Glucagon-Like Peptide-2 Receptor is Involved in Spatial Cognitive Dysfunction in Rats After Chronic Cerebral Hypoperfusion

Abstract

Chronic cerebral hypoperfusion (CCH) affects the aging population and especially patients with neurodegenerative diseases, such as Alzheimer’s disease or Parkinson’s disease. CCH is closely related to the cognitive dysfunction in these diseases. Glucagon-like peptide-2 receptor (GLP2R) mRNA and protein are highly expressed in the gut and in hippocampal neurons. This receptor is involved in the regulation of food intake and the control of energy balance and glucose homeostasis. The present study employed behavioral techniques, electrophysiology, western blotting, immunohistochemistry, quantitative real time polymerase chain reaction (qRT-PCR), and Golgi staining to investigate whether the expression of GLP2R changes after CCH and whether GLP2R is involved in cognitive impairment caused by CCH. Our findings show that CCH significantly decreased hippocampal GLP2R mRNA and protein levels. GLP2R upregulation could prevent CCH-induced cognitive impairment. It also improved the CCH-induced impairment of long-term potentiation and long-term depression. Additionally, GLP2R modulated after CCH the AKT-mTOR-p70S6K pathway in the hippocampus. Moreover, an upregulation of the GLP2R increased the neurogenesis in the dentate gyrus, neuronal activity, and density of dendritic spines and mushroom spines in hippocampal neurons. Our findings reveal the involvement of GLP2R via a modulation of the AKT-mTOR-p70S6K pathway in the mechanisms underlying CCH-induced impairments of spatial learning and memory. We suggest that the GLP2R and the AKT-mTOR-p70S6K pathway in the hippocampus are promising targets to treat cognition deficits in CCH.

Keywords

Chronic cerebral hypoperfusion cognitive dysfunction glucagon-like peptide-2 receptor neural plasticity

INTRODUCTION

Chronic cerebral hypoperfusion (CCH) is a pathological condition with mild cerebral ischemia. In aging subjects, CCH can be a comorbidity of various neurodegenerative diseases such as vascular dementia, Alzheimer’s disease, and Parkinson’s disease [1, 2]. CCH is associated with a pronounced severity of cognitive decline in these diseases [1, 3] and can independently exacerbate cognitive impairment [4]. The degree of cerebral blood flow impairment has been used as a marker to predict the progression of mild cognitive impairment in Alzheimer’s disease [5]. Moreover, stroke-induced CCH can cause in many patients the development of post-stroke dementia namely vascular dementia, which is one of the most common and severe consequences after stroke [6]. Thus, it is very important to uncover the mechanisms of cognitive deficits after CCH.

Continuously reduced blood supply in brains affected by CCH contributes to a decrease in brain metabolism and causes a series of endogenous neuronal changes [7]. CCH has been shown to potentiate amyloid pathology and to induce cerebrovascular demyelination, white matter lesions, microglia activation, and blood-brain barrier disruption [8, 9]. The levels of synaptic proteins, such as N-methyl-D-aspartate (NMDA) receptor subunit 2B and postsynaptic density protein 95 (PSD-95), are decreased as a consequence of CCH leading to impaired neurotransmission [10, 11]. In CCH, autophagy signaling is activated, provoking neuroinflammation and oxidative stress [6 , 12], and amyloid-β protein accumulation and tau hyperphosphorylation are induced [13 –15]. All of these above changes impair cognition. Following CCH, many signaling pathways are either activated or inhibited.

Glucagon-like peptide-2 receptor (GLP2R) mRNA and protein are highly expressed in the gut and in hippocampal neurons [16]. GLP2R has been shown to be involved in the regulation of food intake [17, 18] and controls energy balance and glucose homeostasis [19]. GLP2R can activate intracellular phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathways in neuronal circuits [19] and protects hippocampal neurons from glutamate excitotoxicity [20]. GLP2R has antidepressant-like effects [21] and improves the memory functions in lipopolysaccharide-treated mice [22]. GLP2R stimulates protein synthesis through the PI3K-dependent AKT-mTOR signaling pathway [23]. Reduced activity of the mTOR signaling pathway is closely related to CCH-induced damage in hippocampal neurons [24]. Increased mTOR signaling by autophagy can cause cognitive impairment by CCH-induced [25]. mTOR is at the crossroad of plasticity, memory, and disease [26] and can increase synaptic signaling proteins level [27]. Thus, it is suggested that GLP2R plays a role in the cognitive impairment induced by CCH. However, it remains unclear, whether the expression of GLP2R changes after CCH and whether GLP2R is involved in the cognitive impairment caused by CCH. In the current study, we aim to investigate the GLP2R expression in the brain after CCH, how GLP2R is involved in the development of learning and memory impairment under these conditions, and the underlying mechanisms for these CCH-induced changes.

