GPR75 knockdown alleviates mitochondrial dysfunction via AMPK in diabetic retinal ganglion cells

doi:10.21203/rs.3.rs-5253570/v1

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Research Article

GPR75 knockdown alleviates mitochondrial dysfunction via AMPK in diabetic retinal ganglion cells

https://doi.org/10.21203/rs.3.rs-5253570/v1

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Background

Mitochondrial dysfunction plays a crucial role in retinal ganglion cells (RGCs) injury, the early pathogenesis of diabetic retinopathy (DR). G protein-coupled receptor 75 (GPR75), an orphan receptor, is a novel regulator of metabolic diseases. However, the role and mechanisms of GPR75 underlying diabetic RGCs mitochondrial dysfunction has not been reported.

Methods

High glucose (HG)-treated RGCs and streptozotocin (STZ)-induced C57BL/6 diabetic mice were used in the present study. GPR75-knockdown adeno-associated virus (AAV), GPR75-overexpression (OE) plasmid, and AMPK-activator AICAR were utilized to investigate the role of GPR75 in DR. Retinal thickness and cell number were assessed with optical coherence tomography (OCT) and hematoxylin and eosin (HE) staining. Mitochondrial damage, reactive oxygen species (ROS) levels, and ATP production in the retina and RGCs were assessed with transmission electron microscopy (TEM), Mito-Tracker Red staining, dihydroethidium (DHE) staining, and ATP assay kits. We also assess the expression of GPR75, AMPK, p-AMPK, pyroptosis (NLRP3, Cleaved-Caspase-1, IL-1β, IL-18, GSDMD, N-GSDMD), apoptosis (Cleaved-Caspase-3, Cytochrome C, Bax, Bcl-2) and mitochondrial homeostasis (OPA1, NDUFS3, DRP1). The interaction between GPR75 and AMPK was detected through co-immunoprecipitation (CO-IP) and double immunofluorescence staining.

Results

Compared with control retina and RGCs, diabetic retina and HG-treated RGCs exhibited increased GPR75 expression and AMPK dephosphorylation accompanied by pyroptosis, apoptosis, and a decrease in retinal thickness and RGCs number. Moreover, we observed increased DRP1 expression, decreased expression of OPA1 and NDUFS3, reduced ATP production, abnormal mitochondrial morphology and quantity, and increased ROS accumulation in diabetic retina and HG-treated RGCs, indicating mitochondrial dysfunction. What’s more, GPR75-knockdown reversed these phenomena. Mechanistically, the upregulation of GPR75 inhibits AMPK, leading to mitochondrial dysfunction with increased ROS accumulation, ultimately resulting in RGCs pyroptosis and apoptosis. Additionally, double immunofluorescence demonstrated the presence of both GPR75 and AMPK located in RGCs, and CO-IP revealed an interaction between GPR75 and AMPK in RGCs. Notably, AICAR counteracted the effects of GPR75-OE on pyroptosis, apoptosis and mitochondrial dysfunction in RGCs.

Conclusions

GPR75 induces mitochondrial dysfunction by interacting with AMPK and inhibiting its phosphorylation, which contribute to RGCs pyroptosis and apoptosis in DR. These findings suggest that GPR75 can serve as a therapeutic target in DR treatment.

GPR75

AMPK

Mitochondrial dysfunction

Diabetic retinopathy

Retinal ganglion cells

DR is a common complication linked to diabetes, playing a crucial role in vision loss and blindness in adults of working age worldwide. The progression of DR includes intricate mechanisms, and at present, there are only a few effective treatment options available. RGCs function as the exclusive output neurons within the retina, responsible for integrating and transmitting visual information to the brain [1]. RGCs dysfunction contributes directly to visual impairment in diabetic patients [2, 3].

G protein-coupled receptors (GPCRs) serve as membrane receptor proteins and participate in a variety of physiological functions [4, 5]. Among the GPCR family, GPR75 is significant for its contributions to energy regulation and glycemic control [6–8]. Studies show that GPR75 is highly expressed in adipocytes, vascular endothelial cells, vascular smooth muscle cells, as well as pancreatic beta cells. However, the expression and role of GPR75 in RGCs have not been reported. More recently, it was reported that activated GPR75 can lead to vascular endothelial cell injury by inducing ROS production [9]. In a high-fat diet model, GPR75 induced adipocyte hypertrophy by promoting inflammatory responses and impairing mitochondrial function [7]. Research indicates that disorders in energy metabolism due to mitochondrial dysfunction, along with imbalances in mitochondrial dynamics and the buildup of free radicals, serve as primary contributors to RGC damage and the eventual onset of DR in individuals with diabetes [10–14]. However, the potential relationship between GPR75 and diabetic RGCs mitochondrial dysfunction remains to be revealed.

