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. Author manuscript; available in PMC: 2025 Mar 1.
Published in final edited form as: Exp Neurol. 2023 Dec 17;373:114656. doi: 10.1016/j.expneurol.2023.114656

Intranasal administration of recombinant prosaposin attenuates neuronal apoptosis through GPR37/PI3K/Akt/ASK1 pathway in MCAO rats

Jing Yu a,b,1, Jinlan Li a,1, Nathanael Matei b,c, Wenna Wang a,b, Lihui Tang a,b, Jinwei Pang b, Xue Li a,b, Lili Fang a, Jiping Tang b, John H Zhang b,*, Min Yan a,*
PMCID: PMC10922973  NIHMSID: NIHMS1954131  PMID: 38114054

Abstract

Studies have reported that Prosaposin (PSAP) is neuroprotective in cerebrovascular diseases. We hypothesized that PSAP would reduce infarct volume by attenuating neuronal apoptosis and promoting cell survival through G protein-coupled receptor 37(GPR37)/PI3K/Akt/ASK1 pathway in middle cerebral artery occlusion (MCAO) rats. Two hundred and thirty-five male and eighteen female Sprague-Dawley rats were used. Recombinant human PSAP (rPSAP) was administered intranasally 1 hour (h) after reperfusion. PSAP small interfering ribonucleic acid (siRNA), GPR37 siRNA, and PI3K specific inhibitor LY294002 were administered intracerebroventricularly 48 h before MCAO. Infarct volume, neurological score, immunofluorescence staining, Western blot, Fluoro-Jade C (FJC) and TUNEL staining were examined. The expression of endogenous PSAP and GPR37 were increased after MCAO. Intranasal administration of rPSAP reduced brain infarction, neuronal apoptosis, and improved both short- and long-term neurological function. Knockdown of endogenous PSAP aggravated neurological deficits. Treatment with exogenous rPSAP increased PI3K expression, Akt and ASK1 phosphorylation, and Bcl-2 expression; phosphorylated-JNK and Bax levels were reduced along with the number of FJC and TUNEL positive neurons. GPR37 siRNA and LY294002 abolished the anti-apoptotic effect of rPSAP at 24 h after MCAO. In conclusion, rPSAP attenuated neuronal apoptosis and improved neurological function through GPR37/PI3K/Akt/ASK1 pathway after MCAO in rats. Therefore, further exploration of PSAP as a potential treatment option in ischemic stroke is warranted.

Keywords: Prosaposin, GPR37, PI3K/Akt, Apoptosis, MCAO

1. Introduction

Ischemic stroke is one of the major causes of mortality and morbidity worldwide. It is reported that China has the highest lifetime risk of stroke in the world (Collaborators et al., 2018). Neuronal apoptosis is a key pathological process in early brain injury after ischemic stroke (Cai et al., 2021; Tian et al., 2018). Studies have demonstrated that stroke-induced neuronal apoptosis is potentially recoverable in the acute stage of stroke (Matei et al., 2018; Zhou et al., 2021). However, no therapeutic agents have been successfully translated to ameliorate neuronal apoptosis in stroke. Therefore, our research focused on exploring the neuroprotective agent Prosaposin (PSAP) as a potential means to attenuate apoptosis resulting from ischemic stroke.

PSAP, a neurotrophic factor, regulates important cellular functions including cell survival, neurite outgrowth and differentiation (O'Brien et al., 1994). PSAP can be secreted into the extracellular space, from various cell types, exerting neuroprotective and glioprotective effects in the nervous system; e.g. ameliorating ischemic damage (Morita et al., 2001), rescuing dopaminergic neurons (Gao et al., 2013), facilitating nerve regeneration (Jolivalt et al., 2008), reducing neuropathic pain (Calcutt et al., 2000), protecting myelinating glial cells (Hiraiwa et al., 2001), and serving an important role in cerebellar development (Cove et al., 2006). PSAP expression and secretion was enhanced following conditions of cellular stress such as ischemia (Costain et al., 2010). It was reported that PSAP and prosaptide (an active fragment of PSAP) have neuroprotective and glioprotective effects via the stimulation of G protein-mediated pathways (Meyer et al., 2014); however, the intracellular pathway remains poorly investigated. Studies have confirmed that PSAP activation of the G protein-coupled receptor 37 (GPR37) protected neurons and glial cells via endocytosis of GPR37 and activation of intracellular survival proteins (Jolly et al., 2018; Meyer et al., 2013).

GPR37 is an orphan G protein-coupled receptor that is almost exclusively expressed in the nervous system (Meyer et al., 2013). GPR37 was found to be critically involved in the regulation of neuronal cell death, the modulation of inflammatory responses, and the formation of glial scars within the post-stroke cerebral environment (McCrary et al., 2019). GPR37 expressed at physiological levels has been shown to exert anti-apoptotic effects in neuronal cells induced by dopaminergic toxins, MPP+, rotenone and 6-OHDA (Lundius et al., 2013). The mediation of PSAP's protective effects by GPR37 was evidenced as the knockdown of endogenous astrocytic GPR37 using siRNA reversed PSAP's therapeutic effects in primary astrocytes (Meyer et al., 2013). Congruently, studies have observed that plasma membrane-expressed GPR37 mediated the protective effects of extracellular secreted PSAP in N2a cells (Lundius et al., 2014). Thus, PSAP activation of GPR37 initiated intracellular survival signaling pathways in neurons (Meyer et al., 2013); however, the above studies are mainly in vitro experiments and the downstream targets in response to ischemic stroke remain poorly investigated.

