Pathogenic factor for neurodegenerative diseases and application thereof

Abstract

The invention provides a pathogenic factor for neurodegenerative diseases and application of the pathogenic factor. The pathogenic factor for the neurodegenerative diseases is a PASP-GPR37-IL-6 signal axis. According to the application, the action mechanism of the PASP-GPR37-IL-6 signal axis serving as a neurodegenerative disease (especially Parkinson's disease) pathogenic factor is defined for the first time, the molecular path of neuroinflammation and neurodegenerative diseases regulated and controlled by the oligodendroglia through the signal axis is disclosed, and the research blank of participation of the oligodendroglia in PD pathogenesis in the prior art is filled.

Description

Technical Field

[0001] This invention relates to the pathogenesis and treatment of neurodegenerative diseases, and discloses a pathogenic factor of neurodegenerative diseases and its application. Background Technology

[0002] Neurodegenerative diseases are a class of diseases characterized by the progressive degeneration and death of neurons, among which Parkinson's disease (PD) is one of the most common neurodegenerative diseases. The pathological feature of PD is the degeneration and death of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc), which leads to a decrease in dopamine content in the striatum, resulting in motor dysfunction such as rigidity, bradykinesia, and resting tremor. It is also often accompanied by non-motor dysfunction symptoms such as sensory disturbances and depression. Pain symptoms affect 30% to 85% of patients and worsen as the disease progresses.

[0003] Although interventions targeting specific neural circuits can improve motor and mood disorders in animal models of Parkinson's disease (PD), the highly complex pathogenesis of PD has prevented the development of effective treatments to slow or halt its progression. In recent years, increasing evidence suggests that glial cell dysfunction plays a crucial role in the pathogenesis of PD. Studies have found microglial activation and astrocyte proliferation in the substantia nigra of PD patients, indicating that neuroinflammation is involved in the pathological process of dopamine plexus (DA) neurons. Oligodendrocytes, as an important component of glial cells, have been less studied previously, but recent research has shown their potential involvement in immune regulation, particularly in the initiation phase of the immune process, and a significant association with PD. However, the specific molecular mechanisms by which oligodendrocytes regulate PD pathogenesis remain unclear, limiting the development and application of related therapeutic targets.

[0004] G protein-coupled receptor 37 (GPR37) was initially discovered to be expressed in dopamine neurons in the substantia nigra, and its abnormal accumulation is thought to lead to DA neuron death; however, the cellular expression localization of GPR37 remains controversial. Sphingolipid-activated proteinogen (PSAP), as an endogenous ligand of GPR37, has been suggested to be associated with PD by some genetic studies, but its specific mechanism of action remains unclear. Interleukin-6 (IL-6), an important cytokine, has been shown to be significantly elevated in the peripheral blood and brain of PD patients, but its origin and specific regulatory pathways in PD pathogenesis still need further clarification. Therefore, clarifying the molecular mechanisms by which oligodendrocytes participate in PD pathogenesis and identifying key pathogenic signaling axes are of great significance for developing new diagnostic methods and treatment strategies for PD. Summary of the Invention

[0005] The purpose of this invention is to provide a pathogenic factor for neurodegenerative diseases, clarify its mechanism of action, and provide corresponding diagnostic applications, drug screening methods, treatment methods, and drug compositions based on the pathogenic factor, so as to solve the problems of unclear pathogenesis and poor treatment effects in existing neurodegenerative diseases.

[0006] Therefore, the present invention adopts the following technical solution: In a first aspect, the present invention provides a pathogenic factor of neurodegenerative diseases and its mechanism of action. Specifically, the pathogenic factor of neurodegenerative diseases described in the present invention is the PSAP-GPR37-IL-6 signaling axis, which mediates the occurrence and development of neurodegenerative diseases in the following ways: (1) GPR37 is specifically expressed in oligodendrocytes in the substantia nigra pars compacta (SNpc), and is significantly upregulated in neurodegenerative disease models; (2) PSAP is enriched in dopamine DA neurons and its secretion in cerebrospinal fluid increases in neurodegenerative disease states; (3) Secreted PSAP induces the expression and secretion of IL-6 in oligodendrocytes through the GPR37 / Gαi / MEK pathway; (4) IL-6 secreted by oligodendrocytes activates microglia, forming a positive feedback loop of IL-6 secretion, which exacerbates neuroinflammation, degeneration of DA neurons and behavioral defects.

[0007] Furthermore, the neurodegenerative diseases include, but are not limited to, Parkinson's disease and Alzheimer's disease.

[0008] In a second aspect, the present invention provides a combination of biomarkers for the diagnosis and / or treatment of neurodegenerative diseases, the combination of biomarkers including PSAP, GPR37 and IL-6, and the detection samples of the combination of biomarkers including cerebrospinal fluid and substantia nigra tissue.

[0009] Furthermore, the protein levels of PSAP and IL-6 in the cerebrospinal fluid of patients with neurodegenerative diseases were significantly higher than those in healthy controls, and the expression level of GPR37 in oligodendrocytes of the substantia nigra was significantly higher than that in healthy controls.

[0010] Thirdly, the present invention provides a method for screening therapeutic drugs for neurodegenerative diseases, wherein the method targets any link in the PSAP-GPR37-IL-6 signaling axis and screens compounds that can inhibit the activation of this signaling axis, specifically including the following steps: (1) Construct screening models, including oligodendrocyte models expressing GPR37, cell models or animal models activated by the PSAP-GPR37-IL-6 signaling axis; (2) Apply the candidate compounds to the screening model; (3) To detect the effects of candidate compounds on PSAP secretion, GPR37 expression, IL-6 expression and secretion, neuroinflammation, DA neuron survival or behavioral defects; (4) Screening out candidate compounds that can reduce PSAP secretion, inhibit GPR37 expression, reduce IL-6 expression and secretion, alleviate neuroinflammation, improve DA neuron survival or alleviate behavioral deficits are potential drugs for the treatment of neurodegenerative diseases.

[0011] Furthermore, the animal models include a 6-hydroxydopamine (6-OHDA)-induced PD mouse model, a human A53Tα-synuclein (hA53T-αSyn) transgenic PD mouse model, and an adeno-associated virus-mediated hA53T-αSyn overexpression PD mouse model.

[0012] Fourthly, the present invention provides a medicament for treating neurodegenerative diseases, said medicament inhibiting the activation of the PSAP-GPR37-IL-6 signaling axis, specifically comprising at least one of the following: (1) Inhibit the expression or activity of GPR37 in oligodendrocytes; (2) Reduce PSAP secretion in DA neurons; (3) Inhibit the expression or secretion of IL-6 in oligodendrocytes; (4) Block the binding of PSAP to GPR37; (5) Block the activation of downstream signaling pathways of GPR37.

[0013] Furthermore, the inhibition of GPR37 expression in oligodendrocytes includes knocking out the GPR37 gene in oligodendrocytes in the early stages of neurodegenerative diseases.

[0014] Furthermore, the neurodegenerative diseases include, but are not limited to, Parkinson's disease and Alzheimer's disease.

[0015] Fifthly, the present invention provides a pharmaceutical composition for treating neurodegenerative diseases, comprising an active ingredient capable of inhibiting activation of the PSAP-GPR37-IL-6 signaling axis, said active ingredient comprising at least one of a GPR37 inhibitor, a PSAP inhibitor, an IL-6 inhibitor, a PSAP-GPR37 binding blocker, or a GPR37 downstream signaling pathway inhibitor.

[0016] The beneficial effects of this invention are: 1. This invention clarifies for the first time the mechanism of action of the PSAP-GPR37-IL-6 signaling axis as a pathogenic factor in neurodegenerative diseases (especially Parkinson's disease), and reveals the molecular pathway by which oligodendrocytes regulate neuroinflammation and neurodegenerative lesions through this signaling axis, filling the research gap in the existing technology on the participation of oligodendrocytes in the pathogenesis of PD.

[0017] 2. Based on the combination of biomarkers provided by the PSAP-GPR37-IL-6 signal axis, accurate diagnosis and disease assessment of neurodegenerative diseases can be achieved, providing a basis for early clinical intervention.

[0018] 3. The drug screening method provided by this invention is highly targeted and can efficiently screen potential therapeutic drugs that target the PSAP-GPR37-IL-6 signaling axis, providing a new technical route for the development of drugs for the treatment of neurodegenerative diseases.

[0019] 4. The treatment methods and pharmaceutical compositions provided by this invention can prevent or alleviate the progression of neurodegenerative diseases from the source by directly inhibiting the activation of pathogenic signaling axes, and significantly improve patients' motor and non-motor dysfunctions, showing good prospects for clinical application. Attached Figure Description

[0020] Figure 1 This is a diagram showing the results of Example 1 of the present invention, which verified that GPR37 is specifically expressed in oligodendrocytes and is significantly upregulated in the substantia nigra of a PD model.

[0021] Figure 2 This is a graph showing the expression profile and dynamic changes of GPR37 in the AAV-αSyn-induced PD mouse model in Example 1 of this invention.

[0022] Figure 3 This is a figure showing the experimental results of a PD mouse model mediated by 6-OHDA and AAV-mediated hA53T-αSyn overexpression in Example 2 of this invention.

[0023] Figure 4 This is a diagram showing the strategy and verification results of conditionally knocking out Gpr37 in oligodendrocytes in Example 2 of this invention.