Adenosine-5'-monophosphate (AMP)-activated protein kinase (AMPK) serves as a conserved energy sensor within cells, playing a critical role in maintaining energy balance during cellular stress [15–18]. Research indicates that activating AMPK can initiate autophagy in DR, thereby providing protection for RGCs from undergoing cell death. [19]. Furthermore, AMPK is vital for the preservation of mitochondrial homeostasis. [20–22]. Importantly, recent research has indicated that certain GPCRs contribute to their effects by activating AMPK [23–25]. The International Union of Basic and Clinical Pharmacology classifies GPR75 as an orphan receptor [26]. However, whether GPR75 can interact with AMPK, and then mediate RGCs mitochondrial dysfunction in DR has not been reported.

Therefore, the role of GPR75 in diabetes-induced mitochondrial disorder in RGCs was investigated. Furthermore, the potential mechanisms were also revealed in this study.

Animals

Healthy, male C57BL/6 mice, aged 6-8 weeks and weighing 20±2g, were purchased from Beijing Huafukang Biotechnology Co, Ltd (Beijing, China). All the mice were housed under a specific pathogen-free (SPF) room, given free access standard laboratory diet and water and maintained in a constant environment with standard temperature, standard humidity and a 12h light/12h dark cycle. All animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and the procedures were approved by the Animal Care and Use Committee of Jinzhou Medical University [SYXK•(Liao)•2019–0007].

Experimental procedures

Group design

After 2 w of adaptive feeding, the mice were randomly divided into 4 groups: control group (CON) (mice injected intraperitoneally with citric acid buffer and intravitreally with PBS) (n=10), diabetes group (DM) (mice injected intraperitoneally with STZ and intravitreally with PBS) (n=10), AAV knockdown group (AAV-shGPR75) (mice administered STZ intraperitoneally and AAV2/9-shGPR75 intravitreally) (n=10) and AAV negative control group (AAV-NC) (mice administered STZ intraperitoneally and AAV2/9-NC intravitreally) (n=10).

Preparation of diabetic mouse models

Diabetes was induced in mice by administering an intraperitoneal injection of a 150 mg/kg dose of STZ (Sigma Aldrich, #V900890, USA) following a 12 h fasting period. The STZ was prepared by dissolving it in a freshly prepared 10 mM sodium citrate solution and was stored at low temperature, shielded from light. Mice that presented with two consecutive random blood glucose readings exceeding 16.7 mM were identified as diabetic and subsequently included in this research.

Intravitreal injection for GPR75 inhibition

An AAV serotype 2/9 (AAV2/9), allowing for RNAi against GPR75 (AAV2/9-shGPR75), was constructed and packaged by OBio Technology (Shanghai, China). Detailed AAV2/9-shGPR75 information is presented in Supplementary Material S1. After 4 w of diabetes induction, the vitreous cavities of mice in each group were injected with the appropriate reagents. Mice were deeply anesthetized using 1% sodium pentobarbital at a dosage of 80 mg per kilogram of body weight. Pupils were dilated using tropicamide, and once dilation was achieved, the orientation of the pupils was adjusted to ensure alignment with the corneal limbus. The site for injection was chosen on the temporal aspect of the eye, located 1-2 mm posterior to the corneal limbus. A small hole was first punctured at this location with a 30G insulin needle, and then 2ul of the corresponding reagent was injected with a 10ul microinjector needle (33G) (Hamilton, Reno, NE, USA). To avoid damaging the lens and retina, inject slowly and leave it in for about a minute before removing the needle. Finally, the surface of the eye was covered with gatifloxacin gel. After 8 w of intravitreal injection, the mice were tested for relevant indicators.

Cell culture

Rat RGCs cell line (RGC-5, ATCC, USA) were cultured in DMEM/F12 (Gibco, #11330057, USA) containing 10% foetal bovine blood (FBS, #16140071, Gibco, USA) and 1% penicillin/streptomycin (Hyclone, #SV30010, USA) at 37 °C, 5% CO2.

