
==== Front
Heliyon
Heliyon
Heliyon
2405-8440
Elsevier

S2405-8440(24)12171-8
10.1016/j.heliyon.2024.e36140
e36140
Research Article
GPRASP2 deficiency contributes to apoptosis in the spiral ganglion cells via the AMPK/DRP1 signaling pathway
Huang Kun a
Cai Jing a
Lu Yajie ab
Wang Tianming c
Yue Shen a
Wei Qinjun ab
Yao Jun joelyao@njmu.edu.cn
ab⁎
Chen Zhibin czbnj@163.com
d⁎⁎
Cao Xin caoxin@njmu.edu.cn
ab⁎⁎⁎
a Department of Medical Genetics, School of Basic Medical Science, Nanjing Medical University, Nanjing, China
b Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing, China
c Central Laboratory, Translational Medicine Research Center, the Affiliated Jiangning Hospital of Nanjing Medical University, Nanjing, China
d Department of Otolaryngology, the First Affiliated Hospital with Nanjing Medical University, Nanjing, China
⁎ Corresponding author. Department of Medical Genetics, School of Basic Medical Science, Nanjing Medical University, No.101 Longmian Avenue, Nanjing, 211166, China. joelyao@njmu.edu.cn
⁎⁎ Corresponding author. Department of Otolaryngology, the First Affiliated Hospital with Nanjing Medical University, No.300 Guangzhou Road, Nanjing, 210029, China. czbnj@163.com
⁎⁎⁎ Corresponding author. Department of Medical Genetics, School of Basic Medical Science, Nanjing Medical University, No.101 Longmian Avenue, Nanjing, 211166, China. caoxin@njmu.edu.cn
13 8 2024
30 8 2024
13 8 2024
10 16 e3614026 7 2024
9 8 2024
9 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
G protein-coupled receptor-associated sorting protein 2 (GPRASP2) deficiency has been implicated in immunological inflammation, cancers, and neurological disorders. Our previous work revealed that the pathogenic mutation in GPRASP2 was responsible for X-linked recessive syndromic hearing loss (SHL). Given the specific high expression of GPRASP2 in the spiral ganglion, GPRASP2 likely contributes to the maintenance and functionality of neurons, potentially playing a role in synaptic transmission. The impact of GPRASP2 deficiency on spiral ganglion cells (SGCs) and their underlying pathogenic mechanisms will be investigated in this study. The primary culture of SGCs obtained from mouse cochleae was treated with Gprasp2-targeting short hairpin RNA (Gprasp2-shRNA) via lentivirus infection. The results showed that GPRASP2 deficiency enhanced SGCs apoptosis and decreased cell viability. Meanwhile, a significant abnormality of mitochondrial morphology and decreased membrane potential were observed in GPRASP2-deficient SGCs. These effects could be mitigated by treatment with the mitochondrial division inhibitor 1 (Mdivi-1). In addition to enhancing SGCs apoptosis and decreasing cell viability, GPRASP2 deficiency also inhibited the development of SGCs in mouse cochlear explant culture. Our study further revealed that this deficiency resulted in increased phosphorylation of AMPK and activation of the AMPK/DRP1 pathway, promoting SGCs apoptosis. These findings provide insight into the pathogenic mechanisms by which GPRASP2 deficiency is implicated in auditory dysfunction.

Keywords

AMPK/DRP1 pathway
Mitochondria-mediated apoptosis
Mitochondrial fission
GPRASP2
Spiral ganglion cells
Abbreviations

AMPK 5-AMP activated protein kinase

DRP1 Dynamin-related protein 1

GASP G protein-coupled receptor-associated sorting protein

GPCR G protein-coupled receptor

GPRASP2 G protein-coupled receptor associated sorting protein 2

HL Hearing Loss

Mdivi-1 mitochondrial division inhibitor 1

MMP mitochondrial membrane potential

SGCs spiral ganglion cells

SGNs spiral ganglion neurons

SHL syndromic hearing loss
==== Body
pmc1 Introduction

Hearing loss (HL) is the most prevalent sensory disorder and the fourth leading cause of "Years Lived with Disability" [1]. Genetic factors account for nearly half of all cases of hearing loss [[2], [3], [4]]. About 30 % of genetic HL cases are represented as syndromic HL (SHL) [5]. Our previous study revealed that GPRASP2 was implicated in X-linked recessive SHL [6]. To elucidate the underlying causes of hearing loss associated with GPRASP2 deficiency, further examination of the gene's role and its involvement in biological processes is required.

GPRASP2, part of the G protein-coupled receptor-associated sorting protein (GASP) family, comprises a single exon that codes for a protein comprising 838 amino acids [7,8]. GPCR-associated sorting proteins (GASPs) are crucial for the post-endocytic trafficking of GPCRs, directing them towards degradation or recycling. This process is integral to stem cell properties, embryogenesis, and tissue repair [[9], [10], [11], [12], [13]]. Furthermore, disruptions in GASP function have been associated with the development of various conditions, including neurological disorders, cancer, and hearing impairment [9,[14], [15], [16], [17]]. GPRASP2 is essential for cell proliferation, apoptosis, and carcinogenesis, as well as for the endocytosis and transport of GPCRs [18,19]. Similar to most other GASPs, GPRASP2 expression is primarily observed in the central nervous system [7,8], which plays a vital role in neurite formation and synaptic transmission [[20], [21], [22]]. GPRASP2 deficiency can alter dendritic complexity, spinal density, and synaptic development in the mouse hippocampal regions [22].

It was also noteworthy that GPRASP2 was detected to be highly expressed in mouse spiral ganglion regions, indicating a potential role of GPRASP2 in the development and function of spiral ganglion cells (SGCs) [6]. As the main functional SGCs, spiral ganglion neurons (SGNs) are crucial in transferring intricate auditory data from hair cells to the brainstem [23]. Research indicates that the number of mammalian cells is limited and tends to decrease with age, making it a significant cause of presbycusis, which is age-related hearing loss. In addition, primary factors such as noise, ototoxic drugs, and genetic mutations, as well as secondary damage caused by the death of hair cells, can lead to the degeneration and death of spiral ganglion neurons (SGNs). This ultimately results in permanent hearing loss [[24], [25], [26], [27]]. Many GPRASP/ARMCX family proteins, including GPRASP2, are highly expressed in the central nervous system and are crucial for controlling mitochondrial transport and distribution, as well as maintaining energy synthesis associated with calcium homeostasis and cell viability in neurons [10]. One of the most frequent neuropathic causes is abnormal mitochondria dynamics [28,29]. These findings implied that GPRASP2 deficiency may impair mitochondrial activity and pose harm to auditory nerve cells, resulting in auditory disorder.

