Application of substance taking ANGPTL4 or coding gene thereof as target spot in preparation of product for treating cerebellar ataxia

Abstract

The invention discloses application of a substance taking ANGPTL4 protein or a coding gene thereof as a target spot in preparation of a product for treating cerebellar ataxia. The invention belongs to the technical field of biology, and particularly relates to application of a substance taking ANGPTL4 or a coding gene thereof as a target spot in preparation of a product for treating cerebellar ataxia. The invention provides an application of a substance for reducing ANGPTL4 gene expression, a substance for reducing ANGPTL4 protein activity or a substance for reducing ANGPTL4 protein content in preparation of a product for treating or / and preventing or / and delaying cerebellar ataxia and an application of a product for reducing formation of inhibitory synapses.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and particularly relates to application of a substance taking ANGPTL4 or an encoding gene thereof as a target point in preparation of a product for treating cerebellar ataxia. BACKGROUND

[0002] The cerebrovascular system is critical for brain development and central nervous system (CNS) homeostasis, not only as a key conduit for delivering oxygen, nutrients and removing metabolic waste, but also as a functional integrated neurovascular unit (NVU) through close interactions with glial cells and neurons. In the neurovascular unit, the vasculature exhibits highly precise and regulated signaling capacity, which can actively regulate multiple key developmental processes including neuronal differentiation, maturation, positioning and synaptic plasticity. Among the vascular components, endothelial cells (ECs) are highly specialized and play an active regulatory role through potent paracrine signaling mechanisms.

[0003] As a key center for motor coordination and learning in the central nervous system, the cerebellum integrates sensory and motor information through its highly organized neural circuit. Normal development and sustained functional activity of the cerebellum rely on the support of a well-functioning vascular network. Increasing evidence suggests that cerebellar vascular dysfunction is an important risk factor for motor deficits. In the cerebellar cortex, Purkinje cells (PCs), as the only output neurons, are responsible for integrating cortical information and transmitting it to deep cerebellar nuclei, thereby regulating motor and postural control. Specifically, Purkinje cells receive excitatory synaptic inputs from Climbing fibers (CFs) and Parallel fibers (PFs), as well as inhibitory synaptic inputs from Molecular layer interneurons (MLIs). Purkinje cell degeneration and dysfunction are hallmark pathological features of various neurodegenerative diseases associated with motor impairment. In addition to cell-autonomous damage, an increasing number of studies have revealed that functional changes in cerebellar circuits can also disrupt the normal activity of Purkinje cells. A recent in vivo study found that excessive inhibitory synaptic inputs can impair the intrinsic excitability of Purkinje cells through non-cell-autonomous mechanisms and drive their degeneration, ultimately leading to motor dysfunction. Overall, these findings collectively suggest that improving Purkinje cell function by modulating cerebellar inhibitory circuits has potential therapeutic value. In the cerebellar cortex, blood vessels are closely arranged with Purkinje cells, forming a spatial configuration that facilitates efficient metabolic exchange and signal integration. Previous studies have shown that endothelial cells can secrete signaling molecules such as vascular endothelial growth factor (VEGF) and Angiopoietin-2 (Ang2) to participate in the development process of Purkinje cell dendrites.

[0004] ANGPTL4 (angiopoietin-like protein 4) is a multifunctional molecule secreted by endothelial cells, which plays a role in angiogenesis, metabolic regulation, tumor growth and other physiological processes. SUMMARY

[0005] The main problem to be solved by the present application is how to prevent or treat or delay cerebellar ataxia.

[0006] To solve the above problems, the present application provides the following applications: 1. The use of a substance that reduces the expression of ANGPTL4 gene in the preparation of a product for treating or / and preventing or / and delaying cerebellar ataxia.

[0007] 2. The use of a substance that reduces the expression of ANGPTL4 gene in the preparation of a product for reducing the formation of inhibitory synapses.

[0008] 3. The use of a substance that reduces the activity of ANGPTL4 protein in the preparation of a product for treating or / and preventing or / and delaying cerebellar ataxia.