[0024] Figure 5 This is a diagram showing the verification results of the mechanism linking PSAP with neurodegenerative diseases in Example 3 of the present invention.

[0025] Figure 6 This is a diagram showing the experimental verification results of conditional knockout of DA neurons or PSAP in the substantia nigra in Embodiment 3 of the present invention.

[0026] Figure 7This is a diagram showing the verification results of the mechanism by which PSAP induces upregulation of IL-6 expression and oligodendrocyte-derived IL-6 is associated with neurodegenerative diseases in Example 4 of this invention.

[0027] Figure 8 This is a graph showing the verification results of PSAP-induced upregulation of IL-6 expression in Example 4 of the present invention, and the fact that conditional knockout of IL-6 does not affect basic motor function and pain perception.

[0028] Figure 9 This is a diagram showing the verification results of the use of oligodendrocyte-derived IL-6 in Example 4 of the present invention, which is essential for 6-OHDA-induced neuroinflammation in PD mice.

[0029] Figure 10 This is a related verification of how early knockout of GPR37 in Example 5 of the present invention alleviates neurodegenerative diseases in PD mice.

[0030] Figure 11 This is a schematic diagram showing the results of observing the characteristics of the PSAP-GPR37-IL-6 signal axis in PD patients in Embodiment 6 of the present invention.

[0031] Figure 12 This is a schematic diagram of the supplementary feature analysis results of GPR37 in human PD samples in Embodiment 6 of the present invention.

[0032] Figure 13 This is a schematic diagram illustrating how oligodendrocytes drive neuroinflammation and neurodegenerative changes in PD via the PSAP-GPR37-IL-6 signaling axis, as verified by this invention. Detailed Implementation

[0033] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be described in detail, clearly, and completely below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. Furthermore, based on the embodiments of the present invention, any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art to all other embodiments obtained without creative effort should be included within the protection scope of the present invention.

[0034] First, the experimental materials and methods of the present invention are introduced. In the various embodiments of the present invention, unless otherwise specified, the experimental materials and methods follow the following description. Experimental details not described in detail in the present invention can be implemented by those skilled in the art based on operating habits or conventional operating instructions.

[0035] 1. Laboratory animals The transgenic mouse strain, Gpr37, was purchased from Jackson Laboratory. fl / flThe mice were created by Cyagen Biosciences, using Psap. fl / fl Mice were constructed by GemPharmatech Co., Ltd. The mice were synthesized via Plp1-Cre / ER and Gpr37. fl / fl Gpr37 conditional knockout (CKO) mice and littermate controls were obtained through mating of mice; Psap fl / fl Psap conditional knockout mice and littermate controls were obtained by mating mice with TH-Cre mice. All animals were housed in a 12-hour light / dark cycle environment with free access to food and water. Adult male mice (8-12 weeks old) were used for the experiment. Animals were randomly assigned to different experimental groups.

[0036] 2. Cell Culture Human embryonic kidney cell line (HEK293T, RRID: CVCL_0063) was cultured in DMEM medium (Gibco, #11965092) containing 10% fetal bovine serum, 100 units / mL penicillin, 100 μg / mL streptomycin and 4 mM L-glutamine at 37°C and 5% carbon dioxide.

[0037] Mouse oligodendrocyte precursor cells (OPCs) were isolated from the cerebral cortex of newborn mice, cultured in primary culture, and then induced to differentiate into oligodendrocytes by the addition of T3 and CNTF. In the specific experiment, the cerebral cortex of four newborn mice was placed in ice-cold HBSS (Gibco, #14025092) and washed three times. After mechanical homogenization, the cell suspension was transferred to T75 culture flasks pre-coated with poly-D-lysine (Sigma-Aldrich, #P0899, 0.1 mg / mL). The cells were cultured in DMEM / F12 (Gibco, #11320082) supplemented with 20% FBS at 37°C and 5% CO2. The culture medium was changed every 3 days after 8 days of culture. The culture flasks were then shaken at 200 rpm for 2 hours at 37°C, the medium was replaced with fresh medium, and then shaken at 250 rpm for another 16 hours. The cell suspension was centrifuged at 200 g for 5 minutes, and the cells were cultured in pre-coated culture dishes for 3-4 hours at 37°C and 5% CO2. The culture medium was then replaced with Neurobasal (Gibco, #21103049) medium supplemented with B27 (Gibco, #17504044) and N2 (Gibco, #17502001). OPC differentiation into oligodendrocytes (OLs) was induced by adding T3 (Sigma-Aldrich, #T6397, 40 ng / mL) and CNTF (PeproTech, #450-13, 10 ng / mL). To collect the conditioned medium for oligodendrocytes, the medium was replaced with Opti-MEM medium containing Vehicle or TX-14 and cultured for 12 hours.

[0038] Primary microglia were isolated from the brains of day 1 newborn mouse pups and cultured in primary culture for use in related experiments. In the specific experiment, the entire brain of day 1 newborn (P1) mouse pups was harvested. After removing the meninges, the brain was cut into small pieces (approximately 1 mm). 2 Brain cells were transferred to DMEM / F12 medium supplemented with 8 U / mL papain and 125 U / mL DNase and cultured at 37°C for 20 minutes in a 5% CO2 incubator. Digestion was terminated with DMEM / F12 complete medium. The brain cells were washed three times again with the same medium. The cell suspension was filtered through a 70 μm cell sieve to remove cell debris and clumps. The cells were centrifuged at 200 g for 10 minutes at room temperature, then resuspended and seeded into T75 culture flasks and cultured in a humidified incubator at 37°C with 5% CO2. The complete medium was replaced on day 4. Primary microglia were cultured in conditioned medium after stimulation with Vehicle or TX-14 for 18 hours. The culture medium and cell lysates were collected for further analysis.

[0039] 3. Viral vector AAV9-SYN-SNCA (A53T)-EGFP-3×FLAG-WPRE (AAV-αSyn), AAV9-hSyn-EGFP-3×FLAG-WPRE (AAV-GFP), and AAV9-CAG-mCherry-T2A-Cre (AAV-Cre-mCherry) were synthesized and packaged by ObioTechnology (Shanghai) Co., Ltd. AAV8-pDIO-DSE-mCherry-PSE-shIL-6 was synthesized and packaged by the Vector Center of Zhejiang University, and pDIO-DSE-mCherry-PSE-MCS was provided by Beatriz Rico (Addgene plasmid number #129669, http: / / n2t.net / addgene:129669, RRID: Addgene_129669). The mouse Il-6 shRNA target sequence (5'-GCTCTTCGGCAAATGCTTC-3') was inserted into the AvrII and EcoRI sites. The viral titer used exceeds 10. 12 vg / mL.

[0040] 4. Stereoscopic injection For a unilateral 6-OHDA-induced PD model, adult mice (8-12 weeks old) were anesthetized and fixed in a stereotaxic apparatus (RWD Life Sciences, Ltd.). Half an hour before 6-OHDA injection, mice were intraperitoneally injected with 25 mg / kg desipramine and 5 mg / kg pargeline. 1 μl of 6-OHDA (1 μg / μl dissolved in physiological saline containing 0.02% ascorbic acid, Tocris, #2547) was injected into the MFB according to stereotaxic coordinates (relative to the anterior fontanelle) A / P = -1.2 mm, M / L = -1.2 mm, D / V = -4.75 mm. The control group received the same volume of solvent (0.9% physiological saline containing 0.02% ascorbic acid) into the MFB. Injection was performed at a rate of 0.1 μl / min using a 10 μl Hamilton syringe and a 36-gauge needle. After injection, the needle was left in place for 5 min before being withdrawn. After injection, the mouse wound was sutured and placed on a heating pad until it was fully awakened from anesthesia.

[0041] For AAV-αSyn injection, AAV-GFP or AAV-αSyn was injected unilaterally into the substantia nigra of mice (A / P = -3.1 mm, M / L = -1.2 mm, D / V = -4.3 mm). The injection rate was 0.1 μl / min, and the injection volume was 1 μl. Three weeks after AAV-αSyn injection, mice received intraperitoneal injections of tamoxifen (100 mg / kg, once daily) for 6 days to perform a flipping experiment. Behavioral tests were performed 2 months after injection, and mice were euthanized for pharmacological analysis.

[0042] For the Psap knockdown experiment, AAV-Cre-mCherry was injected into Psap. fl / fl The substantia nigra of mice (0.25 μl each time, 2 injections) was mapped to coordinates of A / P = -3.08 mm, M / L = -1.5 mm, D / V = -4.08 mm and A / P = -3.52 mm, M / L = -1.0 mm, D / V = -4.3 mm. To knock down Il-6, AAV-pDIO-shIL-6 was injected into Plp1-Cre / ER mice 21 days after intraperitoneal injection of tamoxifen, following the same coordinates. WT mice were injected with an equal amount of the corresponding virus as a control. Injections were performed using the same syringe and needle at a rate of 0.05 μl / min. Three weeks later, 1 μl of 6-OHDA (1 μg / μl) was injected into the ipsilateral MFB, and behavioral testing was performed one week later.

