Drug development based on CSF1R polypeptide fragment and application thereof

Abstract

The invention discloses drug development and application based on CSF1R polypeptide fragments, and belongs to the technical field of medicine. The invention provides an application of a CSF1R polypeptide fragment in preparation of a medicine for treating a CSF1R related abnormality (CSF1R-RD) disease. The fact that soluble CSF1R (sCSF1R), namely an extracellular segment generated after colony stimulating factor 1 receptor (CSF1R) is cut, can be used for treating or relieving neurodegenerative change of a CSF1R-RD patient is found for the first time. The invention discloses a medicine based on an sCSF1R polypeptide fragment, and relates to application of the sCSF1R polypeptide fragment and a truncated fragment thereof to preparation of a medicine for supplementing sCSF1R. The medicine can be used for effectively improving the clinical symptoms of CSF1R-RD patients. The medicine is used for improving, relieving or treating the CSF1R-RD. The invention provides a novel CSF1R-RD treatment scheme based on the sCSF1R polypeptide fragment and the truncated fragment thereof, and has a wide clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of CSF1R polypeptide fragments and their truncated fragments in the preparation of drugs for treating CSF1R-RD. Background Art

[0002] Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a subtype of dominantly inherited leukoencephalopathy caused by CSF1R mutations. It usually starts in middle age, with the onset age ranging from 15 to 78 years old, and the average age is 35 to 40 years old; the disease course ranges from 1 to 30 years, with an average of 6.8 years, accompanied by motor symptoms such as cognitive decline, mental symptoms and movement disorders; the cognitive symptoms in ALSP are characterized by frontal lobe dysfunction, such as executive dysfunction, attention deficit and apathy; its main motor disorder symptoms are gait disorder and bradykinesia, which may be the initial symptoms; therefore, ALSP is considered as cognitive impairment and motor disorder; in recent years, several studies have shown that ALSP is related to CSF1R mutants, and CSF1R is the pathogenic gene of ALSP Ikeuchi, T., N. Mezaki, and T. Miura, Cognitive dysfunction and symptoms of movement disorders in adult-onset leukoencephalopathy with axonal spheroids and Pigmented glia. Parkinsonism Relat Disord, 2018.46 Suppl 1: p. S39-S41); the disease is currently uniformly named as CSF1R-related disorder (CSF1R-RD).

[0003] Colony stimulating factor 1 receptor (CSF1R) is a type III tyrosine kinase expressed by phagocytic monocytes (Bencheikh, L., et al., Dynamic gene regulation by nuclear colony-stimulating factor 1 receptor in human monocytes and macrophages.Nat Commun, 2019.10(1): p. 1935), belonging to the platelet-derived growth factor (PDGF) receptor family; CSF1R, also known as macrophage colony-stimulatory factor (M-CSF) or cluster of Differentiation 115 (CD115), is a human cell surface protein encoded by the CSF1R proto-oncogene (Sherr, C.J., et al., The C-Fms Proto-Oncogene Product Is Related to the Receptor for the Mononuclear Phagocyte Growth-Factor, Csf-1. Cell, 1985.41(3): p. 665-676), and the human CSF1R gene is located at 5q32 on chromosome 5; human CSF1R is a transmembrane glycoprotein composed of 972 amino acid residues with a molecular weight of 150 kD, divided into an extracellular region, a transmembrane region, and a cytoplasmic region, consisting of 512, 25, and 435 amino acid residues respectively; the extracellular region contains five immunoglobulin-like regions; CSF1R is mainly expressed in microglia in the central nervous system (Tada, M., et al., Characteristic microglial features in patients with hereditary diffuse leukoencephalopathy with spheroids. Ann Neurol, 2016.80(4):p. 554-65), and CSF1R has a regulatory effect on microglial survival, proliferation, differentiation, and function, as well as on the maintenance of neuronal homeostasis through ligand binding (Mouchemore, K.A. and F.J. Pixley, CSF-1 signaling in macrophages: pleiotrophy through phosphotyrosine-based signaling pathways. Critical Reviews in Clinical Laboratory Sciences, 2012. 49(2): p. 49-61).

[0004] Currently, there is no effective treatment for CSF1R-RD, and symptomatic supportive treatment is usually adopted; the effectiveness of dopaminergic drugs or antidepressants for Parkinson-like symptoms, depression and other mental symptoms has not been confirmed; as the disease progresses, the patient's personality, psychology, and motor function change significantly, affecting the quality of life; therefore, finding effective therapeutic drugs for CSF1R-RD is of great significance for improving the quality of life of patients with CSF1R-RD. Summary of the Invention

[0005] The object of the present invention is to provide the drug development and application based on the CSF1R polypeptide fragment; specifically, to provide a drug or pharmaceutical composition for improving, alleviating or treating CSF1R-RD prepared from the sCSF1R polypeptide fragment and its truncated fragments.

[0006] To achieve the above object of the invention, the present invention provides the following technical solutions.

[0007] The drug based on the CSF1R polypeptide fragment is a drug or pharmaceutical composition for improving, alleviating or treating CSF1R-RD, which can effectively improve the clinical symptoms of patients with CSF1R-RD; the drug or pharmaceutical composition contains a therapeutically effective amount of the sCSF1R polypeptide or its truncated fragment.

[0008] The amino acid sequence of the sCSF1R polypeptide is shown in SEQ ID NO:1; the truncated fragment of the sCSF1R polypeptide only contains the first immunoglobulin-like region of the sCSF1R polypeptide, that is, amino acids 1-86 of the sequence listing SEQ ID NO:1.

[0009] For the therapeutically effective amount of the sCSF1R polypeptide, the concentration of the sCSF1R polypeptide is 0.001-1.0 mg / mL and contains a pharmaceutically acceptable excipient. The concentration can be determined by the BCA protein quantification method.