[0009] 4. The use of a substance that reduces the activity of ANGPTL4 protein in the preparation of a product for reducing the formation of inhibitory synapses.

[0010] 5. The use of a substance that reduces the content of ANGPTL4 protein in the preparation of a product for treating or / and preventing or / and delaying cerebellar ataxia.

[0011] 6. The use of a substance that reduces the content of ANGPTL4 protein in the preparation of a product for reducing the formation of inhibitory synapses.

[0012] In the above applications, the product can be a drug or a vaccine.

[0013] The dosage form of the above-mentioned drug can be tablets, capsules, dripping pills, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, buccal tablets, suppositories or lyophilized powder injections.

[0014] In the above, the drug for treating or / and preventing or / and delaying cerebellar ataxia, in addition to containing a pharmaceutically acceptable salt thereof, can also contain a suitable carrier or excipient. The carrier materials herein include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly water-soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), enteric-soluble carrier materials (such as cellulose acetate phthalate and carboxymethyl cellulose, etc.). Among them, the water-soluble carrier material is preferred. Using these materials, various dosage forms can be prepared, including but not limited to tablets, capsules, drop pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, buccal tablets, suppositories, lyophilized powder injections, etc. They can be ordinary preparations, sustained-release preparations, controlled-release preparations, and various microparticle drug delivery systems. In order to make unit administration dosage forms into tablets, various carriers known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, white clay, microcrystalline cellulose, aluminum silicate, etc.; humectants and binders such as water, glycerol, polyethylene glycol, ethanol, propyl alcohol, starch paste, dextrin, sugar syrup, honey, glucose solution, acacia paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, etc.; disintegrants such as dry starch, alginate, agar powder, alginic acid, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glycerol tri-stearate, cocoa butter, hydrogenated oil, etc.; absorption promoters such as quaternary ammonium salt, sodium dodecyl sulfate, etc.; lubricants such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. The tablets can also be further coated, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets. In order to make unit administration dosage forms into pills, various carriers known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc, etc.; binders such as acacia, tragacanth gum, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrants such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc. In order to make unit administration dosage forms into suppositories, various carriers known in the art can be widely used. Examples of carriers are, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc.For the purpose of preparing a unit dosage form for injection, such as a solution, an emulsion, a lyophilized powder, and a suspension, all diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxyethylated sorbitan fatty acid ester, and the like. In addition, for the purpose of preparing an isotonic injection solution, an appropriate amount of sodium chloride, glucose, or glycerol can be added to the injection preparation, and in addition, a conventional cosolvent, a buffer, a pH adjustor, and the like can be added. In addition, if necessary, a coloring agent, a preservative, a fragrance, a flavoring agent, a sweetening agent, or other materials can be added to the pharmaceutical preparation. The above-mentioned dosage forms can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection, and intracavitary injection, and the like; administration through a body orifice, such as rectal and vaginal administration; administration through the respiratory tract, such as nasal administration; and administration through the mucous membrane. The above-mentioned administration routes are preferably injection administration.

[0015] In the present application, the cerebellar ataxia can be manifested as gait abnormalities, balance and coordination dysfunction, Purkinje cell degeneration, decreased Purkinje cell excitability, and increased inhibitory synapse density of interneurons.

[0016] In the above-mentioned application, the substance that reduces the expression of the ANGPTL4 gene or the substance that reduces the activity or protein content of the ANGPTL4 protein is any one of the following biological materials: B1) an antibody specific to the ANGPTL4 protein; B2) an inhibitor of the activity of the ANGPTL4 protein; B3) a nucleic acid molecule targeting the ANGPTL4 gene to reduce or inhibit or knock out the expression thereof; B4) an expression cassette containing the nucleic acid molecule of B3); B5) a recombinant vector containing the nucleic acid molecule of B3) or the expression cassette of B4); B6) a recombinant microorganism containing the nucleic acid molecule of B3), the expression cassette of B4), or the recombinant vector of B5); B7) a transgenic animal cell line in which the nucleic acid molecule of B3) or the expression cassette of B4) is integrated into the genome; B8) a transgenic animal tissue containing the nucleic acid molecule of B3) or the expression cassette of B4); B9) a transgenic animal organ containing the nucleic acid molecule of B3) or the expression cassette of B4).

