Progranulin promotes hippocampal neurogenesis and alleviates anxiety‐like behavior and cognitive impairment in adult mice subjected to cerebral ischemia

Abstract Aims Cerebral ischemia can lead to anxiety and cognitive impairment due to the loss of hippocampal neurons. Facilitation of endogenous neurogenesis in the hippocampus is a potential therapeutic strategy for alleviating ischemia‐induced anxiety and cognitive impairment. Progranulin (PGRN), a secretory glycoprotein, has been reported to have a mitogentic effect on many cell types. However, it is not clear whether PGRN enhances hippocampal neurogenesis and promotes functional recovery. Methods Adult male C57BL/6 mice were subjected to permanent middle cerebral artery occlusion (pMCAO) and injected intracerebroventricularly with recombinant mouse PGRN 30 min after pMCAO. Anxiety‐like behavior was detected by the open field and the elevated plus maze tests, and spatial learning and memory abilities were evaluated by Morris water maze. Neurogenesis was examined by double labeling of BrdU and neural stem cells or neurons markers. For mechanism studies, the level of ERK1/2 and AKT phosphorylation were assessed by western blotting. Results Progranulin significantly alleviated anxiety‐like behavior and spatial learning and memory impairment induced by cerebral ischemia in mice. Consistent with the functional recovery, PGRN promoted neural stem cells (NSCs) proliferation and neuronal differentiation in the dentate gyrus (DG) after cerebral ischemia. PGRN upregulated the expression of phosphorylated ERK1/2 and Akt in the DG after cerebral ischemia. Conclusions Progranulin alleviates ischemia‐induced anxiety‐like behavior and spatial learning and memory impairment in mice, probably via stimulation of hippocampal neurogenesis mediated by activation of MAPK/ERK and PI3K/Akt pathways. PGRN might be a promising candidate for coping with ischemic stroke‐induced mood and cognitive impairment in clinic.


| INTRODUC TI ON
Ischemia stroke is a cerebrovascular disease which is characterized by high fatality rate and high disability rate. 1 In addition to physical impairments, stroke patients often endure post-stroke anxiety and cognitive impairment including learning and memory deficit, resulting in significant decline in life quality. 2 Previous studies in rodents reported that both middle cerebral artery occlusion (MCAO) and the bilateral common carotid artery occlusion (BCCAO) induced anxietylike behavior and spatial learning and memory impairment. 3,4 The hippocampus has considerable importance for mood and cognition. 5,6 Mood disorders and cognitive impairment caused by cerebral ischemia are closely related to neuronal damage in the hippocampus. 7 In adult mammals, neurogenesis occurs in the subventricular zone (SVZ) of the lateral ventricle and the dentate gyrus (DG) of the hippocampus. A continuous proliferation of neural stem cells (NSCs) was found in the subgranular zone (SGZ) between the granule cell layer and the hilus. 8 The ischemic injury stimulates the neurogenesis of SGZ, and the newly proliferated NSCs migrate and differentiate into mature neurons. 9 Newborn neurons integrate into existing neural circuits and play an important role in functional recovery following cerebral ischemia. 9 Therefore, many therapeutic strategies have been developed to promote hippocampal neurogenesis, which is promising to improve mood disorders and cognitive impairment induced by cerebral ischemia. 3,[9][10][11][12][13] Progranulin (PGRN) is a secretory glycoprotein and abundantly expressed in the brain. 14 PGRN has a mitogentic effect on many cell types including NSCs. 15,16 In recent decades, a few studies have showed that PGRN plays a positive role in hippocampal neurogenesis under several experimental conditions. [16][17][18] PGRN deficiency decreased the proliferation of cultured hippocampal NSCs from embryonic mouse brains, which was rescued by the addition of exogenous PGRN. 16 PGRN deficiency also exacerbated the suppressive effect of lipopolysaccharide on the adult hippocampal neurogenesis. 17 Applying anti-PGRN antibody blocked estrogen-induced proliferation of adult hippocampal NSCs in vitro. 18 PGRN was involved in enhancing hippocampal neurogenesis induced by voluntary exercise. 19 Our previous study has reported that PGRN promotes neurogenesis in the subventricular zone of adult mice subjected to cerebral ischemia. 20 However, whether administration of PGRN could attenuate mood disorder and cognitive impairment by promoting hippocampal neurogenesis after cerebral ischemia has not been investigated.
In this study, adult male C57BL/6 mice were subjected to the permanent middle cerebral artery occlusion (pMCAO) to induce cerebral ischemia, and received an intracerebroventricular (i.c.v.) administration of recombinant mouse PGRN (r-PGRN) at 30 min after pMCAO. First, the effects of PGRN on anxiety-like behavior and spatial learning and memory abilities of ischemic mice were examined. Then, we observed the effects of PGRN on NSCs proliferation and neuronal differentiation in the hippocampus after cerebral ischemia. Additionally, the molecular mechanism of PGRN's effects on neurogenesis after cerebral ischemia was tentatively explored.