[0010] The dosage form of the drug can be made into injection preparations, oral preparations, spray preparations, ointment preparations or patches according to the common methods of drug preparation.

[0011] The injection preparation contains a therapeutically effective amount of the sCSF1R polypeptide and an isotonicity regulator.

[0012] Use of the sCSF1R polypeptide fragment or its truncated fragment in the preparation of a drug for treating diseases related to abnormal CSF1R signaling pathway, the diseases including but not limited to neurodegenerative diseases, autoimmune diseases or demyelinating diseases.

[0013] The treatment mechanism includes inhibiting microglial cell activation, reducing the expression of Tnf-α and IL-1β, or repairing myelin sheath damage, etc.

[0014] The administration mode of the drug or pharmaceutical composition can be intracerebroventricular injection, intravenous injection or oral administration, etc.

[0015] The present invention provides the application of the drug based on the CSF1R polypeptide fragment in the preparation of a drug for treating diseases related to CSF1R-related abnormalities (CSF1R-RD); the present invention for the first time discovers that the extracellular segment, i.e., soluble CSF1R (sCSF1R), generated after the cleavage of colony-stimulating factor 1 receptor (CSF1R) can be used to treat or alleviate the neurodegenerative changes of patients with CSF1R-RD.

[0016] The present invention provides the use of sCSF1R polypeptide fragments in the development of CSF1R-RD drugs; through a large number of experiments, it is confirmed that sCSF1R released by hydrolysis and cleavage of CSF1R can improve the disease phenotypes of mice with CSF1R-RD disease models.

[0017] The application of the sCSF1R polypeptide fragment or its truncated fragment as a lead compound in the development of targeted drugs for CSF1R-related disorders (CSF1R-RD) includes high-throughput screening, structure optimization, or pharmacodynamic evaluation.

[0018] The significant advantages of the present invention are as follows:

[0019] The present invention provides the development and application of drugs based on CSF1R polypeptide fragments, aiming to improve, relieve, or treat CSF1R-RD. The drug or drug composition contains a therapeutically effective amount of the sCSF1R polypeptide or its truncated fragment, and the resulting product can effectively improve the clinical symptoms of patients with CSF1R-RD. Moreover, the drug has good efficacy for CSF1R-RD caused by various reasons. The present invention discovers for the first time that sCSF1R can treat or relieve the neurodegenerative changes of patients with CSF1R-RD, and through experiments, it is confirmed that it can improve the disease phenotypes of mice with CSF1R-RD disease models. The present invention provides a brand-new treatment plan for CSF1R-RD, has good efficacy for CSF1R-RD caused by various reasons, and has important therapeutic significance. Brief Description of the Drawings

[0020] Figure 1Long-term injection of sCSF1R significantly improved the anxiety-like phenotype and learning and memory abilities of CSF1R-RD model mice. Among them, A is a schematic diagram of the animal experiment design for multiple injections of sCSF1R by stereotaxic injection into the cannula; B statistically analyzed the autonomous alternation behavior index of mice in the Y maze, N = 10, two-way ANOVA followed by Tukey’s posthoc test; C is a schematic diagram of the novel arm recognition experiment; D is the statistical analysis of the time of mice entering the novel arm in the novel arm recognition experiment (N = 10); E is a schematic diagram of the novel location recognition experiment design; F statistically analyzed the recognition index of mice recognizing novel location objects in the novel location recognition experiment (N = 10); G statistically analyzed the time of mice recognizing the same object during the training phase (Day1) in the novel location recognition experiment (N = 10); H statistically analyzed the time of mice recognizing novel location objects during the test phase (Day2) in the novel location recognition experiment (N = 10); I statistically analyzed the moving distance of mice entering the central area in the open field experiment (N = 10); J is the statistical analysis of the time of entering the central area in the open field experiment, N = 10, two-way ANOVA followed by Tukey’s post hoc test; K statistically analyzed the total moving distance of mice in the open field experiment, N = 10, two-way ANOVA followed by Tukey’s post hoc test; ; ; ; ; ns: no significant difference.

[0021] Figure 2sCSF1R improved the expression of nerve and myelin-related proteins in CSF1R-RD model mice. Among them, A was the Western Blot detection of the expression of PSD-95, SYN, MBP, and β-actin proteins in mouse cortical tissues (N = 5, Two-way ANOVA followed by Tukey’s post hoc test); B was the quantitative analysis of the gray scale of cortical Psd95 protein with β-actin as the internal reference; C was the quantitative analysis of the gray scale of cortical SYN protein with the same internal reference; D was the quantitative analysis of the gray scale of cortical MBP protein (internal reference β-actin); E was the quantitative analysis of the gray scale of Psd95 protein in the mouse hippocampus (internal reference β-actin); F was the quantitative analysis of the gray scale of SYN protein in the mouse hippocampus (internal reference β-actin); G was the quantitative analysis of the gray scale of MBP protein in the mouse hippocampus (internal reference β-actin); H was the immunofluorescence co-staining detection of GFAP antibody (green), MBP antibody (red), and DAPI (blue) in mouse brain tissues (N = 5, scale bar: 50 μm); I was the quantitative statistical analysis of the MBP fluorescence intensity of the immunofluorescence results. ; ; ; 。

[0022] Figure 3 sCSF1R inhibited the lysosomal activity of microglia in the CA1 region of the hippocampus in CSF1R-RD model mice. Among them, A was the immunofluorescence staining detection of CD68 antibody (red), IBA1 antibody (green), and DAPI (blue) in mouse brain tissues (N = 5, scale bar: 50 μm); B was the statistical analysis of the number of CD68 + / IBA1 + and IBA1 + cells, Two-way ANOVA followed by Tukey’s post hoc test; C was the statistical analysis of the CD68 + area and IBA1 + area in the CA1 region of the mouse hippocampus (N = 5), Two-way ANOVA followed by Tukey’s post hoc test. ; ; ; 。

[0023] Figure 4sCSF1R reduces the transcriptional levels of Tnf-α and Il1β in CSF1R-RD model mice. Among them, A is the detection and analysis of Tnf-α mRNA levels in mouse cortical tissues using RT-PCR (reference gene β-actin, N = 5, two-way ANOVA followed by Tukey’s post hoc test); B is the RT-PCR detection and analysis of Il1β mRNA levels in mouse cortical tissues (reference gene β-actin, N = 5); C is the detection and analysis of Tnf-α mRNA levels in mouse hippocampal tissues using the same method (reference gene β-actin, N = 5); D is the RT-PCR detection and analysis of Il1β mRNA levels in mouse hippocampal tissues (reference gene β-actin, N = 5), two-way ANOVA followed by Tukey’s post hoc test; ; ; ; .