[0017] The amino acid sequence of the ANGPTL4 protein is SEQ ID No: 1 or SEQ ID No: 3 in the sequence listing.

[0018] The coding gene mRNA sequence of the ANGPTL4 protein is SEQ ID No: 2 in the sequence listing.

[0019] Further, the ANGPTL4 protein specific antibody of B1) refers to an immunoglobulin molecule capable of binding to an ANGPTL4 protein epitope and blocking its biological activity.

[0020] Further, the ANGPTL4 protein activity inhibitor of B2) includes a small molecule compound, a polypeptide or an aptamer.

[0021] Further, the expression cassette of B4) comprises a promoter, the ANGPTL4 targeting nucleic acid sequence of B3) and a transcription terminator operably linked, and optionally comprises an enhancer.

[0022] Further, the vector of B5) can be a nucleic acid carrier into which a nucleic acid molecule that reduces or inhibits or knocks out the ANGPTL4 protein is inserted. For example, the vector includes: a plasmid; a phagemid; a cosmid; an artificial chromosome such as a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC) or a P1-derived artificial chromosome (PAC); a bacteriophage such as a lambda phage or a M13 phage; and an animal virus, etc. The animal virus used as the vector includes a retrovirus (including a lentivirus), an adenovirus, an adeno-associated virus, a herpes virus (such as a herpes simplex virus), a poxvirus, a baculovirus, a papillomavirus, a papovavirus (such as SV40). One vector can contain multiple elements for controlling expression.

[0023] Further, the animal can be a mammal, such as: a human, a cynomolgus monkey and a mouse.

[0024] The present application also provides uses of a substance that upregulates the expression of an ANGPTL4 gene, or a substance that upregulates the activity or protein content of an ANGPTL4 protein in the manufacture of a product: a1) promoting cerebellar ataxia; a2) promoting Purkinje cell dysfunction; a3) promoting inhibitory synapse formation; a4) studying a disease associated with cerebellar ataxia; a5) preparing an animal model with a cerebellar ataxia phenotype.

[0025] The substance that upregulates the expression of an ANGPTL4 gene, or the substance that upregulates the activity or protein content of an ANGPTL4 protein can be a substance that expresses the coding gene of an ANGPTL4 protein.

[0026] The substance is a substance containing an ANGPTL4 recombinant protein, which can be rANGPTL4. The amino acid sequence of the rANGPTL4 is UniProtKB: Q9Z1P8 (K167-S41), which is SEQ ID No: 3 in the sequence listing.

[0027] In a specific embodiment, the expression level of ANGPTL4 can also be regulated by a small molecule inhibitor, agonist or PRMT5 recombinant protein that intervenes in the expression and function of the PRMT5 gene or PRMT5 protein.

[0028] In a specific embodiment, the expression level of ANGPTL4 can be significantly increased by knocking out the PRMT5 protein coding gene in brain vascular endothelial cells.

[0029] The amino acid sequence of the PRMT5 protein is UniProtKB: Q8CIG8, updated on January 23, 2007. The coding gene of the PRMT5 protein is Genbank: NM_001313906.1, updated on November 14, 2025.

[0030] The present application also provides the use of ANGPTL4 in the preparation of a product; the function of the product is A1) or A2) or A3) or A4) or A5); A1) promoting cerebellar ataxia; A2) promoting Purkinje cell dysfunction; A3) promoting inhibitory synapse formation; A4) studying diseases related to cerebellar ataxia; A5) preparing an animal model with a cerebellar ataxia phenotype.

[0031] The above-mentioned use or method is a non-disease diagnosis use or method. The above-mentioned use or method is not the direct purpose of obtaining the disease diagnosis result or health status of a living human or animal body.