| Animals
Adult male C57BL/6 mice weighting 20-25 g obtained from the Animal Center of Xi'an Jiaotong University were used in the current study. All animal procedures were licensed by the Shaanxi Normal University Ethics Committee and performed in accordance with the guidelines approved by the Animal Care and Use Committee of Shaanxi Normal University. All mice were maintained under standard conditions of 12 h/12 h light/dark cycle at 20-24°C. Food and water were available ad libitum.

| Experimental design
A schematic of the experimental protocols is depicted in Figure 1.
Briefly, mice were randomly assigned to the following three groups: the sham-operated group, the pMCAO with vehicle-treated group (pMCAO + Vehicle), and the pMCAO with PGRN-treated group (pMCAO + PGRN). Intracerebroventricular injection of r-PGRN (1 ng) or 0.01 M sterile phosphatase buffered saline (PBS) was performed 30 min after the pMCAO procedures. Mice in each group were then randomly divided into two subgroups for behavioral tests and neurogenesis detection, respectively. Experiment 1 was designed to detect the effects of PGRN on mood and cognitive function following cerebral ischemia in mice ( Figure 1A; n = 9 per group). Open-field test and the elevated plus maze were performed to detect mice anxiety-like behavior 14 days after sham or pMCAO surgeries. Morris water maze test was conducted to detect spatial learning and memory abilities 24-28 days after surgeries. Experiment 2 was designed to detect the effect of PGRN on neurogenesis in SGZ and its underlying mechanisms following cerebral ischemia in mice ( Figure 1B). According to previous reported methods, 21

| Mice focal cerebral ischemia model
A mouse pMCAO model was performed as described previously. 22 Briefly, mice were anesthetized intraperitoneally with sodium pentobarbital (50 mg/kg) and fixed on the operating table. A rectal probe was inserted to monitor body temperature, which was maintained at 37.0 ± 0.5°C with a heating pad. A midline skin incision was made in order to expose the right external carotid, internal carotid, and common carotid arteries. A 4-0 nylon monofilament with its tip rounded was carefully advanced into right internal carotid artery, until the origin of the middle cerebral artery was obstructed. Sham-operated mice underwent the same operative procedure, but the middle cerebral artery was not occluded.

| Recombinant mouse PGRN (r-PGRN) administration
r-PGRN (R&D Systems) was dissolved in 0.01 M sterile PBS and i.c.v. administrated using Hamilton syringe according to a previously described method. 23 Briefly, 30 min after the pMCAO surgery, mice were placed on a stereotaxic apparatus (RWD Life Science) under anesthesia condition, and a microsyringe was used to give each mouse a single i.c.v. injection into the right lateral ventricle (0.5 mm posterior to bregma, 1.1 mm lateral to midline, and 2.5 mm vertically from the skull surface). r-PGRN (1 ng in 2 µl PBS) or PBS (2 µl) was injected at a rate of 1 µl/min.

| Behavioral testing
Mice were handled daily for 3 days before the behavioral test. At 14 days after sham or pMCAO surgeries, mice were tested first on the open-field test followed by the elevated plus maze 6 h later.
Before behavioral tests, all mice were allowed to habituate the testing room for at least 30 min. In all behavioral tests, animals' movements were recorded and analyzed using a CCD camera-assisted motion tracking apparatus and software (VideoMot2, TSE Inst).