[0024] Figure 5 sCSF1R inhibits the expression of inflammatory factors in microglia derived from CSF1R-RD model mice. Among them, A is the detection and analysis of Tnf-α mRNA levels in primary microglia of wild-type and CSF1R-RD models treated with sCSF1R using RT-PCR (reference gene β-actin, N = 5, two-way ANOVA followed by Tukey’s post hoc test); B is the RT-PCR detection and analysis of Il1β mRNA levels in WT and HE primary microglia treated with sCSF1R (reference gene β-actin, N = 5); two-way ANOVA followed by Tukey’s post hoc test; ; ; ; .

[0025] Figure 6sCSF1R exerts its function by binding to CSF1R on the cell membrane of microglia and L1CAM on neurons in the central nervous system. Among them, A shows the co-incubation of the sCSF1R protein with a Flag tag and the whole brain protein lysate of adult male mice, followed by immunoprecipitation experiments using an antibody against the Flag tag, an antibody specifically recognizing human CSF1R, and a negative control antibody (rabbit IgG). The figure shows a schematic diagram of the protein immunoprecipitation-mass spectrometry analysis experiment; B shows the overlapping analysis of Flag, hCSF1R, and IgG group proteins using a Venn diagram; C shows the identification statistics and classification analysis of 228 candidate proteins using a pie chart; D shows the display of the names of candidate target proteins; E shows the display and analysis of cell membrane surface proteins analyzed using a scatter plot; F shows the display of secreted proteins analyzed using a scatter plot; G shows the qualitative detection and analysis of murine CSF1R in the endogenous immunoprecipitation experiment using Western Blot; H shows the qualitative detection and analysis of the immunoprecipitation experiment of sCSF1R and L1CAM in HEK293T cells using Western Blot. sCSF1R was used for immunoprecipitation of L1CAM; I shows the qualitative detection and analysis of the immunoprecipitation experiment of sCSF1R and L1CAM in HEK293T cells using Western Blot. L1AM was used for immunoprecipitation of sCSF1R. Detailed implementation mode

[0026] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. All embodiments are only used to illustrate the present invention and in no way limit the protection scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or changed without departing from the spirit and scope of the present invention, but such modifications or changes all fall within the protection scope of the present invention.

[0027] The embodiments of the present invention provide a drug based on a CSF1R polypeptide fragment, which is a drug or drug composition for improving, alleviating, or treating CSF1R-RD and can effectively improve the clinical symptoms of patients with CSF1R-RD; the drug or drug composition contains a therapeutically effective amount of sCSF1R polypeptide or its truncated fragment.

[0028] The amino acid sequence of the sCSF1R polypeptide is shown in SEQ ID NO:1, specifically as follows:

[0029] IPVIEPSVPELVVKPGATVTLRCVGNGSVEWDGPPSPHWTLYSDGSSSILSTNNATFQNTGTYRCTEPGDPLGGSAAIHLYVKDPARPWNVLAQEVVVFEDQDALLPCLLTDPVLEAGVSLVRVRGRPLMRHTNYSFSPWHGFTIHRAKFIQSQDYQCSALMGGRKVMSISIRLKVQKVIPGPPALTLVPAELVRIRGEAAQIVCSASSVDVNFDVFLQHNNTKLAIPQQSDFHNNRYQKVLTLNLDQVDFQHAGNYSCVASNVQGKHSTSMFFRVVESAYLNLSSEQNLIQEVTVGEGLNLKVMVEAYPGLQGFNWTYLGPFSDHQPEPKLANATTKDTYRHTFTLSLPRLKPSEAGRYSFLARNPGGWRALTFELTLRYPPEVSVIWTFINGSGTLLCAASGYPQPNVTWLQCSGHTDRCDEAQVLQVWDDPYPEVLSQEPFHKVTVQSLLTVETLEHNQTYECRAHNSVGSGSWAFIPISAGAHTH。

[0030] The truncated fragment of the sCSF1R polypeptide only contains the first immunoglobulin-like domain of the sCSF1R polypeptide, that is, it is composed of amino acid residues 1 to 86 of the sCSF1R polypeptide SEQ ID NO:1, specifically as follows:

[0031] IPVIEPSVPELVVKPGATVTLRCVGNGSVEWDGPPSPHWTLYSDGSSSILSTNNATFQNTGTYRCTEPGDPLGGSAAIHLYVKDPA。

[0032] Purification is obtained by nickel ion affinity chromatography using eukaryotic protein purification experimental techniques. The experimental principle is roughly described as follows: HEK293T cells are transfected in large quantities using plasmids and the cell culture medium is collected. According to the characteristics that both the 10×His protein tag and imidazole can bind to nickel ions, the non-specific proteins and specific proteins are separated by changing the imidazole concentration in the solution. The purification packing material used in the experiment is nickel ion gel Ni-NTA (QIAGEN, NO. 30210). The specific method is as follows:

[0033] ⑴ A large amount of CSF1R plasmids with 10×His-Flag tags was obtained. The plasmid was transfected into HEK293T cells using the PEI transfection reagent. After 16 h, the cells were washed three times with 1×PBS and then cultured in serum-free DMEM medium for another 48 h.