[0032] The above-mentioned use or method is a non-disease treatment purpose use or method. The above-mentioned use or method is not for the purpose of restoring or obtaining health or reducing pain of a living human or animal body.

[0033] This study found that upregulation of ANGPTL4 expression in brain vascular endothelial cells can promote inhibitory synapse formation and significantly impair cerebellar Purkinje cell function. In a cerebellar ataxia mouse model with PRMT5 deletion in brain vascular endothelial cells, ANGPTL4 expression was significantly increased, leading to an increase in inhibitory synapses and inducing motor coordination disorders.

[0034] The present application provides a treatment method for affecting synapse generation of nervous system by regulating the expression of ANGPTL4, which can effectively treat nervous system diseases caused by abnormal synapse generation, especially movement disorders and neurodegenerative diseases, and provides a new idea and strategy for the treatment of related diseases. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 Analysis of motor defects in Prmt5 knockout mice. Among them, A. Foot grip reflex experiment shows that Prmt5 knockout mice (Prmt5 fl / fl ) have obvious motor defects compared with control mice (Prmt5 fl / + ) at P60; B. Footprint analysis shows that the stride of Prmt5 knockout mice is shortened; C. Rotarod test shows that the fall latency of Prmt5 knockout mice is significantly shortened; D. In the balance beam test, the time for Prmt5 knockout mice to pass through the balance beam is significantly longer than that of the control group.

[0036] Figure 2 Increase of inhibitory synapses from interneurons to Purkinje cells in Prmt5 knockout mice. Among them, A. Immunofluorescence labeling of cerebellar molecular layer of Prmt5 knockout mice and control mice shows that the VGAT (inhibitory synapse marker) positive points on Purkinje cells are significantly increased; B. Quantitative analysis results show that the density of VGAT+ positive points on Purkinje cell dendrites of Prmt5 knockout mice is significantly increased; C. Quantitative analysis results show that the density of VGAT+ positive points around Purkinje cell bodies of Prmt5 knockout mice is higher than that of the control group. D-E. There is no significant difference in VGLUT1 marker of parallel fiber excitatory synapses between Prmt5 knockout mice and control mice. F-H. There is no significant difference in VGLUT2 marker of climbing fiber excitatory synapses between Prmt5 knockout mice and control mice.

[0037] Figure 3 Changes in Purkinje cell spontaneous firing activity in Prmt5 knockout mice. Among them, A. Typical waveform diagram of Prmt5 knockout mice and control mice Purkinje cell sIPSC (spontaneous inhibitory postsynaptic current); B. The frequency of sIPSC of Prmt5 knockout mice Purkinje cells is significantly increased; C. The amplitude of sIPSC of Prmt5 knockout mice is not significantly changed; D-E. Electrophysiological recording shows that the spontaneous action potential (sAP) firing rate of Prmt5 knockout mice Purkinje cells is significantly lower than that of the control mice; F. Statistical analysis shows that the coefficient of variation (CV) of action potential firing interval of Prmt5 knockout mice Purkinje cells is significantly increased.

[0038] Figure 4 Upregulation of Angptl4 in Prmt5 knockout mice. Among them A-E. Single cell RNA sequencing analysis showed that the mRNA level of Angptl4 in cerebellar endothelial cells of Prmt5 knockout mice was significantly upregulated; F-G. qPCR and western blot proved that the expression of ANGPTL4 in brain endothelial cells of Prmt5 knockout mice was increased; H. Elisa detection proved that the secretion of ANGPTL4 in brain endothelial cells of Prmt5 knockout mice was increased.

[0039] Figure 5 Recombinant ANGPTL4 protein promotes inhibitory synapse formation. Among them A. After co-culturing siPrmt5 transfected bEnd.3 cells with primary neurons, the density of VGAT+ positive points on the neuron dendrites was significantly increased; B. Data quantification results showed that the number of VGAT+ synapses in siPrmt5 transfected group was significantly higher than that in control group; C. After rANGPTL4 protein treatment, the density of VGAT+ positive points on the neuron dendrites was significantly increased; D. rANGPTL4 treatment led to upregulation of VGAT protein expression, which showed a dose-dependent change.