Mitochondria within cells display dynamic behaviors. A typical fibroblast contains numerous mitochondria, each containing multiple copies of the mtDNA genome. They exhibit diverse morphologies, including spherical, elongated filaments, or interconnected tubular networks, which are regulated by the balanced processes of fusion and fission [30,31]. Mitochondrial fission and fusion are critical regulatory processes that significantly influence pathways associated with disease, including apoptosis and mitochondrial autophagy [32]. Mitochondrial fission, a process pivotal to cellular health, is facilitated by the dynamin-related protein 1 (Drp1) [33]. Phosphorylation at the serine residue 616 (Ser616) serves as an activating signal for Drp1, thereby promoting mitochondrial fission [34]. In parallel, the orchestration of mitochondrial fusion is contingent upon the coordinated function of mitofusin-1 (Mfn1), mitofusin-2 (Mfn2), and optic atrophy 1 (Opa1), which together mediate the merging of mitochondrial membranes [35,36].

In this study, we demonstrated the impact of GPRASP2 deficiency on SGCs. GPRASP2 deficiency caused abnormal mitochondrial fission in SGCs and showed a significant pro-apoptosis effect on SGCs by inhibiting the AMPK/DRP1 pathway. These findings may help to elucidate the pathogenic mechanisms of GPRASP2 deficiency and lay a theoretical and experimental foundation for the potential therapeutic strategy for SHL.

2 Materials and methods

2.1 Animals and primary culture of SGCs

The C57BL/6 mice used in this study were purchased from the Experimental Animal Center at Nanjing Medical University, China. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University (IACUC number: 2110010) and complied with ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines [37].

The primary culture of SGCs was conducted as described previously [38]. Postnatal day (P)2–3 C57BL/6 mice were subjected to a 30- to 60-s cold anesthesia followed by spraying with 75 % ethanol [39]. The cochlea was immersed in D-Hank's solution (Keygen Biotech, KGL2202-500, Nanjing, China), the brain tissue was excised, and the skull was swiftly severed along the foramen magnum. The organ of Corti, spiral ligament, cochlear shell, cochlear nerve, and cochlear axis were subsequently isolated and removed. Digested tissue 10 min at 37 °C with 0.25 % trypsin/EDTA (Thermo Fisher Scientific, #25200056, Waltham, MA, USA). Centrifuged for 5 min at 800 rpm, repeated three times for cell separation. Cells cultured in 60 mm plate (Thermo Fisher Scientific, #130181, Waltham, MA, USA) coated with collagen, type I rat tail (Corning Incorporated, # 354236, Corning, NY, USA). Cultured in DMEM/F12 (Thermo Fisher Scientific, # 11320033, Waltham, MA, USA) with 5 % FBS (ExCell Bio, FSP500, Suzhou, China), 1 % B27 (Thermo Fisher Scientific, # 17504044, Waltham, MA, USA), 1 % N2 (Thermo Fisher Scientific, # 17502048, Waltham, MA, USA), and 1 % penicillin-streptomycin (Thermo Fisher Scientific, # 15140122, Waltham, MA, USA) at 37 °C, 5 % CO2. Purified SGCs 72 h with 5 μmol/l cytarabine (MedChem Express, HY-13605, Monmouth Junction, NJ, USA). Identified neurons with TUBB3 (1:300, Proteintech, 66375-1-Ig, Wuhan, China).

2.2 Lentivirus infection

The cultured SGCs were grouped following a randomized block design and then infected with lentivirus to establish GPRASP2-deficiency models of SGCs. Recombinant DNA plasmid hU6-MCS-CMV-Puromycin (Shanghai Genechem Co. Ltd.) was used for the expression of shRNAs targeting three different regions of mouse Gprasp2 mRNA, named Gprasp2-shRNA1, Gprasp2-shRNA2, and Gprasp2-shRNA3. None-shRNA treated group was used as the negative control. The shRNA sequences targeting Gprasp2 were listed in Supplementary Table S1. The shRNA plasmid clones, construction of the lentivirus vector, and the packaging of the virus were completed by Shanghai Genechem Company. The SGCs and cochlear explants were grown to a density appropriate before viral infection. The titers of the virus refer to previous studies [40,41]. To increase the infection efficiency, the fresh culture medium containing serum was added after removing the initial cell culture media, and the concentrated lentiviral particles were introduced to the target cells. Cells were cultured under conditions of 37 °C and a 5 % CO2 environment in an incubator. The culture medium was refreshed 4–6 h post-seeding. Expression analysis of the target gene was conducted using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot (WB) at 48–72 h post-infection.

2.3 Cell viability assay

Cell viability was assessed by Cell Counting Kit-8 (Beyotime, C0038, Shanghai, China) per the manufacturer's protocol. Cells plated in triplicate, 3000 cells/well in 96-well plates, incubated overnight. Post-treatment, 100 μl of 10 % diluted CCK-8 solution was added per well, and incubated for 4 h. OD at 450 nm was measured with the microplate reader (Tecan, Switzerland). The untreated group served as the control, while the blank contained media without cells.

2.4 TUNEL assay

The 3'end of DNA fragment of apoptotic cultured spiral ganglion cells was labeled by the TUNEL method [42]. Cells were treated according to the viability assay protocol. They were permeabilized with 0.1 % Triton X-100 on ice for 20 min, then fixed with 4 % paraformaldehyde in PBS at room temperature for 45 min. Subsequently, the cells were TUNEL stained for 60 min at 37 °C in the dark. TUNEL-positive cells were observed under a fluorescent microscope (Zeiss, Oberkochen, Germany). Apoptotic cells were quantitatively analyzed using microscope software.