The PI3K/Akt signaling pathway mediates survival signals in neurons through the inhibition of the cytoplasmic cell death machinery, regulating the expression of genes involved in cell death and survival (Brunet et al., 2001; Zhang et al., 2020). PSAP was shown to stimulate the PI3K/Akt pathway to prevent oxidative cellular death in PC12 cells (Ochiai et al., 2008). Mechanistically, Akt-mediated phosphorylation of Apoptosis signal-regulating kinase 1 (ASK1) on serine 83 activated cell survival factors and reduced apoptosis (Kim et al., 2001).

Although PSAP is known to bind to GPR37, the intracellular pathway within neurons remains poorly investigated. Thus, we hypothesize that intranasal administration of exogenous recombinant PSAP attenuates neuronal apoptosis via activation of the GPR37/PI3K/Akt/ASK1 signaling pathway after middle cerebral artery occlusion (MCAO) in rats.

2. Materials and Methods

2.1. Animals

All experimental protocols were approved by the Institutional Animal Care and Use Committee of Loma Linda University in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Two hundred and thirty-five male and eighteen female Sprague-Dawley (SD) rats (260-280g) were used and housed in a humidity, temperature-controlled room with a 12 h light/dark cycle and free access to water and food.

2.2. Transient MCAO model

Male SD rats were subjected to MCAO as previously described (Liang et al., 2021). Briefly, anesthesia was induced intraperitoneally with a mixture of ketamine (80mg/kg) and xylazine (10mg/kg). Atropine was then administered subcutaneously at a dose of 0.1mg/kg. The right common carotid artery (CCA), internal carotid artery (ICA) and external carotid artery (ECA) were surgically exposed. The ECA was coagulated and a 4-0 nylon suture, with a silicon tip, was inserted into the ICA through the ECA stump to occlude the MCA (approximately 18 to 22 mm). After 2 h, the suture was withdrawn to allow MCA reperfusion. Sham rats underwent the same procedures except for the occlusion of the MCA. Rats were kept at approximately 37°C on an electric heating blanket following closure of the skin incision and housed separately until they fully recovered from the anesthesia.

2.3. Study design

All rats were randomly divided into the following five experiments (Fig. 1).

Fig. 1. Experimental design and animal groups.

Fig. 1.

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DMSO, Dimethyl Sulfoxide; FJC, Fluoro-Jade C; IHC, immunohistochemistry; LY294002, a specific inhibitor of PI3K; MCAO, middle cerebral artery occlusion; NS, normal saline; rPSAP, recombinant human prosaposin; Scr siRNA, scramble siRNA; TTC, 2,3,5-triphenyltetrazolium chloride; TUNEL, Terminal deoxynucleotidyl transferase dUTP nick end labeling; WB, Western blot.

2.3.1. Experiment 1

Thirty rats were divided into five groups (Sham and MCAO after 6 h, 12 h, 24 h and 72 h, n=6) to determine the time course of endogenous PSAP and GPR37 expression via Western blot. Double immunohistochemistry staining was performed to test the colocalization of PSAP and GPR37 on neurons both in sham group and at 24 h after MCAO group (n=2).

2.3.2. Experiment 2

To evaluate the effects of intranasal recombinant human PSAP (rPSAP) on infarct volume and short-term neurobehavioral function at 24 h after MCAO, 30 rats were randomly divided into 5 groups (n=6 per group): (1) Sham; (2) MCAO+Vehicle; (3) MCAO+rPSAP (1μg/kg); (4) MCAO+rPSAP (3μg/kg); (5) MCAO+rPSAP (9μg/kg) (Igase et al., 1999). rPSAP or Vehicle (Normal Saline, NS) was administered intranasally at 1 h after MCAO. Based on the results of above groups, rPSAP (9μg/kg) was chosen for the next study. Then a total of 18 rats were divided to the following 3 groups (n=6 per group): (1) Sham group; (2) MCAO+Vehicle; (3) MCAO+rPSAP (9μg/kg). Infarct volume and short-term neurobehavioral tests (modified Garcia and beam walking scores) were assessed at 24 and 72 h after MCAO. The ipsilateral hemisphere brain samples were collected at 24 h, protein expression was evaluated via Western blots after 2,3,5-triphenyltetrazolium chloride (TTC) stained (n=6 per group). Another 18 rats from Sham, MCAO+Vehicle and MCAO+rPSAP (9μg/kg) group (n=6 per group) were evaluated for Fluoro-Jade C (FJC) staining and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. To assess sexually dimorphic differences after treatment, 18 female rats were divided to the following 3 groups (n=6 per group): (1) Sham group; (2) MCAO+Vehicle; (3) MCAO+rPSAP (9μg/kg). Modified Garcia and beam walking scores were assessed at 24 h after MCAO.

2.3.3. Experiment 3

To study the effects of exogenous rPSAP on long-term neurobehavioral function, 24 rats were randomly divided into 3 groups: Sham, MCAO+Vehicle, and MCAO+rPSAP (9μg/kg) (n=8 per group). rPSAP or Vehicle (NS) were administered intranasally at 1 h after MCAO. Rotarod test was performed on day 7, day 14, and day 21 after MCAO, while Morris water maze was performed on days 22-27 after MCAO.

2.3.4. Experiment 4

To understand the role of PSAP, a specific inhibitor, PSAP small interfering RNA (PSAP siRNA), was administered intracerebroventricularly (i.c.v) at 48 h before MCAO. Neurobehavioral tests and Western blots were performed at 24 h after MCAO. Rats were randomly divided into five groups: Sham, MCAO+Vehicle, MCAO+rPSAP (9μg/kg), MCAO+Scramble siRNA (Scr siRNA) and MCAO+PSAP siRNA. Samples for Sham, MCAO+Vehicle and MCAO+rPSAP (9μg/kg) groups were shared with Experiment 2. Additionally, 24 rats (naive rats, did not undergo any procedure) were randomly divided into four groups: Naive+NS, Naive+rPSAP (9μg/kg), Naive+Scr siRNA, and Naive+PSAP siRNA, to confirm the presence and delivery of intranasal administration rPSAP and siRNA effectiveness. Western blot was performed on the right hemisphere of the brain to measure PSAP expression.

