Pathogenic factor of neurodegenerative disease and use thereof

Abstract

Provided in the present application are a pathogenic factor of neurodegenerative diseases and the use thereof. The pathogenic factor of neurodegenerative diseases is a PSAP-GPR37-IL-6 signaling axis. The present application specifies for the first time the action mechanism of the PSAP-GPR37-IL-6 signaling axis as a pathogenic factor of neurodegenerative diseases (especially Parkinson's disease), and reveals that oligodendrocytes regulate the molecular pathway of neuroinflammation and neurodegenerative changes via the signaling axis, thus filling the research gap in the prior art regarding the participation of oligodendrocytes in the pathogenesis of PD.

Description

A pathogenic factor of a neurodegenerative disease and its application

[0001] This application claims priority to CN202510110114X and CN2026100826531. Technical Field

[0002] This application relates to the pathogenesis and treatment of neurodegenerative diseases, and discloses a pathogenic factor of neurodegenerative diseases and its application. Background Technology

[0003] 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.

[0004] 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.

[0005] 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

[0006] The purpose of this application 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.

[0007] Therefore, this application adopts the following technical solution:

[0008] Firstly, this application provides a pathogenic factor for neurodegenerative diseases and its mechanism of action. Specifically, the pathogenic factor for neurodegenerative diseases described in this application is the PSAP-GPR37-IL-6 signaling axis, which mediates the occurrence and development of neurodegenerative diseases in the following ways:

[0009] (1) GPR37 is specifically expressed in oligodendrocytes in the substantia nigra pars compacta (SNpc), and is significantly upregulated in neurodegenerative disease models.

[0010] (2) PSAP is enriched in dopamine DA neurons and its secretion in cerebrospinal fluid increases in neurodegenerative disease states;

[0011] (3) Secreted PSAP induces the expression and secretion of IL-6 in oligodendrocytes through the GPR37 / Gαi / MEK pathway;

[0012] (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.

[0013] Furthermore, the neurodegenerative diseases include, but are not limited to, Parkinson's disease and Alzheimer's disease.

[0014] Secondly, this application 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.

[0015] 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.

[0016] Thirdly, this application provides a method for screening therapeutic drugs for neurodegenerative diseases. The method targets any step of the PSAP-GPR37-IL-6 signaling axis and screens for compounds capable of inhibiting the activation of this signaling axis. Specifically, the method includes the following steps:

[0017] (1) Construct screening models, including oligodendrocyte models expressing GPR37, cell models or animal models activated by the PSAP-GPR37-IL-6 signaling axis;

[0018] (2) Apply the candidate compounds to the screening model;

[0019] (3) To examine the effects of candidate compounds on PSAP secretion, GPR37 expression, IL-6 expression and secretion, neuroinflammation, DA neuron survival or behavioral defects;

[0020] (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.

[0021] 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.

[0022] Fourthly, this application provides a medicament for treating neurodegenerative diseases, wherein the medicament inhibits the activation of the PSAP-GPR37-IL-6 signaling axis, specifically comprising at least one of the following:

[0023] (1) Inhibit the expression or activity of GPR37 in oligodendrocytes;

[0024] (2) Reduce the secretion of PSAP in DA neurons;

[0025] (3) Inhibit the expression or secretion of IL-6 in oligodendrocytes;

[0026] (4) Block the binding of PSAP to GPR37;

[0027] (5) Block the activation of downstream signaling pathways of GPR37.

[0028] Furthermore, the inhibition of GPR37 expression in oligodendrocytes includes knocking out the GPR37 gene in oligodendrocytes in the early stages of neurodegenerative diseases.

[0029] Furthermore, the neurodegenerative diseases include, but are not limited to, Parkinson's disease and Alzheimer's disease.

[0030] Fifthly, this application provides a pharmaceutical composition for treating neurodegenerative diseases, comprising an active ingredient capable of inhibiting the 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.

[0031] The beneficial effects of this application are:

[0032] 1. This application 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.

[0033] 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.

[0034] 3. The drug screening method provided in this application 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.

[0035] 4. The treatment methods and pharmaceutical compositions provided in this application 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 clinical application prospects. Attached Figure Description

[0036] Figure 1 shows the results of verifying that GPR37 is specifically expressed in oligodendrocytes and significantly upregulated in the substantia nigra of a PD model in Example 1 of this application.

[0037] Figure 2 shows the expression profile and dynamic changes of GPR37 in the AAV-αSyn-induced PD mouse model in Example 1 of this application.

[0038] Figure 3 shows the experimental results of the PD mouse model mediated by 6-OHDA and AAV overexpression of hA53T-αSyn in Example 2 of this application.

[0039] Figure 4 shows the strategy and verification results of conditionally knocking out Gpr37 in oligodendrocytes in Example 2 of this application.

[0040] Figure 5 is a diagram showing the mechanism verification results of PSAP and neurodegenerative diseases in Example 3 of this application.

[0041] Figure 6 shows the experimental verification results of conditional knockout of DA neurons or PSAP in the substantia nigra in Embodiment 3 of this application.

[0042] Figure 7 shows the results of verifying the mechanism of PSAP-induced upregulation of IL-6 expression and the association between oligodendrocyte-derived IL-6 and neurodegenerative diseases in Example 4 of this application.

[0043] Figure 8 shows the verification results of PSAP-induced upregulation of IL-6 expression in Example 4 of this application, and the fact that conditional knockout of IL-6 does not affect basic motor function and pain sensation.