MATERIALS AND METHODS

Antibodies and chemicals

The mouse monoclonal antibody (mAb) against total β-actin was purchased from Abcam (Cambridge, UK). Biotin-labeled goat anti-rabbit IgG, horseradish peroxidase-labeled streptavidin, and the diaminobenzidine chromogenic kit were all purchased from Zhongshan Goldenbridge Biotechnology Co., Ltd. Rabbit polyclonal antibody (pAb) against GLP2R was manufactured by ImmunoStar (Hudson, WI, USA). Rabbit pAb against activity-regulated cytoskeleton-associated protein (Arc) was from Synaptic Systems (Göttingen, Germany). Rabbit mAb against NeuroD1 was from Abcam (Cambridge, CB, UK). Rabbit pAb for doublecortin (DCX) was from Cell Signaling Technology, Inc. (Beverly, MA, USA). Mouse mAb against phosphorylated AKT at Ser473 (pS473-AKT), phosphorylated mTOR (p-mTOR), phosphorylated p70S6K (p-p70S6K), and rabbit pAbs against total AKT, total mTOR, and total p70S6K were from Cell Signaling Technology, Inc. (Beverly, MA, USA). Goat anti-rabbit or anti-mouse IgG conjugated to IRDye™ (800CW) was from LI-COR Biosciences (Lincoln, NE, USA). The BCA protein assay kit was from Pierce Chemical Company (Rockford, IL, USA). TRIzol and synthesized primers were purchased from Invitrogen, Inc. (China). The first strand complementary DNA (cDNA) synthesis kit and the qRT-PCR kit were from Thermo Fisher Scientific (Waltham, MA, USA).

Rat model of chronic cerebral hypoperfusion

Adult Sprague-Dawley rats (72 male, 220–240 g) were purchased from Hunan SJA Laboratory Animal Co., Ltd. and were housed with food and water ad libitum. Rats were kept on a 12-h light/dark cycle with the light on from 7:00 am to 7:00 pm. All animal experiments were approved by the Ethics Committee of the Renmin Hospital of Wuhan University and were conducted in accordance with its guidelines.

The animal randomization and grouping steps were as follows: weighing the animals, numbering the animals in order of weight, and assigning them according to the random number table method to 4 groups, namely the sham group (n = 14), bilateral common carotid artery occlusion (2VO) group (n = 22) injected with control lentiviral vectors, 2VO group injected with Glp2r lentiviral vectors (n = 22), and sham group injected with control lentiviral vectors (n = 14).

To generate 2VO animals, rats were anesthetized with chloral hydrate (0.4 g/kg) intraperitoneally. During surgery, the temperature was maintained at 37°C with a heating pad. After a ventral midline incision, both common carotid arteries were gently separated from the carotid sheath and the vagus nerve [12]. The common carotid arteries were bilaterally occluded just below the carotid bifurcation with double ligation using 4-0 silk suture. In control rats, a similar surgical procedure was performed without blood vessel ligation. After the surgery, the rats were maintained at 37°C until recovery. During the surgery, the brain of the rats did not soften wholly or partly. A successful establishment of the 2VO model was considered when the blood flow was reduced to 70% of the normal value. This was verified using a laser Doppler system to detect the blood flow level in 2VO animals.

Intrahippocampal injection of lentiviral vectors and drug treatment

On the 3rd day after the generation of the CCH model, rats were anesthetized with urethane (1.6 g/kg, intraperitoneally) and subjected to stereotaxic surgery (Fig. 1). Stainless steel needles were used to inject plasmids bilaterally into hippocampal the CA3 area of the rat brain at the following coordinates: 3.4 mm posterior to the bregma, ±3.5 mm lateral to the midline, and 3.5 mm ventral to the skull surface. pGLV-Glp2r lentiviral plasmid or control pGLV lentiviral plasmid (2μl) was injected directly into the CA3 area bilaterally in the rat brain using a microinjector. The injection volume of pGLV lentiviral plasmid or control pGLV lentiviral plasmid was the same as that of the corresponding control. The injection needle was left in place for 5 min to limit the diffusion of the injected agent.

Fig.1

Schematic diagram of the timeline for the experimental procedure. On the 3rd day after the 2VO operation, the lentiviral injection was carried out. From the 30th to the 38th day, the animals were trained in the Morris water maze test. Afterwards, the Y-maze test was carried out.

Morris water maze and Y-maze

After 30-days cerebral hypoperfusion, all rats received a spatial memory training in the Morris water maze. For spatial memory acquisition, rats were trained to find a hidden platform in the water maze on 7 consecutive days, 4 trials per day separated by a 30-s interval from 2:00 to 8:00 pm. For each trial, the rat started with its location being in the middle of the outer round edge in one of the four quadrants facing the wall of the pool and ended when the animal climbed on the platform. Rats that were not able to find the platform within 60 s were guided to the platform. A Morris water maze video tracking analysis system (Shanghai, China) was used to record the activity trajectory of the rats. Their swimming pathways and latencies to find the hidden platform were recorded [28]. The time to arrive at the platform in first trial during 7 days as latency time and ratio between staying time in platform quadrant and total time were recorded to evaluate the learning ability. For the short-term memory retention test, the platform was or was not removed, and rats were released into the 3rd quadrant of the maze to record their swimming pathways to reach the platform area as well as their latencies, the number of times crossing the platform area, and the total time in the platform quadrant.