RNA interference and gene overexpression

Recombinant plasmids for GPR75 overexpression (OE-GPR75), shRNA specifically targeting GPR75 (shRNA-GPR75), as well as the respective negative control plasmids (OE-NC and shRNA-NC) were all constructed by SWS Biotechnology Co (Tianjin, China). Supplementary Material S1 provides detailed information on the recombinant plasmids used in this study. RGCs were grown in 6-well plates with DMEM/F12 medium that contained 10% FBS until they reached 70-90% confluence. The transfection of plasmids into the RGCs was performed utilizing Lipofectamine 2000 (Thermo Fisher, #11668030, USA). Two days post-transfection, we evaluated the impacts of gene knockdown or overexpression via RT-qPCR and Western blotting.

OCT for retina thickness

The structure of the retina was assessed using OCT (Phoenix, USA). Briefly, an administration of 80 mg/kg of body weight of 1% pentobarbital sodium was given to ensure deep anesthesia in the mice. To dilate the pupils, tropicamide was used, while gatifloxacin gel was applied for moisture and protection of the cornea. After achieving full dilation of the pupils, the eyes were aligned directly in front of the anterior lens. The next step involved adjusting the OCT lenses and settings to take images of the retina in mice using a linear transverse scanning mode. After the experiment, place the mice on a reheater and wait for them to wake up. The thickness of the complete retina was assessed using Image J, encompassing several layers: the inner limiting membrane (ILM), nerve fiber layer (NFL), ganglion cell layer (GCL), inner nuclear layer (INL), inner plexiform layer (IPL), outer nuclear layer (ONL), outer plexiform layer (OPL), outer limiting membrane (OLM), along with both the inner and outer segments (IS/OS) and the retinal pigment epithelium (RPE).

Total mRNA extraction and real-time quantitative polymerase chain reaction (RT-qPCR) analysis

RNA was isolated from the retina and RGCs through the use of TRIzol reagent (Tiangen, #DP424, China). Subsequently, the synthesis of cDNA was conducted employing the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher, #K1622, USA). The RT-qPCR analysis took place on a CFX Connect™ Real-Time System (Bio-Rad, USA) utilizing the Taq Pro Universal SYBR qPCR Master Mix (Vazyme, #Q511, China). The sequences of the primers used for the amplification process are provided in Supplementary Material S2. For evaluating expression levels, the threshold cycle (Ct) values were used, and relative expression levels were calculated using the 2^-ΔΔCT method, with β-actin serving as the control gene.

Protein extraction and Western blotting analysis

Proteins from the retina or RGCs were extracted utilizing RIPA lysate (Solarbio, #R0010, China) that was supplemented with 1% PMSF (Solarbio, #R0100, China) and 1% phosphatase inhibitor (KeyGEN, #KGB5101-2, China). First, the protein samples underwent separation through a 10% SDS-PAGE gel (Solarbio, #P1200, China), utilizing a pre-stained protein ladder (UElandy, #P8028L, China) as a molecular weight standard. The isolated proteins were subsequently transferred to PVDF membranes (Sigma-Aldrich, #IPVH00010, USA). After treating the membranes with a 5% BSA solution (Solarbio, #A8020, China) or using a rapid closure solution (Affinibody, #AIWB-004, China) to block impurities, they were incubated with the primary antibody overnight at 4°C. Afterward, goat anti-mouse or rabbit IgG (Proteintech, #SA00001-1/2, China), which was conjugated to horseradish peroxidase (HRP), was incubated for 2 h. Lastly, the detection of signals was performed utilizing the ChemiDoc™ Touch Imaging System (Bio-Rad, USA) and employing ECL Light Emitting Reagent. The protein expression levels were determined through densitometric analysis of the bands via Image J software, with normalization against β-actin or Na+/K+ ATPase. The primary antibodies utilized in this study included: GPR75 (1:2000, Bioss, #bs-16263R, China), AMPK (1:2000, Affinity, #AF6423, China), p-AMPK (Thr172) (1:2000, Affinity, #AF3423, China), Bax (1:2000, Wanleibio, #WL01637, China), Bcl-2 (1:1500, Wanleibio, #WL01556, China), Cytochrome C (1:2000, Bioss, #bs-0013R, China), Cleaved-Caspase-3 (1:1000, Affinity, #AF7022, China), NLRP3 (1:1000, Abcam, #ab214185, USA), Cleaved-Caspase-1(1:1000, Wanleibio, #WL02996a, China), IL-1β (1:2000, Bioss, #bs-6319R, China), IL-18 (1:2000, Bioss, #bs-0529R, USA), GSDMD (1:2500, Abcam, #ab219800, USA), NDUFS3 (1:1000, Affinity, #DF4210, China), OPA1 (1:1000, Bioss, #bs-11764R, China), DRP1 (1:2000, Boster, #A00556-2, China), Na+/K+ ATPase (1:8000, Abmart, #T55159, China), β-actin (1:8000, Bioss, #bs-0061R, China). Antibodies were diluted using Antibody Dilution Sensitisation Protection Solution (Affinibody, #AIWB-009, China).