[0034] ⑵ After collecting the cell culture medium, it was aliquoted into 50 mL centrifuge tubes and centrifuged at 12,000 rpm / min for 20 minutes to remove cell debris in the culture medium.

[0035] ⑶ The centrifuged culture medium was filtered again through a 0.22 μm filter membrane using a suction filtration device to remove impurities in the culture medium.

[0036] ⑷ The culture medium was concentrated in volume by centrifuging at 4000 g for 30 minutes at 4℃ using an ultrafiltration tube with a 10 kDa filter membrane.

[0037] ⑸ The concentrated culture medium was collected and mixed with purification buffer I (20 mM PBS, 0.5 M NaCl, pH 7.4) in a ratio of 1:3 for standby.

[0038] ⑹ The purification packing material was shaken well in a bottle, and 5 mL was pipetted into a filtration tube using a dropper. After the liquid had dripped until the liquid level was close to the upper layer of the gel, the gel column was equilibrated with three times the volume of the packing material of ultrapure water and then purification buffer I for standby.

[0039] ⑺ The sample mixture was slowly added dropwise to the packing material using a dropper (the packing material plane should not be blown away), and the sample was loaded three times repeatedly to allow the protein to bind to the gel column as much as possible.

[0040] ⑻ Three times the volume of the packing material of purification buffer I was added to the upper end of the packing material to initially wash away non-specific binding components.

[0041] ⑼ Elution buffers containing 40 mM and 500 mM (Flow through) imidazole concentrations were prepared using purification buffer I and purification buffer II (20 mM PBS, 0.5 M NaCl, 1 M imidazole, pH 7.4) respectively, and were added dropwise to the packing material successively, and the corresponding eluates were collected.

[0042] ⑽ The 500 mM eluate was added to a prepared semi-permeable membrane with a pore size of 500 MW (the semi-permeable membrane pretreatment requires sequential use of 1 mM EDTA, 5% Na 2 CO 3 and 5% Na 2 CO 3During the boiling treatment for 10 minutes), the imidazole and NaCl in the eluate were dialyzed out using PBS buffer solution with a gradient of high to low concentration. Finally, the eluate was dialyzed twice with 1×PBS to change the system in the eluate to a 1×PBS buffer solution system suitable for biological experiments (the number of dialysis times and time should be sufficient to remove the toxic substances and impurities in the culture solution as much as possible, otherwise it will have a greater impact on the treatment effect).

[0043] ⑾ The dialyzed liquid was concentrated again using a 10 kDa ultrafiltration tube, the residual liquid was collected, and the protein was filtered through a 0.22 μm filter membrane to complete the purification.

[0044] The purified sCSF1R protein was identified using silver ion staining and Western Blot techniques. Among them, the silver ion staining technique was used to detect the protein purification efficiency, and the primary antibody recognizing human sCSF1R was used in the Western Blot technique for detection to confirm the correctness of the purified protein.

[0045] The following describes the effect of sCSF1R in treating CSF1R-RD based on experiments.

[0046] 1. Casing embedding and sCSF1R treatment

[0047] Compared with stereotaxic injection in the mouse brain, the cannula positioning injection in the mouse brain can inject experimental samples into specific brain regions of the mouse repeatedly. At the same time, because the anesthetic used during cannula injection is the inhaled anesthetic isoflurane, the damage to animals and the impact on experimental results are relatively small; compared with conventional stereotaxic injection in the brain, the additional equipment and devices required for cannula injection are as follows: cannula base, cannula nut, fixing screw, glass ionomer cement, injection inner tube, injection hose, isoflurane, gas anesthesia machine, etc.

[0048] ⑴ Weigh the target mice and inject anesthetic, and remove the hair on the mouse's head.

[0049] ⑵ Fix the anesthetized mice to the stereotaxic apparatus by the three-point fixation method. After disinfecting the mouse's head with iodine, use surgical scissors to cut open the mouse's head skin to expose the skull.

[0050] ⑶ Use a cotton swab dipped in 3% hydrogen peroxide to ablate the subcutaneous membrane tissue of the mouse and fully expose the anterior and posterior fontanelle points of the skull. Keeping the height error of the anterior and posterior fontanelles of the mouse within 0.02 mm is regarded as accurate fixation of the mouse skull.

[0051] ⑷ Fix the microinjector needle to the injection arm of the apparatus, point the tip of the needle at the anterior fontanelle point of the mouse skull, and set the X-axis and Y-axis values to zero to set the coordinate origin.

[0052] ⑸ According to the coordinates found in the mouse brain atlas, move the needle to the corresponding position and mark it (the positioning injection position in this embodiment is the lateral ventricle, and the coordinates are X, Y, Z = 1.0, -0.5, 1.8).

[0053] ⑹ After marking the site, use a cranial drill to drill through the skull at the specific site. At the same time, use a cranial drill to drill a small pit similar to the diameter of the fixing screw 2 mm away from the specific site in the lateral direction of the mouse body for fixing the screw to the mouse skull.

[0054] ⑺ After fixing the screw, assemble the cannula base and cannula nut, fix the combination with a cannula holder, and then fix the cannula holder to the stereotaxic apparatus.

[0055] ⑻ Rotate the X, Y, and Z axes to place the lower metal tube of the cannula against the previously marked brain hole, set the Z-axis value to zero, and slowly rotate the Z-axis to insert the cannula into the mouse brain to the specified depth.

[0056] ⑼ After mixing the liquid and powder of glass ionomer cement in a ratio of 1:1, use small forceps to gradually apply the mixture to the bottom of the cannula and gradually wrap the fixing screw.

[0057] ⑽ Wait for more than 10 minutes. After the cement has completely solidified, remove the holder, take the mouse out and place it on a warming blanket to wait for the mouse to wake up.