[0043] For microinjection of recombinant IL-6 and TX-14, a 0.41 mm diameter stainless steel guide cannula (RWD Life Sciences, #62004) was implanted into the substantia nigra (A / P = -3.0 mm, M / L = -1.2 mm, D / V = -4.5 mm). The guide cannula was secured to the skull with two anchor screws and dental cement. During the microinjection, mice were briefly anesthetized with 5% isoflurane and maintained under 1% isoflurane. 0.5 μl of IL-6 (100 ng / μl, R&D Systems, #406-ML) was injected at a rate of 0.1 μl / min using the microinjection cannula for 12 consecutive days. After injection, the microinjection cannula was left in place for 5 minutes before removal. Control mice were injected with an equal volume of saline. For TX-14 injection, 0.5 μl of TX-14 (1 μg / μl, QYAOBIO) was injected at a rate of 0.1 μl / min using a microinjection cannula for 12 consecutive days. Behavioral tests were performed the day after the last injection, and mice were euthanized for pharmacological analysis.

[0044] 5. Immunofluorescence staining and in situ hybridization Mice were perfused with PBS via the cardiac cavity, followed by perfusion with 4% paraformaldehyde. The entire brain was removed, fixed overnight with 4% paraformaldehyde at 4°C, and then transferred to 30% sucrose. Immunofluorescence staining was performed on 30 μm thick sections. For staining, brain slices were blocked in PBS containing 2% BSA and 0.3% Triton X-100 at room temperature for 1 hour, then incubated overnight with primary antibody at 4°C. After washing three times with PBS, the brain slices were incubated with the corresponding fluorescently conjugated secondary antibody at room temperature for 2 hours. If antigen retrieval was required, the brain slices were incubated in sodium citrate buffer at 95°C for 20 minutes before staining. Detection and imaging were performed using a CCD SPOT camera (SPOT imaging). For high-resolution images, sections were photographed using an Olympus FluoView FV1000. The primary antibody dilutions used were as follows: chicken anti-β-galactosidase (1:5000), goat anti-Iba1 (1:1000), mouse anti-GFAP (1:1000), mouse anti-APC (1:200), mouse anti-olig2 (1:200), rabbit anti-ATF3 (1:200), goat anti-TH (1:1000), and rabbit anti-TH (1:1000). Secondary antibodies with different fluorescent labels were diluted 1:500. The number of DA neurons was determined to be 72 using stereotactic counting as previously described. To analyze fluorescence intensity, at least three coronal sections were selected from each brain, and three mice were measured per group. Fluorescence intensity was analyzed using NIH Image J software.

[0045] In situ hybridization was performed according to the instructions of the RNAscope Multiplex Fluorescence Manual Detection Kit (Advanced Cell Diagnostics). Brain tissue was prepared under RNase-free conditions, and 18 μm thick sections were used for in situ hybridization. Prehybridization, hybridization, and washing were all performed according to the kit instructions.

[0046] 6. Plasmid construction and transfection The entire coding region of the Gpr37 gene (#NM_010338) was cloned by PCR. The primers used for PCR cloning are as follows: Upstream primer: 5'-ATGAAGCTTATGAGCTACCTGCTGCTG-3' Downstream primer: 5'-ATGGTACCCTAGTCTTCAGCTTCTTGG-3'.

[0047] PCR products were inserted into the HindIII and KpnI restriction sites of the mammalian expression vectors pCMV-Flag and pEGFP-N1, respectively. All constructed plasmids were sequenced by Hangzhou Qingke Biotechnology Co., Ltd. to verify sequence and orientation. The GPR37 plasmid was transfected into HEK293T cells using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. Cells were used for subsequent experiments 48-72 hours post-transfection.

[0048] 7. Real-time quantitative PCR and protein detection Total RNA was extracted from tissues or cells, reverse transcribed, and then subjected to real-time quantitative PCR to detect the mRNA expression levels of genes such as Gpr37, Psap, and IL-6. The expression or secretion levels of proteins such as PSAP, IL-6, and p-ERK1 / 2 in tissues, cell culture media, or cerebrospinal fluid were detected by immunoblotting or ELISA.

[0049] 8. Behavioral testing Adult male mice were allowed to acclimatize to their environment for at least two days before the tests began. All behavioral tests were conducted in a double-blind manner.

[0050] Rotary bar test: Motor function in mice was assessed using a rotary bar system (IITC Life Science Inc.). Before testing, mice were trained on a rotary bar rotating at 6 rpm until they could sustain the rotation for 5 minutes, with training continuing for two consecutive days. During testing, the rotary bar started at 4 rpm and accelerated to 40 rpm within 5 minutes. The duration of the mouse's movement on the rotary bar and the rotational speed at the moment of fall were recorded. The test was repeated three times, with 20-minute intervals between each attempt, and the average value was taken.

[0051] Mine Field Experiment: Under normal lighting conditions, mice were placed in the center of a mine field (40 cm long × 40 cm wide × 30 cm high). The distance the mice traveled in the mine field over 30 minutes was measured using an Open Field Video Tracking System.

[0052] Apomorphine-induced rotation assay: Two weeks after the 6-OHDA-induced PD model, an apomorphine-induced rotation assay was performed. Mice were subcutaneously injected with apomorphine (US Pharmacopeia, #R08440, 1 mg kg-1) and placed in a circular container. After acclimatization for 5 minutes, the number of rotations to the contralateral side was recorded, and the recording time was 30 minutes.

[0053] Von Frey experiment: Mice were placed in a 10 cm square plastic box for 2 hours to acclimatize and enter a quiet state before the experiment began. Von Frey fibers (0.02-2.56 g, stoelting) were used to stimulate the mid-paw of the mouse's hind paw. Starting with 0.16 g, each mouse was tested 6 times, and Dixon's up-down method 75 was used to assess the paw withdrawal threshold. When the mouse exhibited paw withdrawal behavior, a finer fiber was used; if there was no response, a thicker fiber was used.

[0054] Tail-flick test: Immerse the tail (one-third of its length) of a mouse in water bath at 48℃, 50℃, or 52℃, and record the reaction time for the mouse's tail to shake off water. The duration of the mouse's tail in the water should not exceed 20s, 15s, or 10s, respectively. Repeat the test 3 times, with an interval of 1 minute between each test, and take the average value.

[0055] Pole climbing experiment: Mice were placed head-up at the top of a vertical pole with a diameter of 9 mm and a height of 75 cm, wrapped with gauze. The total time it took for them to climb to the bottom of the pole was recorded. Before the test, the mice were trained for two consecutive days, three times a day.

[0056] 9. Observation using transmission electron microscopy Rats were anesthetized with sodium pentobarbital (50 mg / kg), and after cardiac perfusion with phosphate buffer (0.1 M, pH 7.4) containing 4% glutaraldehyde, the brains were removed and fixed in 4% glutaraldehyde at 4°C for at least one week. The corpus callosum (CC) and striatum (CPu) (1 mm) were then sectioned and collected. 3The samples were placed in 4% glutaraldehyde and incubated overnight at 4°C. They were then rinsed three times with 0.1 M sodium dimethylarsenate (CAS) at 4°C for 10 min each time. Afterward, they were fixed on ice for 1 hour with OsO4 (2%) containing 3% K3Fe(CN)6. Next, the samples were rinsed four times with deionized water at 4°C for 5 min each time, incubated on ice with 4% uranium acetate for 1 hour, and then rinsed with deionized water at room temperature for 5 min. Next, the samples were dehydrated sequentially with 50%, 70%, 90%, and 95% ethanol for 15 min each, followed by dehydration with 100% ethanol for 30 min. Subsequently, the samples were placed in different ratios of acetic acid:Epon embedding agent mixtures (1:3, 1:1, 3:1) for 2 hours each, and then embedded overnight with 100% Epon embedding agent. During the polymerization reaction, samples embedded in 100% Epon were placed in a 45°C oven for 12 hours, followed by a 65°C oven for 48–72 hours. Ultrathin sections of 60 nm were prepared and stained with uranyl acetate and lead citrate. Images were taken using a Thermo Scientific Talos L120C 120 kV transmission electron microscope. Axon diameter and myelin thickness were calculated using ImageJ software.

[0057] 10. Cell cluster analysis We obtained snRNA-seq data from the GSE178265 database, screened samples from PD patients and healthy individuals, and analyzed the expression distribution of GPR37 in different cell types after processing such as standardization, scaling, batch effect removal, and dimensionality reduction clustering.

[0058] 11. Statistical Analysis All data were statistically analyzed using GraphPad Prism 6.1 software. Data are presented in mean ± SEM format. Differences between two groups were analyzed using a two-tailed Student's unpaired t-test. For data with more than two groups, one-way ANOVA was used, followed by Bonferroni multiple comparison correction. For comparisons of multiple mixed groups, two-way ANOVA was used, followed by Bonferroni multiple comparison correction. The slope of the G-ratio and axon diameter was compared using simple linear regression analysis. Statistical criteria for differences were: ns (no significant difference), p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; statistical methods used in all experiments can be found in the legend.

[0059] The core technical point of this invention is that the PSAP-GPR37-IL-6 signaling axis is a key pathogenic factor in neurodegenerative diseases (especially Parkinson's disease). Several examples are provided below to verify this conclusion.

[0060] Example 1: Validation of GPR37 specific expression in oligodendrocytes in SNpc and significant upregulation in a PD model.

