in IH. As Fig. 8c-d shown, cleaved Caspase3 expression was decreased in PC12 cells with treatment of PD98059 after IH, compared with IH group (p < 0.001), but there was no statistical difference in the level of CaSR (p = 0.489). Chelerythrine chloride (CHE) is a potent inhibitor of PKC. In our previous study, CHE (10µM) was selected in IH-induced PC12 cell injury [33]. In order to identify PKC was involved in the IH-induced cell apoptosis, the cells in C + CHE and IH + CHE group were pretreated with CHE (10µ mol/L) for 0.5 h and respectively cultured in an intermittent air or intermittent hypoxia incubator. As Fig. 8c-e and 8h shown, cleaved Caspase3 expression was decreased in PC12 cells with treatment of CHE after IH, while the level of synaptic associated protein Syn was increased compared with IH group (p < 0.001; p = 0.001), but there was no statistical difference in the level of CaSR (p = 0.751). In addition, compared with IH group, p-ERK1/2 and PKC expression was down-regulated (p = 0.012, p < 0.001) in IH + CHE group, while the level of PKC was no change (p = 0.706) and p-ERK1/2 was down-regulated in IH + PD98059 group (p < 0.001, Fig. 8f-g). All the data indicated that p-ERK1/2 and PKC were involved in the IH-induced cell apoptosis and synaptic plasticity deficit.

Our present study was the first to investigate that CaSR held a critical function on CIH-induced cognitive dysfunction of mice. Herein, we provided the evidence that CaSR exerted neuronal apoptosis and synaptic plasticity deficit by elevating PKC and ERK1/2 phosphorylation levels in hippocampus. Therefore, these data not only revealed the underlying mechanisms of CIH-induced neuronal apoptosis and synaptic plasticity in hippocampus but also further demonstrated the important role of CaSR in CIH. This study maybe provide a new target for drug selection in the treatment of OSAHS.

Spatial memory, responsible for recording a course to a location and recalling the location of an object, is a manifestation of cognitive function [34]. A variety of studies have demonstrated that mice showed deficits in spatial memory after IH exposure [5, 35]. In our present study, we simulated memory impairment caused by OSAHS via a model of spatial memory dysfunction following CIH exposure in mice. The spatial memory was examined via an 8-Arm radial maze on growing mice subjected to CIH. Consistent with previous studies [36], our results suggested that CIH impaired the mice spatial memory function, evidenced by more working and reference memory errors in CIH mice (Fig. 1a). Hippocampus and superior frontal gyrus were identified as the main brain area of learning and memory [37]. Our recent study has demonstrated that the hippocampus of rats was significantly damaged by CIH exposure, especially the hippocampus CA1 region [38], and the present study also supports the result. These findings were in agreement with previous studies [14]. Our previous study found that CIH-induced cognitive dysfunction was associated with hippocampal apoptosis and we detected the neuronal viability in hippocampus CA1 region via Nissl staining and the apoptosis cells by TUNEL assay for further confirmation, and the present results were consistent with other studies [39, 40]. Furthermore, We found CIH exposure increased cleaved Caspase-3 protein expression and decreased Bcl-2 protein expression of the mice. Thus, we suggested that CIH induced memory dysfunction of mice by promoting neuronal apoptosis in hippocampal CA1 region.

Besides, except for significantly increased neuronal apoptosis in hippocampus, we also found that CIH result in hippocampal synaptic plasticity deficit, manifested by increased mean density of Syn and up-regulated protein levels. At present, many evidences have shown that synaptic plasticity in hippocampus is closely related to cognitive function. Consequently, we infered that CIH leaded to synaptic plasticity deficit in hippocampus, thereby impairing cognitive function.