| Open-field test
The apparatus was made of a polyvinyl chloride box

| Morris water maze
A large circular pool (diameter 100 cm, depth 60 cm, and filled to a depth of 45 cm with water at 25 ± 1°C) was divided into four quadrants. A hidden circular platform (diameter 10 cm) at 1 cm below the water surface was placed in the center of one quadrant, providing the escape area. Mice were trained to discover the hidden platform for 4 consecutive days (spatial acquisition training days) with 4 training trials per day. During the training trials, mice were allowed to start from one random location in each of 4 quadrants and find the hidden platform within 60 s. If the mouse failed to find the hidden platform within 60 s, the experimenter guided them to reach it. Swimming speed, the escape latency, and the distance traveled to find the hidden platform were detected. On the fifth day, mice were subjected to a 60 s test (probe trial), in which the platform was removed. The time in the target quadrant (the location where the platform had been placed during the training tests) was recorded.

| Immunohistochemistry and immunoflurescence
Mice were deeply anaesthetized and transcardially perfused with PBS, followed by 4% ice-cold paraformaldehyde (PFA) at 1, 3, 7, 14, and 28 days after sham or pMCAO surgeries. Sixteen-micrometer coronal brain sections were cut using a cryotome. To stain BrdU, the sections were treated with 2 mol/L HCl at 37°C for 30 min, and neutralized with 0.1 mol/L sodium borate (pH 8.5) for 10 min before blocking. After rinsing in PBS three times, sections were incubated in a blocking solution containing 0.5% Triton X-100 and 5% bovine serum albumin for 1 h at room temperature.

| Cell counts and quantification
All images were acquired with a Zeiss microscope imaging system and a confocal laser scanning microscope (Olympus). Image

| Statistical analysis and quantification
All data are presented as mean ±standard error of mean (SEM).
Statistical analyses were conducted using SPSS 10.0 (SPSS Inc.). The normality test was performed by the Shapiro-Wilk test. The data in MWM training trial were analyzed using a two-way repeated measure analysis of variance (ANOVA) with group and day. Statistical comparisons between two groups were performed using the independent two-sample t-test. Statistical comparisons among three groups were performed using one-way ANOVA followed by the least significant difference (LSD) post hoc test. Differences were considered significant when p < 0.05.

| Progranulin alleviates anxiety-like behavior induced by cerebral ischemia in mice
To determine whether administration of PGRN affects ischemiainduced anxiety in mice, the anxiety-like behavior was evaluated using the open-field and the elevated plus maze tests (Figure 2A

| Progranulin alleviates spatial learning and memory impairment induced by cerebral ischemia in mice
To investigate the effect of PGRN on cognitive deficits after cerebral ischemia, we conducted Morris water maze test to evaluate mouse spatial learning and memory abilities. Because swimming abilities impact the performance of Morris water maze test, swimming speed during the test was evaluated. The result showed there was no significant difference in swimming speed among the three groups, which indicated equivalent swimming abilities in Morris water maze test (all p > 0.05; Figure 3A). In the training session, group (F group = 34.157, p < 0.001) and day (F day = 98.992, p < 0.001) exerted significant effects on escape latency and the distance traveled to find the hidden platform during training days. The pMCAO + Vehicle group showed spatial learning and memory deficit compared to the sham group as indicated by the longer escape latencies (F (2, 24) = 2.915, p < 0.05;  Figure 3D,E). Overall, the results suggest that PGRN treatment alleviated the spatial learning and memory impairment induced by cerebral ischemia.