[0061] To confirm the expression pattern of GPR37 in SNpcs, this embodiment used in situ hybridization to detect the expression of Gpr37 mRNA in SNpcs of Plp1-Cre / ER;Ai14 oligodendrocyte reporter mice. Figure 1 Figure A shows the co-localization detection of Gpr37 mRNA with tdTomato and TH in the substantia nigra of Plp1-Cre / ER;Ai14 mice, scale bar = 50 μm; inset represents the corresponding magnified area, scale bar = 10 μm; Figure 1 B shows Gpr37 + Percentage of cells colocalized with tdTomato or TH (n = 9). Figure 1 A and Figure 1 The results of B show that, unlike the publicly available research in the prior art, Gpr37 in tdTomato + It is specifically expressed in oligodendrocytes, but not in TH (a marker of dopamine neurons) positive dopamine neurons.

[0062] To determine the expression distribution of Gpr37 mRNA in SNpc, this example uses Gpr37-LacZ reporter mice containing the bacterial β-galactosidase (β-Gal) reporter gene at the Gpr37 site. Figure 1 Figure C shows the colocalization detection of β-Gal with Gpr37 mRNA, APC, TH, GFAP and Iba1 in the substantia nigra of Gpr37-LacZ reporter mice, scale bar = 50 μm and 10 μm (inset). Figure 1 D shows Gpr37 in the substantia nigra. + With β-Gal + Percentage of cell colocalization (n = 9); Figure 1 E represents β-Gal. + Percentage of colocalization with each cell marker (n = 5). From Figure 1 C Figure 1 D and Figure 1 As shown in E, Gpr37 (labeled by β-Gal) is mainly expressed in APC. +It is expressed in oligodendrocytes, but not in cells that are positive for TH, Iba1 (microglia marker), and GFAP (astrocytosis marker).

[0063] To determine the potential role of GPR37 in PD development, this study examined the expression of Gpr37 mRNA in 6-hydroxydopamine (6-OHDA)-induced PD mice, human A53T α-synuclein (hA53T-αSyn) transgenic PD mice, and adeno-associated virus-mediated hA53T-αSyn (AAV-αSyn) overexpression PD mice (SNpc). Figure 1 F and Figure 1 G shows elevated Gpr37 mRNA expression in the substantia nigra of 6-OHDA-induced PD mice (F) and human A53T α-Syn transgenic mice (G), n=4-7; Figure 2 Figure A shows the qPCR detection of Gpr37 mRNA in AAV-αsyn-induced PD model mice, analyzed using Tow-way ANOVA followed by Bonferroni's post hoc test. P = 0.0011, n = 4–6. Figure 1 As shown in Figure F, one week after 6-OHDA injection, the expression of Gpr37 mRNA in SNpc significantly increased. Similarly, as... Figure 1 As shown in G and 2A, in A53T transgenic PD mice ( Figure 1 G) and AAV-αSyn-induced PD mice ( Figure 2 A) In the early stages, the expression of Gpr37 mRNA was also significantly upregulated.

[0064] Figure 1 H shows the colocalization detection of Gpr37 mRNA, Plp1 mRNA, and TH in the substantia nigra of WT or A53T transgenic mice (6 months old), with scale bars of 100 μm and 20 μm. Figure 2 Figure B shows the co-localization detection of Gpr37 and Plp1 mRNA in the substantia nigra of WT mice injected with AAV-GFP or -αSyn, scale bar = 100 μm; the right side is a magnified image, scale bar = 20 μm. Figure 1 H and Figure 2 As shown in B, in hA53T transgenic mice ( Figure 1 H) and AAV-αSyn-induced PD mice ( Figure 2 B) In SNpc, Gpr37 mRNA expression is also mainly concentrated in Plp1. + Oligodendrocytes.

[0065] These results indicate that GPR37 is specifically expressed in oligodendrocytes in SNpc and is significantly upregulated in the PD model.

[0066] All data in this embodiment are mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001. Data were obtained through a two-tailed Student's unpaired t-test. Figure 1 F) and Bonferroni's two-way ANOVA ( Figure 1 G and Figure 2 A) Statistical analysis.

[0067] Example 2: Verification of the correlation between GPR37 deficiency and the alleviation of neurodegenerative diseases and behavioral deficits induced by 6-OHDA or hA53T-αSyn.

[0068] To investigate whether GPR37 in oligodendrocytes is involved in regulating toxin-induced DA neuronal death, this embodiment constructs a PD model by unilaterally injecting 6-OHDA into the medial forebrain tract (MFB) of WT, Gpr37 knockout (gKO), and Gpr37 CKO mice.

[0069] Figure 3 A and Figure 3 C represents the TH staining of substantia nigra DA neurons (scale bar = 200 μm) and striatal DA nerve fibers (scale bar = 400 μm) 3 weeks after injection of saline or 6-OHDA. Figure 3 B is the substantia nigra TH. + Cell count (n = 3-5); Figure 3 D is the striatum TH + Quantitative analysis of the relative optical density of DA nerve fibers was performed using 3-4 mice per group, yielding 9-12 slides in total. The figures show that, compared to WT PD mice, Gpr37 gKO and CKO PD mice exhibited significantly reduced DA neuron death. Figure 3 A and 3B); the density of DA nerve fibers in the striatum of Gpr37 gKO and CKO mice was also significantly restored (A and 3B); Figure 3 C and 3D).

[0070] Figure 3 E and Figure 3 F represents the apomorphine (APO)-induced rotation and rotarod tests performed 2 weeks after injection of saline or 6-OHDA (n = 8-17). The results in the figure show that, through APO-induced rotation and rotarod tests, Gpr37 deficiency significantly alleviated 6-OHDA-induced motor dysfunction. Figure 3 E and 3F).

[0071] Pain is a common and increasingly recognized non-motor symptom in PD, affecting 30-85% of patients. Figure 3 G and Figure 3 H represents the detection of mechanoreachia and thermal reachia in WT, Gpr37 gKO, and CKO PD mice induced by 6-OHDA (n = 5-17). The results in the figure show that, as previously reported, 6-OHDA induces mechanoreachia and thermal reachia in WT mice, while in Gpr37 gKO and CKO PD mice, mechanoreachia (…) Figure 3 G) and hot pain ( Figure 3 H) was relieved.

[0072] To better understand the role of GPR37 in PD patients, this embodiment constructs a PD mouse model mediated by AAV-mediated hA53T-αSyn overexpression, which better reflects the clinical and pathological characteristics of PD. Figure 3 I is a representative TH staining image of substantia nigra DA neurons 3 months after injection of AAV-GFP (control) or AAV-αSyn (scale bar = 100 μm). Figure 3 J is the substantia nigra TH + Cell number (left) and striatal TH + Quantitative statistics of relative optical density of DA nerve fibers (right), n = 6-10; Figure 3 K is the substantia nigra NeuN + The number of neurons (n ​​= 4-6); Figure 3 L- Figure 3 O were used to test AAV-αSyn induced PD mice in rotarod test (L), pole climbing test (M), von Frey test (N) and tail flicking test (O) (n = 6-11); Figure 3 P is an immunoblot of human αSyn and β-actin proteins in the substantia nigra; Figure 3 Q was used to quantify αSyn using β-actin as an internal control (n = 4). The results in the figure show that, as previously described, unilateral injection of AAV-αSyn led to a reduction in DA neurons in the SNpc of WT mice and a decrease in TH neurons in the ipsilateral striatum. + Decreased nerve fiber density ( Figure 3 In contrast, Gpr37 gKO mice showed significant remission of AAV-αSyn-induced neurodegeneration (I-3K). Figure 3 I-3K); In addition, compared with WT PD mice, Gpr37 gKO PD mice showed motor impairment ( Figure 3 L and 3M) as well as chronic mechanical hyperalgesia and thermal hyperalgesia (L and 3M) Figure 3N and 3O) significantly alleviated, and the expression level of total αSyn in SNpc of WT and gKO mice was comparable ( Figure 3 P and 3Q).

[0073] To determine whether GPR37 regulates the formation of pathological αSyn, this embodiment examined the phosphorylation (p-αSyn) of the typical pathological form of αSyn Ser129. Figure 3 R is an immunofluorescence staining image of p-αSyn and TH in the substantia nigra 3 months after AAV-GFP and AAV-αSyn injection, scale bar = 200 μm; Figure 3 S represents the quantitative measurement of the relative optical density of p-αSyn in the substantia nigra (n = 4-5). The results in the figure show that AAV-αSyn-induced accumulation of p-αSyn in SNpc of WT PD mice is significantly increased, while p-αSyn is significantly decreased in Gpr37gKO PD mice.

[0074] in, Figure 3 B. Figure 3 DF, Figure 3 JM, Figure 3 Q and Figure 3 S was analyzed using Bonferroni's one-way ANOVA. Figure 3 G, Figure 3 H, Figure 3 N and Figure 3 Bonferroni's two-way ANOVA analysis was performed in O. All data are expressed as mean ± SEM. ns: no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0075] To clarify the regulatory function of GPR37 in oligodendrocytes in PD, we constructed mice with conditional knockout of Gpr37 in oligodendrocytes (CKO). Figure 4 A is a schematic diagram of the strategy for conditionally knocking out Gpr37 (Gpr37 CKO) in oligodendrocytes. Figure 4 B is Gpr37 fl / fl In situ hybridization detection of Gpr37 in the substantia nigra of (WT) and Gpr37 CKO mice, scale bar = 200 μm.