Large amounts of evidence demonstrated that CaSR played an critical role in neuronal proliferation and differentiation during early neural development [22, 41]. In our present study, CaSR up-regulated in CIH-induced neuronal apoptosis and synaptic plasticity deficit in hippocampus, contributing to the cognitive dysfunction in mice. To gain further insights into the association between the activation of CaSR and neuronal apoptosis and synaptic plasticity during IH exposure, NPS-2143, a specific CaSR inhibitor, was used to inhibit the expression of CaSR in mice subjected to IH. We found that CaSR inhibition attenuated neuronal apoptosis via Nissl and TUNEL staining, following down-regulated cleaved Caspase-3 level and up-regulated Bcl-2. Meanwhile, CaSR inhibition also improved synaptic plasticity in the hippocampus, as evidenced by increased Syn expression levels. The present study demonstrated that CaSR inhibition contributed to the protective effect on CIH-induced neuronal injury. Moreover, CaSR was up-regulated in CIH-induced apoptosis and synaptic plasticity deficit of hippocampus, accompanying the increasing p-ERK1/2 and PKC, while inhibition of CaSR down-regulated p-ERK1/2 levels and PKC, we suggested that phosphorylation of ERK1/2 and PKC may act as downstream products of CaSR and be involved in neuronal injury and synaptic plasticity deficit induced by CIH, which was consistent with our results in vitro.

To explore the mechanism of CaSR in CIH-induced cognitive impairment of the mice, we selected highly differentiated PC12 cell lines expressing nerve growth factor (NGF) receptors that can induce neurophenotypes. Studies of NGF-stimulated differentiation in the PC12 cells were conducted as in the previous reports [42]. PC12 line, a useful model system for the study of numerous problems in neurobiology and neurochemistry, is widely used for neuronal development. So in others and our researches, NGF-stimulated differentiation of PC12 cells were used to replace hippocampal primary cultures [27, 33]. We successfully established the intermittent hypoxia PC12 model to identify that CaSR up-regulation contributed to the apoptosis in PC12 cells and further research the downstream signaling pathway of CaSR.

The ERK1/2 cascade, as the primary pathway of the MAPK family, was involved in cell proliferation and cell survival [43]. Previous research indicated that the phosphorylation of ERK1/2 plays an important role in cell apoptosis, but had a bidirectional regulation to apoptosis at different disease models [44, 45]. Recently, some studies revealed a significantly increased p-ERK1/2 expression in the hippocampus of rats undergoing cerebral ischemia, as underlying apoptotic machinery of hippocampus injury [46]. In intermittent hypoxia condition, we further investigated the role of ERK1/2 in PC12 cells. We found that CIH elevated ERK1/2 phosphorylation levels in vitro and vivo experiments, while p-ERK1/2 inhibition via a selective MEK inhibitor attenuated cell injury, following decreased cleaved Caspase-3 level and increased cell viability. Therefore we suggested that ERK1/2 phosphorylation held a critical function on cell apoptosis following CIH. Of note, we found that the expression of CaSR showed no significant difference via treatment with MEK inhibitor following IH exposure, while PC12 cells, exposed IH and treated with CaSR inhibitor showed significantly decreased phosphorylation of ERK1/2. These results suggested that p-ERK1/2 played a key role in IH-induced apoptosis and maybe a downstream product of CaSR. ERK1/2 signaling pathways play a significant part in synaptic plasticity [47]. We also found that CIH-induced synaptic plasticity deficit, manifested by a decrease in Syn, following up-regulated p-ERK1/2, while p-ERK1/2 was down-regulated and Syn expression was increased after MEK inhibitor intervention. The data revealed that p-ERK1/2 held on a critical function on synaptic plasticity deficit induced by CIH.