[0040] Figure 6 Construction of Angptl4 knockout mice of brain vascular endothelial cells. Among them A. Construction of Angptl1 flox / flox mouse strategy schematic diagram; B. PCR and electrophoresis detection of Loxp1 and Loxp2 site insertion; C. qPCR detection of P20 days Prmt5 fl / + ;Angptl4 fl / + and Prmt5 fl / + ;Angptl4 fl / fl expression level in primary brain endothelial cells of mice.

[0041] Figure 7 Knockout of Angptl4 restores inhibitory synaptic input of Purkinje cells and restores excitability of Purkinje cells. Among them A-C. Angptl4 gene knockout restored the density of VGAT+ positive points on the dendrites and cell bodies of Purkinje cells in Prmt5 knockout mice; D-F. The frequency of sIPSC returned to a similar level as that of control mice. Figure 8To improve the motor function of Prmt5 knockout mice by knocking out Angptl4. Among them, A. Footprint analysis showed that after deleting Angptl4, the stride of double knockout mice was restored, close to the level of the control group; B. In the rotarod test, after deleting Angptl4, the double knockout mice significantly improved the latency to fall; C. In the balance beam test, the performance of double knockout mice was significantly better than that of Prmt5 knockout mice after deleting Angptl4. DETAILED DESCRIPTION

[0042] The application will be further described in detail below in conjunction with the specific embodiments. The examples given are only to illustrate the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the application.

[0043] The experimental methods in the following examples are all routine methods, unless otherwise specified, according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.

[0044] Unless otherwise specified, the quantitative tests in the following examples are all set up with three repeated experiments, and the results are averaged.

[0045] Prmt5 in the following examples flox / flox The mice have been described in: Li Z, Xu J, Song Y, Xin C, Liu L, Hou N, Teng Y, Cheng X, Wang T, Yu Z, Song J, Zhang Y, Wang J, Yang X. PRMT5 Prevents Dilated Cardiomyopathy via Suppression of Protein O-GlcNAcylation. Circ Res. 2021 Oct 15;129(9):857-871. doi: 10.1161 / CIRCRESAHA.121.319456. Epub 2021 Sep 10. PMID: 34503365. The biological material can be obtained from the applicant, which is only used for repeating the experiments of the application and cannot be used for other purposes. Prmt5 flox / flox The mouse shows that under the action of Cre recombinase, the Prmt5 gene sequence between the two loxp sequences will be deleted.

[0046] The SP-A-Cre transgenic mice used in the following examples have been described in: Meng F, Shi L, Cheng X, Hou N, Wang Y, Teng Y, Meng A, Yang X. Surfactant protein A promoter directs the expression of Cre recombinase in brain microvascular endothelial cells of transgenic mice. Matrix Biol. 2007 Jan;26(1):54-7. doi: 10.1016 / j.matbio.2006.09.003. Epub 2006 Sep 16. PMID: 17059883. The biological material is available from the applicant for the sole purpose of repeating the experiments of the present application and cannot be used for other purposes. The SP-A-Cre transgenic mice show: Cre activity is detectable in brain microvascular endothelial cells, lung alveolar type II cells and gastric gland epithelial cells.

[0047] The following examples use statistical software to process data, and the experimental results are expressed as mean ± standard deviation. Student's t test is used for comparison between groups, and the significance determination criteria are: P < 0.05 (significant), P < 0.01 (very significant), P < 0.001 (highly significant), and P < 0.0001 (extremely highly significant).

[0048] Example 1, Construction of a brain microvascular endothelial cell Prmt5 knockout mouse model By mating Prmt5 flox / flox mice with SP-A-Cre transgenic mice to generate SP-A-Cre;Prmt5 flox / flox mutant mice and SP-A-Cre;Prmt5 fl / fl control mice. flox / + fl / + Example 2, Behavioral study of Prmt5

[0049] 1, Behavioral study of mice Prmt5 fl / fl mice showed gradually worsening motor defects, manifested as a shaking posture and circular walking pattern at P60, and eventually at 5 months of age, they were unable to maintain a straight posture with their hind limbs.