The Y-maze is used to evaluate the spatial working memory facilitating the tendency of rodents to explore new environments [29]. In our study, the Y-maze was constructed of gray plastic arms (50 cm length, 25 cm height, 10 cm width) with three arms at a 120° angle from each other. Before the test, the rats were allowed to adapt to the experimental environment for 15 min. Then, the animals were placed in the center of the Y-maze, which they freely explored for 8 min. An arm entry was counted when their hind paws were completely within an arm. A successive entry into the three arms was counted as one alternation, and the maximum possible alternations was the total number of arm entries minus two. To evaluate the spatial working memory, alternation percent was calculated as (alteration/maximum possible alternations)*100%). The total number of arm entries was also used to evaluate the locomotive activity of the rats [30].

Electrophysiology

After the spatial memory retention test, rats were anesthetized with urethane (1.6 g/kg, intraperitoneally). Electrodes were implanted using the following coordinates: 3.4 mm posterior to the bregma and 3.5 mm lateral to the midline for the recording electrode, and 7.0 mm posterior to the bregma and 4.1 mm lateral to the midline for the stimulating electrode. The ground electrode was connected to the neck muscle contralateral to the electrode sites. Field excitatory postsynaptic potentials (fEPSPs) were recorded from pyramidal neurons of the CA3 region in response to stimulation of the perforant path. The data acquisition system was triggered simultaneously to record all events. fEPSP recordings were sampled at 3 kHz. The high-frequency stimulation protocol for inducing long-term potentiation (LTP) consisted of 10 trains of 15 stimuli (200 Hz, 0.5 mA) with 5-s intervals. This rather weak LTP induction protocol was chosen to prevent saturation of LTP and to allow the possibility to detect improvements or impairments. The low-frequency stimulation protocol for inducing long-term depression (LTD) consisted of 900 pulses of 1 Hz for 15 min [31, 32]. LTP or LTD was measured as % of the baseline fEPSP slope recorded over a 15 min period prior to the application of high- or low-frequency stimulation. This value was taken as 100% of the fEPSP slope, and all recorded values were normalized to this baseline value. Data were analyzed with Igor Pro 6.1 (WaveMetrics, Lake Oswego, Oregon) software.

Immunohistochemistry

For immunohistochemical studies, rats were sacrificed with an overdose of chloral hydrate (1 g/kg) and perfused through the aorta with 100 mL 0.9% NaCl followed by 400 mL phosphate buffered saline (PBS) containing 4% paraformaldehyde. The brains were removed and post-fixed in perfusate overnight and then cut into sections (5μm thickness). The sections were collected consecutively in PBS for immunohistochemistry staining. The sections were blocked with 0.3% H₂O₂ in absolute methanol for 30 min, and nonspecific sites were blocked with bovine serum albumin for 30 min at room temperature. Sections were then incubated overnight at 4°C with the primary antibodies, pAb NeuroD (1:200) or pAb DCX (1:200). After washing with PBS, the sections were subsequently incubated with biotin-labeled secondary antibodies for 1 h at 37°C. The immunoreaction was detected using horseradish peroxidase-labeled antibodies for 1 h at 37°C and visualized using the diaminobenzidine tetrachloride system (brown color). For each primary antibody, 3–5 consecutive sections from each brain were used. The images were observed using a microscope (Olympus BX60, Tokyo, Japan). Image Pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA) software was used to select the regions of interest (ROI), count the number of positively stained cells, measure the ROI area, and calculate the density of positively stained cells.

Total RNA extraction and quantitative RT-PCR analysis

Hippocampal tissue was removed, frozen in liquid nitrogen, and stored at –80°C. Total RNA was extracted from the 50 mg hippocampal tissue using TRIzol reagent according to the manufacturer’s protocol (Invitrogen, Singapore). First strand cDNA was synthesized from total RNA using a first strand cDNA synthesis kit. The SYBR GREEN Mix reaction system was used for the RT-PCR along with the forward primer, the reverse primer, and the cDNA. The reaction process included a pre-incubation step at 95°C for 3 min, an amplification step of 45 cycles at 94°C for 35 s, at 56.8°C for 30 s and at 72°C for 60 s, and an further elongation step at 72°C for 10 min. A melting curve was recorded to verify the absence of primer dimers. Glyceraldehyde 3-phosphate dehydrogenase was the endogenous control. GLP2R and Arc mRNA levels were assayed using the “ΔΔ Ct method” for relative expression.

Golgi staining

For Golgi staining, rats were anesthetized and perfused through the aorta with 100 mL 0.9% NaCl containing 0.5% NaN₂O, followed by 250 mL 4% paraformaldehyde solution and 250 ml 4% paraformaldehyde solution containing 5% potassium bichromate as well as 5% chloral hydrate. Brains were removed, cut to a volume of 5 mm³, post-fixed in Golgi stain for 3 days and then cut into 45-μm thick sections. The sections were collected for 3-days silver staining. Two to three dendrites from each of 10 cells per animal were analyzed. Spines were counted while manually changing the focus in order to identify all spines on a particular stretch of a dendrite. Spine density was defined as the density of all spines counted per animal divided by the total length of the dendrite and expressed as spine number per 100μm. Additionally, the numbers of mushroom spines were counted and divided by the total length of the dendrite. The mushroom spine density was also expressed as spine number per 100μm.