2.3.5. Experiment 5

To evaluate the effects of rPSAP treatment and in vivo knockdown of GPR37 on neuronal apoptosis, rPSAP was administered intranasally at 1 h after MCAO, while GPR37 siRNA was administered i.c.v at 48 h before MCAO. Rats were randomly divided into five groups (n= 6 per group): Sham, MCAO+Vehicle, MCAO+rPSAP (9μg/kg), MCAO+rPSAP+Scr siRNA and MCAO+rPSAP+GPR37 siRNA. Samples for Sham, MCAO+Vehicle and MCAO+rPSAP (9μg/kg) groups were shared with Experiment 2. Neuronal degeneration (FJC staining) and neuronal apoptosis (TUNEL staining) were measured at 24 h after MCAO.

To explore the downstream molecular mechanisms of GPR37, GPR37 siRNA and PI3K inhibitor LY294002 were used. Rats were randomly assigned to seven groups (n=6 per group): Sham, MCAO+Vehicle, MCAO+rPSAP (9μg/kg), MCAO+rPSAP+Scr siRNA, MCAO+rPSAP+ GPR37 siRNA, MCAO+rPSAP+DMSO and MCAO+rPSAP+LY294002. Neurobehavioral tests and Western blots were examined at 24 h after MCAO. Samples for Sham, MCAO+Vehicle and MCAO+rPSAP (9μg/kg) groups were shared with Experiment 2 and 4. Additionally, to evaluate the knockdown efficiency of GPR37 siRNA, 12 rats were randomly divided into two groups: Naive+GPR37 siRNA and MCAO+GPR37 siRNA. The expression of GPR37 was evaluated by Western blot in the right hemisphere of the brain.

2.4. Intranasal administration of rPSAP

Intranasal administration at 1 h after MCAO was performed as previously described (Wu et al., 2017). The dosage of rPSAP was based on a previous study (Igase et al., 1999). Briefly, rats were placed in a supine position under isoflurane anesthesia (4% induction, 2.5% maintenance). Sterile normal saline (NS) or rPSAP (1μg/kg, 3μg/kg, 9μg/kg) (Creative BioMart, NY, USA) was administered intranasally. A total volume of 12 μl was delivered into left and right nares, alternating one naris at a time, every 5 min over a period of 10 min.

2.5. Intracerebroventricular injection

Intracerebroventricular (i.c.v) drug administration was performed as previously described (Liang et al., 2020). Briefly, rats were placed in a stereotaxic apparatus under isoflurane anesthesia (4% induction, 2.5% maintenance). The tip of a 10 μl Hamilton syringe (Microliter 701; Hamilton Company, USA) was inserted into the right lateral ventricle through a burr hole. The stereotactic i.c.v injection site was relative to bregma: 1.5 mm posterior, 0.9 mm right lateral, and 3.3 mm depth. PSAP siRNA, GPR37 siRNA and scrambled siRNA (OriGene Technologies, Rockville, MD, USA) were prepared at 500 pmol in RNAse free suspension buffer and administered (5 μl of the siRNAs) 48 h before MCAO (Zhou et al., 2018). LY294002 (50 mmol/L, Selleck Chemicals, Houston, USA) was dissolved in 25% DMSO in PBS and 10 μl was infused 30 min before MCAO (Zhou et al., 2021). The injection rate was 1μl/min, and the needle was left in place for an additional 5 min after injection, to prevent possible leakage, and then withdrawn over 5 min. After the needle was removed, the burr hole was sealed with bone wax. The incision was closed with sutures and the rat was allowed to recover.

2.6. Short-term neurological score evaluation

Short-term neurobehavioral deficits were assessed by a blinded investigator at 24 h or 72 h after MCAO, using both the modified Garcia and beam walking tests, as previously described (Garcia et al., 1995). The modified Garcia scoring system consisted of 6 tests (spontaneous activity, symmetry in the movement of four limbs, forepaw outstretching, climbing, body proprioception, response to vibrissae touch) with a maximum score of 18, higher scores indicating better performance. The beam walking test was performed as previously described (Goldstein and Davis, 1990) and based on a 0-5-point scale.

2.7. Long-term neurobehavioral evaluation

To evaluate long-term neurological function, rotarod and Morris water maze were conducted by a blinded investigator as previously described (Yu et al., 2018).

Briefly, the rotarod (Columbus Instruments, Columbus, OH) test was performed weekly after MCAO to assess sensorimotor coordination and balance. In detail, the cylinder started at 5 revolutions per minute (RPM) and 10 RPM, respectively, and accelerated by 2 RPM every 5 s. Latency to fall off was recorded and analyzed. Morris water maze was used to evaluate spatial learning and memory abilities on days 22 to 27 after MCAO. The procedures of Morris water maze were performed in a blinded manner as previously described (Xu et al., 2018). Briefly, each animal performed 5 trials per day over 6 days. On day 1 (cued test), the platform was made visible above water surface where the rats remained on the platform for 10 s (diameter: 10 cm, block 1) after finding or being guided to it. On days 2-5, spatial water maze test measured the latency to find the submerged platform (1 cm below the water, blocks 2-5, 1 min each trial). On day 6 (probe trial), the platform was removed and the time the rat spent in the platform quadrant was evaluated over 60 s. Swim path, escape latency, and swim distance were recorded by a computerized tracking system (Noldus Ethovision; Noldus, Tacoma, WA, USA).