[0044] Figure 9 shows the verification results of oligodendrocyte-derived IL-6 being essential for 6-OHDA-induced neuroinflammation in PD mice in Example 4 of this application.

[0045] Figure 10 is a verification of the effect of early knockout of GPR37 in alleviating neurodegenerative diseases in PD mice in Example 5 of this application.

[0046] Figure 11 is a schematic diagram of the results of observing the characteristics of the PSAP-GPR37-IL-6 signal axis in PD patients in Example 6 of this application.

[0047] Figure 12 is a schematic diagram of the supplementary feature analysis results of GPR37 in human PD samples in Embodiment 6 of this application.

[0048] Figure 13 is a schematic diagram of how oligodendrocytes drive neuroinflammation and neurodegenerative changes in PD through the PSAP-GPR37-IL-6 signaling axis as verified in this application. Detailed Implementation

[0049] To enable those skilled in the art to better understand and implement the technical solutions of this application, the following detailed, clear, and complete description of this application is provided in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are merely some embodiments of this application, and not all embodiments. Furthermore, any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art based on the embodiments of this application without creative effort to obtain all other embodiments should be included within the protection scope of this application.

[0050] First, the experimental materials and methods of this application are introduced. In each embodiment of this application, unless otherwise specified, the experimental materials and methods follow the following description. Experimental details not described in detail in this application can be implemented by those skilled in the art based on operating habits or conventional operating instructions.

[0051] 1. Laboratory animals

[0052] The transgenic mouse strain, Gpr37, was purchased from Jackson Laboratory. fl / fl The 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 by mating 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.

[0053] 2. Cell Culture

[0054] 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.

[0055] 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, cerebral cortex samples from four newborn mice were 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). 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 200g 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.

[0056] 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 in 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.

[0057] 3. Viral vector

[0058] 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 Obio Technology (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.

[0059] 4. Stereoscopic injection

[0060] 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 minutes before being withdrawn. After injection, the mouse wound was sutured and placed on a heating pad until it was fully awakened from anesthesia.

[0061] 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 were given 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.

[0062] 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: 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, 21 days after intraperitoneal injection of tamoxifen, AAV-pDIO-shIL-6 was injected into Plp1-Cre / ER mice at the same coordinates. WT mice were injected with the corresponding viral load 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.

[0063] 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 the same 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.

[0064] 5. Immunofluorescence staining and in situ hybridization

[0065] 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 ImageJ software.

[0066] 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.

[0067] 6. Plasmid construction and transfection

[0068] The entire coding region of the Gpr37 gene (#NM_010338) was cloned by PCR. The primers used for PCR cloning are as follows:

[0069] Upstream primer: 5'-ATGAAGCTTATGAGCTACCTGCTGCTG-3'

[0070] Downstream primer: 5'-ATGGTACCCTAGTCTTCAGCTTCTTGG-3'.

[0071] 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.

[0072] 7. Real-time quantitative PCR and protein detection

[0073] 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.

[0074] 8. Behavioral testing

[0075] 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.

[0076] 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 test, and the average value was taken.

[0077] Mine Field Experiment: Under normal lighting conditions, mice were placed in the center of a mine field (40cm long × 40cm wide × 30cm high). The distance the mice traveled in the mine field over 30 minutes was measured using an Open Field Video Tracking System.

[0078] Apomorphine-induced rotation experiment: Two weeks after the 6-OHDA-induced PD model, the apomorphine-induced rotation experiment was performed. Mice were subcutaneously injected with apomorphine (USPharmacopeia, #R08440, 1 mg kg-1) and placed in a circular container. After acclimatization for 5 minutes, the number of times the mice rotated to the opposite side was recorded, and the recording time was 30 minutes.

[0079] Von Frey experiment: Mice were placed in a 10cm square plastic box for 2 hours to acclimatize and enter a calm state before the experiment began. 0.02-2.56g (stoelting) of Von Frey fibers were used to stimulate the mid-paw of the mouse's hind paw. Starting with 0.16g, each mouse was tested 6 times, and Dixon's up-down method 75 was used to assess the mouse's 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.

[0080] 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.

[0081] 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.

[0082] 9. Observation using transmission electron microscopy

[0083] 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. 3 The sample was placed in 4% glutaraldehyde and incubated overnight at 4°C. It was then washed three times with 0.1M sodium dimethyl arsenate (CAS) at 4°C for 10 min each time. Afterward, it was fixed on ice for 1 hour with OsO4 (2%) containing 3% K3Fe(CN)6. Next, the sample was washed 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 washed with deionized water at room temperature for 5 min. Next, the sample was dehydrated sequentially with 50%, 70%, 90%, and 95% ethanol for 15 min each, followed by dehydration with 100% ethanol for 30 min. Subsequently, the sample was 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 an oven at 45°C for 12 hours, followed by an oven at 65°C 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 120kV transmission electron microscope. Axonal diameter and myelin thickness were calculated using ImageJ software.

[0084] 10. Cell cluster analysis

[0085] 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.

[0086] 11. Statistical Analysis

[0087] 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 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.

[0088] The core technical point of this application 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.

[0089] Example 1: Validation of GPR37 specific expression in oligodendrocytes in SNpc and significant upregulation in a PD model.

[0090] 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 1A shows the colocalization detection of Gpr37 mRNA with tdTomato and TH in the substantia nigra of Plp1-Cre / ER; Ai14 mice, scale bar = 50 μm; the inset represents the corresponding magnified area, scale bar = 10 μm; Figure 1B shows Gpr37... + Percentage of cells co-localized with tdTomato or TH (n=9). Figures 1A and 1B show that, unlike previously disclosed research, Gpr37 co-localizes with tdTomato.+ It is specifically expressed in oligodendrocytes, but not in TH (a marker of dopamine neurons) positive dopamine neurons.