Western blots

For western blotting, rats were decapitated, and the hippocampi were rapidly removed and homogenized. The extract was mixed with sample buffer, boiled for 10 min and then centrifuged at 12,000× g for 10 min at 25°C. The protein concentration was estimated by a BCA Kit, and proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat milk and probed overnight with primary antibody at 4°C, then incubated with anti-rabbit or anti-mouse IgG conjugated to IRDye™ for 1 h at 4°C and visualized using the Odyssey Infrared Imaging System.

Statistical analysis

Data were expressed as mean±SEM and analyzed using the statistical software SPSS 12.0 (SPSS Inc., Chicago, Illinois, USA). The repeated-measures analysis of variance procedure was used to determine the statistical significance of differences in the means of learning latency among the three groups. The one-way analysis of variance procedure followed by Dunnett’s t-test was used to determine the statistical significance of differences in the means for all other experiments. p < 0.05 was considered a statistically significant difference.

RESULTS

CCH downregulates GLP2R levels in the hippocampus

To investigate the effects of CCH on the GLP2R expression in rat brains, we detected by western blots GLP2R levels in the hippocampal tissue from 2VO rats, which is an animal model of CCH. Our data show that CCH significantly decreased hippocampal GLP2R expression levels in comparison with the control group (p < 0.01; Fig. 2A, B). Its detection at the mRNA level demonstrates that CCH also significantly decreased GLP2R mRNA expression in hippocampal tissues of 2VO animals compared to those of the control group (p < 0.01; Fig. 2C). Immunohistochemical stainings show that CCH significantly decreased the numbers of GLP2R-positive neurons in the cortex as well as in hippocampal sub-regions such as CA1, CA3, and dentate gyrus (p < 0.01; Fig. 2D, E). Our results reveal that CCH significantly decreases hippocampal GLP2R levels, suggesting that this may be related to cognitive impairment induced by CCH.

Fig.2

CCH downregulates GLP2R expression in the hippocampus of rats. The brain tissues of animals exposed to CCH were analyzed. A) Western blot of GLP2R expression in the hippocampus. B) The relative intensities of the GLP2R bands as shown in (A) (n = 4). C) mRNA analysis of GLP2R expression in the hippocampus by qRT-PCR (n = 3). D) Immunohistochemical staining of GLP2R. E) Number of GLP2R-positive cells in brain slices (n = 3). Bar = 50μm. Con, control group; 2VO, group with bilateral common carotid artery ligation; DG, dentate gyrus. Data are expressed as mean±SEM. ^**p < 0.01 compared with Con.

Upregulation of GLP2R can rescue the cognition impairment after CCH

In order to investigate whether GLP2R downregulation was involved in the cognitive dysfunction caused by CCH, we stereotactically injected into the hippocampus a lentiviral vector containing the Glp2r gene to partly restore the expression of GLP2R (Fig. 3A, B) and tested the spatial learning and memory abilities of these rats after CCH in the Morris water maze.

Fig.3

GLP2R upregulation can improve CCH-induced dysfunction of spatial cognition in the Morris water maze test. A) Western blot demonstrating GLP2R upregulation in the hippocampal tissue. B) The relative intensities of the GLP2R bands as shown in (A) (n = 4). C) The training track of rats in a Morris water maze was recorded and analyzed. The latency time to reach the platform and the relative time of staying in the platform quadrant during the last training are shown in (D) and (E), respectively (n = 12). After a 1-day rest, the platform was removed and the crossing times near the platform area (F), the time in the platform quadrant (G), and the latency to reach the platform area (H) were analyzed (n = 12). Con, sham-operated group; 2VO, group with bilateral common carotid artery ligation; 2VO+GLP2R, 2VO group with GLP2R; GLP2R, sham-operated group with upregulated GLP2R. Data are expressed as mean±SEM. ^**p < 0.01 compared with Con; ^# #p < 0.01 compared with 2VO.