Extraction of Membrane and Cytoplasmic Proteins

Proteins in membranes and cytoplasm were extracted according to the guidelines provided in the Membrane and Cytoplasmic Protein Extraction Kit (Beyotime, #P0033, China).

Immunohistochemistry for GPR75

The sections that were embedded in paraffin were first subjected to deparaffinization and then underwent antigen retrieval, followed by a 10-minute incubation in 3% fresh H2O2. After this step, the sections were blocked using 5% BSA at 4°C for a duration of 2 h. Next, they were incubated with GPR75 at a dilution ratio of 1:100. Subsequently, the sections were incubated with biotin-conjugated secondary antibody and avidin-biotin peroxidase complex (Vector Laboratories, USA). The sections were later stained with DAB (Zsbio, China), then dehydrated, cleared, and sealed. Lastly, fluorescence images were captured using a fluorescence microscope (Olympus, Japan).

Immunofluorescence assay

Expression of GPR75 and Brn3a was assessed using immunofluorescence staining. The secondary antibodies employed were goat anti-rabbit IgG Alexa Fluor 488 (1:200, Abcam, #ab150077, USA) and goat anti-mouse IgG Alexa Fluor 594 (1:200, Abcam, #ab150116, USA). In addition, the slides underwent counterstaining with DAPI (Abcam, #ab104139, USA). Fluorescence microscope (Nexcope, China) was used for the visualization of the fluorescent images. Brn3a is a specific biomarker of RGCs. Brn3a-positive nuclei were counted with the Image J software.

RGCs were fixed in 4% paraformaldehyde for 30 min. After the fixation process, RGCs were incubated at 4°C overnight either GPR75 rabbit antibody (1:100) or AMPK mouse antibody (1:50, Santa Cruz, #sc-74461, USA). Subsequently, RGCs were treated with both secondary antibodies for 2 h. To stain the nuclei, DAPI was applied for 5 minutes. Lastly, fluorescence images were captured using a fluorescence microscope (Nexcope, China).

HE staining

HE staining on retinal paraffin sections was carried out following the guidelines provided (Solarbio, #G1120, China). The quantification of cell nuclei within the GCL was conducted utilizing Image J software.

TEM imaging

Mitochondrial morphological changes in retinal tissue were assessed by TEM. Sample processing and follow-up experiments were sent to Beijing Zhongke Baisi Technical Service Co.

Detection of ROS production in the retinal tissue

DHE interacts with DNA after being oxidized by O2·–, leading to the formation of fluorescent ethidium bromide; the level of this fluorescence corresponds to the amount of ROS generated. Therefore, we analyzed ROS production in retinal tissues utilizing DHE. In summary, retinal tissue sections, measuring 5μm in thickness and embedded in paraffin, were prepared from each group and exposed to DHE (10 mM, MCE, #HY-D0079, China) in darkness at 37°C for 1 h. Following DAPI staining, observations were conducted using a fluorescence microscope (Nexcope, China) and DHE fluorescence intensity was assessed through Image J software

Measurement of cellular ROS in cultured RGCs

The assessment of ROS generation in RGCs cultured in vitro was conducted utilizing DHE staining. RGCs were treated with a 10μM DHE solution at 37°C for 1 h. Once the staining process was completed, the samples were examined using a fluorescence microscope (Nexcope, China), and the DHE fluorescence intensity was assessed with Image J software.

ATP assay

Assessment of ATP Levels in Mouse Retina and RGC Using ATP Chemiluminescent Assay Kits (Elabscience, #E-BC-F002, China). The CytationTM5 (BioTek, USA) was utilized to gather the chemiluminescent signals.

Staining of mitochondrial number in RGCs

RGCs were cultured in glass-bottomed cell culture dishes (Nest, China) for 24 h. Following exposure to various stimuli, the number of mitochondria was evaluated through red staining with Mito-Tracker. Confocal microscopy (Olympus, Japan) was utilized to analyze immunofluorescence images. The quantification of total fluorescence intensity for Mito-Tracker Red was performed with Image J software. Additionally, the total fluorescence intensity was again measured with the confocal microscope (Olympus, Japan).