2.5 Detection of ROS production

Reactive oxygen species (ROS) levels were measured utilizing the ROS Detection Kit (Keygen Biotech, KGT010-1, Nanjing, China). Cells were treated with DCFH-DA and incubated for 20 min at 37 °C, followed by washing with PBS. Analysis of ROS was performed using a flow cytometer (BD Biosciences, San Jose, CA, USA), and data were processed with FlowJo software (Version 10.9.0; Tree Star, Ashland, OR, USA).

2.6 Annexin V-FITC/PI assay

The Annexin V-FITC/PI apoptosis detection kit (Vazyme Biotech, A211-01, Nanjing, China) was utilized for the quantitative measurement of apoptosis by the manufacturer's recommendations. In summary, Cells were treated with trypsin (EDTA-free), followed by centrifugation at 1000 rpm for 5 min at 4 °C, after which the supernatant was removed. The cell pellet was washed with cold PBS, then resuspended in 100 μl of Binding Buffer and incubated for 10 min at room temperature in darkness. Subsequently, 5 μl of Annexin V-FITC and PI were added, and the mixture was incubated before being diluted with 400 μl of Binding Buffer. The cells were analyzed using flow cytometry (BD Biosciences, San Jose, CA, USA) with 488 nm excitation, and the data were processed using FlowJo software (Version 10.9.0; Tree Star, Ashland, OR, USA).

2.7 Caspase-3 activation assay

The caspase-3 activation assay kit (Beyotime, C1115, Shanghai, China) was used. In brief, 10 ml of caspase-3 substrate solution (5 min in the dark at 37 °C) was used, followed by detection at 450 nm using a microplate reader (Tecan, Switzerland). The proportion of activated caspase-3 was determined based on the absorbance measurements of cells undergoing apoptosis.

2.8 5-Ethynyl-2-deoxyuridine (EdU) cells proliferation assay

Cell proliferation was detected using the EdU kit (RiboBio, Guangzhou, China). In brief, SGCs were grown to a suitable density, and EdU dissolved in buffer was added to the cell culture at the recommended concentration. Treated 2–4 h, removed medium, fixed with 4 % paraformaldehyde for 10–15 min, treated with 0.5 % Triton X-100 (Sigma Aldrich, St. Louis, MO, USA) for 5–10 min, washed 2–3 times with PBS for 5 min each. Added fluorescent EdU-conjugated antibodies, incubated for 1 h at room temperature. Washed cells with PBS to remove excess. Stained nuclei with DAPI for 5 min, and mounted samples. Assessed EdU-labeled cell proliferation using fluorescence microscopy (Zeiss, Oberkochen, Germany). Analyzed EdU red fluorescence and DAPI blue nuclear fluorescence with ImageJ software.

2.9 Mitochondrial membrane potential (MMP) evaluation

The JC-1 mitochondrial membrane potential detection kit (Beyotime, C2006, Shanghai, China) assessed MMP. After PBS treatment, cells were incubated with 1 ml of fresh media followed by the addition of 1 ml JC-1 staining solution. The staining was performed for 20 min at 37 °C in darkness. The supernatant was then removed, and cells were resuspended in 1 ml JC-1 buffer. Subsequent centrifugation was carried out at 600 g for 3–4 min at 4 °C. Fluorescence intensity was assessed at 514 nm/529 nm for JC-1 monomers and 585 nm/590 nm for JC-1 aggregates using a flow cytometer (BD Biosciences, San Jose, CA, USA). Data analysis was conducted using FlowJo software (Version 10.9.0; Tree Star, Ashland, OR, USA).

2.10 Mitochondrial mass assay

The manufacturer's recommendations determined the mass of cell mitochondria using the Mito-Tracker Green kit (Keygen Biotech, KGMP007, Nanjing, China). Cells were briefly rinsed with PBS, after which the original culture medium was removed. Subsequently, the cells were treated with the pre-warmed Mito-Tracker Green staining solution and incubated at 37 °C for 60 min. Observations were continued under a fluorescence microscope (Olympus, Tokyo, Japan) following the replacement with fresh, warmed media.

2.11 RNA isolation and quantitative PCR

Primary cochlear SGCs' total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA), following manufacturer protocol.RNA integrity was assessed with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), ensuring an absorbance ratio of 1.8–2.1 at 260/280 nm. Subsequently, Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) facilitated the synthesis of cDNA from the total RNA. Quantitative PCR (qPCR) was performed on the Step One Plus Real-time PCR System, utilizing the TransStart® Tip Green qPCR SuperMix (+ Dye I) kit (TransGen Biotech, Beijing, China). Supplementary Table S2 contains a list of all primers used in this study. GAPDH served as the endogenous reference gene. Triplicate PCR reactions were conducted for each sample, and gene expression levels were determined relative to GAPDH using the 2−ΔΔCt method.

2.12 ATP production assay

ATP levels were measured using an ATP assay kit (Beyotime, S0026, Shanghai, China) according to the manufacturer's instructions. Cells treated in 6-well plates were lysed with 200 μl of lysis buffer. Then, 20 μl of the lysate was added to a 96-well black, clear-bottom plate containing 100 μl of detection reagent. The chemiluminescence was read with a microplate reader.

2.13 Transmission electron Microscope(TEM)

Cells were seeded into 6-well plates and cleaned with PBS. They were fixed for 2 h in 1 % OsO4, followed by an overnight fixation in 2 % glutaraldehyde at 4 °C. The cells were then stained for 2 h with 2 % uranyl acetate. Dehydration was carried out through a series of alcohol gradients (50 %, 70 %, 90 %, 100 %). Infiltration was performed for 2 h in a mixture of 100 % acetone and embedding agent. Samples were kept overnight at 30 °C in pure embedding media. Polymerization was allowed to progress in a temperature-controlled box. Ultrathin sections were imaged using a transmission electron microscope (TEM) (JEOL, Tokyo, Japan).