[0076] Figure 4 C- Figure 4 F shows the results obtained through the rotating bar experiment ( Figure 4 C, n = 15-18), Open Field Experiment (OFT) ( Figure 4 D, n = 10⁻⁸), von Frey experiment ( Figure 4 E, n = 9-14) and tail-flick experiment ( Figure 4 F, n = 12-14) was used to detect motor function and basic pain sensation in mice. Among them, Figure 4 C Figure 4 D and Figure 4 E uses a two-tailed student's unpaired t-test for analysis. Figure 4 In F, tow-way ANOVA followed by Bonferroni's post hoc test was used for analysis. The above experiments revealed the motor function of Gpr37 CKO mice (…). Figure 4 C and 4D) and baseline pain threshold ( Figure 4 E and 4F mice were not different from wild-type (WT) mice.

[0077] To investigate potential changes in myelin sheath and related axons in Gpr37 CKO mice, we examined the integrity of myelin sheath and related axons using transmission electron microscopy (EM). Figure 4 G and Figure 4 H represents the corpus callosum of WT and Gpr37 CKO mice, respectively. Figure 4 G, CC) and striatum (G, CC) and striatum Figure 4 Transmission electron microscopy (TEM) images of myelin sheath in H, CPU, scale bar = 500 and 250 μm; Figure 4 I is a statistical analysis of the diameter of a single axon in the CC and CPU of WT and Gpr37 CKO mice. There were 4 mice in each group. The number of axons in the CC was 355 and 370, respectively, and the number of axons in the CPU was 200 and 212, respectively. Figure 4 J represents a statistical analysis of the mean axonal diameter in the CC and CPU of WT and Gpr37 CKO mice, with 4 mice in each group; Figure 4 K and Figure 4 M represents the CC (coronavirus) levels in WT and Gpr37 CKO mice, respectively. Figure 4 K) and CPU Figure 4 Statistical analysis of the average G-Ratio of myelin sheath in M), n = 4; Figure 4 L and Figure 4 N represents the CC of WT and Gpr37 CKO mice ( Figure 4 L) and CCu ( Figure 4 Detection of G-Ratio distribution of individual axons in N), Figure 4 In L, n = 4, there are 356 and 370 axons respectively. Figure 4 In N, n = 4, and there are 200 and 212 axons respectively; Figure 4O is the average number of myelinated axons per square millimeter of CC in WT and Gpr37 CKO mice (n = 4). Figure 4 P represents the proportion of myelinated axons of different diameters in the CC of WT and Gpr37 CKO mice (n = 4). Figure 4 I- Figure 4 K, Figure 4 M and Figure 4 O was analyzed using a two-tailed student's unpaired t-test. Figure 4 P was analyzed using Tow-way ANOVA followed by Bonferroni's post hoctest. Figure 4 L and Figure 4 N was analyzed using simple linear regression of the slope. ns: no significant difference. All data are expressed as mean ± SEM.

[0078] EM data showed that the axonal diameter in the corpus callosum (CC) and striatum (CPu) of WT and Gpr37 CKO mice was significantly different. Figure 4 G- Figure 4 J), myelin G-Ratio ( Figure 4 K- Figure 4 N) and the number of myelinated axons ( Figure 4 O and Figure 4 There were no significant differences in P values. These data indicate that conditional knockout of Gpr37 in oligodendrocytes does not impair the integrity of myelin sheath and axons, motor function, or baseline pain threshold in mice.

[0079] These data suggest that GPR37 deficiency can alleviate DA neuronal damage, motor dysfunction, and chronic pain induced by toxins and αSyn aggregates.

[0080] Example 3: The effect and mechanism of PSAP on PD.

[0081] Known research has found that PSAP is an endogenous ligand of GPR37. Based on this disclosure, this embodiment further explores the potential regulatory function of PSAP on PD.

[0082] Figure 5 A shows the co-localization detection of Psap mRNA with TH or Gpr37 mRNA in SNpc, with scale bars of 100 μm and 20 μm (inset). This demonstrates that Psap co-localizes with TH or Gpr37 mRNA in SNpc. + Selective enrichment in DA neurons. Furthermore, from... Figure 5Western blot analysis of B showed that PSAP levels were significantly increased in the cerebrospinal fluid of PD mice one week after 6-OHDA injection, meaning that the PSAP protein content was significantly increased in the CSF of the 6-OHDA-induced PD mouse model.

[0083] To explore the role of PSAP in PD, this embodiment utilizes PSAP. fl / fl Wt mice (WT mice) were crossed with TH-Cre mice to create mice with conditional knockout of Psap in DA neurons (Psap CKO). Figure 5 C is a schematic diagram of the strategy of conditionally knocking out Psap (Psap CKO) in DA neurons.

[0084] Figure 6 A and Figure 6 B represents the in situ hybridization of Psap in SNpc of WT and Psap CKO mice, respectively. Figure 6 A) and qPCR ( Figure 6 B) Detection, scale bar = 20 μm. As can be seen from the figure, compared with WT, the expression of Psap in DA neurons of Psap CKO mice was significantly reduced.

[0085] Figure 6 C and Figure 6 D represents the TH staining of SNpc dopamine neurons (scale bar = 200 μm) in WT and Psap CKO mice, respectively. Figure 6 C) and quantitative statistical results of SNpc dopaminergic neurons and striatal dopaminergic fibers ( Figure 6 (D, n=4-5), as can be seen from the figure, the number of DA neurons and the density of striatal DA nerve fibers in the Psap CKO mouse SNpc are comparable to those in the WT mouse.

[0086] Figure 6 E and Figure 6 F represents the baseline motor function of WT and Psap CKO mice as determined by open field test, rotarod test, von Frey test, and tail-flick test. Figure 6 E) and sensation ( Figure 6 F) Function (n = 8-9), as shown in the figure, there was no significant difference in motor function and baseline pain threshold between Psap CKO mice and WT mice.

[0087] Figure 5 D is a representative TH staining image of SNpc dopamine neurons (left, scale bar = 200 μm) and striatal dopamine nerve fibers (right, scale bar = 500 μm) 3 weeks after 6-OHDA injection. Figure 5 E is TH+ Quantitative statistics of cell number (left) and dopamine nerve fiber optical density (right), n = 4. As can be seen from the figure, the death of DA neurons in Psap CKO mice was significantly reduced after injection of 6-OHDA.

[0088] Figure 5 F- Figure 5 I show the use of the rotator experiment ( Figure 5 F), APO-induced rotation experiment ( Figure 5 G), von Frey experiment ( Figure 5 H) and tail-flick experiment ( Figure 5 I) The test results showed that the behavioral disorders of Psap CKO PD mice were significantly alleviated compared with WT mice (n = 6-7).

[0089] Figure 6 G shows the accumulation of DA neurons and p-αSyn in SNpc of WT and Psap CKO mice 3 months after AAV-αSyn injection, detected by immunofluorescence staining, scale bar = 200 μm; Figure 6 H is TH + Quantitative statistics of cell count; Figure 6 I is a quantitative statistic of the relative optical density of p-αSyn in SNpc; Figure 6 J is the striatum TH + Quantitative analysis of the relative optical density of dopaminergic fibers. Among them, Figure 6 H- Figure 6 J uses a two-tailed student's unpaired t-test for analysis, n = 4, P = 0.0032 ( Figure 6 H), 0.0010 ( Figure 6 I), 0.0111 ( Figure 6 J); Figure 6 K and Figure 6 L represents the results of rotarod and pole climbing experiments (K and L) performed after AAV-αSyn-induced PD model. Analysis was performed using tow-way ANOVA followed by Bonferroni's post hoctest (n = 9-10, *P < 0.05). The results showed that the accumulation of pathological αSyn and the loss of DA neurons were significantly reduced in the SNpc of AAV-αSyn-induced Psap CKO PD mice. Figure 6 G- Figure 6 J), and the movement disorder was also significantly alleviated ( Figure 6 K and Figure 6 L).

[0090] To further confirm whether PD-related degenerative changes depend on PSAP at the SNpc site, such as Figure 5 As shown in J, this embodiment achieves this by sending data to Psap. fl / fl AAV-Cre-mCherry was injected into mouse SNpcs to specifically knock out Psap (Psap cKO) in SNpcs.

[0091] Figure 6 In the image M, left, immunostaining shows co-localization of mCherry (Cre) and TH in AAV-Cre-injected mouse SNpcs (scale bar = 200 μm). The boxed area in the image is magnified at the bottom (scale bar = 20 μm). Right, TH in cells co-expressing tdTomato. + Percentage in cells, n = 9 slices from 3 mice; Figure 6 N represents the in situ hybridization detection of Psap in SNpc of WT and Psap cKO mice, scale bar = 25 μm; Figure 6 O is qPCR analysis showing that Psap expression in SNpc of Psap cKO mice was significantly reduced; Figure 6 P- Figure 6 R represents the basic motor function of WT and Psap cKO mice, respectively. Figure 6 P and Figure 6 Q) and feelings ( Figure 6 R) functional assay (n = 13). Results showed that Psap expression was significantly reduced in SNpc of Psap cKO mice (n = 13). Figure 6 M- Figure 6 O), however its basic motor function ( Figure 6 P and Figure 6 Q) and pain threshold were not significantly different from those in WT mice. Figure 6 R).