[0091] To determine the expression distribution of Gpr37 mRNA in SNpc, this example utilizes Gpr37-LacZ reporter mice containing the bacterial β-galactosidase (β-Gal) reporter gene at the Gpr37 locus. Figure 1C 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 1D shows the expression distribution of Gpr37 in the substantia nigra. + With β-Gal + Percentage of cellular colocalization (n=9); Figure 1E shows β-Gal + Percentage of colocalization with each cell marker (n=5). As shown in Figures 1C, 1D, and 1E, Gpr37 (labeled by β-Gal) is mainly expressed in APCs. + It is expressed in oligodendrocytes, but not in cells that are positive for TH, Iba1 (microglia marker), and GFAP (astrocytosis marker).

[0092] To determine the potential role of GPR37 in the development of PD, this study examined the expression of Gpr37 mRNA in SNpcs of 6-hydroxydopamine (6-OHDA)-induced PD mice, human A53Tα-synuclein (hA53T-αSyn) transgenic PD mice, and adeno-associated virus-mediated hA53T-αSyn (AAV-αSyn) overexpression PD mice. Figures 1F and 1G show the increased Gpr37 mRNA expression in the substantia nigra of 6-OHDA-induced PD mice (F) and human A53Tα-Syn transgenic mice (G), respectively (n = 4-7). Figure 2A 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). As shown in Figure 1F, Gpr37 mRNA expression in SNpcs increased significantly one week after 6-OHDA injection. Similarly, as shown in Figures 1G and 2A, Gpr37 mRNA expression was also significantly upregulated in the early stages of A53T transgenic PD mice (Figure 1G) and AAV-αSyn-induced PD mice (Figure 2A).

[0093] Figure 1H shows the colocalization of Gpr37 mRNA with 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 2B shows the colocalization of Gpr37 and Plp1 mRNA in the substantia nigra of AAV-GFP or -αSyn injected WT mice, with a scale bar of 100 μm. The right side is a magnified view with a scale bar of 20 μm. As shown in Figures 1H and 2B, in hA53T transgenic mice (Figure 1H) and AAV-αSyn-induced PD mice (Figure 2B) SNpc, Gpr37 mRNA expression is also mainly concentrated in Plp1. + Oligodendrocytes.

[0094] These results indicate that GPR37 is specifically expressed in oligodendrocytes in SNpc and is significantly upregulated in the PD model.

[0095] All data in this embodiment are mean ± SEM. *P<0.05, **P<0.01, ****P<0.0001. Data were statistically analyzed using two-tailed Student's unpaired t-test (Figure 1F) and Bonferroni's two-way ANOVA (Figures 1G and 2A).

[0096] 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.

[0097] 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.

[0098] Figures 3A and 3C are representative TH staining images 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, respectively; Figure 3B is a TH staining image of substantia nigra. + Cell count (n=3-5); Figure 3D shows the striatal TH. +Quantitative analysis of the relative optical density of DA nerve fibers was performed using 3-4 mice per group, with a total of 9-12 slices. As shown in the figures, compared with WT PD mice, Gpr37 gKO and CKO PD mice showed significantly reduced DA neuron death (Figures 3A and 3B); the density of DA nerve fibers in the striatum of Gpr37 gKO and CKO mice also showed significant recovery (Figures 3C and 3D).

[0099] Figures 3E and 3F show the apomorphine (APO)-induced rotation and rotarod experiments (n=8-17) performed 2 weeks after injection of saline or 6-OHDA. The results in the figures show that Gpr37 deficiency significantly alleviated 6-OHDA-induced motor dysfunction, as detected by the APO-induced rotation and rotarod experiments (Figures 3E and 3F).

[0100] Pain is a common and increasingly recognized non-motor symptom in Parkinson's disease (PD), affecting 30-85% of patients. Figures 3G and 3H show the detection of mechanical and thermal analgesia in WT, Gpr37 gKO, and CKO PD mice induced by 6-OHDA (n=5-17). The results in the figures show that, as previously reported, 6-OHDA induces mechanical and thermal analgesia in WT mice, while mechanical pain (Figure 3G) and thermal pain (Figure 3H) are both alleviated in Gpr37 gKO and CKO PD mice.

[0101] 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 3I shows a representative TH staining map of substantia nigra dorsalis neurons (scale bar = 100 μm) 3 months after injection of AAV-GFP (control) or AAV-αSyn; Figure 3J shows the TH staining map of substantia nigra. + Cell number (left) and striatal TH + Quantitative statistics of relative optical density of DA nerve fibers (right), n = 6-10; Figure 3K shows the substantia nigra NeuN. + Neuron count (n=4-6); Figures 3L-3O show the rotarod test (L), pole climbing test (M), von Frey test (N), and tail flick test (O) of AAV-αSyn induced PD mice (n=6-11); Figure 3P shows the immunoblotting of human αSyn and β-actin in the substantia nigra; Figure 3Q shows the quantification of αSyn using β-actin as an internal control (n=4). The results in the figures show that, as previously mentioned, unilateral injection of AAV-αSyn leads to a decrease in DA neurons in the SNpc of WT mice and a decrease in TH neurons in the ipsilateral striatum. +Nerve fiber density was reduced (Fig. 3I-3K). In contrast, Gpr37 gKO mice showed significant relief from AAV-αSyn-induced neurodegeneration (Fig. 3I-3K). Furthermore, compared with WT PD mice, Gpr37 gKO PD mice showed significant relief from motor impairment (Fig. 3L and 3M) as well as chronic mechanical and thermal hyperalgesia (Fig. 3N and 3O). The expression levels of total αSyn in SNpc of WT and gKO mice were comparable (Fig. 3P and 3Q).