[0058] The injection through the cannula can be carried out one week after the cannula is implanted. The method is as follows:

[0059] ⑴ Assemble the injection unit. Combine the inner injection tube, a certain length of injection hose, and a microsyringe in sequence. Inject a certain length of sterile PBS into the injection hose (there is a certain resistance inside the brain. During the pushing process of the microsyringe, the air in the hose will be compressed due to too much air, which will affect the actual volume of the liquid injected into the mouse brain. Therefore, the air in the inner tube needs to be minimized).

[0060] ⑵ Add isoflurane to the gas anesthesia machine and turn on the machine. Adjust the air pressure to the range of 1.5 - 2.0, adjust the anesthetic to 2.0, turn the knob to the "Chamber" position to fill the anesthesia chamber with isoflurane. Place the mouse in the anesthesia chamber. After the mouse is anesthetized, take out the mouse, put the anesthesia mask on the mouse's mouth and nose, and turn the knob to the "Mask" position.

[0061] ⑶ Aspirate the experimental liquid into the injection unit, unscrew the cannula nut, insert the inner injection tube into the cannula base, and adjust the injection parameters (the volume in this experiment is still 1 μL, and the speed is 0.2 μL / min) for injection.

[0062] ⑷ After the injection is completed, wait for 5 min to allow the drug to diffuse in the brain as much as possible, then pull out the injection inner tube and reinstall the cannula nut onto the cannula base.

[0063] ⑸ Remove the mouse anesthesia mask and place the mouse back in the cage to complete the cannula injection.

[0064] 2. Animal behavior observation

[0065] The mouse behavior experiments were strictly carried out in accordance with animal ethics and were all conducted between 14:00 and 20:00 in the afternoon. Three days before the start of the experiment, the mice were gradually touched to reduce their stress behavior in the behavior experiments.

[0066] (I) Y-maze experiment

[0067] The Y-maze is used to detect the instantaneous spatial working ability of mice. The method is as follows: Place the mouse at the center of the Y-maze and allow the mouse to freely explore for 5 min. During the exploration, use a small animal behavior detection and analysis system to record various data of the mouse and calculate the spontaneous alternation percentage of the mouse. After each experiment on each maze, clean the mouse feces and urine, then spray 75% alcohol with a spray bottle and wipe it clean with absorbent paper. Finally, use a fan to completely dry the residual alcohol to remove the smell left by the previous mouse and avoid affecting the subsequent mouse.

[0068] (II) Novel-arm recognition experiment

[0069] The novel-arm recognition experiment is used to detect the spatial memory ability of mice. The Y-maze device is used in the experiment, which is divided into two experimental stages:

[0070] (1) Training stage: Seal one arm of the Y-maze device with a baffle and then place the mouse in the Y-maze and allow the mouse to freely explore for 5 min.

[0071] (2) Testing stage: After 3 h, open the sealed arm, place the mouse back in the same Y-maze and allow the mouse to freely explore for 5 min, and use a small animal behavior detection and analysis system to record the time the mouse enters the novel arm.

[0072] (III) Open field experiment

[0073] The open field experiment is used to detect the anxiety-like emotional performance of mice. Mice that are overly anxious show less time and distance in the center of the open field box.

[0074] The mice were placed in a square open box measuring 40 cm × 40 cm × 40 cm. A square area 10 cm away from the surrounding walls was marked as the central area in the square box. The mice were allowed to freely explore for 10 min, and during the exploration, a small animal behavior detection and analysis system was used to record the time and distance the mice entered the open area, as well as other data.

[0075] (4) Novel location recognition experiment

[0076] The novel location recognition experiment focuses more on detecting functional changes in the hippocampus of mice compared to the novel object recognition experiment (the novel object recognition is used for detecting complex circuits in the hippocampus and cortical projections). The experiment also consists of two stages, and the methods are as follows:

[0077] (1) On the first day, two identical objects were placed at opposite positions in the open field box, with a distance of 10 cm from both walls. A circular area with a radius of 5 cm outside the objects was set as the recognition area. The mice were placed in the open field box facing the wall at a corner at the same distance from the two objects (ensuring that the objects were tall enough to prevent the mice from climbing to the top of the objects), and the mice were allowed to freely explore for 10 min. A small animal behavior detection and analysis system was used to record the time and number of times the mice sniffed the objects with their noses.

[0078] (2) On the second day, one of the objects was moved to the same side. The mice were placed in the open field box facing the wall at the same position as on the first day, and the mice were allowed to freely explore for 10 min. A small animal behavior detection and analysis system was used to record the time and number of times the mice sniffed the objects with their noses.

[0079] 3. Western blotting

[0080] Cells were collected using lysis buffer for immunoprecipitation-free assay (Boster) and protease inhibitor mixture without EDTA (Roche). The concentration of total protein was determined by BCA (Bicinchoninic Acid Assay) Protein Assay Kit (Boster). Equal amounts of protein samples or supernatant samples were tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to polyvinylidene fluoride membranes (PVDF, Millipore). After incubation with specific antibodies at 1:1000 human CSF1R (RRID: AB_2799725, Cell Signaling Technology) or 1:1000 / 1:6000 Anti-MCSF Receptor (RRID: AB_2927559, Abcam) or protein tag antibodies at 1:1000 / 1:2000 GFP-tag or 1:10000 Flag-tag), they were combined with horseradish peroxidase (HRP)-labeled secondary antibodies and developed by ECL. The immunoreactive bands were quantitatively analyzed using ImageJ software.