[0050] Tail suspension experiments showed that Prmt5 fl / fl mice exhibited limb grasping reflexes at P60 (60 days after birth) Figure 1 ​A), which is a typical symptom of neurodegenerative defects.

[0051] To quantify the above-mentioned movement defects, male Prmt5 fl / fl The mice and control mice were subjected to the following behavioral tests.

[0052] A. Mouse Rotarod Test 1) Before the formal test, the mice were adapted to the 4 r / min slow speed for 3 min.

[0053] 2) During the test, the speed of the rotating rod was set to accelerate from 4 r / min to 40 r / min within 5 min, and the time for the mouse to fall off the rod was recorded.

[0054] 3) If a mouse holds the rotating rod and rotates it completely for 3 turns, it is considered to have fallen off. Each mouse was tested 4 times a day, with an interval of 30 min between each test. Before each test, the mice were adapted to the slow speed.

[0055] B. Mouse Gait Analysis 1) The forepaws of the mice were dyed with red ink, and the hindpaws were dyed with green ink.

[0056] 2) The grid was placed on A1 paper to form a narrow runway, and a dark box was placed at one end. The mice were placed in the dark box for 20 min before the experiment to familiarize themselves with the environment and develop dependence.

[0057] 3) During the test, the mice were placed at the starting point of the runway on the opposite side of the dark box, and they ran in the narrow tunnel covered with white paper.

[0058] 4) During recording, the distance between the consecutive initial contact points of the same hindpaws or forepaws was measured and recorded as the step length for statistical analysis.

[0059] C. Mouse Balance Beam Test 1) The balance beam was placed on a 50 cm high platform, inclined at 30° compared to the ground, and a dark box was placed on the higher side. The mice were placed in the dark box for 20 min before the experiment to familiarize themselves with the environment.

[0060] 2) Before recording, the mice were trained to climb from the low platform to the high platform dark box along the balance beam. Each mouse was trained twice.

[0061] 3) During the formal experiment, the mice were tested 3 times on a balance beam with a diameter of 11 mm, and the time required for the mice to cross the 75 cm balance beam was recorded.

[0062] The results of the behavioral tests are as follows: 1) Footprint analysis showed that Prmt5 fl / fl The step length of the mice was significantly shorter than that of the control group ( Figure 1 B), which is consistent with the ataxic movement pattern.

[0063] 2) In the rotarod test, Prmt5 fl / fl The fall latency of mice was significantly shortened ( Figure 1 C).

[0064] 3) In the balance beam test, Prmt5 fl / fl Mice took longer time to pass through the 11 mm wide balance beam ( Figure 1 D).

[0065] In summary, the deletion of Prmt5 gene in brain endothelial cells leads to progressive motor defects in mice.

[0066] 2, Prmt5 fl / fl Changes in inhibitory synapses on Purkinje cells in mice Cerebellum-mediated fine motor control relies on the integration of excitatory and inhibitory synaptic inputs by Purkinje cells. To explore the effects of Prmt5 deletion on synaptic inputs, inhibitory and excitatory synapses on Purkinje cells of P60 Prmt5 fl / fl and control mice were analyzed. The specific analysis steps are as follows: A, Prmt5 fl / fl Preparation of paraffin sections of Prmt5 1. Sample collection 1) The mouse was deeply anesthetized with anesthetic and the limbs were fixed on the dissection table.

[0067] 2) The mouse chest was opened using ophthalmic scissors.

[0068] 3) The perfusion needle was inserted into the left ventricle of the mouse, and the right auricle was cut.

[0069] 4) Start the perfusion pump and perfuse the whole body with 30 mL of normal saline.

[0070] 5) Replace the perfusion fluid with paraformaldehyde and perfuse the whole body with 15 mL.