[0102] To determine whether GPR37 regulates the formation of pathological αSyn, this study examined the phosphorylation (p-αSyn) of the typical pathological form of αSyn, αSyn Ser129. Figure 3R shows the immunofluorescence staining of p-αSyn and TH in the substantia nigra three months after AAV-GFP and AAV-αSyn injections, scale bar = 200 μm; Figure 3S shows the quantitative quantification of the relative optical density of p-αSyn in the substantia nigra (n = 4-5). The results show that AAV-αSyn-induced accumulation of p-αSyn was significantly increased in SNpc of WT PD mice, while p-αSyn was significantly decreased in Gpr37 gKO PD mice.

[0103] Figures 3B, 3D-F, 3J-M, 3Q, and 3S were analyzed using Bonferroni's one-way ANOVA. Figures 3G, 3H, 3N, and 3O were analyzed using Bonferroni's two-way ANOVA. All data are expressed as mean ± SEM. ns: no significant difference, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0104] To clarify the regulatory function of GPR37 in oligodendrocytes in PD, we constructed mice with conditional knockout of Gpr37 in oligodendrocytes (CKO). Figure 4A is a schematic diagram of the strategy for conditional knockout of Gpr37 in oligodendrocytes (Gpr37 CKO), and Figure 4B is a diagram of Gpr37... fl / fl In situ hybridization detection of Gpr37 in the substantia nigra of (WT) and Gpr37 CKO mice, scale bar = 200 μm.

[0105] Figures 4C-4F show the motor function and baseline pain threshold of mice assessed using the rotarod test (Figure 4C, n=15-18), open field test (OFT) (Figure 4D, n=10-8), von Frey test (Figure 4E, n=9-14), and tail-flick test (Figure 4F, n=12-14), respectively. Figures 4C, 4D, and 4E used a two-tailed Student's unpaired t-test for analysis, while Figure 4F used a two-way ANOVA followed by Bonferroni's post-hoc test. These experiments showed that the motor function (Figures 4C and 4D) and baseline pain threshold (Figures 4E and 4F) of Gpr37 CKO mice were not different from those of wild-type (WT) mice.

[0106] 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). Figures 4G and 4H are transmission electron microscopy (TEM) images of myelin sheaths in the corpus callosum (Figure 4G, CC) and striatum (Figure 4H, CPU) of WT and Gpr37 CKO mice, respectively, with scale bars of 500 and 250 μm; Figure 4I is a statistical analysis of the diameter of a single axon in the CC and CPU of WT and Gpr37 CKO mice, with 4 mice in each group, showing 355 and 370 axons in the CC and 200 and 212 axons in the CPU, respectively; Figure 4J is a statistical analysis of the average diameter of axons in the CC and CPU of WT and Gpr37 CKO mice, with 4 mice in each group; Figures 4K and 4M are statistical analyses of the average G-Ratio of myelin sheaths in the CC (Figure 4K) and CPU (Figure 4M) of WT and Gpr37 CKO mice, respectively, with n=4; Figures 4L and 4N are images of the diameter of axons in the CC and CPU of WT and Gpr37 CKO mice, respectively. The distribution of individual axons' G-Ratio in CC (Fig. 4L) and CCu (Fig. 4N) of CKO mice was detected. In Fig. 4L, n=4, there were 356 and 370 axons, respectively; in Fig. 4N, n=4, there were 200 and 212 axons, respectively. Fig. 4O shows the average number of myelinated axons per square millimeter of CC in WT and Gpr37 CKO mice (n=4). Fig. 4P shows the proportion of myelinated axons of different diameters in CC of WT and Gpr37 CKO mice (n=4). Figs. 4I-4K, 4M, and 4O were analyzed using Two-tailed Student's unpaired t-test; Fig. 4P was analyzed using Two-way ANOVA followed by Bonferroni's post hoc test; and Figs. 4L and 4N were analyzed using simple linear regression analysis of slope. ns: no significant difference. All data are expressed as mean ± SEM.

[0107] EM data showed no significant differences between WT and Gpr37 CKO mice in axonal diameter (Fig. 4G-4J), myelin G-Ratio (Fig. 4K-4N), and the number of myelinated axons (Fig. 4O and 4P) in the corpus callosum (CC) and striatum (CPu). These data indicate that conditional knockout of Gpr37 in oligodendrocytes does not impair myelin and axonal integrity, motor function, or baseline pain threshold in mice.

[0108] These data suggest that GPR37 deficiency can alleviate DA neuronal damage, motor dysfunction, and chronic pain induced by toxins and αSyn aggregates.

[0109] Example 3: The effect and mechanism of PSAP on PD.

[0110] 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.

[0111] Figure 5A 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, Western blot analysis as shown in Figure 5B revealed a significant increase in PSAP levels in the cerebrospinal fluid of PD mice one week after 6-OHDA injection, indicating a significant increase in PSAP protein levels in the CSF of the 6-OHDA-induced PD mouse model.

[0112] To explore the role of PSAP in PD, this embodiment utilizes PSAP. fl / fl Wt mice were crossed with TH-Cre mice to create mice with conditional knockout of Psap in DA neurons (Psap CKO). Figure 5C is a schematic diagram of the strategy for conditional knockout of Psap in DA neurons (Psap CKO).