2.8. Assessment of cerebral infarct volume

Animals were anesthetized and perfused with cold PBS (0.1 M, pH 7.4) as previously described (Pang et al., 2022). Brains were rapidly removed and sectioned coronally into 2 mm thick slices. Brain slices were incubated in 2 % 2,3,5 triphenyltetrazolium chloride (TTC, Sigma-Aldrich, St. Louis, MO, USA) for 15min at room temperature. The infarct and total hemispheric areas of each slice were traced and analyzed using ImageJ (ImageJ, NIH). The area of each slice was calculated using the formula: ((Area of Contralateral - Area of noninfarcted Ipsilateral tissue)/2* (Area of Contralateral)) *100%. The area was calculated for each slice and the average was taken to represent the Percentage of Infarcted Area for that animal (McBride et al., 2016).

2.9. Immunofluorescence Staining

Double immunofluorescence staining was performed at 24 h after MCAO as previously described (Kang et al., 2023). Briefly, Rats were deeply anesthetized and transcardially perfused with cold PBS followed by 10% formaldehyde solution (PFA) at 24 h after MCAO. The brains were removed and fixed with 10% PFA for 24 hours, and then transferred into 30% sucrose solution for three days. Brains were cut into 10 μm thick coronal sections on a cryostat (LM3050S; Leica Microsystems, Bannockburn, III, Germany). Coronal frozen slices were blocked with 5% donkey serum for 1 h and incubated at 4°C overnight with primary antibodies: mouse anti-NeuN (1:200, ab104224, Abcam), rabbit anti-PSAP (1:50, 10801-1-AP, ProteinTech) and rabbit anti-GPR37 (1:50, 14820-1-AP, ProteinTech) followed by incubation with appropriate fluorescence-conjugated secondary antibodies (1:100) for 2 h at room temperature. The sections were visualized and photographed with a fluorescence microscope (Leica DMi8, Leica Microsystems, Wetzlar, Germany).

2.10. Fluoro-Jade C staining

Degenerating neurons were evaluated by Fluoro-Jade C (FJC) staining as previously described (Tang et al., 2020). A modified FJC ready-to-dilute staining kit was used (Biosensis, USA). Slides were immersed in 1% sodium hydroxide in 80% ethanol for 5 min, followed by a 2 min rinse in 70% ethanol, then 2 min in distilled water. Slides were incubated in 0.06% potassium permanganate solution for 10 min, after 2 min in distilled water, and transferred into a 0.0001% solution of FJC (Millipore, USA), which was dissolved in 0.1% acetic acid. Slides were rinsed with distilled water for 1 min, three times. Slides were dried, immersed in xylene for 1 min, and a cover slip was added with DPX (Sigma-Aldrich, USA). The sections were visualized in blinded manner with a fluorescence microscope, Leica DMi8 (Leica Microsystems, Germany). FJC-positive neurons were manually counted in the peri-ischemic regions in six sections per brain with ImageJ software (ImageJ 1.5, NIH, USA). The data was presented by the average number of FJC-positive neurons in the field as cells/mm2.

2.11. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining

Neuronal apoptosis was evaluated by double staining of NeuN and TUNEL with in situ “Apoptosis Detection Kit” (Roche, USA) at 24 h after MCAO as previously described (Mo et al., 2019). TUNEL positive neurons and NeuN-stained positive cells were individually counted in peri-infract area of ipsilateral cortex (6 sections per brain). Data was expressed as ratio of TUNEL-positive neurons (%).

2.12. Western blots analysis

After TTC staining and imaging at 24 h after MCAO, brain slices were separated into the contralateral and ipsilateral hemispheres, flash frozen in liquid nitrogen, and then stored at −80°C. Western blot was performed as previously described (Kerr et al., 2022). Equal amounts of protein samples from the ipsilateral hemisphere were separated by SDS-PAGE gel electrophoresis and then transferred onto nitrocellulose membranes. Membranes were blocked and incubated at 4 °C overnight with the following primary antibodies: rabbit polyclonal anti-PSAP (1:500, 10801-1-AP, ProteinTech), rabbit polyclonal anti-GPR37 (1:500, 14820-1-AP, ProteinTech), rabbit monoclonal anti-PI3K (1:1000, ab151549, Abcam), rabbit anti-Akt (1:1000, #9272, Cell Signaling), rabbit anti-phospho-Akt (Ser473) (p-Akt, 1:1000, #9271, Cell Signaling), mouse monoclonal anti-ASK1 (1:500, sc-390275, Santa Cruz Biotechnology), rabbit monoclonal anti-phospho-ASK1 (Ser83) (p-ASK1, 1:1000, ab278647, Abcam), rabbit monoclonal anti-JNK (1:1000, ab179461, Abcam), rabbit monoclonal anti-phospho-JNK (p-JNK, 1:1000, ab124956, Abcam), rabbit polyclonal anti-Bcl-2 (1:1000, ab194583, Abcam), rabbit monoclonal anti-Bax (1:1000, ab182733, Abcam). Goat anti-β-actin (1:3000, Santa Cruz Biotechnology) was used as an internal loading control. Appropriate secondary antibodies (Santa Cruz Biotechnology) were used for 1 h at room temperature. Immunoblots were probed via an ECL Plus kit (American Bioscience, Little Chalfont, UK). Blot bands were quantified by densitometry using ImageJ software (ImageJ 1.4; NIH, Bethesda, MD).

2.13. Statistical Analysis

Data was expressed as mean ± standard deviation (SD). All analyses were performed with GraphPad Prism (GraphPad Software, San Diego, CA, USA). Statistical differences among groups were analyzed using one-way analysis of variance (ANOVA) followed by multiple comparisons using post-hoc Tukey tests. Mortalities were expressed as frequency (percentage), and the significance of difference among groups was assessed by Pearson Chi-square test or Fisher's exact test. A p-value less than 0.05 was considered statistically significant.