[0081] 4. RT-PCR method

[0082] ㈠ RNA extraction

[0083] A certain amount of tissue samples were transferred to EP tubes after tissue grinding, 500 μL of TRIzol lysis buffer was added, and they were placed at room temperature for 5 min. Further, to lyse the cells more thoroughly, the EP tubes could be placed in an -80°C refrigerator for one freeze-thaw cycle. 100 μL of chloroform (one-fifth of TRIzol) was added, inverted and mixed evenly for 30 s, placed at room temperature for 3 min, and centrifuged at 4°C, 12000 rpm / min for 15 min. After centrifugation, the liquid was divided into three layers. The colorless upper layer was the aqueous phase where RNA existed (the middle layer was protein, and the lower layer was the organic phase). The supernatant was aspirated into a new RNA-free EP tube (the protein should not be aspirated), and about 200 μL could be aspirated. 250 μL of isopropanol was added, gently mixed for 10 s, and left at room temperature for 10 min to precipitate the RNA. It was centrifuged at 4°C, 12000 rpm / min for 10 min, and the supernatant was discarded leaving the precipitate. 500 μL of 75% ethanol prepared with enzyme-free water was added, placed at room temperature for 2 min, and centrifuged at 4°C, 12000 rpm / min for 10 min, and the supernatant was discarded leaving the precipitate. After drying the residual ethanol in the EP tube in a laminar flow hood, 20 - 50 μL of enzyme-free water preheated at 60°C was added to dissolve the RNA. The concentration and purity of the RNA were detected using a NanoDrop instrument.

[0084] ㈡ RNA reverse transcription

[0085] According to the RNA concentration obtained in the previous step, aspirate 1 μg of RNA for reverse transcription. The reverse transcription procedure refers to the instructions in the reverse transcription kit. Store the reverse-transcribed cDNA at 4°C for standby, and store it at -80°C for long-term preservation.

[0086] (3) Real-time fluorescence quantitative PCR

[0087] Design and calculate the volumes of each component in the quantitative PCR according to the experimental requirements, add them to the 96-well plate according to the experimental design, and seal the 96-well plate with a film. Use a fluorescence quantitative PCR instrument for detection. The PCR reaction is amplified by a two-step method, and the reaction program is set according to the instructions of the Novoprotein SYBR kit.

[0088] The primers involved are as follows:

[0089] Table 1 Primer sequences for real-time fluorescence quantitative PCR

[0090]

[0091] Example 1. Long-term injection of sCSF1R significantly improves the anxiety-like phenotype and learning and memory ability of CSF1R-RD model mice

[0092] The experimental mice were divided into four groups: the CSF1R-RD model mice injected with the control Vehicle group (HE-Vehicle group), the CSF1R-RD model mice injected with sCSF1R group (HE-sCSF1R group), the wild-type mice injected with the control Vehicle group (WT-Vehicle group), and the wild-type mice injected with sCSF1R group (WT-sCSF1R group). All the experimental mice were male.

[0093] In this example, the implantation of a cannula was used to locate the injection site in the lateral ventricle region of CSF1R-RD model mice ( , HE) and their wild-type controls ( , WT) mice. Six days after the completion of cannula implantation, the injection experiments of sCSF1R and the control Vehicle were carried out. In the following 120 days, the mice were injected once every 5 days. After the mice reached the onset age, a series of behavioral tests were performed on the mice. The schematic diagram of the animal experiment of multiple stereotactic injections of sCSF1R into the brain through the cannula is shown as Figure 1 shown in A of

[0094] In the Y-maze test, it was found that the mice in the HE-Vehicle group showed a decrease in spontaneous alternation behavior compared with the WT-Vehicle group, while after treatment with sCSF1R, the spontaneous alternation behavior of the mice in the HE-sCSF1R group was significantly restored compared with the HE-Vehicle group ( Figure 1 B in

[0095] In the novel arm maze experiment ( Figure 1 C in Figure 1 ), the mice in the HE-Vehicle group showed less time entering the novel arms compared with the WT-Vehicle group, suggesting impaired short-term spatial memory ability in HE mice; while the time of the HE mice entering the novel arms was restored after receiving sCSF1R treatment ( D in

[0096] The open field experiment was used to evaluate the anxiety-like behavior of the mice. It was found that the mice in the HE-Vehicle group showed significant anxiety-like behavior compared with the WT-Vehicle group, specifically manifested as shorter moving distance entering the central area and less time staying in the central area; while after treatment with sCSF1R, the anxiety-like behavior of the mice in the HE-sCSF1R group was effectively alleviated ( Figure 1 J, K in

[0097] Finally, the novel location recognition experiment was used to detect the function of the hippocampal region of the mice. This novel location recognition experiment focused more on detecting the functional changes in the hippocampal region of the mice compared with the novel object recognition experiment that detects the functions of multiple brain regions of the mice ( Figure 1 E in Figure 1 ). In the novel location recognition experiment, the mice in the HE-Vehicle group showed lower recognition ability for the moved object compared with the WT-Vehicle group, suggesting damage to the function of the hippocampal region in HE-Vehicle mice; while after treatment with sCSF1R, the mice in the HE-sCSF1R group were able to show a recognition ability level close to that of the WT-Vehicle (

[0098] In summary, the behavioral tests in this example confirmed that sCSF1R treatment can effectively alleviate the impaired learning and memory ability and anxiety behavior in CSF1R-RD model mice. In the statistical analysis of all experimental data, the sample size N = 10, and two-way ANOVA followed by Tukey’s post hoc test was used for analysis (unless otherwise specified). In the training phase (Day 1) of the novel location recognition test, unpaired Student’s t test was used for the statistical analysis of the time that mice recognized the same object (G in Figure 1), and in the test phase (Day 2), unpaired Student’s t test was also used for the statistical analysis of the time that mice recognized the object in the novel location (H in Figure 1). The data results were expressed by ; ; ; indicating significant differences, and ns indicating no significant differences.