[0071] 6) Take out the brain and fix it in paraformaldehyde at 4°C for 12 h.

[0072] 2. Tissue waxing and sectioning 1) Take out the fixed brain tissue and dehydrate it in the following order: 70% ethanol overnight; 80% ethanol for 2 h; 90% ethanol for 2 h; 95% ethanol for 2 h; anhydrous ethanol for 1 h; anhydrous ethanol for 1 h; xylene for 30 min; xylene for 30 min; xylene for 30 min.

[0073] 2) Melt the paraffin in advance, and place the dehydrated tissue in a 15 mL centrifuge tube. Pour in enough paraffin according to the size of the tissue, and place it in a 60°C incubator for 30 min. Pour out the paraffin and replace it with new paraffin. Repeat this process 3 times.

[0074] 3) Embed the paraffinized brain tissue into a suitable shape using a tissue embedding machine, and cool it at room temperature for more than 6 hours.

[0075] 4) Use a paraffin microtome to slice the brain tissue in the sagittal plane, with a thickness of 6 μm.

[0076] 5) Place the slices in a 40°C water bath and attach them to the glass slide with adhesive, and then wax them at 55°C to make them firmly adhere. Then proceed with the subsequent immunofluorescence experiment.

[0077] B, Prmt5 fl / fl and control mouse paraffin sections Select Prmt5 fl / fl and control mouse cerebellar vermis paraffin sections, and rehydrate them in the following order: xylene for 10 min; xylene for 20 min; xylene for 20 min; anhydrous ethanol for 15 min; anhydrous ethanol for 15 min; 95% ethanol for 5 min; 90% ethanol for 5 min; 80% ethanol for 5 min; and 70% ethanol for 5 min.

[0078] C, paraffin section antigen repair 1) Microwave the rehydrated paraffin sections for antigen repair. Place the paraffin sections in a citrate buffer and heat them in a microwave oven at high heat until boiling. Keep them boiling for 15 seconds, and then remove them and let them cool to room temperature.

[0079] D, staining of paraffin sections after repair 1) Add a peroxidase inhibitor to the tissue and incubate it at room temperature for 15 min to remove peroxidase.

[0080] 2) Add blocking serum and incubate it at room temperature for 30 min to block non-specific binding sites.

[0081] 3) Add the primary antibody (VGAT / VGLUT2 / VGLUT1) and incubate it at 37°C for 2 hours.

[0082] 4) Wash it with PBS 3 times, each time for 5 min.

[0083] 5) Add the secondary antibody corresponding to the species of origin of the primary antibody and incubate it at room temperature for 30 min.

[0084] 6) Prepare the color developing solution with a specific wavelength at a ratio of 1:100.

[0085] 7) Incubate the color developing solution for 20 seconds.

[0086] 8) The binding of the antigen to the primary antibody is disrupted using the elution buffer, the fluorescence signal is preserved due to the binding to the tyrosine sites in the vicinity of the antigen, the next isotype antibody can be incubated.

[0087] 9) The primary antibody (Calbindin) is added dropwise and incubated at 37°C for 2 h.

[0088] 10) Washed 3 times with PBS for 5 min each time.

[0089] 11) The secondary antibody corresponding to the isotype of the primary antibody is added dropwise and incubated at room temperature for 30 min.

[0090] 12) The chromogenic solution of a specific wavelength is prepared at a ratio of 1:100.

[0091] 13) Incubate the chromogenic solution for 10 s.

[0092] 14) DAPI is used to stain the nucleus.

[0093] 15) Mount and observe.

[0094] E. Confocal imaging 1) Confocal imaging is performed at room temperature using a Zeiss LSM 880 laser scanning confocal microscope.

[0095] 2) The following objectives are used for imaging: EC Plan-Neofluar lOx / 0.30 M27, EC Plan-Neofluar 20x / 0.50 M27, EC Plan-Neofluar 40x / 0.75 M27 and Plan-Apochromat 63x / 1.4 Oil DIC M27.