CO-IP assay

The interaction between GPR75 and AMPK was detected using the CO-IP kit (Beyotime, #P2181S, China). RGCs that overexpress GPR75 were collected 48 hours following transient transfection. The kit's lysis buffer was applied to break down the cells, and following centrifugation, the resulting supernatant was gathered. For immunoprecipitation (IP), proteins were treated with either Mouse IgG Magnetic Beads or Anti-Flag Magnetic Beads. After washing, proteins were eluted using 3X Flag Peptide elution buffer followed by immunoblotting (IB) with appropriate antibodies.

Statistical analysis

The data are expressed as the means ± standard deviations (SD). Two groups of the data were compared using the two-tailed Student's t test. Data from more than two groups were compared using one-way analysis of variance (ANOVA) followed by Turkey's post-test. Statistical analyses were evaluated using GraphPad Prism 9 (GraphPad Software, USA). All experiments were replicated independently a minimum of three times. A value of p < 0.05 was considered statistically significant.

GPR75 expression is elevated in the retina of diabetic mice and HG-treated RGCs

To assess the role of GPR75 in the progression of DR, we examined the expression of GPR75 both in vivo and in vitro. Immunofluorescence staining of GPR75 and the RGCs marker Brn3a showed that GPR75 is predominantly localized at the membrane of RGCs in the retina (Fig.1A). Moreover, our results indicated that GPR75 was upregulated in diabetic retina and HG-treated RGCs compared with control group respectively (Fig.1A-I). The findings indicated that GPR75 in RGCs is involved in DR.

Suppressing GPR75 ameliorates retina and RGCs injury in DR

To investigate whether increased expression of GPR75 contributes to retina and RGCs injury in DR, we used AAV-shGPR75 (AAV-shGPR75) to knock down GPR75 in diabetic retina. The effectiveness of GPR75 knockdown was verified using Western blotting and RT-qPCR analyses. The results revealed that the expression levels of GPR75 were significantly reduced in the retina of mice from AAV-shGPR75 group when contrasted with DM group (Fig.2A-C). Subsequently, we assessed the role of GPR75 in pathological retinal damage. Retina OCT showed increased thickness in AAV-shGPR75 group compared with DM group (Fig.2D-E). HE staining indicated that the cell count in the GCL was restored in AAV-shGPR75 group compared with DM group (Fig.2F-G). Moreover, the density of RGCs was evaluated through immunofluorescent staining of Brn3a, a specific biomarker of RGCs. The findings indicated a reduction in the number of RGCs within the retinas of DM group. Conversely, the density of RGCs was found to be higher in the retinas of diabetic mice with GPR75 knockdown (Fig. 2H-I). Thus, these results indicate that blocking GPR75 provides a safeguard for the retina and RGCs against damage in DR.

GPR75 mediates pyroptosis and apoptosis in both diabetic retina and HG-treated RGCs

Cell death in RGCs is involved in DR including pyroptosis and apoptosis [2, 27, 28]. In order to elucidate the mechanisms of GPR75 deficiency protecting against DR, we initially analyzed the expression of proteins related to pyroptosis and apoptosis. The analysis using Western blot demonstrated an increase in the levels of NLRP3, cleaved Caspase-1, IL-1β, IL-18, GSDMD, and N-GSDMD in DM group compared with CON group, whereas GPR75 knockdown decreased the expression of pyroptosis-related proteins (Fig.3 A-G). In terms of apoptosis, we noted an increase in the levels of Cleaved-Caspase-3 and Cytochrome C in DM group compared with CON group, whereas GPR75 knockdown reduced the expression of these proteins (Fig.3G-K). Furthermore, GPR75 knockdown also significantly decreased the diabetes-induced increase in the Bax/Bcl2 ratio, which was an indicator of apoptosis (Fig.3I). These data suggest that GPR75 inhibition alleviates pyroptosis and apoptosis in diabetic retina. Given that HG induced GPR75 activation in RGCs (Fig.1G-F), we further confirmed whether GPR75 participates in HG-induced RGCs injury. For this purpose, we used small hairpin RNA (shRNA) and OE plasmid to inhibit or increase GPR75 expression (Fig.3L-N). Western blot analysis indicated an elevation in the levels of NLRP3, Cleaved-Caspase-1, IL-1β, IL-18, GSDMD, N-GSDMD, Cleaved-Caspase-3, and Cytochrome C in OE group relative to HG group. This finding implies that the overexpression of GPR75 intensified pyroptosis and apoptosis induced by HG. Conversely, the levels of proteins associated with pyroptosis and apoptosis were reduced in shRNA-GPR75 group compared with HG group, suggesting that GPR75 knockdown inhibited HG-induced RGCs injury (Fig.3O-X). Moreover, the Bax/Bcl-2 ratio was elevated in OE group compared with HG group, whereas GPR75 knockdown significantly mitigated the HG-induced increase in Bax/Bcl-2 ratio (Fig.3Y). Collectively, these findings indicate that GPR75 inhibition offers protection to the retina and RGCs from injury in DR by decreasing both pyroptosis and apoptosis.