2.14 Mouse cochlear explants

P2-3 C57BL/6 mice were anesthetized on ice and sacrificed on the floor of the foramen magnum with surgical scissors. The cranium was opened along the sagittal suture, followed by a bisection of the forebrain [43]. The forebrain, cerebellum, and brain stem were removed by blunt dissection. The head was washed in D-Hank's solution (Keygen Biotech, KGL2202-500, Nanjing, China) to locate the cochlea, which was then detached from the vestibular system. The organ of Corti was dissected in D-Hank's solution, and the bony labyrinth was separated. The Corti organs were fixed to hooks and transferred to Petri dishes containing collagen, type I rat tail (Corning Incorporated, # 354236, Corning, NY, USA). They were cultured in serum-free DMEM/F12 (Thermo Fisher Scientific, # 11320033, Waltham, MA, USA) supplemented with 10 % FBS (ExCell Bio, FSP500, Suzhou, China), 1 % N2 (Thermo Fisher Scientific, # 17502048, Waltham, MA, USA), 2 % B27 (Thermo Fisher Scientific, # 17504044, Waltham, MA, USA), and 1 % penicillin-streptomycin (Thermo Fisher Scientific, # 15140122, Waltham, MA, USA). The samples were placed in the incubator for 3–5 min at room temperature, and half of the medium was replaced the next day.

2.15 Immunofluorescence staining

The SGCs and cochlear explants are prepared as stated above. In a nutshell, cells were cultured in 24-well plates and processed as follows: After two rinses with PBS, they were fixed with 4 % paraformaldehyde for 15 min at room temperature. Cells were then permeabilized with 0.1 % Triton X-100 (Sigma Aldrich, St. Louis, MO, USA) for 10 min, followed by three washes with PBS. A blocking step was performed for 1 h at room temperature using goat serum/PBS (1:10). The primary antibody, TUBB3 (1:300, Proteintech, 66375-1-Ig, Wuhan, China), was applied and incubated overnight at 4 °C. Subsequently, cells were stained with the secondary antibody, Alexa Fluor 488 goat anti-mouse IgG (H + L) (Thermo Fisher Scientific, A-10667, Waltham, MA, USA), for 2 h at room temperature in darkness. After three additional washes with PBS, nuclei were counterstained with DAPI. Finally, cells were examined using confocal microscopy. The prepared cochlear explants were cultured and randomly assigned to different treatment groups, with each mouse cochlea serving as an experimental unit.

2.16 Western blot assay

The cell sample was combined with the appropriate volume of RIPA lysate (Beyotime, P0013B, Shanghai, China) and a specific amount of phosphatase inhibitor (MedChem Express, HY-K0021, Monmouth Junction, NJ, USA), and it was lysed on ice for 30 min. After sonicating the cell fragments to lyse them, the supernatant was centrifuged for 15 min at 4 °C and 12,000 g. After adding the β-mercaptoethanol-containing protein loading buffer, the protein was denatured in a metal bath at 100 °C to collect the lysate. The proteins were transferred to the polyvinylidene fluoride (PVDF) membrane after being separated by electrophoresis on a 10–12 % SDS-polyacrylamide gel. After blocking the membrane for 2 h with 5 % skim milk powder dissolved in Tris-buffered saline and Tween-80 (TBST), the membrane was incubated with various primary antibodies for the entire night at 4 °C. In this study, primary antibodies included GAPDH (1:1000, Abcam, ab8245, Cambridge, MA, USA), Bax (1:1000, Abcam, ab32503, Cambridge, MA, USA), Bcl-2 (1:1000, Abcam, ab32124, Cambridge, MA, USA), Caspase-3 (1:1000, Abcam, ab32351, Cambridge, MA, USA), Cleaved-Caspase-3 (1:1000, Affinity Biosciences, AF7022, OH, USA), p-AMPKα (Thr172) (1:1000, Affinity Biosciences, AF3422, OH, USA), AMPKα (1:1000, Proteintech, 10929-2-AP, Wuhan, China), p-LKB1 (Ser428) (1:1000, Abcam, ab63473, Cambridge, MA, USA), LKB1 (1:1000, Abcam, ab15095, Cambridge, MA, USA), DRP1 (1:1000, Affinity Biosciences, DF7037, OH, USA), p-DRP1 (Ser616) (1:1000, Affinity Biosciences, AF8470, OH, USA), MFN1 (1:1000, Biogot, BS79959, Nanjing, China), FIS1 (1:1000, Biogot, BS7881, Nanjing, China), OPA1 (1:1000, Proteintech, 27733-1-AP, Wuhan, China). The membrane was washed three times with TBST, followed by a 2-h incubation with an HRP-conjugated secondary antibody.The binding region of a particular antibody and protein on the membrane was visible during incubation with the ECL luminous solution (Biosharp, BL520A, Hefei, China), allowing for the detection of protein expression and presence. The relative density was examined using ImageJ software, and each protein's strength was adjusted to GAPDH.

2.17 Statistical analysis

Statistical evaluations were conducted with GraphPad Prism Software (Version 8.0.2, GraphPad Software, San Diego, CA, USA). The data underwent analysis via one-way or two-way ANOVA, with post hoc tests using the Bonferroni correction to determine significant mean differences. A p-value of less than 0.05 was set as the threshold for statistical significance.

3 Results

3.1 GPRASP2 deficiency induces SGCs apoptosis and inhibits cell viability

The cochlear SGCs of C57BL/6 wild-type mice at 2–3 days postnatal were used for primary culture and identified by neuronal marker TUBB3 (Supplementary Fig. 1A) to investigate the impact of GPRASP2 deficiency on SGCs. Three distinct shRNA sequences were able to knock down the Gprasp2 gene. RT-qPCR and Western blot analysis revealed that the mRNA and protein levels of GPRASP2 in cells transfected with shRNA vectors targeting Gprasp2 (vectors 1, 2, and 3) were significantly reduced compared to the control group (Supplementary Figs. 1B and C). Given the comparable knockdown efficiency among the three shRNAs, we selected Gprasp2-shRNA1 for further investigation in our study.