[0113] Figures 6A and 6B show the in situ hybridization (Figure 6A) and qPCR (Figure 6B) detection of Psap in SNpc of WT and Psap CKO mice, respectively, with a scale bar of 20 μm. As can be seen from the figures, the expression of Psap in DA neurons of Psap CKO mice is significantly reduced compared with WT mice.

[0114] Figures 6C and 6D are representative TH staining images of SNpc dopaminergic neurons (scale bar = 200 μm) in WT and Psap CKO mice, respectively (Figure 6C) and quantitative statistical results of SNpc dopaminergic neurons and striatal dopaminergic fibers (Figure 6D, n = 4-5). As can be seen from the figures, the number of DA neurons in SNpc and the density of striatal DA nerve fibers in Psap CKO mice are comparable to those in WT mice.

[0115] Figures 6E and 6F show the basic motor (Figure 6E) and sensory (Figure 6F) functions of WT and Psap CKO mice, respectively, tested using the open field test, rotarod test, von Frey test, and tail-flick test (n=8-9). As can be seen from the figures, there was no significant difference in motor function and basic pain threshold between Psap CKO mice and WT mice.

[0116] Figure 5D shows representative TH staining images 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 5E shows TH staining. + 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.

[0117] Figures 5F-5I show that, based on the rotarod test (Figure 5F), APO-induced rotation test (Figure 5G), von Frey test (Figure 5H), and tail-flick test (Figure 5I), the behavioral disorders in Psap CKO PD mice were significantly alleviated compared to WT mice (n=6-7).

[0118] Figure 6G shows the accumulation of DA neurons and p-αSyn in SNpc of WT and Psap CKO mice 3 months after AAV-αSyn injection, as detected by immunofluorescence staining (scale bar = 200 μm); Figure 6H shows the TH... + Quantitative statistics of cell number; Figure 6I shows the quantitative statistics of relative optical density of p-αSyn in SNpc; Figure 6J shows the striatal TH +Quantification of the relative optical density of dopaminergic fibers. Figures 6H-6J were analyzed using a two-tailed Student's unpaired t-test, n=4, P=0.0032 (Figure 6H), 0.0010 (Figure 6I), 0.0111 (Figure 6J). Figures 6K and 6L were performed using the rotarod test (K) and pole climbing test (L) after AAV-αSyn-induced PD model, respectively, analyzed using Tow-way ANOVA followed by Bonferroni's post hoc test, n=9-10, *P<0.05. The results showed that the accumulation of pathological αSyn and the loss of DA neurons in the SNpc of AAV-αSyn-induced Psap CKO PD mice were significantly reduced (Figures 6G-6J), and motor dysfunction was also significantly alleviated (Figures 6K and 6L).

[0119] To further confirm whether PD-related degenerative lesions depend on local PSAP at the SNpc, as shown in Figure 5J, this embodiment involves directing PSAP to the Psap fl / fl AAV-Cre-mCherry was injected into mouse SNpcs to specifically knock out Psap (Psap cKO) in SNpcs.

[0120] Figure 6M shows, on the left, immunostaining revealing the co-localization of mCherry (Cre) and TH in AAV-Cre-injected mouse SNpcs (scale bar = 200 μm). The boxed area in the figure is magnified at the bottom (scale bar = 20 μm). On the right, TH is shown in cells co-expressing tdTomato. + The percentage of Psap in cells, 9 slices from n=3 mice; Figure 6N shows the in situ hybridization detection of Psap in SNpcs of WT and Psap cKO mice, scale bar=25μm; Figure 6O shows qPCR analysis showing a significant reduction in Psap expression in SNpcs of Psap cKO mice; Figures 6P-6R show the basal motor (Figure 6P and Figure 6Q) and sensory (Figure 6R) function detection of WT and Psap cKO mice, respectively (n=13). The results showed that Psap expression in SNpcs of Psap cKO mice was significantly reduced (Figures 6M-6O), however, their basal motor function (Figures 6P and Figure 6Q) and pain threshold were not significantly different from those of WT mice (Figure 6R).

[0121] Figures 5K and 5L show the death of DA neurons in the substantia nigra of Psap cKO PD mice (Figure 5K, n=4) and TH neurons in the striatum, respectively. +The reduction in fluorescence density (Fig. 5L, n = 9-12 brain slices, from 3-4 mice) was significantly reduced. Figs. 5M-5P showed that behavioral disorders in Psap cKO PD mice were significantly alleviated using the rotarod test (Fig. 5M), APO-induced rotation test (Fig. 5N), von Frey test (Fig. 5O), and tail-flick test (Fig. 5P), respectively (n = 11-13). The results in the figures indicate that 6-OHDA-induced DA neuron death in the SNpc of Psap cKO PD mice was significantly reduced (Figs. 5K and 5L), and the accompanying motor disorders were also significantly improved (Figs. 5M-5P).

[0122] 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.

[0123] In this embodiment, B, EG, and KN in Figure 5 were analyzed using a two-tailed Student's unpaired t-test, while H, I, O, and P were analyzed using a two-way ANOVA followed by Bonferroni's post-hoc test. F and R in Figure 6 were analyzed using a two-way ANOVA followed by Bonferroni's post-hoc test, while B, D, E, F, and OR were analyzed using a two-tailed Student's unpaired t-test. In B, P = 0.0005, n = 4; in O, P < 0.0001, n = 5. ns: no significant difference. All data are mean ± SEM.