3. Results

3.1. Mortality and exclusion

All sham rats survived, but the overall mortality of MCAO was 16.8% (29/173). The mortality rate was not significantly different among MCAO groups (p > 0.05). Seven animals were excluded from this study due to no infarction (Tab1e S1).

3.2. Experiment 1: Expression of endogenous PSAP and GPR37 after MCAO

The endogenous protein levels of PSAP and GPR37 in the ipsilateral hemisphere were measured by Western blot. Expression of PSAP and GPR37 acutely increased after ischemia, peaking at 24 h, but showed a significant decrease by 72 h after MCAO when compared to sham (p<0.05, Fig. 2A-B). Congruently, double immunofluorescence staining of PSAP and GPR37 with neurons (NeuN) showed strong immunoreactivity in the penumbra at 24 h after MCAO (Fig. 2C-D).

Fig. 2. Expression of endogenous prosaposin (PSAP) and GPR37 after MCAO.

Fig. 2.

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(A-B) Representative western blot band of time course and quantitative analysis of PSAP and GPR37 after MCAO. * p<0.05 vs Sham. Error bars are represented as mean ± SD. One-way ANOVA, Tukey’s test, n=6 per group. (C-D) Representative microphotographs of double immunofluorescence staining showed the localization of PSAP and GPR37 with NeuN in sham and MCAO (24 h) groups. DAPI marked Nuclei. Arrows indicate the cell shown in the higher magnification box. n=2 per group. Scale bar=50μm. NeuN, neuronal nuclei.

3.3. Experiment 2: Effects of rPSAP on infract volume and short-term neurological function at 24 and 72h after MCAO

In the 24 and 72 h MCAO vehicle group, infarct volume was significantly increased and neurological scores were significantly decreased compared to the sham group (p < 0.05, Fig. 3). There was a significant reduction in infarct volume and improvement in neurological deficits with high dose of rPSAP (9μg/kg) at 24 h after MCAO compared to vehicle group (p < 0.05, Fig. 3A-D). No significant differences were observed in both 1μg/kg and 3μg/kg rPSAP groups compared to the vehicle group (p > 0.05, Fig. 3A-D). Intranasal rPSAP (9μg/kg) significantly reduced infarct volume and restored neurological function for modified Garcia and beam walking scores at 72 h after MCAO compared to the vehicle group (p<0.05, Fig.3E-H). Based on these results, we used the 9μg/kg dose for rPSAP in the subsequent studies.

Fig. 3. The effects of recombinant PSAP (rPSAP) on infract volume and neurological function at 24 and 72 h after MCAO.

Fig. 3.

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(A, E) Representative images of TTC staining brain slices indicating brain infarction at 24 and 72 h after MCAO. (B, F) Quantified infarct volume, (C, G) Modified Garcia score, and (D, H) Beam walking score showed that intranasal administration of rPSAP 9μg/kg significantly reduced infarct volume and improved neurological function compared to vehicle at both 24 and 72 h after MCAO. * p<0.05 vs. Sham, # p<0.05 vs. Vehicle, & p<0.05 vs. rPSAP 1μg/kg. Error bars are represented as mean ± SD. One-way ANOVA, Tukey’s test, n=6 per group.

To assess potential sexually dimorphic responses to treatment, neurological scores were assessed at 24 h in female rats. Intranasal rPSAP (9μg/kg) significantly improved Modified Garcia score and Beam walking score at 24 h after MCAO compared to the vehicle group (p < 0.05, Fig. S1. A-B). rPSAP treatment after MCAO had significantly therapeutic effects in both sexes, with no major intersex differences in functional outcomes.

3.4. Experiment 3: rPSAP improved long-term neurological function at four weeks after MCAO

In the Rotarod test, animals in the MCAO+Vehicle group had a significantly shorter latency to fall in both the 5 RPM and 10 RPM tests compared to the sham group on days 7, 14 and 21 after MCAO. This effect was reversed, significantly improving sensorimotor coordination and balance with intranasal rPSAP (9μg/kg) treatment (p < 0.05, Fig. 4A).

Fig. 4. The effects of rPSAP on long-term neurological function after MCAO.

Fig. 4.

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(A) Treatment with rPSAP significantly increased falling latency in the rotarod test on days 7, 14, and 21 after MCAO. rPSAP (9ug/kg) treatment group showed significant improvement in spatial memory loss in terms of decreasing escape latency (B), exhibiting a smaller swim distance to find the platform (C), and spending more time in the target quadrant (SW quadrant) (E) when compared to the vehicle group. (D) Representative picture of swim track in Probe Trial. Block1-5 represented days 22-26 after MCAO, respectively. * p<0.05 vs. Sham; # p<0.05 vs. Vehicle. Error bars are represented as mean ± SD. One-way ANOVA, Tukey’s test, n=8 per group.

In the Morris water maze test, the escape latency and swim distance to find the platform significantly increased in the vehicle group compared to the sham group (p<0.05, Fig. 4B-C). rPSAP (9ug/kg) treatment significantly decreased the escape latency and swim distance in blocks 3 to 5 (days 24 to 26 after MCAO) compared to the vehicle group (p < 0.05, Fig. 4B-C). In the probe trial, the vehicle group spent less time in the target quadrant compared to sham; however, rPSAP (9ug/kg) treatment significantly increased the time spent in the platform quadrant compared to the vehicle group (p <0.05, Fig. 4D-E).