Our data show that the rats in the 2VO group took a longer time than the control animal to reach the platform during the 3rd–7th trial day (3rd day, p < 0.05; 4th–7th day, p < 0.01; Fig. 3D). However, the 2VO rats with GLP2R upregulation spent a shorter time than the 2VO rats during the 4th–7th trial day (4th and 6th–7th day, p < 0.01; 5th day, p < 0.05; Fig. 3C, D). During the 3rd–7th day, the 2VO rats had a significantly lower ratio of time staying in the platform quadrant over total exercising time in a quadrant than the control animals (3rd day p < 0.05, 4th–7th day p < 0.01; Fig. 3E), whereas the 2VO rats with GLP2R upregulation had a higher ratio of time staying in the platform quadrant than the 2VO rats (3rd day p < 0.05, 4th–7th day p < 0.01; Fig. 3E). After the 7-day training, the short-term memory test showed that 2VO rats spent significantly less time crossing the target area compared with control rats (p < 0.01; Fig. 3F), while the 2VO rats with GLP2R upregulation crossed the objective area more times compared with 2VO rats (p < 0.01; Fig. 3F). The 2VO rats spent significantly less time in the platform quadrant compared with control rats (p < 0.01; Fig. 3G), while the 2VO rats with GLP2R upregulation stayed noticeably longer in the platform quadrant compared with 2VO rats (p < 0.01; Fig. 3G). In addition, the 2VO rats had significantly longer latency times to reach the platform compared with control rats (p < 0.01; Fig. 3H), while the 2VO rats with GLP2R upregulation exhibited decreased latency times to reach the platform compared with 2VO rats (p < 0.01; Fig. 3H).

In order to further investigate the effects of GLP2R upregulation on spatial learning and memory dysfunction caused by CCH, we used a Y-maze to test the spatial recognition and memory of rats. Our findings indicate that the alternation percent in 2VO rats was significantly lower than that in control rats (p < 0.01; Fig. 4A), while the alternation percent in the 2VO rats with GLP2R upregulation was significantly higher than that in 2VO rats (p < 0.01; Fig. 4A). There was no difference in the total number of arm entries between control, 2VO animals, and 2VO rats with GLP2R upregulation (p > 0.05; Fig. 4B).

Fig.4

GLP2R upregulation can improve CCH-induced dysfunction of spatial cognition in the Y-maze test. All rats were further tested for spatial learning and memory ability in a Y-maze test. The walking tracks were recorded and alteration percent (A) and total times of arm entries (B) were analyzed (n = 12). Con, sham-operated group; 2VO, group with bilateral common carotid artery ligation; 2VO+GLP2R, 2VO group with GLP2R upregulation; GLP2R, sham-operated group with GLP2R upregulation. Data are expressed as mean±SEM. ^**p < 0.01 compared with Con; ^# #p < 0.01 compared with 2VO.

These findings suggest that CCH may cause cognitive impairment and that upregulation of GLP2R expression can rescue the cognitive impairment induced by CCH.

Upregulation of GLP2R can improve the LTP dysfunction after CCH

LTP and LTD are facilitated by hippocampus-dependent learning and memory processes [33, 34] and required for the formation and retrieval of spatial hippocampus-dependent learning and memory consolidation [35]. LTP and LTD are important cellular mechanisms for synaptic plasticity and cognition [36, 37]. A dysfunction of LTP or LTD can impair spatial learning and memory [38 –40]. In order to investigate the mechanisms of GLP2R in cognitive impairment induced by CCH, we studied whether GLP2R regulates LTP and LTD after CCH. Generally, the parallel excitatory pathways are referred to as the trisynaptic pathway (which carries information as follows: entorhinal cortex ⟶ dentate gyrus ⟶ CA3 ⟶ CA1 ⟶ entorhinal cortex) and the monosynaptic pathway (entorhinal cortex ⟶ CA1 ⟶ entorhinal cortex) [41]. The trisynaptic pathway containing CA3 is required for spatial learning and memory recall [41, 42]. For this pathway, inducible and reversible inhibition of synaptic transmission can impair learning and memory [42]. In the present study, to uncover the underlying electrophysiological mechanisms of cognitive dysfunction after CCH, we recorded and evaluated effect of GLP2R downregulation, on the circuit entorhinal cortex ⟶ CA3. To achieve this, fEPSPs were recorded and measured. After high-frequency stimulation, the fEPSPs were 206.18±12.36%, 143.41±10.42%, and 173.76±10.96% for the control, 2VO, and 2VO+GLP2R groups, respectively (Fig. 5A, C). The fEPSPs were significantly lower in the 2VO group than those in the control group (p < 0.01), however, the fEPSPs were higher in the 2VO+GLP2R group than those in the 2VO group (p < 0.01). After low-frequency stimulation, the fEPSPs were 68.87±4.05%, 94.56±5.34%, and 80.00±5.73% for the control, 2VO, and 2VO+GLP2R groups, respectively (Fig. 5B, D). The fEPSPs were significantly higher in the 2VO group than those in control (p < 0.01), but the fEPSPs were lower in the 2VO+GLP2R group than that in the 2VO group (p < 0.05).

Fig.5

GLP2R upregulation can improve LTP and LTD impairments after CCH. Following the behavioral tests, the rats were investigated for LTP and LTD using electrophysiological recordings. A, B) Field potential ratio before and after HFS (A) and LFS (B) (n = 12). C, D) The relative field excitatory postsynaptic potentials were analyzed (n = 12). HFS, high frequency stimulation; LFS, low frequency stimulation; Con, sham-operated group; 2VO, group with bilateral common carotid artery ligation; 2VO+GLP2R, 2VO group with GLP2R upregulation; GLP2R, sham-operated group with GLP2R upregulation. Data are expressed as mean±SEM. ^**p < 0.01 compared with Con; ^#p < 0.05, ^# #p < 0.01 compared with 2VO.