[0124] Example 4: PSAP induces upregulation of IL-6 in oligodendrocytes via GPR37 and promotes PD-like dysfunction.

[0125] 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 SNpcs of WT and Gpr37 gKO mice one week after 6-OHDA injection. The results are shown in Figure 8A. Although Apc mRNA expression was higher in SNpcs 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 myelin formation may not be involved in 6-OHDA-induced PD-like neurodegeneration.

[0126] To investigate the role of GPR37 in the immunomodulatory function of oligodendrocytes, this study used primary cultured oligodendrocytes for detection. Figure 8B shows the expression of Apc, Iba1, and TH mRNA in primary cultured WT oligodendrocytes (n=5) detected by qPCR. Figure 7A 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 8C 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 WT oligodendrocytes after 2 hours of TX-14 stimulation by qPCR. As shown in Figures 7A and 8C, the PSAP-derived peptide (TX-14) can significantly upregulate the expression of IL-6 in primary cultured oligodendrocytes in a concentration-dependent manner. Conversely, the qPCR detection of IL-6 mRNA in primary cultured oligodendrocytes in Figure 7B (n=9-11) showed that TX-14 could not mediate the expression of IL-6 in oligodendrocytes derived from Gpr37 gKO mice.

[0127] Figure 8D shows that the Gαi inhibitor PTX (100 ng / ml) inhibits TX-14-induced ERK1 / 2 phosphorylation in HEK293T cells. Figure 8E is the quantification of phosphorylated ERK1 / 2. Tow-way ANOVA followed by Bonferroni's post hoc test analysis. These results indicate that GPR37 can couple with Gαi to mediate downstream ERK phosphorylation, which is inhibited by the Gαi inhibitor PTX.

[0128] Figure 8F shows the effect of qPCR detection of PTX (100 ng / ml) and MEK inhibitor U0126 (1 μM) on TX-14-induced IL-6 mRNA expression in primary cultured oligodendrocytes. Before TX-14 (50 nM) stimulation, cells were pretreated with inhibitors for 8 hours (PTX) or 1 hour (U0126). As shown in Figure 8F, both PTX and MEK inhibitor U0126 can inhibit TX-14-mediated upregulation of IL-6 expression.

[0129] 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.

[0130] Regarding whether TX-14 treatment is sufficient to induce neurodegeneration and PD-like dysfunction, Figure 7C shows immunofluorescence staining of DA neurons in the substantia nigra of mice injected with saline or TX-14, scale bar = 200 μm; Figure 7D shows the TH neurons in the substantia nigra.+ Neuron count (n=4-5), Figure 7E 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 unilaterally injected with TX-14 by SNpc had significantly fewer DA neurons.

[0131] Figure 7F shows the motor function of mice detected by the rotarod test (n=8-10), and Figure 8G shows the mechanorexia of mice detected by the von Frey test. The 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). It can be seen that TX-14 also caused significant impairment of motor coordination in WT mice (Figure 7F) and abnormal mechanorexia, while Gpr37 gKO mice did not show this (Figure 8G). This indicates that PSAP-derived peptides can induce neurodegenerative diseases and PD-like dysfunction in a GPR37-dependent manner.

[0132] To verify the elevated IL-6 levels in peripheral blood and brain during PD, this embodiment used 6-OHDA injection as the detection protocol. Figure 7G shows the qPCR detection of IL-6 mRNA in the substantia nigra (n=6-7). Figures 7H and 7I show the ELISA detection of IL-6 protein levels in the substantia nigra (Figure 7H) and cerebrospinal fluid (Figure 7I) of mice one week after saline or 6-OHDA injection (n=4-6). The results show that IL-6 mRNA expression increased in the SNpc of WT mice, while no increase in IL-6 expression was detected in Gpr37 gKO mice. Similarly, IL-6 protein levels increased in the SNpc (Figure 7H) and CSF (Figure 7I) of WT PD mice treated with 6-OHDA, but no increase was observed in Gpr37 gKO mice treated with 6-OHDA.

[0133] 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 (Figure 7J). As shown in Figure 8H, the left image shows immunofluorescence staining revealing co-localization of mCherry (shIL-6) with olig2, with scale bars of 50 μm and 10 μm; the right image shows mCherry... + The percentage of cells co-expressing olig2 and olig2+ The percentage of cells co-expressing mCherry (n = 10 slices from 3 mice). Figures 8I-8K show the baseline motor function and pain threshold of IL-6 knockdown mice as detected by rotarod assay (Figure 8I), von Frey assay (Figure 8J), and tail-flick assay (Figure 8K), respectively. The results in the figures show that mCherry-shIL-6 is mainly expressed in olig2 cells. + Oligodendrocytes (Fig. 8H), and knockdown of IL-6 in oligodendrocytes had no significant effect on basic motor function and pain sensation (Fig. 8I-Fig. 8K).

[0134] This embodiment utilizes a PD model constructed using 6-OHDA. Figure 7K shows the immunofluorescence staining of TH neurons in the substantia nigra (scale bar = 200 μm) and the statistical count of TH+ neurons (n ​​= 3) three weeks after 6-OHDA injection. Figures 7L and 7M, using rotarod experiments (Figure 7L) and APO-induced rotation experiments (Figure 7M), respectively, showed that IL-6 knockdown in oligodendrocytes significantly improved motor dysfunction in PD mice (n = 7-10). The results in the figures show that DA neuron damage in IL-6 knockdown PD mice was significantly reduced (Figure 7K), and motor dysfunction (Figures 7L and 7M) and mechanorepression (Figure 8L) were significantly alleviated. These data indicate that IL-6 in oligodendrocytes is crucial for dopamine neuron death, motor dysfunction, and chronic pain in PD mice.