3.5. Experiment 4-1: Effects of rPSAP and PSAP siRNA on neurobehavioral functions at 24 h after MCAO

Western blot analysis was used to check the delivery and presence of the intranasally administrated rPSAP (9μg/kg) and knockdown efficiency of PSAP siRNA. Following intranasal administration of rPSAP, the overall PSAP expression in the ipsilateral hemisphere significantly increased compared to non-treated groups at 23 h after rPSAP administration (p < 0.05, Fig. 5A), which suggests that the intranasal administration of rPSAP was effectively delivered to the brain. While PSAP siRNA pretreatment significantly reduced the PSAP expression in the ipsilateral hemisphere compared to the Scr siRNA group at 72 h after injection (p < 0.05, Fig. 5B). Knockdown of endogenous PSAP by pretreatment with PSAP siRNA exacerbated neurological deficits at 24 h post MCAO compared to the Scr siRNA group (p < 0.05, Fig. 5C-D).

Fig. 5. The effects of rPSAP and PSAP siRNA on neurobehavioral functions at 24 h after MCAO.

Fig. 5.

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(A) PSAP expression in the ipsilateral hemisphere was significantly increased in the rPSAP treatment group, which indicated that intranasal administration of rPSAP (9 μg/kg) was successfully delivered into the brain. * p<0.05 vs. Naive+NS (normal saline), # p<0.05 vs. MCAO+NS, @ p<0.05 vs. Naive+NS, $ p<0.05 vs. Naive+rPSAP 9μg/kg. (B) PSAP siRNA significantly inhibited PSAP expression. * p<0.05 vs. Naive+Scr siRNA, # p<0.05 vs. MCAO+Scr siRNA, @ p<0.05 vs. Naive+Scr siRNA, $ p<0.05 vs. Naive+PSAP siRNA. (C-D) Treatment with rPSAP improved the neurological functions, while knockdown of endogenous PSAP by pretreatment with PSAP siRNA exacerbated neurological deficits. * p < 0.05 vs. Sham, # p < 0.05 vs. Vehicle, & p < 0.05 vs. Scr siRNA. Scr siRNA, scramble siRNA. Error bars are represented as mean ± SD. One-way ANOVA, Tukey’s test, n=6 per group.

3.6. Experiment 4-2: Effects of rPSAP and PSAP siRNA on changes of downstream signaling molecules of Akt/ASK1 expression after MCAO

In the MCAO+Vehicle group, protein expression of phosphorylated-Akt (p-Akt), phosphorylated-ASK1 (p-ASK1) and Bcl-2 were significantly decreased compared to sham, while phosphorylated-JNK (p-JNK) and Bax were significantly increased compared to the sham group (p < 0.05, Fig. 6A-F). Additionally, rPSAP (9μg/kg) treatment significantly increased the expression of p-Akt, p-ASK1 and Bcl-2, whereas the expression of p-JNK and Bax were decreased compared to vehicle group at 24 h after MCAO (p < 0.05, Fig. 6A-F). Pretreatment with PSAP siRNA significantly suppressed p-Akt, p-ASK1 and Bcl-2 expression compared to the Scr siRNA control group, whereas, p-JNK and Bax expression were increased compared to the Scr siRNA control group at 24 h after MCAO (p < 0.05, Fig. 6A-F).

Fig. 6. The effects of rPSAP and PSAP siRNA on changes of downstream signaling molecules of Akt/ASK1 expression at 24 h after MCAO.

Fig. 6.

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Exogenous rPSAP significantly increased the expression of p-Akt, p-ASK1 and Bcl-2 (A-C, E), while p-JNK and Bax were reduced (A, D, F). However, following the knockdown of endogenous PSAP using PSAP siRNA, there was a further decrease in the expression of p-Akt, p-ASK1, and Bcl-2 (A-C and E), while the expression of p-JNK and Bax showed a further increase. * p < 0.05 vs. Sham, # p < 0.05 vs. Vehicle, & p < 0.05 vs. Scr siRNA. Error bars are represented as mean ± SD. One-way ANOVA, Tukey’s test, n=6 per group.

3.7. Experiment 5-1: rPSAP attenuated neuronal apoptosis via GPR37 signaling at 24 h after MCAO

Since MCAO results in neuronal degeneration and apoptosis, FJC and TUNEL staining were used at 24 h after MCAO to test whether rPSAP treatment will provide therapeutic benefits and attenuate neuronal apoptosis. Higher levels of FJC positive neurons were observed in the vehicle group when compared to the sham group (p < 0.05, Fig. 7A and C). rPSAP significantly reduced the FJC positive neurons in the penumbra compared to vehicle (p < 0.05, Fig. 7A and C); however, knockdown of GPR37 by pretreatment with GPR37 siRNA increased FJC positive neurons compared to MCAO+rPSAP or MCAO+rPSAP+Scr siRNA groups (p < 0.05, Fig. 7A and C), which means GPR37 siRNA reversed the protective effects of rPSAP. Also, higher levels of TUNEL positive neurons were observed in the vehicle group compared to the sham group (p < 0.05, Fig. 7B and D); however, treatment with rPSAP reduced the number of TUNEL positive neurons in the penumbra compared to the vehicle group (p < 0.05, Fig. 7B and D). Similar to FJC staining, pretreatment with GPR37 siRNA increased TUNEL positive neurons compared to MCAO+rPSAP or MCAO+rPSAP+Scr siRNA groups (p < 0.05, Fig. 7B and D).

Fig. 7. Effects of rPSAP and GPR37 siRNA on neuronal apoptosis at 24 h after MCAO.

Fig. 7.