[0092] Figure 5 K and Figure 5 L shows the death of DA neurons in the substantia nigra of Psap cKO PD mice. Figure 5 K, n = 4) and TH in the striatum + Decrease in fluorescence density ( Figure 5 L, n = 9-12 brain slices (from 3-4 mice) were significantly reduced. Figure 5 M- Figure 5 P represents the results of the rotating bar experiment ( Figure 5 M), APO-induced rotation experiment ( Figure 5 N), von Frey experiment ( Figure 5 O) and tail-flick experiment ( Figure 5P) detection revealed a significant reduction in behavioral disorders in Psap cKO PD mice (n = 11-13). The results in the figure indicate that 6-OHDA-induced DA neuron death in the SNpc of Psap cKO PD mice was significantly reduced. Figure 5 K and 5L), and the accompanying motor impairments were also significantly improved ( Figure 5 M-5P).

[0093] These results indicate that PSAP is primarily expressed in dopaminergic neurons in the substantia nigra, and that PD-related neurodegenerative diseases of dopaminergic neurons depend on PSAP.

[0094] In this embodiment, Figure 5 Two-tailed Student's unpaired t-test was used to analyze B, EG, and KN, while two-way ANOVA followed by Bonferroni's post hoc test was used to analyze H, I, O, and P. Figure 6 The free and positive (F) and reverse (R) results were analyzed using two-way ANOVA followed by Bonferroni's post-hoc test. The results for B, D, E, F, and the relative odds (OR) were analyzed using a two-tailed Student's unpaired t-test. For B, P = 0.0005 (n = 4); for O, P < 0.0001 (n = 5). ns: no significant difference. All data are presented as mean ± SEM.

[0095] Example 4: PSAP induces upregulation of IL-6 in oligodendrocytes via GPR37 and promotes PD-like dysfunction.

[0096] Previous studies have found that osteocalcin can negatively regulate oligodendrocyte differentiation and myelin formation through GPR37. In this example, qPCR was used to detect the expression of Apc, Mbp, and olig2 mRNA in SNpc of WT and Gpr37 gKO mice one week after 6-OHDA injection. The results are as follows: Figure 8 As shown in Figure A, although Apc mRNA expression was higher in the SNpc of Gpr37 gKO mice, there was no significant difference in the expression of Apc, Mbp, and olig2 mRNA between WT and Gpr37 gKO mice injected with 6-OHDA. These experiments and data suggest that the function of GPR37 in mediating oligodendrocyte differentiation and myelination may not be involved in 6-OHDA-induced PD-like neurodegeneration.

[0097] To investigate the role of GPR37 in the immunomodulatory function of oligodendrocytes, this example uses primary cultured oligodendrocytes for testing. Figure 8 Figure B shows the expression of Apc, Iba1, and TH mRNA in primary WT oligodendrocytes detected by qPCR (n = 5). Figure 7 A shows that the PSAP-derived peptide TX-14 mediates the increase in IL-6 mRNA expression in primary oligodendrocytes in a dose-dependent manner; Figure 8 C shows the expression of cytokines and chemokines (IL-6, Tnfα, Tgfβ1, IL-18, IL-1β, IL-10, IL-12A) and olig2, Mog, and Plp1 mRNA in primary cultured oligodendrocytes of WT after TX-14 stimulation for 2 hours, as detected by qPCR. Figure 7 A and Figure 8 C indicates that the PSAP-derived peptide (TX-14) significantly upregulated IL-6 expression in primary cultured oligodendrocytes in a concentration-dependent manner. Conversely, Figure 7 qPCR detection of IL-6 mRNA in primary cultured oligodendrocytes (n = 9-11) showed that TX-14 could not mediate IL-6 expression in oligodendrocytes derived from Gpr37 gKO mice.

[0098] Figure 8 D shows that the Gαi inhibitor PTX (100 ng / ml) inhibits TX-14-induced ERK1 / 2 phosphorylation in HEK293T cells. Figure 8 E represents the quantification of phosphorylated ERK1 / 2. Tow-way ANOVA followed by Bonferroni's post hoctest analysis showed that GPR37 can couple with Gαi to mediate downstream ERK phosphorylation, which is inhibited by the Gαi inhibitor PTX.

[0099] Figure 8 F represents the effect of qPCR on Il-6 mRNA expression in TX-14-induced primary oligodendrocytes, detected by PTX (100 ng / ml) and MEK inhibitor U0126 (1 μM). Cells were pretreated with the inhibitor for 8 hours (PTX) or 1 hour (U0126) before TX-14 (50 nM) stimulation. Figure 8 As shown in F, both PTX and the MEK inhibitor U0126 can inhibit TX-14-mediated upregulation of IL-6 expression.

[0100] All of the above results show that PSAP-derived peptides mediate the upregulation of IL-6 expression in oligodendrocytes through the GPR37 / Gαi / MEK pathway.

[0101] Regarding whether TX-14 treatment is sufficient to induce neurodegenerative diseases and PD-like dysfunction, Figure 7 C shows immunofluorescence staining of DA neurons in the substantia nigra of mice injected with saline or TX-14, scale bar = 200 μm. Figure 7 D shows the TH in the substantia nigra. + Neuron count (n = 4-5) Figure 7 E shows the quantitative statistics of the relative optical density of DA nerve fibers in the striatum (n = 5-6). The results in the figure show that, compared with the WT control group and Gpr37 gKO mice, WT mice with unilateral injection of TX-14 in SNpc mice had a significant reduction in DA neurons.

[0102] Figure 7 In F, the motor function of mice was assessed using a rotarod test (n = 8-10). Figure 8 In G, mechanorexia in mice was assessed using the von Frey assay. Analysis was performed using tow-way ANOVA followed by Bonferroni's post-hoc test. P < 0.0001 (WT+Vehicle vs. WT+TX-14) and P = 0.0373 (WT+TX-14 vs. gKO+TX-14) (n = 9-10) showed that TX-14 significantly impaired motor coordination in WT mice. Figure 7 F) and abnormal mechanical pain, which were not observed in Gpr37 gKO mice ( Figure 8 (G), indicating that PSAP-derived peptides can induce neurodegenerative diseases and PD-like dysfunction in a GPR37-dependent manner.

[0103] To verify the elevated IL-6 levels in peripheral blood and brain during PD, this embodiment uses 6-OHDA injection as the detection method. Figure 7 G is the qPCR detection of IL-6 mRNA in the substantia nigra (n = 6-7). Figure 7 H and Figure 7 I represents the substantia nigra of mice one week after injection of physiological saline or 6-OHDA. Figure 7 H) and cerebrospinal fluid ( Figure 7 ELISA analysis of IL-6 protein levels in WT PD mice (n = 4-6) showed increased IL-6 mRNA expression in SNpcs, while no increase in IL-6 expression was detected in Gpr37 gKO mice. Similarly, 6-OHDA-treated WT PD mice SNpcs... Figure 7 H) and CSF ( Figure 7I) IL-6 protein levels were elevated, but no elevation was observed in Gpr37 gKO mice treated with 6-OHDA.

[0104] To determine the specific regulatory role of IL-6 in oligodendrocytes in 6-OHDA-induced neurodegeneration, this study injected Cre-dependent IL-6 shRNA virus (AAV-DIO-shIL-6-mcherry) into the substantia nigra of Plp1-Cre / ER mice to conditionally knock down IL-6 in oligodendrocytes of the substantia nigra. Figure 7 J). For example Figure 8 As shown in Figure H, the left image shows immunofluorescence staining revealing co-localization of mCherry (shIL-6) and olig2, with scale bars of 50 μm and 10 μm. The right image shows mCherry. + The percentage of cells co-expressing olig2 and olig2 + Percentage of cells co-expressing mCherry (n = 10 slices from 3 mice). Figure 8 I- Figure 8 K was tested using a rotating rod experiment ( Figure 8 I), von Frey experiment ( Figure 8 J) and tail-flick experiment ( Figure 8 K) The baseline motor function and pain threshold of IL-6 knockdown mice were detected. The results in the figure show that mCherry-shIL-6 is mainly expressed in olig2. + Oligodendrocytes ( Figure 8 H), and knockdown of IL-6 in oligodendrocytes has no significant effect on basic motor function and pain sensation. Figure 8 I- Figure 8 K).

[0105] This embodiment utilizes 6-OHDA to construct a PD model for detection. Figure 7 K represents the immunofluorescence staining of TH neurons in the substantia nigra (scale bar = 200 μm) and the number of TH+ neurons (n ​​= 3) 3 weeks after 6-OHDA injection. Figure 7 L and Figure 7 M was tested using a rotating bar experiment ( Figure 7 L) and APO-induced rotation experiment ( Figure 7 M) assays revealed that IL-6 knockdown in oligodendrocytes significantly improved motor dysfunction in PD mice (n = 7-10). The results in the figure show a significant reduction in DA neuronal damage in IL-6 knockdown PD mice. Figure 7 K), movement disorders ( Figure 7 L and Figure 7 M) and mechanical pain sensitivity ( Figure 8The IL-6 levels significantly alleviated symptoms. These data indicate that IL-6 in oligodendrocytes is crucial for dopamine neuron death, motor dysfunction, and chronic pain in PD mice.

[0106] To investigate whether and how IL-6 can induce PD-like symptoms, this study involved unilateral injection of recombinant IL-6 protein into WT mice (SNpc) and comparison with a control group of mice injected with saline. Figure 7 N-stereoscopic injection and timeline diagram Figure 7 O and Figure 7 P represents TH in SNpc. + Quantitative analysis of the number of dopamine neurons (O) and the optical density of dopamine fibers in the striatum (P) (n = 6-7). Figure 7 Q refers to rotarod experiments performed 12 days after injection of saline or recombinant IL-6 protein into the substantia nigra (n = 13-15). Results showed that DA neurons in the SNpc of IL-6-injected mice were significantly reduced, and the optical density of TH-positive fibers in the striatum was significantly decreased. Figure 7 O and Figure 7 Furthermore, IL-6 injection also caused motor coordination impairment in mice (P). Figure 7 Q) and mechanical pain sensitivity ( Figure 8 M).