Flow cytometry analysis indicated that apoptosis occurred in both groups of supporting cells (SGCs) following transfection with shRNA (Fig. 1A and B). Further assessment of apoptosis using TUNEL staining confirmed an increase in the proportion of apoptotic SGCs in response to GPRASP2 deficiency (Fig. 1C and D). Furthermore, an increase in intracellular Caspase-3 activity was brought on by GPRASP2 deficiency (Fig. 1E). The results of the CCK-8 assay indicated that GPRASP2 deficiency decreased the viability of SGCs (Fig. 1F). Supplementary data also revealed that GPRASP2 deficiency negatively affected the proliferative capacity of SGCs (Supplementary Figs. 2A and B). Collectively, these observations imply that GPRASP2 deficiency not only diminishes cellular viability but also promotes apoptotic processes within SGCs.Fig. 1 GPRASP2 deficiency induces SGC apoptosis and inhibits cell viability. A. SGCs apoptosis was analyzed by Annexin V-FITC and PI staining and detected by flow cytometry. B. FlowJo was used to analyze the apoptosis ratio of SGCs. C. Apoptosis ratio was determined by TUNEL assay. TUNEL was used to label in situ terminal transferase, that is, the broken DNA chain (green) in apoptotic cells, and DAPI was used to label the nucleus (blue) (scale bar = 100 μm). D. The proportion of apoptotic cells was analyzed by ImageJ. E. Caspase-3 activity assay kit was used to detect the Caspase-3 activity of SGCs after GPRASP2 deficiency. F. The cell viability of SGCs was determined by the CCK-8 method. (n = 6; Data are presented as Mean ± SEM; ***p < 0.001).

Fig. 1

3.2 GPRASP2 deficiency activates the mitochondrial apoptotic pathway in SGCs

The JC-1 assay was employed to assess the impact of GPRASP2 deficiency, induced by lentiviral infection, on the mitochondrial membrane potential in spiral ganglion cells (SGCs). Flow cytometric analysis revealed a significant collapse in mitochondrial membrane potential, indicating a decrease attributable to the absence of GPRASP2 (Fig. 2A and B). ROS overproduction and mitochondrial apoptosis are tightly associated. Flow cytometry was used to detect the ROS content of SGCs after GPRASP2 deficiency. The results showed that ROS levels were significantly higher than those in the control group (Fig. 2C and D). The expression of the protein level was then examined in further detail. The amount of pro-apoptotic protein Bax rose in SGCs with GPRASP2 deficiency, whereas the level of anti-apoptotic protein Bcl2 significantly decreased (Fig. 2E, F and G). GPRASP2 deficiency resulted in a significant increase in Cleaved-Caspase-3 protein levels (Fig. 2E and H). In short, GPRASP2 deficiency activates the mitochondrial apoptotic pathway in SGCs.Fig. 2 GPRASP2 deficiency activates the mitochondrial apoptotic pathway in SGCs. A. SGCs' mitochondrial membrane potential was analyzed by JC-1 staining and detected by flow cytometry. B. Mitochondrial membrane potential was analyzed by FlowJo. C. ROS levels in SGCs were measured using DCFH-DA staining and flow cytometry. D. FlowJo was used to analyze ROS levels. E: The protein expression levels of Caspase-3, Cleaved Caspase-3, and Bcl-2 family members (Bcl-2, Bax) were analyzed by WB, and GAPDH was used as the internal reference. F. The level of anti-apoptosis protein Bcl-2 was quantified by ImageJ; G: The level of pro-apoptotic protein Bax was quantitatively analyzed by ImageJ. H. Quantitative statistics were performed on protein Caspase-3 and Cleaved Caspase-3 protein levels by ImageJ. (n = 3; Data are presented as Mean ± SEM; **p < 0.01, ***p < 0.001).

Fig. 2

3.3 GPRASP2 deficiency induces DRP1-mediated mitochondrial fission and mitochondrial dysfunction in SGCs

Transmission electron microscopy was utilized to view the mitochondria and further investigate the impact of GPRASP2 deficiency on the mitochondria in SGCs. The results indicated that, relative to the control group, the mitochondria exhibited signs of swelling, and the internal cristae either diminished in number or vanished entirely (Fig. 3A). Mitochondrial quality control serves not only as an essential endogenous cell protection mechanism critical for the preservation of mitochondrial homeostasis and functionality but also constitutes a comprehensive surveillance network dedicated to monitoring mitochondrial integrity. Consequently, our investigation has focused on the biological processes of mitochondrial fission and fusion, key regulators of mitochondrial dynamics. The results of RT-qPCR demonstrated that GPRASP2 deficiency raised the expression of genes associated with mitochondrial fission (Drp1, Fis1), while Opa1 and Mfn1—related to mitochondrial fusion—had lower expression (Fig. 3B). Proteins involved in mitochondrial fission and fusion were measured using Western blot. The findings demonstrated decreased mitochondrial fusion proteins OPA1 and MFN1 levels and increased mitochondrial fission proteins DRP1 and FIS1 (Fig. 3C, D, E, F, and G).Fig. 3 GPRASP2 deficiency induces DRP1-mediated mitochondrial fission and mitochondrial dysfunction in SGCs. A. Transmission electron microscopy images of SGCs. The yellow frame is mitochondria, and the right side is a local enlargement map in the left frame (scale bar = 1 μm). B. After GPRASP2 deficiency, SGCs were extracted for RT-qPCR to verify the statistical map of mitochondrial quality control-related gene expression. C. WB was used to verify the protein levels of mitochondrial quality control proteins (DRP1, FIS1, MFN1, OPA1) in SGCs, and GAPDH was used as an internal reference. D-G. Quantitative analysis of mitochondrial quality control protein (DRP1, FIS1, MFN1, OPA1) protein levels by ImageJ. H. After GPRASP2 deficiency, SGCs were stained with Mito-Tracker Green, a mitochondrial-specific fluorescent probe. The mitochondria were labeled green, and the red box was a large part of the mitochondria (scale bar = 20 μm). I. The ATP content determination kit was used to determine the statistical energy map in SGCs after GPRASP2 deficiency. (n = 3; Data are presented as Mean ± SEM; *p < 0.05, **p < 0.01).

Fig. 3

Mitochondrial-specific fluorescent probe Mito-Tracker Green was used to detect SGCs after GPRASP2 deficiency. The results showed that the mitochondria in Gprasp2-deficient SGCs presented as small scattered dots. In the control group, the mitochondria showed a long strip of polymerization (Fig. 3H). To learn more about its mechanism, Mdivi-1 was utilized to block DRP1, the primary regulator of mitochondrial fission specifically. Following GPRASP2 deficiency, it was discovered that Mdivi-1 markedly decreased the levels of DRP1 and FIS1 in SGCs while modestly raising the levels of OPA1 and MFN1 proteins (Fig. 3C, D, E, F, and G). Furthermore, the mitochondrial fission of SGCs resulting from GPRASP2 deficiency was mitigated by Mdivi-1 (Fig. 3H). We ran cell viability assays for 24 h at various drug concentrations to rule out the medication's effect on the cells. The results showed that the drug concentration of 0, 1, 5, and 10 μM did not affect cell viability (Supplementary Fig. 3A). Consequently, 5 μM was employed in the following tests. In addition, after GPRASP2 deficiency, the energy production and function of SGCs mitochondria changed. The results showed that ATP production was significantly reduced after Gprasp2 knockdown (Fig. 3I). These data suggest that GPRASP2 deficiency leads to mitochondrial fission and dysfunction in SGCs.