GPR75 mediates mitochondrial dysfunction in diabetic retina and HG-treated RGCs

A growing body of evidence highlights the important role of ROS-induced oxidative damage in the onset of pyroptosis and apoptosis [27, 29]. Consequently, we investigated the possible regulatory function of GPR75 in the production of ROS within the diabetic retina. DHE staining indicated that the knockdown of GPR75 led to a decrease in ROS levels in diabetic retina, indicating that GPR75 may play a role in RGCs injury in DR through a ROS-dependent mechanism (Fig.4 A-B). Furthermore, recent findings suggest that dysfunctional or compromised mitochondria serve as the main contributors to ROS generation [30, 31]. Therefore, we investigated the effects of GPR75 activation on mitochondrial function in more detail. TEM micrographs revealed that mitochondrial structures in the retina showed significant swelling, formation of vacuoles, and fractured or absent mitochondrial cristae in DM group compared with CON group, whereas GPR75 knockdown restored the normal morphology of mitochondria (Fig.4C). Additionally, we observed that GPR75 knockdown resulted in a reduction of the mitochondrial fission protein DRP1 in diabetic retina, while simultaneously increasing the levels of the mitochondrial fusion protein OPA1 and the mitochondrial respiratory chain complex I protein NDUFS3, along with an increase in ATP content (Fig.4D-H). These findings suggest that GPR75 knockdown ameliorated mitochondrial kinetic imbalances and disturbances in energy metabolism in the diabetic retina. Subsequently, we examined how GPR75 influences mitochondrial dysfunction induced by HG in RGCs, similar to our in vivo observations. DHE staining indicated that ROS levels were significantly elevated in OE group when contrasted with HG group. However, the knockdown of GPR75 led to a marked decrease in ROS accumulation in RGCs treated with HG (Fig.4I-J). Mito-Tracker Red staining indicated that the number of mitochondria in RGCs was considerably reduced in OE group when contrasted with HG group. Conversely, GPR75 knockdown restored the mitochondrial number of HG-treated RGCs (Fig.4K-L). Additionally, when compared to HG-treated RGCs, the introduction of GPR75-OE resulted in increased protein expression of DRP1, while simultaneously decreasing the levels of OPA1 and NDUFS3, as well as reducing ATP content. However, GPR75 knockdown decreased DRP1 protein expression while increasing OPA1 and NDUFS3 expression and increasing ATP content compared with HG group (Fig.4M-Q). Altogether, these data illustrate that GPR75 mediates mitochondrial dysfunction in diabetic retina and HG-treated RGCs, ultimately leading to increased ROS generation.

GPR75 physically interacts with AMPK in RGCs

Previous studies have shown that AMPK activation is vital for maintaining mitochondrial equilibrium, which helps safeguard retinal cells from damage in DR [32-34]. Therefore, we evaluated the relationship between GPR75 and AMPK in RGCs。We conducted protein-protein docking experiments of GPR75 with AMPK using pymol software. The results indicated a good binding affinity between GPR75 and AMPK (Fig.5A). CO-IP analysis showed that both GPR75 and AMPK were present in the eluent labelled with Flag-GPR75, suggesting their interaction in RGCs (Fig.5B). Additionally, immunofluorescence co-localization studies further supported that GPR75 interacts with AMPK in RGCs (Fig.5C). Collectively, these findings suggest that GPR75 physically interacts with AMPK in RGCs.

GPR75 activation mediates mitochondrial dysfunction and cell death in RGCs by inhibiting AMPK activity