[0107] Activation of microglia and astrocytes in the substantia nigra is a pathological feature of neuroinflammation in Parkinson's disease (PD). This example demonstrates the detection of microglia and astrocyte proliferation using immunofluorescence staining and qPCR. Figure 7 R and Figure 7 S represents the immunofluorescence staining (R, scale bar = 100 μm) and qPCR analysis of Iba1 and GFAP in the substantia nigra, respectively. Figure 7 (S, n = 5) As shown in the figure, Iba1 and GFAP in the brain of mice injected with IL-6 were significantly increased, indicating enhanced neuroinflammation in SNpc.

[0108] also, Figure 9 A and Figure 9 C represents the number of microglia in the substantia nigra of WT, Gpr37 gKO, and CKO mice detected by immunofluorescence staining two weeks after 6-OHDA injection. Figure 9 A) and astrocytes ( Figure 9 C) activation, scale bar = 200 μm, Figure 9 B and Figure 9 D represents Iba1 in SNpc. + Microglia ( Figure 9 B) and GFAP + Astrocytes ( Figure 9D) Quantitative statistical analysis of relative optical density revealed that 6-OHDA-induced activation of microglia and astrocytes in ipsilateral SNpc of WT PD mice was increased, while the absence of Gpr37 and significantly reduced IL-6 expression in oligodendrocytes were observed. Figure 7 G- Figure 7 In Gpr37 gKO and CKO PD mice (I), glial cell activation was significantly reduced.

[0109] Figure 9 E represents the activation of microglia and astrocytes in the substantia nigra of WT and Psap CKO mice, detected by immunofluorescence staining two weeks after 6-OHDA injection. (Scale bar = 200 μm) Figure 9 F is Iba1 in SNpc + Microglia and GFAP + Quantitative statistics of relative optical density in astrocytes. Figure 9 G was detected by immunofluorescence staining of microglia and astrocytes in the substantia nigra of WT and IL-6 knockdown mice two weeks after 6-OHDA injection. (Scale bar = 200 μm) Figure 9 H is Iba1 in SNpc + Microglia and GFAP + Quantitative statistical analysis of the relative optical density of astrocytes. The results in the figure show that 6-OHDA-induced microglia and astrocyte proliferation occurred in Psap CKO mice (…). Figure 9 In E and 9F) and oligodendrocyte IL-6 knockdown mice ( Figure 9 G and Figure 9 The symptoms of both H) were relieved, indicating that the PSAP-GPR37-IL6 signaling axis plays an important role in neuroinflammation in Parkinson's disease mice.

[0110] All of these data indicate that IL-6 in SNpc is sufficient to induce neuroinflammation, dopamine neuron loss, motor dysfunction, and chronic pain.

[0111] To detect whether IL-6 can directly cause DA neuron death, this embodiment uses in situ hybridization to detect whether DA neurons express IL-6R and gp130. Figure 9 As shown in Figure I, gp130 mRNA expression in TH + In DA neurons, IL-6R mRNA was not expressed, while both IL-6R and gp130 mRNA were expressed in Iba1. + Microglia express ( Figure 9I), based on qPCR analysis (n = 5-7) of gp130 and IL-6R mRNA in the substantia nigra one week after saline or 6-OHDA injection, showed that the expression of IL-6R and gp130 mRNA was significantly increased in 6-OHDA-induced WT and Gpr37 gKO PD mice SNpc. Figure 7 T).

[0112] To investigate the potential role of microglia in the increase of IL-6 in the substantia nigra induced by IL-6 derived from initial oligodendrocytes, this study further investigated whether the conditioned medium (CMO) for oligodendrocytes could induce cultured microglia to release more IL-6. Figure 7 U represents an ELISA analysis of IL-6 protein levels in oligodendrocyte conditioned medium (TX-14-CMO) after TX-14 stimulation (n = 4). Figure 7 V's qPCR analysis revealed a significant increase in Il-6 mRNA in microglia cultured with TX-14-CMO compared to those cultured with Vehicle-CMO (n = 4-5). Figure 7 W showed that IL-6 protein levels were significantly increased in TX-14-CMO after co-culturing with microglia (n = 5). Figure 7 X shows that, compared with the Vehicle, recombinant IL-6 promotes the expression of IL-6 mRNA (left) and IL-6 protein (right) in primary cultured microglia (n = 4–6). Figure 7 Y represents the qPCR analysis of Caspase 3 and Bax mRNA in SNpc (n = 7-8). The results indicate that TX-14 stimulation induces increased Il-6 release in CMO. Figure 7 U), TX-14-stimulated CMO can induce a significant upregulation of Il-6 expression in cultured microglia ( Figure 7 V) and release ( Figure 7 Similarly, direct stimulation with recombinant IL-6 protein also significantly induced the upregulation and release of IL-6 in cultured microglia. Figure 7 X), more importantly, injecting IL-6 into the substantia nigra can also promote the expression of apoptosis-related proteins Caspase 3 and Bax (X). Figure 7 These data suggest that IL-6 from oligodendrocytes can induce microglia activation and the production of more IL-6, thereby promoting enhanced neuroinflammation and the degeneration of dopaminergic neurons.

[0113] The above findings suggest that oligodendrocyte-derived IL-6 is crucial for neuroinflammation, dopamine neuron damage, motor dysfunction, and chronic pain in 6-OHDA-induced Parkinson's disease mice.

[0114] In this embodiment, Figure 7 A, DF, H, I, L, M, and X (right) were analyzed using One-way ANOVA followed by Bonferroni's post hoc test; B, G, and T were analyzed using Bonferroni's two-way ANOVA; and K, OQ, S, UX (left), and Y were analyzed using Two-tailed Student's unpaired t-test. Figure 8 In categories A, C, E, G, K, L, and M, a two-way ANOVA followed by Bonferroni's post hoc test was used for analysis; in A, n = 4–6, *P = 0.0002 (saline) and 0.0003 (6-OHDA) (WT vs. gKO); in C, n = 5–7, P < 0.0001; in E, n = 3, *P < 0.0001 (0 nM vs. 300 nM), *P = 0.0017 (Vehicle vs. PTX); in L, n = 8–11, *P = 0.006 for WT vs. 3-week Il-6 knockdown, and *P < 0.0001 for the rest; in M, *P < 0.0001, n = 11–12; in I and J, a two-tailed student's unpaired t-test was used for analysis; in F, a one-way ANOVA followed by Bonferroni's post hoc test was used for analysis. Bonferroni's post hoc test analysis showed *P < 0.001, n = 4-6. Figure 9 B, D, F, and H were analyzed using One-way ANOVA followed by Bonferroni's post hoc test. All data are expressed as mean ± SEM.

[0115] Example 5: Verification of how early knockout of GPR37 in oligodendrocytes can alleviate neurodegenerative diseases in PD mice.

[0116] Based on the verification of the above embodiments, this embodiment further investigates whether inhibiting GPR37 in oligodendrocytes in the early stage of PD can alleviate the loss of dopamine neurons in Parkinson's disease mice. This embodiment uses AAV-αSyn to construct a PD model. When hA53T-αSyn is widely expressed in the substantia nigra (3 weeks after AAV-αSyn injection), plp1-Cre / ER and Gpr37 are selectively knocked out by intraperitoneal injection of tamoxifen (TAM). fl / flGpr37 in (CKO) mouse oligodendrocytes ( Figure 10 A: Timeline pattern diagram).

[0117] Figure 10 B is a representative TH staining map of substantia nigra dorsalis neurons (scale bar = 100 μm) and striatal dorsalis nerve fibers (scale bar = 500 μm). Figure 10 C is TH in the substantia nigra. + Neuron count (n = 4) Figure 10 D is a quantitative statistical representation of the relative optical density of DA nerve fibers in the striatum (n = 5-6). Figure 10 Three months after injection of AAV-GFP or αSyn, WT and CKO mice injected with TAM were subjected to rotator experiments. Figure 10 F and Figure 10 G represents mechanical and thermal hyperalgesia detection, respectively. Figure 10 H represents the representative immunoblotting results of human αSyn and β-actin in the substantia nigra. Figure 10 I used β-actin as an internal reference to quantify αSyn (n = 3-4). Figure 10 J is the quantitative measure of the relative optical density of p-αSyn in the substantia nigra (n = 4-6). Figure 10 The results showed that, compared with TAM-treated Gpr37 fl / fl Compared to WT mice, TAM-induced reduction in DA neurons and nerve fibers was significantly reduced in Gpr37 CKO PD mice. Figure 10 B-10D). Furthermore, TAM treatment significantly alleviated motor dysfunction, mechanical pain, and thermal pain in CKO PD mice (B-10D). Figure 10 E-10G). Although the expression levels of total αSyn were comparable ( Figure 10 H and 10I), but the accumulation of pathological αSyn was significantly reduced in Gpr37 CKO mice ( Figure 10 J).

[0118] These results indicate that knocking out GPR37 in oligodendrocytes in the early stages of PD can treat neurodegenerative diseases induced by hA53T-αSyn in PD mice.