[0099] Example 2. sCSF1R improves the expression of nerve and myelin-related proteins in CSF1R-RD model mice

[0100] Based on the behavioral changes in mice treated with sCSF1R by cannula injection, the mouse brain tissues were collected and detected by Western Blot and immunohistochemical fluorescence staining. In the Western Blot detection, experiments were conducted with a sample size of N = 5. The results showed that compared with WT-Vehicle mice, the expressions of synaptic-related proteins Psd95 and SYN and myelin-related protein MBP in the cortical tissues of HE-Vehicle mice were significantly decreased. The gray scale quantification analysis of Psd95, SYN, MBP and β-actin as the internal reference protein in the cortex was performed using the statistical method of Two - way ANOVA followed by Tukey’s post hoc test, which further confirmed this expression difference, indicating that HE mice had impaired neuronal function, synaptic function and myelin-related function; after treatment with sCSF1R, the protein expression levels of Psd95, SYN and MBP in HE mice were significantly restored ( Figure 2(A-E in). Similarly, in the mouse hippocampal tissue, the protein expression levels of Psd95, SYN, and MBP in HE mice were significantly lower than those in WT mice; after gray scale quantification analysis by Two-way ANOVA followed by Tukey’s post hoc test with β-actin as the internal reference protein, this change was clearly presented. After treatment with sCSF1R, it was significantly restored ( Figure 2 A, F-I in).

[0101] In the immunohistochemical fluorescence staining experiment, mouse brain slices obtained from frozen sections were detected by immunohistochemical fluorescence staining using GFAP antibody (green), MBP antibody (red), and DAPI (blue). The sample size N = 5, and the scale bar was 50 . The experiment found that in the CA1 region of the mouse hippocampus, the fluorescence intensity and area of MBP protein in HE-Vehicle mice were significantly lower than those in WT-Vehicle mice. Consistently with the Western Blot results, after treatment with sCSF1R, the immunofluorescence intensity level of MBP protein in the CA1 region of the hippocampus in HE-sCSF1R mice was restored ( Figure 2 J-K in). The data results were obtained through ; ; ; indicates a significant difference. These experimental results fully demonstrate that sCSF1R can improve the expression of nerve and myelin-related proteins in CSF1R-RD model mice and repair related functional damage.

[0102] Example 3. sCSF1R inhibition reduces the lysosomal activity of microglia in the CA1 region of the hippocampus in CSF1R-RD model mice

[0103] This example aims to explore the effect of sCSF1R on the lysosomal activity of microglia in the hippocampal CA1 region of CSF1R-RD model mice, and an immunofluorescence staining detection method is used to detect the immunofluorescence of microglia in the mouse brain tissue. During the experiment, the mouse brain tissue was immunofluorescently stained and detected using CD68 antibody (red), IBA1 antibody (green), and DAPI (blue). The sample size N = 5, and the scale for observing the staining results was set at 50μm. The results showed that in the hippocampal CA1 region of HE-Vehicle mice, compared with WT-Vehicle mice, microglia showed an over-activated state with enlarged cell bodies and reduced branches. At the same time, the fluorescence of lysosomes labeled by CD68 was significantly enhanced, and the fluorescence area was significantly increased. After treatment with sCSF1R, the cell bodies of microglia in the HE-sCSF1R group of mice decreased, the branches increased, the fluorescence intensity of lysosomes weakened, and the area decreased, showing a resting state ( Figure 3 as shown in A-C of . indicates a significant difference. The results fully suggest that sCSF1R can effectively inhibit the over-activation of microglia in CSF1R-RD model mice and simultaneously reduce the over-enhancement of lysosomal activity, which has a positive effect on maintaining the normal state of microglia.

[0104] Example 4. sCSF1R reduces the transcriptional levels of Tnf-α and Il1β in the brains of CSF1R-RD model mice

[0105] It has been reported in the literature that the inflammatory level in the brain tissues of CSF1R-RD patients and model mice is significantly enhanced. Based on this background, this example focuses on exploring the effect of sCSF1R on the transcriptional levels of inflammatory factors Tnf-α and Il1β in the brains of CSF1R-RD model mice, and the research is carried out by RT-PCR detection.

[0106] The experiment was carried out with a sample size of N = 5 to perform RT-PCR detection on the inflammatory factors Tnf-α and Il1β in the cerebral cortex and hippocampus of mice. During the RT-PCR experiment, β-actin was used as a reference gene to ensure the accuracy and comparability of the experimental data. For the mouse cerebral cortex tissue, RT-PCR was used to detect Tnf-α (Figure 4A) and Il1βThe mRNA levels of (Figure 4B) were detected and analyzed, and the data were processed using the two-way ANOVA followed by Tukey’s post hoc test statistical method; similarly, for mouse hippocampal tissues, when detecting and analyzing the mRNA levels of Tnf-α Figure 4C Il1β and (Figure 4D), β-actin was also used as the reference gene, and the same statistical method was applied.

[0107] The experimental results showed that and were significantly highly expressed in HE-Vehicle mice compared with WT-Vehicle, further confirming the abnormal increase in the brain inflammation level of CSF1R-RD model mice. After treatment with sCSF1R, the transcriptional levels of HE-sCSF1R mice were restored compared with HE-Vehicle mice and ( Figure 4 ). The data results were presented through ; ; ; indicating significant differences. sCSF1R can effectively reduce the transcriptional levels of and in the brains of CSF1R-RD model mice, inhibit the over-activation of microglia and lysosomes in brain tissues, and thus play a positive role in inflammation regulation, which is of great significance for improving the brain microenvironment of CSF1R-RD model mice. Therefore, sCSF1R inhibits the over-activation of microglia and lysosomes in the brain tissues of CSF1R-RD model mice.

[0108] Example 5. sCSF1R inhibits the expression of inflammatory factors in primary microglia derived from CSF1R-RD model mice

[0109] This example aims to deeply explore the effect of sCSF1R on the expression of inflammatory factors in primary microglia derived from CSF1R-RD model mice, and the research was carried out through RT-PCR experiments and immunocytochemical fluorescence staining experiments.