To evaluate the relationship between GPR75 and AMPK in RGCs in DR, we examined the effect of GPR75 on AMPK activity in diabetic retina and HG-treated RGCs. Western blotting showed that retinal AMPK phosphorylation was elevated in AAV-shGPR75 group compared with DM group (Fig.6A-B). Furthermore, overexpression of GPR75 further reduced AMPK activity in HG-treated RGCs, whereas knockdown of GPR75 restored AMPK activity (Fig.6C-D). These results suggest that GPR75 is a key regulator for inhibiting AMPK phosphorylation. To elucidate the role of AMPK activation in GPR75-mediated mitochondrial dysfunction and cellular damage in RGCs, the impact of an AMPK activator (5-aminoimidazole-4-carboxamido-ribonucleotide, AICAR) on the mitochondrial dysfunction and cellular injury mediated by GPR75 in RGCs was assessed by us (Fig.6E-F). The cultured RGCs were transfected with OE-NC or OE-GPR75 plasmids. After transfection for 48h, cells were treated with Vehicle or AICAR (10uM, 24h) and then exposed to HG (50mM, 48h). Subsequently, we examined the expression of proteins related to pyroptosis and apoptosis. Western blotting showed that AICAR treatment reduced the expression of NLRP3, Cleaved-Caspase-1, IL-1β, IL-18, GSDMD, N-GSDMD, Cleaved-Caspase-3, and Cytochrome C in RGCs treated with HG and OE-GPR75 plasmid, as well as reducing the Bax/Bcl-2 ratio (Fig.6G-Q). This suggests that activation of AMPK ameliorated GPR75-mediated pyroptosis and apoptosis in HG-treated RGCs. Additionally, we observed that activation of AMPK reduced ROS accumulation and concomitantly restored numbers of mitochondrial (Fig.6R-U). Meanwhile, the findings indicated that AICAR decreased the expression of DRP1 of RGCs treated with HG and OE-GPR75 plasmid, while simultaneously increasing the expression of OPA1 and NDUFS3, along with an increase in ATP content (Fig.6V-Z). Taken together, activation of AMPK ameliorated GPR75-mediated mitochondrial dysfunction and ROS accumulation, thereby protecting RGCs from injury in DR.

Retinal neurodegeneration is a significant factor contributing to the development of DR [35–38]. In this research, we clarify the contributory role of GPR75 up-regulation in promoting retina pathological alterations including RGCs pyroptosis and apoptosis. Mechanistically, we elucidated that elevated GPR75 results in decreased AMPK, contributing to mitochondrial homeostasis imbalance, and then leading to disturbance of mitochondrial dynamics, imbalance of energy metabolism, and excessive ROS production. These ROS, in turn, participates in triggering pyroptosis and apoptosis, as illustrated in Fig. 7.

Recent developments suggest that GPR75 represents a potential therapeutic target for conditions linked to metabolic syndrome, such as obesity, dyslipidemia, hypertension, and vascular disorders [39, 40]. In a pulmonary hypertension mouse model, the expression of GPR75 was observed to be increased in smooth muscle cells of the pulmonary artery. The deletion of GPR75 led to relaxation of the pulmonary artery and reduced hypoxia-driven pulmonary hypertension [41]. Additionally, in a high-fat diet model, GPR75 knockout in mice led to resistance to weight gain and improved glycemic control [6]. Furthermore, hypertension induces endothelial dysfunction and vascular inflammation through activation of GPR75 signaling [9]. However, the function of the GPR75 pathway in DR remains unreported. In our current investigation, we discovered that GPR75 is predominantly located in RGCs of the retina and that its expression is significantly increased in diabetic retina and HG-treated RGCs. Furthermore, inhibition of GPR75 improved pyroptosis and apoptosis in diabetic retina and HG-treated RGCs, which emphasizes the potential therapeutic benefits of targeting GPR75 in DR studies.

Dysfunction of mitochondria is involved in facilitating both cell pyroptosis and apoptosis, and its significance in the development of DR has been well documented [42–45]. Diabetes activates retinal matrix metalloproteinase 2 (MMP2) and MMP9, destroying retinal mitochondria and triggering an increase in ROS, which leads to cellular dysfunction and promotes apoptosis in retinal capillary cells [46]. In addition, sustained hyperglycemia stimulates TXNIP migration to mitochondria, leading to mitochondrial dysfunction and causing ROS accumulation, which activates NLPR3 inflammatory vesicles and promotes pyroptosis in retinal cells [47]. Targeting mitochondria for interventions against pyroptosis and apoptosis presents a promising avenue for the treatment of DR. Recent studies indicate that GPR75 leads to adipocyte hypertrophy and insulin resistance by inducing inflammation and impairing mitochondrial function in diet-induced obesity. Our data suggest that knockdown of GPR75 restores mitochondrial dynamic homeostasis and respiratory chain complexⅠexpression, resulting in increased ATP production and reduced ROS accumulation. In addition, TEM and Mito Tracker results showed that knockdown of GPR75 in the retina of diabetic mice and in HG-cultured RGCs exhibited normal mitochondrial structure and number. These findings highlight the essential function of GPR75 in mitochondrial impairment and provide valuable perspectives on possible pathways for therapeutic strategies aimed at the loss of RGCs and DR.