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(A) Representative microphotographs and (C) quantitative analysis of Fluoro-Jade C staining (FJC)-positive neurons at the ischemic penumbra cortex. (B) Representative images and (D) quantitative analysis of TUNEL staining with neurons in the ipsilateral cortex. (C, D) Data showed that there were more FJC-positive and TUNEL-positive neurons observed in the vehicle group compared to the sham group; whereas, rPSAP treatment significantly decreased positively stained neurons. However, GPR37 siRNA reversed the effects of rPSAP, increasing the number of positive stained neurons. * p < 0.05 vs. Sham, # p < 0.05 vs. MCAO+Vehicle, & p < 0.05 vs. MCAO+rPSAP or MCAO+rPSAP+Scr siRNA. Scale bar=100μm. Error bars are represented as mean ± SD. One-way ANOVA, Tukey’s test, n=6 per group.

3.8. Experiment 5-2: GPR37 siRNA and LY294002 reversed the effects of rPSAP on neurobehavior function at 24 h after MCAO

The knockdown efficacy of GPR37 siRNA was demonstrated by western blot. In vivo knockdown of GPR37 by using GPR37 siRNA significantly decreased GPR37 expression in the ipsilateral hemisphere (p < 0.05, Fig. 8A). Also, GPR37 siRNA significantly decreased the neurological scores of Modified Garcia and beam walking compared to MCAO+rPSAP or MCAO+rPSAP+Scr siRNA groups at 24 h after MCAO (p < 0.05, Fig. 8B-C). Similarly, neurological scores in the MCAO+rPSAP+LY294002 group were significantly lower compared to MCAO+rPSAP or MCAO+rPSAP+DMSO groups at 24 h after MCAO (p < 0.05, Fig. 8B-C).

Fig. 8. GPR37 siRNA and LY294002 reversed the effects of rPSAP on neurobehavior function at 24 h after MCAO.

Fig. 8.

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(A) GPR37 siRNA efficiently knocked down GPR37 expression in the ipsilateral hemisphere. * p<0.05 vs. Naive+Scr siRNA, # p<0.05 vs. MCAO+Scr siRNA, @ p<0.05 vs. Naive+Scr siRNA, $ p<0.05 vs. Naive+GPR37 siRNA. Pretreatment with GPR37 siRNA and LY294002 significantly decreased Modified Garcia score (B) and beam walking score (C). * p<0.05 vs. Sham, # p<0.05 vs. Vehicle, & p<0.05 vs. rPSAP+Scr siRNA, @ p<0.05 vs. rPSAP+DMSO. Error bars are represented as mean ± SD. One-way ANOVA, Tukey’s test, n=6 per group.

3.9. Experiment 5-3: rPSAP attenuated neuronal apoptosis via the GPR37/PI3K/Akt/ASK1 signaling pathway at 24 h after MCAO

Western blot results showed that pretreatment with GPR37 siRNA significantly reduced the expression of PI3K, p-Akt, p-ASK1 and Bcl-2 compared to MCAO+rPSAP or MCAO+rPSAP+Scr siRNA groups, while p-JNK and Bax were increased compared to MCAO+rPSAP or MCAO+rPSAP+Scr siRNA groups (p < 0.05, Fig. 9A-G). Consistently, the PI3K inhibitor LY294002 significantly decreased the protein expression of PI3K, p-Akt, p-ASK1 and Bcl-2, while p-JNK and Bax were increased compared to both MCAO+rPSAP and MCAO+rPSAP+DMSO groups (p < 0.05, Fig. 9A-G).

Fig. 9. rPSAP attenuated neuronal apoptosis via the GPR37/Akt/ASK1 signaling pathway at 24 h after MCAO.

Fig. 9.

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(A) Representative Western blot images and quantitative analyses of (B) PI3K, (C) p-Akt/Akt, (D) p-ASK1/ASK1, (E) p-JNK/JNK, (F) Bcl-2 and (G) Bax. rPSAP increased PI3K, p-Akt, p-ASK1, Bcl-2, while p-JNK and Bax were decreased; these effects were reversed with GPR37 siRNA and LY294002. * p<0.05 vs. Sham, # p<0.05 vs. Vehicle, & p<0.05 vs. rPSAP+Scr siRNA, @ p<0.05 vs. rPSAP+DMSO. Error bars are represented as mean ± SD. One-way ANOVA, Tukey’s test, n=6 per group.

4. Discussion

In the present study, we reported the following observations: (1) endogenous PSAP and GPR37, expressed on neurons, exhibited an increase in expression during the acute phase of MCAO; (2) intranasal administration of rPSAP (9μg/kg) not only reduced infarct volume but also improved short- and long-term neurological functions, mediated through the upregulation of PI3K, p-Akt, p-ASK1, and Bcl-2, and the downregulation of p-JNK and Bax; (3) immunohistochemical analysis revealed that rPSAP decreased the count of degenerating (FJC positive) and apoptotic (TUNEL positive) neurons following MCAO; (4) knockdown of endogenous PSAP further aggravated neurological deficits after MCAO; (5) GPR37 siRNA abolished the anti-apoptotic effects of rPSAP; the reliance of rPSAP on the PI3K/Akt pathway was established when the PI3K inhibitor LY294002 reversed its neuroprotective effects post-MCAO. Taken together, these findings demonstrate that intranasal administration of exogenous rPSAP reduced neuronal apoptosis via the GPR37/PI3K/Akt/ASK1 pathway in rats after MCAO. (Fig. 10).

Fig. 10. Proposed pathway for rPSAP via GPR37/PI3K/Akt/ASK1 after MCAO.

Fig. 10.

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In the acute stage of cerebral artery occlusion in stroke, a hypoxic state is created that activates neuronal apoptotic pathways (Deng et al., 2020). Therefore, a reasonable approach for treatment would involve enhancing neuronal survival. Studies have descriptively shown PSAP to be anti-apoptotic on neurons, yet the intracellular mechanisms involved remain insufficiently explored (Morita et al., 2001). Consistent with literature, we confirmed the interaction of PSAP on neurons using immunohistochemistry and noticed an increased expression of PSAP in the penumbra after MCAO (Costain et al., 2010). Western blot analyses confirmed that intranasal administration of rPSAP significantly upregulated the expression of survival proteins, p-Akt and Bcl-2, and concurrently downregulated the apoptotic marker Bax. Congruently, the number of FJC positive and TUNEL positive neurons were reduced after rPSAP treatment, leading to improved neurological outcomes.