3.4 Mdivi-1 attenuated GPRASP2 deficiency-induced apoptosis of SGCs

The results showed that Mdivi-1 partially rescued the apoptosis induced by GPRASP2 deficiency (Fig. 4A–B) and also attenuated the decrease of cell viability induced by GPRASP2 deficiency (Fig. 4C). WB results showed that Mdivi-1 reduced the cleavage of Caspase-3 caused by GPRASP2 deficiency (Fig. 4D and G). Mdivi-1 increased the anti-apoptotic protein Bcl-2's expression level and decreased the pro-apoptotic protein Bax (Fig. 4D, E, and F). In addition, flow cytometry analysis showed that Mdivi-1 alleviated the decrease of mitochondrial membrane potential caused by GPRASP2 deficiency (Fig. 4H and I).Fig. 4 Mdivi-1 attenuated GPRASP2 deficiency-induced apoptosis of SGCs. A. After adding Mdivi-1, the apoptosis of SGCs was analyzed by Annexin V-FITC and PI staining, and detected by flow cytometry. B. FlowJo was used to analyze the apoptosis ratio of SGCs. C. After adding Mdivi-1, the cell viability of SGCs was determined by the CCK-8 method. D. After adding Mdivi-1, the protein expression levels of Caspase-3, Cleaved-Caspase-3, and Bcl-2 family members (Bcl-2, Bax) in SGCs were analyzed by WB, and GAPDH was used as the internal reference. E-G. The protein levels of Caspase-3, Cleaved-Caspase-3, and Bcl-2 family members (Bcl-2, Bax) were quantitatively analyzed by ImageJ. (n = 3; Data are presented as Mean ± SEM; **p < 0.01, ***p < 0.001).

Fig. 4

3.5 GPRASP2 deficiency induces DRP1-mediated mitochondrial fission via the AMPK pathway

AMP-activated protein kinase (AMPK) is recognized as the central metabolic sensor that becomes activated in response to mitochondrial damage. In our quest to elucidate the mechanisms underlying the apoptosis of SGCs triggered by mitochondrial fission consequent to GPRASP2 deficiency, we have concentrated our efforts on examining the pivotal role of the AMPK pathway in mitochondrial energy metabolism. AMPK regulation is closely related to mitochondrial biogenesis, cell growth, metabolism, and apoptosis. WB analysis was used to quantify AMPK and p-AMPKα (Thr172) protein levels after transfecting primary SGCs with the Gprasp2-shRNA lentivirus. The findings indicated that p-AMPKα (Thr172) expression was elevated in SGCs due to the GPRASP2 deficiency (Fig. 5A and B). Compound C, a specific inhibitor of AMPK, was introduced to study whether the activation of AMPK in SGCs occurs upstream or downstream of mitochondrial dynamics abnormalities after GPRASP2 deficiency. To rule out the effect of drugs on cells, SGCs were treated with different drug concentrations for 24 h, and then cell viability was evaluated. The results showed that the drug concentrations of 0,1,5 and 10 μM did not affect cell viability (Supplementary Fig. 3B), so 5 μM was used in subsequent studies. WB results showed the application of mitochondrial division inhibitor Mdivi-1 to p-LKB1 (Ser428) and p-AMPKa proteins. The level was unaffected (Fig. 5C, D, and E). On the contrary, inhibition of AMPK phosphorylation can significantly eliminate the increase of mitochondrial fission proteins DRP1 and FIS1, and the decrease of fusion protein MFN1 is slightly weakened (Fig. 5F, G, H, and I). Mito-Tracker green staining analysis showed that Compound C treatment partially alleviated Gprasp2-induced mitochondrial fission (Fig. 5K). In summary, these results indicate that LKB1/AMPK signaling activation occurs upstream of mitochondrial dynamics disorders by promoting mitochondrial fission and that GPRASP2 deficiency induces DRP1-mediated mitochondrial fission through the AMPK pathway.Fig. 5 GPRASP2 deficiency induces DRP1-mediated mitochondrial fission via the AMPK pathway. A. WB was used to analyze the activation of AMPK protein in SGCs after infection with different Gprasp2-shRNA sequences, and GAPDH was used as the internal reference. B. The protein levels of AMPK and its phosphorylated form were quantified by ImageJ. C. After adding Mdivi-1, the activation of key proteins in the AMPK pathway in SGCs was analyzed by WB, and GAPDH was used as an internal reference. D-E. The protein levels of AMPK, LKB1, and their phosphorylated forms were quantified by ImageJ. F. WB analyzed the expression of key proteins in the mitochondrial quality control system in SGCs after adding Compound C, and GAPDH was used as the internal reference. G-J. The protein levels of key proteins of the mitochondrial quality control system (DRP1, FIS1, MFN1) and AMPK pathway proteins (AMPK, p-AMPK (Thr172)) were quantified by ImageJ. K. After SGCs were infected with Gprasp2 lentivirus, Compound C was added, and the mitochondrial-specific fluorescent probe Mito-Tracker Green was used to stain the fluorescence map. The mitochondria were labeled as green, and the red box was the local majority of the mitochondria (scale bar = 20 μm). (n = 3; Data are presented as Mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ns means no statistical significance).