[0135] To investigate whether and how IL-6 can induce PD-like symptoms, this study unilaterally injected recombinant IL-6 protein into the SNpc of WT mice and compared it with a control group of mice injected with saline. Figure 7N shows a stereotactic injection and timeline diagram. Figures 7O and 7P show the TH levels in the 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 7Q shows the rotarod experiment performed on the substantia nigra 12 days after injection of saline or recombinant IL-6 protein (n=13-15). The results showed that IL-6 injection significantly reduced DA neurons in the SNpc of mice and significantly decreased the optical density of TH-positive fibers in the striatum (Figures 7O and 7P). In addition, IL-6 injection also caused motor coordination impairment in mice (Figure 7Q) and mechanorexia (Figure 8M).

[0136] Activation of microglia and astrocytes in the substantia nigra is a pathological feature of neuroinflammation in PD. In this example, immunofluorescence staining and qPCR were used to detect the proliferation of microglia and astrocytes. Figures 7R and 7S show the immunofluorescence staining (R, scale bar = 100 μm) and qPCR analysis (Figure 7S, n = 5) of Iba1 and GFAP in the substantia nigra, respectively. The figures show that Iba1 and GFAP were significantly increased in the midbrain of mice injected with IL-6, indicating enhanced neuroinflammation in the substantia nigra cells (SNpc).

[0137] In addition, Figures 9A and 9C show the activation of microglia (Figure 9A) and astrocytes (Figure 9C) in the substantia nigra of WT, Gpr37 gKO, and CKO mice, respectively, two weeks after 6-OHDA injection, using immunofluorescence staining (scale bar = 200 μm). Figures 9B and 9D show the Iba1 in SNpc. + Microglia (Fig. 9B) and GFAP + Quantitative statistical analysis of the relative optical density of astrocytes (Fig. 9D) revealed that 6-OHDA-induced activation of microglia and astrocytes increased in ipsilateral SNpc of WT PD mice, while glial cell activation was significantly reduced in Gpr37 gKO and CKO PD mice with Gpr37 deficiency in oligodendrocytes and significantly reduced IL-6 expression (Fig. 7G-Fig. 7I).

[0138] Figure 9E shows 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 9F shows Iba1 in SNpc. + Microglia and GFAP + Quantitative statistical analysis of the relative optical density of astrocytes. Figure 9G shows the activation of microglia and astrocytes in the substantia nigra of WT and IL-6 knockdown mice, detected by immunofluorescence staining two weeks after 6-OHDA injection. Scale bar = 200 μm. Figure 9H shows 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 were alleviated in Psap CKO mice (Fig. 9E and 9F) and oligodendrocyte IL-6 knockdown mice (Fig. 9G and 9H), indicating that the PSAP-GPR37-IL6 signaling axis plays an important role in neuroinflammation in Parkinson's disease mice.

[0139] All of these data indicate that IL-6 in SNpc is sufficient to induce neuroinflammation, dopamine neuron loss, motor dysfunction, and chronic pain.

[0140] To investigate 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. As shown in Figure 9I, gp130 mRNA is expressed in TH... + In DA neurons, IL-6R mRNA was not expressed, while both IL-6R and gp130 mRNA were expressed in Iba1. + Expression in microglia (Fig. 9I). Based on qPCR analysis (n=5-7) of gp130 and IL-6R mRNA in the substantia nigra after 1 week of saline or 6-OHDA injection, the expression of IL-6R and gp130 mRNA was significantly increased in 6-OHDA-induced WT and Gpr37gKO PD mice SNpc (Fig. 7T).

[0141] 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 oligodendrocyte conditioned medium (CMO) could induce cultured microglia to release more IL-6. Figure 7U shows the ELISA analysis (n=4) of IL-6 protein levels in oligodendrocyte conditioned medium (TX-14-CMO) after TX-14 stimulation. Figure 7V shows the results of qPCR detection, revealing the difference between IL-6 levels in microglia cultured with those in Vehicle-CMO. Compared with microglia, TX-14-CMO cultured microglia showed a significant increase in Il-6 mRNA (n=4-5). Figure 7W shows that after co-culturing with microglia, TX-14-CMO significantly increased IL-6 protein (n=5). Figure 7X shows that compared with Vehicle, recombinant IL-6 promoted the expression of IL-6 mRNA (left) and Il-6 protein (right) in primary cultured microglia (n=4-6). Figure 7Y is the qPCR analysis of Caspase3 and Bax mRNA in SNpc (n=7-8). From the above results, it can be seen that TX-14 stimulation induces increased Il-6 release in CMO (Figure 7U), and TX-14-stimulated CMO can induce a significant upregulation of Il-6 expression (Figure 7V) and release (Figure 7W) in cultured microglia. Similarly, direct stimulation with recombinant IL-6 protein significantly induced the upregulation and release of IL-6 in cultured microglia (Fig. 4X). More importantly, injection of IL-6 into the substantia nigra also promoted the expression of apoptosis-related proteins Caspase 3 and Bax (Fig. 4Y). These data indicate 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.

[0142] 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.

[0143] In this embodiment, A, DF, H, I, L, M, and X (right) in Figure 7 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 shows that A, C, E, G, K, L, and M were analyzed using two-way ANOVA followed by Bonferroni's post hoc test; 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 others; in M, *P < 0.0001, n = 11–12; I and J were analyzed using a two-tailed student's unpaired t-test; and F was analyzed using one-way ANOVA followed by Bonferroni's post hoc test. Hoc test analysis, *P<0.001, n=4–6. Figures 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.