Our findings indicate that the upregulation of GLP2R could improve the impairment of LTP and LTD induced by CCH, which may be involved in the cognitive impairment induced by CCH.

GLP2R regulates after CCH the AKT-mTOR-p70S6K pathway in the hippocampus

GLP2R can regulate the PI3K-AKT-mTOR-p70S6K pathway in the central nervous system [19, 43], and PI3K-AKT-mTOR can modulate the synapse-related protein synthesis, which is involved in synaptic plasticity, LTP, LTD, neurogenesis, and memory formation. In order to further elucidate the molecular mechanisms of GLP2R in cognitive impairment induced by CCH, we investigated whether CCH causes changes in the AKT-mTOR-p70S6K pathway. We performed a western blot analysis, to detect the hippocampal levels of AKT and pS473-AKT. This showed that the relative levels of pS473-AKT (/total AKT) in the hippocampus of 2VO animals were decreased to 53.9±6.56% of those in control (p < 0.01), whereas the relative levels of pS473-AKT in the hippocampus in the 2VO rats with GLP2R upregulation were 91.7±7.9% of those in 2VO rats (p < 0.01; Fig. 6A, B). The relative levels of p-mTOR (/total mTOR) in the hippocampus declined after 2VO to 57.9±6.1% of the control (p < 0.01), whereas the relative levels of p-mTOR in the hippocampus in the 2VO rats with GLP2R upregulation were 92.1±5.1% of those in 2VO rats (p < 0.01; Fig. 6A, C). The relative levels of p-p70S6K (/total p70S6K) in the hippocampus after 2VO were decreased to 59.5±6.5% of the control values (p < 0.01), whereas the relative levels of p-p70S6K in the hippocampus in the 2VO rats with GLP2R upregulation were 93.8±4.2% of those in 2VO rats (p < 0.01; Fig. 6A-D).

Fig.6

GLP2R upregulation regulates after CCH the AKT-mTOR-p70S6K pathway in the hippocampus. The hippocampi were removed and homogenized for western blot analysis of pS473-AKT, AKT, p-mTOR, mTOR, p-p70S6K, p70S6K, and β-actin expression (A). The relative intensities of (B) pS473-AKT (/total AKT), (C) p-mTOR (/total mTOR), and (D) p-p70S6K (/total p70S6K) are shown (n = 4). Con, sham-operated group; 2VO, group with bilateral common carotid artery ligation; 2VO+GLP2R, 2VO group with upregulated GLP2R; GLP2R, sham-operated group with upregulated GLP2R. Data are expressed as mean±SEM. ^**p < 0.01 compared with Con; ^# #p < 0.01 compared with 2VO.

Upregulation of GLP2R can increase neurogenesis in the dentate gyrus and neuronal activity in the hippocampus after CCH

Neurogenesis in the dentate gyrus of the adult mammalian hippocampus is important for learning and memory [44 –46]. A reduction in newborn neurons is closely associated with cognitive dysfunction [47, 48]. To further explore the underlying mechanisms of CCH-induced cognitive impairment, we investigated the effect of GLP2R on the neurogenesis in the dentate gyrus of the hippocampus after CCH treatment. The cytoskeletal proteins doublecortin (DCX) and NeuroD are two important markers for neurogenesis [49]. Therefore, we stained brain slices with DCX and NeuroD antibodies and counted the number of newborn neurons. Our findings show that the numbers of both DCX- and NeuroD-positive neurons in the dentate gyrus of 2VO rats were significantly decreased compared to those of control rats (p < 0.01; Fig. 7A, B). Nevertheless, the numbers of both DCX- and NeuroD-positive neurons in the dentate gyrus of 2VO rats with GLP2R were significantly higher compared to those of 2VO rats (p < 0.01; Fig. 7C, D).

Fig.7

GLP2R upregulation can improve neurogenesis in the dentate gyrus after CCH. The brains were perfused and fixed by polyoxymethylene, then cut into slices (5μm; n = 3). Slices were stained with NeuroD (A) and DCX (C) antibody, and the cells positive for NeuroD (B) and DCX (D) were quantified. Bar = 50μm. Con, sham-operated group; 2VO, group with bilateral common carotid artery ligation; 2VO+GLP2R, 2VO group with GLP2R upregulation; GLP2R, sham-operated group with GLP2R upregulation. Data are expressed as mean±SEM. ^**p < 0.01 compared with Con; ^# #p < 0.01 compared with 2VO.