The AMPK consists of three subunits: α, β, and γ, and it is essential for preserving the homeostasis of mitochondria in cells and for balancing redox states [20, 48, 49]. Strong evidence indicates that the activation of AMPK can modulate mitochondrial homeostasis, reduce ROS, and mitigate cell death of various pathological processes [50–52]. AMPK activators and inhibitors have been used to illustrate that AMPK serves a protective function in reducing imbalances in mitochondrial dynamics and injuries caused by oxidative stress in the myocardium [53], brain [54] and liver [55]. We found that GPR75 interacts with AMPK in RGCs by molecular docking, immunoprecipitation and fluorescence co-localization. Additionally, our results show that mitochondrial damage occurs in both diabetic retina and HG-treated RGCs, and that elevated GPR75 expression is accompanied by AMPK dephosphorylation. In addition, molecular docking, immunoprecipitation and fluorescence co-localization results indicated an interaction between GPR75 and AMPK. We hypothesized that AMPK could serve as a downstream signaling molecule of GPR75, playing a role in diabetes-induced mitochondrial disorder in RGCs. To investigate the relationship between GPR75 and AMPK signaling, we utilized the AMPK activator AICAR. We found that AICAR treatment partially counteracted GPR75-mediated mitochondrial dysfunction, thereby attenuating pyroptosis and apoptosis in RGCs. This suggests that the AMPK signalling pathway is involved in GPR75-mediated mitochondrial damage in HG-treated RGCs.

Although our study demonstrated that knockdown of GPR75 alleviated retinal and RGCs injury in DR by restoring AMPK activity, certain limitations exist within our study. First, the use of a transgenic mouse model appears to effectively eliminate numerous background variations. Second, the interaction between GPR75 and AMPK has not been studied in depth. In a recent study, Pascal et al. demonstrated that 20-HETE is a high-affinity ligand for the GPR75 receptor using surface plasmon resonance techniques [56]. However, we did not examine the impact of the 20-HETE-GPR75 interaction on the retinal effects linked to GPR75. The significance of the 20-HETE-GPR75 interaction in retinal function is an area deserving further investigation.

In summary, this research significantly enhances our comprehension of how GPR75 contributes to mitochondrial dysfunction in RGCs and the progression of DR. Our data clearly show that elevated GPR75 expression is accompanied by AMPK inactivation during DR progression. Furthermore, we demonstrated that GPR75 deficiency inhibits mitochondrial dysfunction by restoring AMPK activity, which attenuates pyroptosis and apoptosis in RGCs, thereby delaying DR progression. Therefore, the down-regulation of GPR75 expression to enhance AMPK activity presents a promising therapeutic strategy for addressing RGC injury and metabolic disorders associated with mitochondrial dysfunction.

AAV	adeno-associated virus
AMPK	adenosine-5'-monophosphate (AMP)-activated protein kinase
CO-IP	co-immunoprecipitation
DHE	dihydroethidium
DR	diabetic retinopathy
GPCRs	G protein-coupled receptors
GPR75	G protein-coupled receptor 75
OCT	optical coherence tomography
RGCs	retinal ganglion cells
ROS	reactive oxygen species
shRNA	small hairpin RNA
STZ	streptozotocin
TEM	transmission electron microscopy

Acknowledgements

We wish to express our gratitude to all individuals who took part in the research. We also thank the drawing tools provided by Figdraw.

Authors’ contributions

MRL, XC, WQL, XZL and ZFZ participated in the research design. MRL, YLW, XYC, QLM, YRY, WD, JWH, NZ and JHS conducted the experiments. MRL, HDY and SXY contributed to data analysis and interpretation. MRL, XC, WQL, XZL and ZFZ drafted the paper. WRL, XZL and ZFZ supervised the study and revised the paper. All authors contributed to the paper and approved the final manuscript.

Funding

Support for this research was provided by the Foundation of Education Department of Liaoning Province (LJKMZ20221241) and the Natural Science Foundation of Liaoning Province of China (2023-MS-312).

Availability of data and materials

The information backing the conclusions of this research can be found within this article and its supplementary files. Any other pertinent data can be obtained from the corresponding authors upon a reasonable request.

Ethics approval and consent to participate

All experimental procedures described in this article were approved by the Animal Care and Use Committee of Jinzhou Medical University.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no conflict of interest.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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GPR75 knockdown alleviates mitochondrial dysfunction via AMPK in diabetic retinal ganglion cells

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