Various researches demonstrated PKC was an important regulator in the process of ERK1/2 phosphorylation, and ERK1/2 activation has been reported to be mediated by PKC in hippocampal neural stem cells [48]. Recently, several studies have shown that ERK phosphorylation regulates different disease processes under the activation of PKC [49]. we examined whether the CaSR/PKC/ERK1/2 pathway is involved in apoptosis and synaptic plasticity deficit of PC12 cells via the three corresponding inhibitors pretreated with PC12 cells after CIH. we further found p-ERK1/2 and PKC expression were down-regulated with PKC inhibitor precondition, while the level of PKC was no change and p-ERK1/2 was down-regulated treated with MEK inhibitor, along with CaSR as upstream, regulated p-ERK1/2 and PKC expression. Therefore, our results demonstrated that p-ERK1/2 was regulated via PKC while both are regulated by CaSR in cell apoptosis and synaptic plasticity deficit following CIH, which was consistent with the previous researches [18]. Strictly speaking, we did not demonstrate whether there was a direct or indirect relationship among CaSR, PKC and ERK1/2. In structure, CaSR contains PKC consensus sequences and the phosphorylation site of PKC protein. Therefore, there can be a direct interaction between CaSR and PKC Co-immunoprecipitation studies showed that ERK interacted indirectly with PKC via SGLT1 or Raf-1 [50, 51]. No evidence of direct interaction between ERK1/2 and PKC has been reported so far.

In summary, the present study demonstrated CaSR hold a critical function on CIH-induced cognitive dysfunction. The activation of CaSR induced up-regulated PKC, phosphorylation of ERK1/2, and finally resulted neuronal apoptosis and the synaptic plasticity deficit in hippocampus. In addition, we speculated that the increased p-ERK1/2 and PKC expression may be the underlying downstream of CaSR in CIH-induced cognitive dysfunction of mice (Fig. 9). Our study provides an alternative signaling pathway in terms of OSAHS-induced cognitive impairments and may contribute to a theoretical basis for the treatment of OSHAS.

Acknowledgment

This work was supported by Natural Science Foundation of China (81870073), Medical Science and Technology Project of Zhejiang Province (2021KY209), Science and Technology Plan Project of Wenzhou, China (Y20210009). We sincerely thank the staff of the Department of Science and Research Center at the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University for their constant and unselfish help.

Authors’ contributions Xiucui Li, Wei Wang and Xiaohong Cai participated in the research design. Huiya Ying, Zilong Zhang, Zijing Yang, Yuanai Li and Cancan You conducted the experiments and synthesized the data. Zilong Zhang, Huiya Ying, Xiucui Li and Wei Wang contributed to the writing of the manuscript.

Statements and Declarations

Competing Interests The authors declare no competing financial interest.

Funding This work was supported by Natural Science Foundation of China (81870073), Medical Science and Technology Project of Zhejiang Province (2021KY209), Science and Technology Plan Project of Wenzhou, China (Y20210009).

Data Availability The datasets generated during the current study are available from the corresponding author on reasonable request.

Ethics Approval All animals used in the experiment were cared for in accordance with the ethical guidelines on animal experimentation of Laboratory Animals of China National Institutes of Health.

Consent to Participate This study did not include the human subjects.

Consent for Publication This study did not include the human subjects

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Inhibition of Calcium-sensing Receptor Alleviates Chronic Intermittent Hypoxia-induced Cognitive Dysfunction via CaSR-PKC-ERK1/2 Pathway

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Experimental animals and grouping

2.2. Animal models

2.3. 8-Arm radial maze test

2.4. Animal sample collection

2.5. Nissl staining

2.6. Terminal transferase-mediated dUTP nick end labeling (TUNEL) staining

2.7. Immunohistochemistry

2.8. Tissue protein extraction

2.9. Western blot

2.10. Cell culture, CIH model and chemical treatment

3. Results

3.1. Inhibition of CaSR alleviated CIH-induced cognitive dysfunction of mice.

3.3. Inhibition of CaSR attenuated synaptic plasticity deficit in the hippocampus of mice following CIH.

3.5. IH induced Caspase3-dependent apoptosis in PC12 cells.

3.6. CaSR mediated PC12 cell apoptosis induced by IH.

3.7. CaSR modulated the synaptic plasticity in PC12 cells with IH exposure.

3.8. Phosphorylated ERK1/2 and PKC were involved in the IH-induced cell apoptosis and synaptic plasticity deficit.

4. Discussion

5. Conclusion

Declarations

References

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