[0144] Example 5: Verification of how early knockout of GPR37 in oligodendrocytes can alleviate neurodegenerative diseases in PD mice.

[0145] 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 (Fig. 10A: Schematic diagram of timeline pattern).

[0146] Figure 10B 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 10C is a TH staining map of substantia nigra. + The neuron count (n=4) is shown in Figure 10D, which shows the quantitative statistics of the relative optical density of DA nerve fibers in the striatum (n=5-6). Figure 10E shows the rotarod experiment performed on WT and CKO mice injected with TAM 3 months after AAV-GFP or αSyn injection. Figures 10F and 10G show the detection of mechanoreactivity and thermal reactivity, respectively. Figure 10H shows the representative immunoblotting results of human αSyn and β-actin in the substantia nigra. Figure 10I shows the quantification of αSyn using β-actin as an internal control (n=3-4). Figure 10J shows the quantitative quantification of the relative optical density of p-αSyn in the substantia nigra (n=4-6). The results in Figure 10 show that, compared with TAM-treated Gpr37... fl / fl Compared to WT mice, TAM-induced reductions in DA neurons and nerve fibers were significantly reduced in Gpr37 CKO PD mice (Fig. 10B-10D). Furthermore, TAM treatment significantly alleviated motor dysfunction, mechanical pain, and thermal pain in CKO PD mice (Fig. 10E-10G). Although total αSyn expression levels were comparable (Fig. 10H and 10I), the accumulation of pathological αSyn was significantly reduced in Gpr37 CKO mice (Fig. 10J).

[0147] 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.

[0148] In this embodiment, Figures 10C-10E, 10I, and 10J were analyzed using One-way ANOVA followed by Bonferroni's post hoc test; Figures 10F and 10G were 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.

[0149] Example 6: Validation of PSAP-GPR37-IL-6 signal axis characteristics in PD patients.

[0150] This embodiment explores whether the research results of the above embodiments have translational potential for the clinical treatment of PD. To investigate the possible relevance of GPR37 in human PD, this embodiment analyzes the snRNA-seq database of SNpc from PD patients. Through cluster analysis, combined with cell type-specific genes, seven different cell populations in SNpc, including oligodendrocytes and DA neurons, were identified. Figure 12A is the UMAP diagram of clustering 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 12B is a violin diagram of the marker genes of the seven major cell types. Figure 11A is the UMAP plot of GPR37 expression after reanalysis of the snRNA-seq database published by Tushar Kamath et al. 21. Figure 11B, a violin plot, shows that GPR37 expression in oligodendrocytes of the substantia nigra of PD patients was significantly increased compared with the control group (CTL). Figure 11C is the colocalization detection of GPR37 mRNA with TH and PLP1 mRNA in human substantia nigra (scale bar = 20 μm). Figure 11D is the in situ hybridization detection of GPR37 mRNA and PLP1 mRNA in human substantia nigra (scale bar = 20 μm). Figure 11E is the quantitative statistical analysis of the optical density 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, but not in DA neurons. Consistent with the findings in mice described in the previous examples, GPR37 was significantly elevated in SNpc oligodendrocytes of PD patients (Figs. 11B, 11D, and 11E). Furthermore, as shown in Fig. 12E, analysis of GPR37 mRNA expression in the lateral substantia nigra of healthy controls and sporadic PD patients from the GSE8397 database (n=7–9) revealed a significant increase in GPR37 expression in a separate PD patient substantia nigra transcriptome sequencing database. These findings suggest that GPR37 in oligodendrocytes may have a potential association with human PD.

[0151] In human brain slices, Figure 11F shows the co-localization detection of PSAP mRNA and TH in the substantia nigra (SDN), scale bar = 20 μm. Figure 11G shows representative immunoblot maps of PSAP and TTR in human cerebrospinal fluid (CSF). The figures show that PSAP mRNA is enriched in DA neurons, and PSAP protein is significantly increased in the CSF of PD patients. Subsequently, we detected IL-6 levels in CSF samples using ELISA. Figure 11H shows the ELISA analysis of IL-6 protein levels in the CSF of PD patients and age-matched controls (n = 5). Figure 11I shows the analysis of GP130 mRNA expression in the lateral substantia nigra of healthy controls and sporadic PD patients from the GSE8397 database (n = 7-9). Figure 11J shows representative immunofluorescence staining of Iba1 and GFAP in the SDN, scale bar = 100 μm. Figure 11K shows the quantitative statistics of the relative optical density of Iba1 and GFAP in the SDN (n = 3). The results showed that IL-6 protein in the CSF of PD patients was significantly higher than that in the control group (Fig. 11H), GP130 mRNA expression was significantly increased in the SNpc of sporadic PD patients (Fig. 11I), and abnormal activation of microglia and astrocytes was detected in the substantia nigra of PD patients (Fig. 11J and 11K).

[0152] 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.

[0153] 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.

[0154] The pathogenic mechanism proposed in this application is shown in Figure 13. In Parkinson's disease (PD), GPR37 expression is upregulated in oligodendrocytes. 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. This proposed pathogenic factor and its associated validation mechanism suggest the PSAP-GPR37-IL-6 signaling axis as a potential detection, screening, and therapeutic agent for PD and other neurodegenerative diseases.

[0155] Although this application 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 this application should be included within the protection scope of this application.

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 examine 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 A53Tα-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 the secretion of PSAP 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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