[0110] In the RT-PCR experiment section, primary microglia from wild-type (WT) treated with sCSF1R and from the CSF1R-RD model source (HE) were detected with a sample size of N = 5. During the experiment, β-actin was used as a reference gene to ensure the accuracy and reliability of the experimental data. The mRNA level of Tnf-α was detected and analyzed using the RT-PCR method (A in Figure 5), and the data were processed using the statistical method of two-way ANOVA followed by Tukey’s post hoc test; similarly, when detecting and analyzing the mRNA level of (B in Figure 5), the same experimental conditions and statistical methods were followed.

[0111] The experimental results showed that the transcription levels of inflammatory factors and were significantly upregulated in HE microglia compared to WT microglia. After treatment with sCSF1R, the transcription levels of inflammatory factors and in both WT and HE microglia were significantly inhibited (A-B in Figure 5 ). The data results were presented as ; ; ; , indicating significant differences.

[0112] The experimental results showed that sCSF1R could effectively inhibit the expression of inflammatory factors in primary microglia from CSF1R-RD model mice, which plays an important role in improving the inflammatory state of CSF1R-RD model mice.

[0113] Example 6. sCSF1R exerts its effects by binding to CSF1R on the cell membrane of microglia and L1CAM on neurons in the central nervous system.

[0114] In this example, we explored which proteins sCSF1R binds to after entering the central nervous system to produce a series of biological effects. Based on the plasmid used for purifying sCSF1R, it was modified again, and a Flag-tag was inserted after the purification protein tag 6×His, and then protein purification was carried out again. As Figure 6As shown in A of [Figure 0], after co-incubating the sCSF1R protein with a Flag tag and the whole brain protein lysate of adult male mice, immunoprecipitation experiments were performed using an anti-Flag tag antibody, an antibody specifically recognizing human CSF1R, and a negative control antibody (rabbit IgG); then the bound proteins were eluted with an 8M urea solution and subjected to protein mass spectrometry. The mass spectrometry results showed ( Figure 6 in B of [Figure 1]), 1612 proteins were immunoprecipitated by the anti-Flag tag antibody; 1315 proteins were immunoprecipitated by the human CSF1R (hCSF1R) antibody; 1083 proteins were immunoprecipitated by the negative control antibody. Overlapping analysis of the Flag group and hCSF1R yielded 1076 proteins, and then subtracting the proteins detected in the IgG group gave 228 specific proteins. Further, these 228 proteins were entered into the UniProt protein database (website: https: / / www.uniprot.org / ), and the subcellular localization of these proteins was queried based on the existing information. Among the 228 proteins: 186 proteins were located in the cytoplasm or nucleus of the cell; 22 proteins were located inside the cell membrane; 5 proteins were located on the cell membrane surface and 15 were secreted proteins ( Figure 6 in C of [Figure 2]). Since the sCSF1R protein is free outside the cell and secreted proteins are also worthy of attention ( Figure 6 in F of [Figure 3]), but because there are also a series of binding targets for secreted proteins that are difficult to clarify the line and require more in-depth analysis and research, it is tentatively presumed that the protein targets bound by sCSF1R in the central nervous system should be located on the cell membrane surface. The 5 proteins located on the cell membrane surface were found to be Colony stimulating factor 1 receptor (CSF1R), L1 cell adhesion molecule (L1CAM), Thy1 Cell Surface Antigen (Thy1, also known as CD90), Contactin 1 (CNTN1), and H2-K1 (Histocompatibility 2, K1, K region) ( Figure 6 in F of [Figure 4]). Then, based on the ALSP disease-related phenotypes and literature review and analysis of these 5 candidate proteins, CSF1R specifically expressed on the surface of microglia and L1CAM specifically expressed at the axon initial segment of neurons and involved in action potential formation were worthy of special attention.

[0115] For CSF1R and L1CAM screened by mass spectrometry, immunoprecipitation verification was performed separately. First, sCSF1R was co-incubated with the lysate of primary microglial cells of WT, and then the endogenous CSF1R immunoprecipitation was detected using the Flag-tag antibody to confirm the binding of sCSF1R to CSF1R ( Figure 6 G in). Next, a human L1CAM plasmid with an HA protein tag inserted at the carboxyl terminus (C-terminus) and a plasmid of the extracellular domain of CSF1R with a Flag protein tag at the amino terminus (N-terminus) were constructed and transfected into HEK293T cells. Through co-immunoprecipitation experiments, it was found that Flag-tagged sCSF1R binds to L1CAM ( Figure 6 H, I in). The above results indicate that sCSF1R exerts its function by binding to CSF1R on the cell membrane of microglia and L1CAM on neurons in the central nervous system.

[0116] The above embodiments are only preferred embodiments of the present invention and cannot be considered as limiting the scope of implementation of the present invention. All equivalent changes and improvements made according to the scope of the present invention application should still fall within the scope covered by the patent of the present invention. Those skilled in the art can modify and process based on the CSF1R polypeptide fragment (sCSF1R) described in the present invention. As long as the modified drug is related to the sCSF1R polypeptide fragment, it should fall within the scope to be protected by the present invention.

Claims

1. A drug based on a CSF1R polypeptide fragment, characterized in that: The drug comprises sCSF1R polypeptide or a truncated fragment thereof; the amino acid sequence of the sCSF1R polypeptide is shown in SEQ ID NO: 1 in the sequence table, and the truncated fragment is the first immunoglobulin-like domain of the sCSF1R polypeptide.

2. A drug based on a CSF1R polypeptide fragment as claimed in claim 1, characterized in that The dosage form of the drug is an injection preparation, an oral preparation, a spray preparation, an ointment preparation or a patch.

3. Use of the sCSF1R polypeptide fragment or its truncated fragment as claimed in claim 1 in the preparation of a medicament or a pharmaceutical composition for improving, alleviating or treating a disease related to abnormal CSF1R.

4. Use of the sCSF1R polypeptide fragment or a truncated fragment thereof according to claim 1 in the development of CSF1R-RD drugs.

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