Fig. 5

3.6 GPRASP2 deficiency induces apoptosis by activating the AMPK pathway

Subsequently, this investigation assessed the potential involvement of the AMPK signaling pathway in SGC apoptosis that is mediated by mitochondrial fission. The results showed that GPRASP2 deficiency caused SGC apoptosis, which could be partially offset by Compound C treatment (Fig. 6A and B). Compound C could mitigate the loss of SGCs viability resulting from GPRASP2 deficiency, according to the CCK-8 cell viability experiment (Fig. 6C). Western blot analysis further demonstrated that treatment with Compound C could decrease the levels of the pro-apoptotic protein Bax, increase the levels of the anti-apoptotic protein Bcl-2, and prevent the cleavage of Caspase-3 triggered by GPRASP2 deficiency. (Fig. 6D, E, F, and G). The JC-1 kit was used to detect the mitochondrial membrane potential by flow cytometry. It was found that Compound C attenuated the decrease of mitochondrial membrane potential induced by GPRASP2 deficiency (Fig. H–I).Fig. 6 GPRASP2 deficiency induces apoptosis by activating the AMPK pathway. A. After Compound C was added, the apoptosis of SGCs was analyzed by Annexin V-FITC and PI staining, and detected by flow cytometry. B. FlowJo was used to analyze the apoptosis ratio of SGCs. C. After adding Compound C, the cell viability of SGCs was determined by the CCK-8 method. D. After adding Compound C, the protein expression levels of Caspase-3, Cleaved-Caspase-3, and Bcl-2 family members (Bcl-2, Bax) in SGCs were analyzed by WB, and GAPDH was used as an internal reference. E-G. The protein levels of Caspase-3, Cleaved-Caspase-3, and Bcl-2 family members (Bcl-2, Bax) were quantitatively analyzed by ImageJ. H. After SGCs were treated with Compound C, the mitochondrial membrane potential of SGCs was analyzed by JC-1 staining and detected by flow cytometry. I. Mitochondrial membrane potential was analyzed using FlowJo. (n = 6; Data are presented as Mean ± SEM; **p < 0.01, ***p < 0.001).

Fig. 6

In summary, Compound C rescued SGCs apoptosis and membrane potential collapse caused by GPRASP2 deficiency by inhibiting the phosphorylation level of AMPK. This suggests that the AMPK pathway plays a role in mitochondrial fission-mediated apoptosis of SGCs after GPRASP2 deficiency.

3.7 GPRASP2 deficiency mediates apoptosis and inhibits SGCs growth in cochlear explants by regulating the AMPK/DRP1 pathway

As previously mentioned, the AMPK pathway is upstream of mitochondrial diseases. When Compound C was added, TUNEL labeling of SGCs revealed a considerable decrease in the rate of SGCs apoptosis (Fig. 7A and B). A cochlear explant culture system was set up to more thoroughly investigate the impact of GPRASP2 deficiency on SGCs (Supplementary Figs. 4A and B). After lentivirus-mediated Gprasp2 gene knockdown of Gprasp2, immunofluorescence labeling (TUBB3 labeled SGNs, DAPI labeled nucleus) was performed. The staining results show that the deficiency of GPRASP2 leads to a general disarray in the arrangement of the spiral ganglion fibers (Supplementary Fig. 5). To further investigate the changes in the spiral ganglion, a count of the fibers and cells of the spiral ganglion was conducted. The count results indicate that both the number of fibers and cells in the spiral ganglion are significantly reduced after the deficiency of GPRASP2 (Fig. 7C, D, E, and F).The above results indicate that GPRASP2 deficiency induces apoptosis and inhibits the growth of cochlear SGCs via the AMPK/DRP1 pathway.Fig. 7 GPRASP2 deficiency mediates apoptosis and inhibits SGCs growth in cochlear explants by regulating the AMPK/DRP1 pathway. A. SGCs were treated with Compound C, and the apoptosis of SGCs was determined by the TUNEL method. TUNEL-labeled in situ terminal transferase, that is, the broken DNA chain in apoptotic cells (green), DAPI-labeled nucleus (blue) (scale bar = 100 μm). B. The proportion of apoptotic cells was analyzed by ImageJ. C. The cochlear explants were stained with immunofluorescence. TUBB3 was used to label the microtubules of spiral neurons (green), and DPAI was used to label the nucleus (blue) (scale bar = 200 μm). D. Quantitative statistics of the number of spiral ganglion fibers by ImageJ software. E. The cochlear explants were stained with immunofluorescence. TUBB3 was used to label the microtubules of spiral neurons (green), and DPAI was used to label the nucleus (blue) (scale bar = 100 μm). F. Quantitative statistics on the number of spiral ganglion cells were performed using ImageJ software. (n = 6; Data are presented as Mean ± SEM; ***p < 0.001).

Fig. 7

4 Discussion

GPRASP2 belongs to the family of GPCR-associated sorting proteins (GASPs), playing a role in the post-endocytic trafficking of G protein-coupled receptors (GPCRs). This involvement is crucial for initiating a range of cellular processes such as the regulation of gene expression, trafficking of receptors, and their subsequent degradation [44]. GPCRs play a crucial role in various physiological activities, including regulating the immune system, regulating the autonomic nervous system, and maintaining the homeostasis of organisms. Owing to their multifaceted roles, GPCRs consistently represent a focal point in scientific inquiry and a prime target for pharmaceutical innovation [45]. As an essential protein family that interacts with GPCRs, GASPs are pivotal in modulating the diverse biological functions of GPCRs. They govern the activity of GPCRs, ensuring the efficacious transmission of signals. Additionally, GASPs participate in the endocytic and sorting processes of GPCRs, which are vital for receptor recycling and degradation, as well as for regulating the receptor density on the cell surface. Consequently, investigating GPRASP2 and its family members is of significant importance in understanding the intricate regulatory mechanisms of GPCRs.

Current studies highlight the critical function of GPRASP2 in regulating neurite elongation and diverse synaptic activities. It has been implicated in the pathophysiology of neurodevelopmental disorders, including Huntington's disease and autism [[20], [21], [22]]. Despite the growing recognition of GPRASP2's importance, there is a paucity of research examining its role in audiological diseases. Our earlier work demonstrated robust expression of GPRASP2 in the spiral ganglion of the cochlea, suggesting a potential involvement in the modulation of cochlear SGCs and a significant impact on auditory function regulation. However, the underlying mechanisms await elucidation. Guided by these scientific premises, the present study aimed to establish a GPRASP2-deficient SGCs model. Our findings indicate that the deficiency of GPRASP2 leads to pronounced apoptosis and diminished viability in SGCs.