| Progranulin promotes ischemia-induced NSCs proliferation in the SGZ
To explore the effect of PGRN on NSCs proliferation in the SGZ following cerebral ischemia, we used BrdU to label newly prolif-

| Progranulin promotes ischemia-induced NSCs neuronal differentiation in DG
To assess whether PGRN treatment regulates the NSCs differentiation in the hippocampus, double labeling of BrdU and cell markers (DCX, marker of immature neurons; NeuN, marker of mature neurons) was performed to label newborn immature and mature neurons.
We first detected the temporal patterns of DCX expression in SGZ among the three groups. Although no significant statistical difference was observed in DCX expression between the pMCAO + Vehicle group and the sham group 7 days after cerebral ischemia (p > 0.05; Figure 5A Figure 6). In DG, a few BrdU + /NeuN + cells were observed in pMCAO+ Vehicle group 28 days after cerebral ischemia ( Figure 6D). Compared to the pMCAO + Vehicle group, the number of BrdU + /NeuN + cells in the pMCAO + PGRN group was significantly increased (p < 0.01; Figure 6E). In summary, these results suggest that PGRN promotes the differentiation of NSCs into neurons in DG after ischemia.

| Progranulin treatment activates the MAPK/ERK and PI3K/Akt signaling pathways after cerebral ischemia
Activation of MAPK/ERK and PI3K/Akt signaling pathways in NSCs is crucial for the induction of adult neurogenesis. 24 To explore whether PGRN's effect on ischemia-induced neurogenesis in DG was through activation of MAPK/ERK and PI3K/Akt signaling pathways, we tested phosphorylated ERK1/2 and Akt in DG 3 days after F I G U R E 3 Progranulin (PGRN) administration alleviates cerebral ischemia-induced spatial learning and memory impairment. (A) Swimming speed in the Morris water maze test for evaluating swimming abilities. No difference in swimming speed was observed among the sham group, pMCAO + Vehicle group, and pMCAO + PGRN group. Escape latency (B) and the distance traveled (C) to find the hidden platform measured during 24 to 27 days after cerebral ischemia in training trials. # p < 0.05, ## p < 0.01 vs. sham group; * p < 0.05, ** p < 0.01 vs.  Figure 7A,B and Figure   S1) or Akt (F (2, 6) = 17.352, p < 0.05; Figure 7C,D and Figure S1) compared to the pMCAO + Vehicle group.