[0119] In this embodiment, Figure 10 C- Figure 10 E, Figure 10 I and Figure 10 J was analyzed using One-way ANOVA followed by Bonferroni's post hoc test; Figure 10 F and Figure 10G was analyzed using two-way ANOVA followed by Bonferroni's post hoc test. All data are expressed as mean ± SEM. ns: no significant difference, *P < 0.05, ****P < 0.0001. ip: intraperitoneal injection; TAM: tamoxifen.

[0120] Example 6: Validation of PSAP-GPR37-IL-6 signal axis characteristics in PD patients.

[0121] This embodiment explores whether the research results of the above embodiments have translational potential for the clinical treatment of PD. To investigate the potential relevance of GPR37 in human PD, this embodiment analyzes the snRNA-seq database of SNpcs from PD patients. Through cluster analysis, combined with cell type-specific genes, seven different cell populations in SNpcs, including oligodendrocytes and DA neurons, were identified. Figure 12 A is a clustering UMAP plot of 78,110 cells (42,407 oligodendrocytes; 670 dopaminergic neurons; 12,439 non-dopaminergic neurons; 5,047 microglia; 8,371 astrocytes; 4,326 oligodendrocyte precursor cells; and 4,850 endothelial cells). Figure 12 B is a violin diagram of marker genes for seven major cell types. Figure 11 A is the GPR37 expression UMAP diagram after reanalysis of the snRNA-seq database published by Tushar Kamath et al. 21. Figure 11 The violin plot in B shows that, compared with the control group (CTL), GPR37 expression in oligodendrocytes of the substantia nigra was significantly increased in PD patients. Figure 11 C represents the co-localization detection of GPR37 mRNA with TH and PLP1 mRNA in the substantia nigra of human tissue, scale bar = 20 μm. Figure 11 D represents the in situ hybridization detection of GPR37 mRNA and PLP1 mRNA in human substantia nigra, with a scale bar of 20 μm. Figure 11 E represents the optical density quantitative statistics of GPR37 mRNA in PLP1+ oligodendrocytes (n = 199-220). In situ hybridization using freshly frozen midbrain sections confirmed that GPR37 mRNA is mainly expressed in PLP1+ cells. + In oligodendrocytes, not DA neurons. Consistent with the findings in mice described in the previous examples, GPR37 was significantly elevated in SNpc oligodendrocytes of PD patients ( Figure 11 B, 11D, and 11E). Furthermore, such as Figure 12Analysis of GPR37 mRNA expression in the lateral substantia nigra of healthy controls and sporadic PD patients (n = 7–9) shown in Figure E reveals a significant increase in GPR37 expression in a separate PD patient substantia nigra transcriptome sequencing database. These findings suggest a potential association between GPR37 in oligodendrocytes and human PD.

[0122] In human brain slices Figure 11 F represents the detection of co-localization of PSAP mRNA and TH in the substantia nigra of human tissue, with a scale bar of 20 μm. Figure 11 G is a representative immunoblot map of PSAP and TTR in human cerebrospinal fluid. As shown in the figure, PSAP mRNA is enriched in DA neurons, and PSAP protein is significantly increased in the cerebrospinal fluid of PD patients. Subsequently, we detected IL-6 levels in CSF samples using ELISA. Figure 11 H represents an ELISA analysis of IL-6 protein levels in the cerebrospinal fluid of PD patients and age-matched controls (n = 5). Figure 11 I is an analysis of GP130 mRNA expression in the lateral substantia nigra of healthy controls and sporadic PD patients in the GSE8397 database (n = 7–9). Figure 11 J is a representative immunofluorescence staining image of Iba1 and GFAP in human substantia nigra, scale bar = 100 μm. Figure 11 K is a quantitative statistical measure of the relative optical density of Iba1 and GFAP in the substantia nigra of human tissue (n = 3). The above results show that IL-6 protein in the CSF of PD patients is significantly higher than that in the control group. Figure 11 H), the expression of GP130 mRNA in SNpc was significantly increased in sporadic PD patients (H). Figure 11 I), and abnormal activation of microglia and astrocytes was detected in the substantia nigra of PD patients (I ...). Figure 11 J and 11K).

[0123] In the figures of this embodiment, all data were analyzed using a two-tailed Student's unpaired t-test. All data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. CTL: Control; DA: Dopamine; Non-DA: Non-dopamine; OPC: Oligodendrocyte precursor cells; Olig: Oligodendrocytes; Endo: Endothelial cells.

[0124] In summary, we confirmed the expression distribution of PSAP and GPR37 in the human substantia nigra and the elevated levels of PSAP and IL-6 in the cerebrospinal fluid of PD patients, suggesting that the PSAP-GPR37-IL-6 signaling axis may be related to the clinical symptoms of Parkinson's disease.

[0125] The pathogenic mechanism proposed in this invention is as follows: Figure 13 As shown, GPR37 expression is upregulated in oligodendrocytes in Parkinson's disease (PD). Increased PSAP secretion from dopamine neurons, mediated by the GPR37 / Gαi / MEK pathway, leads to upregulation of IL-6 expression in oligodendrocytes. IL-6 secreted by oligodendrocytes activates microglia, causing them to express even more IL-6, thereby exacerbating neuroinflammation and further leading to dopamine neuron death and behavioral disorders. The proposed pathogenesis and related validation mechanisms suggest the PSAP-GPR37-IL-6 signaling axis as a potential pathway for the detection, screening, and treatment of neurodegenerative diseases such as Parkinson's disease.

[0126] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of the PSAP-GPR37-IL-6 signaling axis as a pathogenic factor in neurodegenerative diseases, characterized in that... The PSAP-GPR37-IL-6 signaling axis mediates the development and progression of neurodegenerative diseases in the following ways: (1) GPR37 is specifically expressed in oligodendrocytes in the substantia nigra pars compacta (SNpc), and is significantly upregulated in neurodegenerative disease models; (2) PSAP is enriched in dopamine (DA) neurons and its secretion in cerebrospinal fluid increases in neurodegenerative disease states; (3) Secreted PSAP induces the expression and secretion of IL-6 in oligodendrocytes through the GPR37 / Gαi / MEK pathway; (4) IL-6 secreted by oligodendrocytes activates microglia, forming a positive feedback loop of IL-6 secretion, which exacerbates neuroinflammation, degeneration of DA neurons and behavioral defects.

2. The application according to claim 1, characterized in that, The neurodegenerative diseases mentioned include, but are not limited to, Parkinson's disease and Alzheimer's disease.

3. A combination of biomarkers for the diagnosis and / or treatment of neurodegenerative diseases, characterized in that, The biomarker combination includes PSAP, GPR37, and IL-6, and the samples for testing the biomarker combination include cerebrospinal fluid and substantia nigra.

4. The biomarker combination according to claim 3, characterized in that, The protein levels of PSAP and IL-6 in the cerebrospinal fluid of patients with neurodegenerative diseases were significantly higher than those in healthy controls, and the expression level of GPR37 in oligodendrocytes of the substantia nigra was significantly higher than that in healthy controls.

5. A method for screening therapeutic drugs for neurodegenerative diseases, characterized in that, Using any step of the PSAP-GPR37-IL-6 signaling axis as the target, compounds that can inhibit the activation of this signaling axis are screened, specifically including the following steps: (1) Construct screening models, including oligodendrocyte models expressing GPR37, cell models or animal models activated by the PSAP-GPR37-IL-6 signaling axis; (2) Apply the candidate compounds to the screening model; (3) To detect the effects of candidate compounds on PSAP secretion, GPR37 expression, IL-6 expression and secretion, neuroinflammation, DA neuron survival or behavioral defects; (4) Screening out candidate compounds that can reduce PSAP secretion, inhibit GPR37 expression, reduce IL-6 expression and secretion, alleviate neuroinflammation, improve DA neuron survival or alleviate behavioral deficits are potential drugs for the treatment of neurodegenerative diseases.

6. The screening method according to claim 5, characterized in that, The animal models include a 6-hydroxydopamine (6-OHDA)-induced PD mouse model, a human A53T α-synuclein (hA53T-αSyn) transgenic PD mouse model, and an adeno-associated virus-mediated hA53T-αSyn overexpression PD mouse model.

7. A medicament for treating neurodegenerative diseases, characterized in that, The drug inhibits the activation of the PSAP-GPR37-IL-6 signaling axis, specifically including at least one of the following methods: (1) Inhibit the expression or activity of GPR37 in oligodendrocytes; (2) Reduce PSAP secretion in DA neurons; (3) Inhibit the expression or secretion of IL-6 in oligodendrocytes; (4) Block the binding of PSAP to GPR37; (5) Block the activation of downstream signaling pathways of GPR37.

8. The medicament according to claim 7, characterized in that, The inhibition of GPR37 expression in oligodendrocytes includes knocking out the GPR37 gene in oligodendrocytes in the early stages of neurodegenerative diseases.

9. The drug according to claim 7, characterized in that, The neurodegenerative diseases mentioned include, but are not limited to, Parkinson's disease and Alzheimer's disease.

10. A pharmaceutical composition for treating neurodegenerative diseases, characterized in that, It contains an active ingredient capable of inhibiting the activation of the PSAP-GPR37-IL-6 signaling axis, said active ingredient including at least one of a GPR37 inhibitor, a PSAP inhibitor, an IL-6 inhibitor, a PSAP-GPR37 binding blocker, or a GPR37 downstream signaling pathway inhibitor.

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