Apoptosis, a form of programmed cell death, can be categorized into three primary pathways: the intrinsic (mitochondrial) pathway, the extrinsic (death receptor) pathway, and the crosstalk between these two pathways [46]. The intrinsic (mitochondrial) apoptotic pathway serves as a principal mechanism underlying the cellular response to stress and the execution of programmed cell death. By studying this pathway, we can understand how cells respond to internal damage and stress and how to maintain tissue homeostasis. Therefore, we conducted a multi-dimensional experimental study on the mitochondria of GPRASP2-deficient SGCs. The study's observations indicated notable changes in mitochondrial structure, a decrease in mitochondrial membrane potential, an increase in the levels of proteins that promote apoptosis, and a decrease in the levels of proteins that inhibit apoptosis in SGCs lacking GPRASP2. Moreover, the engagement of the apoptotic executioner Caspase was detected in these cells. These pieces of evidence collectively suggest that GPRASP2 deficiency triggers the mitochondrial apoptotic pathway in SGCs.

In addition, this study found that the mitochondrial fission state was apparent after GPRASP2 deficiency, which meant that the mitochondrial quality control system was unbalanced. Mitochondrial morphology is a determinant of apoptotic susceptibility, as mitochondrial fission mediated by DRP1 is a pivotal and early step in the apoptotic cascade. Prior research has documented that mitochondrial fragmentation and clustering precede the release of cytochrome c, a hallmark of apoptosis [47,48]. The research findings about mitochondrial fission-fusion proteins further validated an increase in the expression of proteins associated with mitochondrial fission, coupled with a decrease in those related to fusion, in SGCs after GPRASP2 deficiency.

To further understand the mechanism of GPRASP2 deficiency leading to SGCs apoptosis, the DRP1 inhibitor Mdivi-1 was introduced for rescue experiments. The results showed that Mdivi-1 successfully rescued SGCs apoptosis, GPRASP2 deficiency-induced apoptosis, by mediating DRP1-dependent mitochondrial fission. Previous studies have shown that inhibition of DRP1 inhibits mitochondrial fission and delays the process of MMP collapse, Caspase activation, and cell death itself [48], which is consistent with our results. Furthermore, this study evaluated the cytotoxic effects of the compounds Mdivi-1 and Compound C at concentrations ranging from 0 to 10 μM. The findings indicated that there was no significant difference in cell viability across these concentrations. In a thorough assessment conducted within the 1–10 μM range, we considered factors such as drug efficacy, cellular activity, and experimental costs. Additionally, we referenced the drug concentrations utilized by Yao Chen and Qiong Xu et al. in their respective studies on neuronal and tumor cells [49,50], as well as the potential cellular risks. Consequently, a concentration of 5 μM was identified as the optimal balance, integrating these considerations.

To have a deeper understanding of its molecular mechanism, this study attempts to explore the related pathways mediating this biological event and explore potential upstream and downstream molecules. AMP-activated protein kinase (AMPK) serves as a key metabolic regulator, capable of initiating responses to diverse forms of mitochondrial stress, such as mitochondrial fission. AMPK plays a central role in cell metabolism, including glucose metabolism, lipid metabolism, and energy production. Mitochondria are the metabolic center of cells, and their division and function are closely related to the overall metabolic state of cells. Previous studies have shown that activation of AMPK can promote apoptosis [51,52]. This study also demonstrates that the deficiency of GPRASP2 activates the AMPK pathway in SGCs. The AMPK inhibitor partially rescues the observed apoptosis and mitochondrial damage in SGCs by inhibiting the phosphorylation of AMPK at Thr172. However, whether AMPK is located upstream or downstream of DRP1-dependent mitochondrial division is still unclear. Therefore, this study used inhibitors corresponding to AMPK and DRP1 to detect related proteins. The results showed that inhibition of AMPK phosphorylation could reduce the increase of DRP1 and other fission proteins after GPRASP2 deficiency and increase the expression of MFN1 and other mitochondrial fusion proteins at a certain level. On the contrary, the DRP1 inhibitor did not affect AMPK and its upstream LKB1 protein levels. Therefore, this study concluded that AMPK is upstream of DRP1-dependent mitochondrial division; after GPRASP2 deficiency, DRP1 promotes mitochondrial division by activating the AMPK pathway, thereby inducing SGCs apoptosis. This study also established a cochlear explant culture model to study the effect of GPRASP2 deficiency on the growth of spiral neurons. The results showed that GPRASP2 deficiency reduced the number of neuronal fibers in spiral neurons, disordered arrangement, and reduced the number of neuronal cells.

In summary, this study found the relationship between GPRASP2 and mitochondria in SGCs by studying the apoptosis of cochlear spiral ganglion cells caused by GPRASP2 deficiency. It elucidated the potential molecular mechanism, laying a theoretical and experimental foundation for studying GPRASP2 in SHL.

5 Conclusions

In conclusion, this study demonstrates that GPRASP2 deficiency activated the AMPK signaling pathway, thereby enhancing DRP1-mediated mitochondrial fission and precipitating mitochondrial dysfunction and apoptosis. Our findings elucidated the potential mechanistic role of GPRASP2 in HL.

Ethics statement

This study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Nanjing Medical University, with the assigned IACUC protocol number: 2110010.

Consent for publication

All authors agree to submit for consideration for publication in the journal.

Data availability

The authors confirm that the data supporting the findings of this study are available within the article and/or its supplementary materials.

CRediT authorship contribution statement

Kun Huang: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. Jing Cai: Methodology, Investigation. Yajie Lu: Methodology, Investigation. Tianming Wang: Methodology, Investigation, Funding acquisition. Shen Yue: Methodology, Investigation, Formal analysis. Qinjun Wei: Methodology, Investigation. Jun Yao: Writing – review & editing, Supervision, Investigation, Funding acquisition, Formal analysis. Zhibin Chen: Supervision, Resources, Methodology, Investigation, Formal analysis. Xin Cao: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A Supplementary data

The following is the Supplementary data to this article:Multimedia component 1

Multimedia component 1

Acknowledgments

This work was supported by the grants from the grants from the 10.13039/501100001809 National Natural Science Foundation of China (32070587 ) to XC, the 10.13039/501100001809 National Natural Science Foundation of China (82071052 ) to JY and the 10.13039/501100001809 National Natural Science Foundation of China (82200642 ) to TW.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2024.e36140.
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