Neuronal activity regulates synaptic plasticity through transcription and translation [50]. Arc is an important marker of neuronal activity and is required for protein-synthesis-dependent synaptic plasticity and spatial memory [51 –54]. To investigate whether GLP2R can regulate neuronal activity, we detected Arc expression levels using western blots and investigated the Arc distribution with immunohistochemical staining. Our findings show that the relative levels of Arc in the hippocampus declined after 2VO to 46.4±3.85% of those in control animals (p < 0.01), whereas the relative levels of Arc in the hippocampus of 2VO rats with GLP2R upregulation were 82.7±6.89% compared to those of 2VO rats (p < 0.01; Fig. 8A, B). Immunohistochemical stainings demonstrated that the Arc expression in CA1, CA3, dentate gyrus (DG), and in the cortex of 2VO rats was significantly reduced compared to that in control rats (p < 0.01), whereas the Arc expression in the hippocampus sub-regions and the cortex of 2VO animals with GLP2R upregulation was increased compared to that of 2VO rats (p < 0.01; Fig. 8C, D).

Fig.8

GLP2R upregulation can upregulate Arc expression in the brain after CCH. The hippocampal homogenates were analyzed by western blots (A) and the relative intensities of Arc bands were analyzed (B). C) Murine brain slices (n = 3) were stained with an anti-Arc antibody. D) The Arc-positive cells were quantified. Bar = 50μm. Con, sham-operated group; 2VO, group with bilateral common carotid artery ligation; 2VO+GLP2R, 2VO group with GLP2R upregulation; GLP2R, sham-operated group with GLP2R upregulation. Data are expressed as mean±SEM. ^**p < 0.01 compared with Con; ^#p < 0.05, ^# #p < 0.01 compared with 2VO.

Upregulation of GLP2R can increase the density of dendritic spines and mushroom spines in hippocampal neurons

Dendritic spines are important network structures of neural circuits, and their normal distribution and morphological structure are critical for maintaining synaptic plasticity and hippocampus-dependent spatial memory functions [55, 56]. To investigate whether GLP2R can regulate the density and morphology of dendritic spines after CCH, we employed Golgi staining to label dendritic spines. Our findings show that the density of dendritic spines in 2VO rats was decreased compared to that in control rats (p < 0.01), but the density of dendritic spines in 2VO rats with GLP2R upregulation was increased compared to that in 2VO rats (p < 0.01; Fig. 9A, B). The density of mushroom spines in 2VO rats was lower than that in control rats (p < 0.01), whereas the density of mushroom spines in 2VO rats with GLP2R upregulation was significantly higher than that in 2VO rats (p < 0.01; Fig. 9A, C).

Fig.9

GLP2R upregulation increases the density of dendritic spines and mushroom spines in hippocampal neurons after CCH. Tissue blocks of murine brains (n = 3) were immersed into a Golgi staining kit for 2 months, and then these brains were cut into 100-μm slices. A) The slices were observed with a microscope using an oil immersion lens with 1,000x magnification. The dendritic spines were counted and sorted to analyze the density of dendritic spines (B) and mushroom spines (C). Bar = 50μm. Con, sham-operated group; 2VO, group with bilateral common carotid artery ligation; 2VO+GLP2R, 2VO group with GLP2R upregulation; GLP2R, sham-operated group with GLP2R upregulation. Data are expressed as mean±SEM. ^**p < 0.01 compared with Con; ^# #p < 0.01 compared with 2VO.

DISCUSSION

GLP2R is a nutrient-responsive neuropeptide that directly stimulates protein synthesis in a dose-dependent manner with activation of the AKT-mTOR signaling pathway [23]. mTOR is a central regulator of translational initiation and can regulate two critical translation initiation proteins, p70 ribosomal S6 kinase and the eIF4E binding proteins, to regulate mRNA translation [57]. mTOR is at the crossroad of plasticity, memory, and disease [26] and can increase the number and activity of new spine synapses [27] as well as the expression of synaptic signaling proteins including NMDA receptors, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, and brain-derived neurotrophic factor proteins. mTOR knockout mice show hippocampus-dependent learning and memory deficits and impairments in social behavior [58]. In our study, we found that GLP2R expression as well as the activity in the AKT-mTOR signaling pathway are decreased after CCH and that GLP2R upregulation improves the AKT-mTOR signaling. Therefore, it can be concluded that GLP2R downregulation may be involved in CCH-induced cognitive impairment by inhibiting synaptic plasticity via decreased AKT-mTOR signaling. How GLP2R changes dynamically in the CCH-induced decline of cognitive function has not been investigated in this study. The time-dependent changes in GLP2R expression could be of interest to study the role of GLP2Rs in the development of cognitive dysfunctions after CCH. In future studies, we will address this aspect.

Glucagon-like peptide-2 (GLP-2) is a 33-amino-acid peptide derived from the proglucagon gene expressed in the intestines, the pancreas, and the brain [17 , 60]. The chronic administration of GLP-2 affects hippocampal neurogenesis and exhibits antidepressant-like effects [61]. Additionally, GLP-2 has protective effects in ischemia-reperfusion models [62]. In the current study, we detected decreased GLP2R levels, but we did not investigate GLP-2 levels in brain tissue. It is not clear how GLP-2 levels change in the brain after CCH induction. Since GLP-2 acts mainly via binding to GLP2Rs, the GLP2R pathways are inhibited even if GLP-2 changes were not detectable because of a decrease in GLP2R levels. In future studies, we will examine GLP-2 levels to determine whether CCH also regulates the GLP-2 expression.