Interestingly, PSAP has been demonstrated to facilitate neuroprotection by interacting with two orphan G protein-coupled receptors, GPR37 and GPR37L1 (Meyer et al., 2013). These G protein-coupled receptors are mainly expressed in the nervous system on neurons and glia (Cahoy et al., 2008; Imai et al., 2001; Liu et al., 2018; Valdenaire et al., 1998). Specifically, GPR37 is concentrated on neuronal cells in the hippocampus of the brain, including pyramidal cells of Ammon’s horn and granule cells of the dentate gyrus (Zeng et al., 1997); however, GPR37L1 is expressed exclusively on glial cells (Valdenaire et al., 1998). In primary astrocytes, rPSAP was shown to be neuroprotective against oxidative stress through the activation of GPR37 (Meyer et al., 2013). PSAP’s neuroprotective effects were preserved in GPR37L1 knockout mice (Jolly et al., 2018). It can be inferred that PSAP’s anti-apoptotic actions are mediated primarily through GPR37 instead of GPR37L1. In our current study, we focused on exploring the downstream signaling pathways of GPR37. Paralleling literature, our immunofluorescence data showed GPR37 co-localization with neurons and an increase in endogenous expression after MCAO. Moreover, silencing GPR37 using siRNA led to reduced expression of GPR37 and negated the neuroprotective effects, resulting in an increase in the number of degenerating and apoptotic neurons post-rPSAP treatment. Based on these results, it can be concluded that rPSAP mediates its neuroprotective action through the GPR37 receptor.

The Akt serine/threonine kinases promote cellular survival via phosphorylation of downstream effectors, which inhibit death-inducing proteins (Kim et al., 2001). Other studies have found that PSAP attenuates apoptosis, but the mechanistic understanding is limited (Ochiai et al., 2008). To explore the potential of an alternate pathway, we employed siRNA to inhibit GPR37. In the group pretreated with GPR37 siRNA, we observed a decrease in the levels of PI3K, p-Akt, p-ASK1, and Bcl-2, while levels of p-JNK and Bax increased. Similarly, inhibiting the downstream PI3K pathway blocked the activation of PI3K, p-Akt, and p-ASK1, leading to increased neuronal apoptosis post-MCAO. These mechanistic insights indicate a connection between PSAP's anti-apoptotic effects and the activation of the GPR37/PI3K/Akt/ASK1 signaling pathway.

There are some limitations in the present study. First, our research focused solely on the neuroprotective effects of PSAP in neuronal apoptosis following MCAO in rats. However, it's important to acknowledge that other potential protective roles of PSAP, such as preserving the blood-brain barrier and reducing neuroinflammation post-MCAO, were not examined and thus cannot be ruled out. Thus, additional studies are warranted to investigate these other functions and their possible underlying mechanisms. Second, previous studies have reported that PSAP activation of extracellular signal-regulated kinase (ERK) phosphorylation may be crucial in the regulation of neuronal apoptosis (Meyer et al., 2013; Ochiai et al., 2008); therefore, additional research is needed to elucidate the relationship between PSAP and ERK. Third, since previous studies have reported that the activation of GPR37L1 is neuroprotective for pyramidal neurons of the hippocampus in an in vitro ischemic model, PSAP activation of GPR37L1 may be responsible for additional neuroprotective effects. Further research is required to elucidate the cognitive effects of PSAP.

In conclusion, intranasal administration of exogenous rPSAP reduced infarct volume, were mediated through the activation of GPR37/PI3K/Akt/ASK1 signaling pathway and subsequent inhibition of apoptotic proteins. Therefore, activation of GPR37 via rPSAP may be a promising therapeutic strategy for the treatment of ischemic stroke patients.

Supplementary Material

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Highlights.

  • Intranasal administration of exogenous rPSAP reduced infarct volume, attenuated neuronal apoptosis, and improved short-term and long-term neurological functions.

  • Knockdown of endogenous PSAP further aggravated neurological deficits after MCAO.

  • rPSAP alleviated neuronal apoptosis by binding and activating GPR37.

  • Novelty lies in the intracellular pathway after treatment with rPSAP in MCAO rats. rPSAP attenuates neuronal apoptosis via activation of the GPR37/PI3K/Akt/ASK1 signaling pathway after MCAO in rats.

Funding

This work was supported by the National Institutes of Health [grant numbers NS081740, NS082184]; the National Natural Science Foundation of China [grant number 82101531]; and the Natural Science Foundation of Zhejiang Province [grant numbers Y21H090049, LQ19C090006 and LQ19H090015].

Abbreviations

PSAP

Prosaposin

rPSAP

recombinant human prosaposin

GPR37

G protein-coupled receptor 37

siRNA

small interfering ribonucleic acid

IHC

Immunohistochemistry

PI3K

Phosphoinositide 3-kinase

Akt

Protein kinase B

ASK1

Apoptosis signal-regulating kinase 1

MCAO

Middle Cerebral Artery Occlusion

NS

Normal Saline

SD

Sprague–Dawley

TTC

2,3,5-triphenyltetrazolium chloride

FJC

Fluoro-Jade C

TUNEL

Terminal deoxynucleotidyl transferase dUTP nick end labeling

DMSO

Dimethyl Sulfoxide

WB

Western blot

Footnotes

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Conflicts of interest

The authors declare that they have no conflict of interests.

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