| DISCUSS ION
In this study, we provided the first evidence that PGRN treatment alleviated anxiety-like behavior and spatial learning and memory impairment in adult mice subjected to pMCAO. Meanwhile, we demonstrated that PGRN treatment promoted the proliferation and neuronal differentiation of NSCs in hippocampus after cerebral ischemia.
Additionally, PGRN treatment enhanced ischemia-induced activation In the present study, we used i.c.v. administration of 1 ng r-PGRN after pMCAO to assess the effect of PGRN on ischemia-induced behavioral deficits and hippocampal neurogenesis. Previous studies found that r-PGRN at a dose of 1 ng via i.c.v. injection resulted in a reduction in the infarct volume and an improvement in neurological function in ischemic mice, and administration of r-PGRN at lower doses (0.1 and 0.3 ng) or higher dose (5 ng) did not show the desired protective effect. 20,23 Our previous study also demonstrated that i.c.v. administration of 1 ng r-PGRN promoted ischemia-induced neurogenesis and neuronal differentiation in the SVZ. 20 Based on these studies, we chose administration of r-PGRN at a dose of 1 ng in this study, and found that 1 ng r-PGRN significantly enhanced hippocampal neurogenesis and alleviated anxiety-like behavior and spatial learning and memory impairment in ischemic mice.
The hippocampus is a key brain region for mood and cognition in mammals. 5,6 As a potential endogenous compensatory mechanism, cerebral ischemia has been shown to stimulate the proliferation of NSCs residing in SGZ, newly generated neurons form functional synapses and integrate into the existing hippocampal circuitry by 28 days, which contribute to improving functional recovery after cerebral ischemia. 9,25 As previous study reported, we also demonstrated that cerebral ischemia alone induced an increase in hippocampal NSCs proliferation ( Figure 4) and neuronal differentiation ( Figure 6). Several putative diffusible mitogens including growth factors, cytokines, and cell division modulators are known to be upregulated in the ischemic brain. 25 These diffusible factors play a major role in promoting post-ischemia neurogenesis. 25 However, endogenous neurogenesis fails to produce adequate amounts of newborn neurons to facilitate the functional recovery. 9 In this study, we found that PGRN administration has an improve- neurons and integrated into hippocampal circuitry to play a functional role. 9 Notable, we found that exogenous administration of PGRN enhanced ischemia-induced neurogenesis in the SGZ, and increased the number of NSCs ( Figure 4) and immature neurons in SGZ by post-ischemic day 14 ( Figure 5). NSCs has been reported to secrete growth factors (e.g., brain-derived neurotrophic factor and vascular endothelial growth factor), which contribute to neuron repair and functional recovery by a broad range of neurotropic and neuroprotective effects. 12,26 Therefore, it could be concluded that PGRN facilitates cerebral ischemia-induced NSCs proliferation in the SGZ, and NSCs could secrete growth factors which contribute to the reduction in anxiety-like behavior after cerebral ischemia. In ite growth, and synaptic plasticity. [30][31][32][33] A recent study has reported that PGRN not only attenuated sleep-deprived-induced memory impairment and anxiety-like behavior through enhancing neurogenesis in the SGZ but also through suppressing inflammation and restoring dendritic spine density in the hippocampus. 30 Therefore, more studies are required to fully understand the mechanism behind it.
To study possible mechanisms through which PGRN induced increases in neurogenesis, selected proteins in the MAPK/ERK and PI3K/Akt signaling pathways were studied using western blotting.
The MAPK/ERK and PI3K/Akt signaling pathways play a key role in adult hippocampal neurogenesis. 24 The activation of MAPK/ ERK and PI3K/Akt signaling pathways markedly enhances the cerebral ischemia-induced hippocampal neurogenesis. 24 PI3K inhibitor LY294002 and ERK kinase inhibitor U0126 inhibited hypoxia/ reoxygenation-induced increases in the proliferation in cultured mouse NSCs. 34 Phosphorylation of ERK and Akt was significantly enhanced in the hippocampus and hemisphere on days 3 and 7 after the cerebral ischemia. 35,36 Consistent with these findings, we demonstrated that cerebral ischemia upregulated phosphorylation of ERK1/2 and Akt in the hippocampus (Figure 7). It has been reported that PGRN could activate these two pathways, resulting in increased expression of cyclin D1 and cyclin B and an enhanced proliferation rate in cancer cell. 37,38 Previous study showed that PGRN treatment increased the proliferation of cultured hippocampal NSCs, and PI3K inhibitor blocked this effect, suggesting that PGRN enhances NSCs proliferation and neurogenesis through activating the PI3K/Akt signaling pathway. 16 Our previous study showed that PGRN may promote SVZ neurogenesis by activating the MAPK/ERK and PI3K/Akt signaling pathways. 20 The current study revealed that administration of PGRN increased the level of phosphorylation of ERK1/2 and Akt induced by cerebral ischemia (Figure 7), suggesting that PGRN may promote ischemia-induced hippocampal neurogenesis by activating the MAPK/ERK and PI3K/Akt signaling pathways.
Overall, this work is the first to demonstrate that PGRN enhances ischemia-induced hippocampal neurogenesis through activation of MAPK/ERK and PI3K/Akt signaling pathways, thereby alleviating anxiety-like behavior and the spatial learning and memory impairment following cerebral ischemia. Because of such biological functions, PGRN could be applied as a potential therapeutic agent to improve ischemic stroke-induced mood disorder and cognitive impairment.

ACK N OWLED G EM ENTS
This work was supported by the National Natural Science Foundation of China (81971285), and the Fundamental Research Funds for the F I G U R E 7 PGRN increased the level of ERK1/2 and Akt phosphorylation in DG 3 days after cerebral ischemia. Representative images of immunoblots using antibodies against p-ERK1/2 (Thr-202/Tyr-204)/t-ERK1/2 and p-Akt (Thr-308)/t-Akt are shown (A and C), and quantitative analyses of ERK1/2 and Akt phosphorylation by densitometry are shown (B and D). Immunoblotting for actin reveals that equal amounts of proteins were loaded in each lane. Data are expressed as the ratio to the optical density value of sham-operated animals. Data are presented as mean ± SEM, n = 3 per group. * p < 0.05 Central Universities (GK202005001, GK201803044), Shaanxi Normal University.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.