Arc/Arg3.1, located in the postsynaptic density, is a protein of an immediate-early gene implicated in memory consolidation. Arc/Arg3.1 expression has been used as a marker of neuronal activity because of its rapidly increased expression in dendrites after neuronal activation [63]. In our previous study investigating the changes in neuron numbers one month after the 2VO surgery, the neuron loss in 2VO rats was not more pronounced than that in sham-operated rats [64, 65]. Therefore, we think that the upregulation of GLP2R probably does not have an effect on the neuron number in the brains of 2VO model rats and that the decreases in Arc expression partly reflect the decreased neuronal activity in these animals. The Arc mRNA induction, distribution, and accumulation in dendrites as well as Arc translation require NMDA receptor activation and participate in information processing and storage [66]. The AKT-mTOR signaling pathway is involved in the regulation of Arc expression [67]. Because of the reduction in AKT-mTOR signaling after CCH in our study, we suggest that the AKT-mTOR signaling pathway is involved in the reduction of Arc expression after CCH. Changes in GLP2R activity are important for understanding the role of GLP2R in the mechanism of CCH-induced cognitive impairment. Since previous studies have demonstrated that GLP2R can regulate the AKT-mTOR pathway, the changes of GLP2R activity partly can reflect the changes of GLP2R activity from the side. GLP2R, is a member of the G protein-coupled receptor superfamily, so in the future, we will elucidate the changes in GLP2R activity by investigating in more detail the pathways downstream of this G protein-coupled receptor.

LTD is caused mainly by a decrease in the postsynaptic receptor density at the synaptic surface. LTD, as a form of long-term synaptic plasticity, is as significant as LTP [68, 69]. LTD selectively weakens the continued LTP-induced strengthening of specific synapses and made LTP ultimately avoid reaching an oversaturated level of efficiency, induced excitatory toxicity, and inhibited the encoding of new information [69, 70]. LTP and LTD are both critical for maintaining normal learning and memory processes [71]. The results of our study show that CCH inhibits both LTP and LTD and that an upregulation of GLP2R can reverse this, suggesting that a downregulation of GLP2R may modulate the synaptic plasticity of hippocampal neural circuits and thereby play a role in the development of cognitive dysfunction after CCH.

The dendritic spine is one element of the neural circuit. Its structural plasticity is closely in line with synaptic function and plasticity, memory and cognition [72, 73]. When a long-term potentiation or depression is induced, the spine size increases or decreases, respectively decreases, respectively [74]. There are different classes of spine shape such as filopodia, thin, stubby, and mushroom spines. Filopodia and thin spines are immature spines and lack functional synapses, whereas mature spines, called mushroom, are stable and have an enlarged spine head. These spine heads contain neurotransmitter receptors and postsynaptic density proteins, which is essential for the normal function of the neural circuit [75]. Synaptic enhancement leads to an enlargement of thin spines into mushroom spines and the mobilization of subcellular resources to potentiated synapses [76, 77]. Arc overexpression or disruption can increase or decrease the spine density, respectively [78], and the mTOR pathway can regulate the expression of many synaptic proteins and influence the dendritic spine morphology [79]. In our study, we found that after CCH the spine density and the number of mature spines as well as the activity levels of Arc and the mTOR pathway were decreased, while GLP2R upregulation treatment was able to reverse these effects. We that GLP2R probably regulate the density and maturation of dendritic spines by regulating Arc expression and mTOR pathway activation.

Adult hippocampal neurogenesis is important for the normal function of the dentate gyrus as well as for spatial learning and memory [80]. Enhancing neurogenesis can improve spatial learning and memory performance and neural plasticity, such as LTP [81, 82], whereas ablated neurogenesis impairs spatial learning and memory and fear conditioning memory [83, 84]. On the basis of these roles of neurogenesis in cognition, a reduced neurogenesis after CCH in the present study should play an important role in the development of spatial learning and memory deficits. An activated mTOR signaling pathway can promote neurogenesis [85], and the present study shows that GLP2R upregulation can improve neurogenesis and activate the mTOR signaling pathway after CCH. Thus, it is conceivable that in our model GLP2R-activated mTOR mediates the neurogenesis in the dentate gyrus.

Our study demonstrates that CCH led to a downregulation of hippocampal GLP2R and AKT-mTOR signaling. CCH also caused deficits in hippocampal neurogenesis, structural plasticity of dendritic spines, LTP, LTD, and spatial learning and memory; however, GLP2R upregulation partly could rescue these changes. Our findings reveal the mechanisms underlying an impairment in spatial learning and memory in our CCH model. Moreover, our findings also indicate that GLP2R and the AKT-mTOR-p70S6K pathway may be promising targets to treat cognitive deficits after CCH.

Footnotes

ACKNOWLEDGMENTS

This work was supported in part by grants from the National Natural Science Foundation of China (NSFC) (81400891).

Authors’ disclosures available online ().

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