[0096] 3) For colocalization analysis of cellular markers, three-dimensional Z-stack images are acquired. Z-axis alignment is processed using ZEN software (Carl Zeiss).

[0097] 4) For imaging the whole cerebellum, tiled scanning is performed at 20x magnification with 10% spatial overlap between adjacent tile areas. Tiled images are automatically stitched and assembled using ZEN software.

[0098] The results are as follows: In Prmt5 fl / fl mice, the number of VGAT (marker of inhibitory synapses) positive puncta significantly increased in the molecular layer and cell bodies around Purkinje cells, indicating an increase in inhibitory synaptic inputs from molecular layer interneurons (MLIs) Figure 2 A-C); in contrast, excitatory synaptic markers such as VGLUT1 (marker of parallel fiber terminals) and VGLUT2 (marker of climbing fiber terminals) decreased in Prmt5fl / fl Molecular layer of mice and control mice exhibited similar spatial spread and quantitative density of Prmt5 Figure 2 D-H). This selective enhancement of MLI-Purkinje cell inhibitory synaptic connectivity provides structural evidence for a functional imbalance of Purkinje cell activity, which can be a consequence of Prmt5 fl / fl The origin of motor coordination defects in mice.

[0099] 3、Prmt5 fl / fl Effects of Prmt5 on Purkinje cell activity in mice To further investigate the functional consequences of increased inhibitory input, Prmt5 fl / fl Purkinje cell spontaneous inhibitory postsynaptic currents (sIPSCs) and Purkinje cell spontaneous action potentials (sAPs) were recorded from mice and control mice. The detailed analysis steps are as follows: A, Preparation of cerebellar slices 1) Animals were rapidly decapitated after anesthesia with 5% (v / v) isoflurane inhalation. The whole cerebellum was removed and sectioned (300 μm).

[0100] 2) The brain slices were incubated in oxygenated artificial cerebrospinal fluid (aCSF, containing 5 mM KCl, 124 mM NaCl, 1.25 mM NaH2PO4, 10 mM glucose, 26 mM NaHCO3, 2.4 mM CaCl2and 1.2 mM MgCl2, pH 7.3-7.5, osmolarity 292-320 mOsm) continuously perfused with 95% O2and 5% CO2.

[0101] 3) All recordings were obtained from Purkinje cell bodies in the cerebellar ependymal slices. Purkinje cells were visualized using a standard upright microscope.

[0102] B, Whole-cell spontaneous inhibitory postsynaptic current (sIPSC) recording 1) The electrodes were filled with internal solution containing 140 mM KCl, 10 mM HEPES, 4 mM MgCl2, 0.1 mM EGTA, 2 mM Na2ATP and 0.3 mM Na2GTP.

[0103] 2) 10 μM NBQX was added to the aCSF to block AMPA receptor-mediated currents.

[0104] 3) After a stable gigaseal was formed between the electrode and the Purkinje cell membrane, negative pressure was applied to break the membrane and establish a whole-cell configuration (access resistance <20 MΩ, leak current <-30 pA).

[0105] 4) After the membrane stabilizes, clamp it at -70 mV for recording.

[0106] C. Cell attachment spontaneous action potential (sAP) recording 1) Apply negative pressure to gradually increase the resistance between the electrode and the cell membrane to 200 to 300 MΩ.

[0107] 2) After a brief period of stabilization, cell attachment recording begins.

[0108] D. Data Acquisition and Analysis Electrophysiological data were acquired using Clampex 10.7 software and analyzed using Clampfit 10.7 software.

[0109] The results are as follows: 1) Prmt5 fl / fl Purkinje cells from mice showed a higher frequency of sIPSCs, while the amplitude of sIPSCs remained unchanged. Figure 3 (AC) 2) Spontaneous action potential (sAP) firing in Purkinje cells was recorded using a cell patch clamp. The results showed that Prmt5... fl / fl The spontaneous firing rate of Purkinje cells in mice was significantly lower than that in the control group. Figure 3 (D and E in the middle).

[0110] 3) The coefficient of variation (CV) of spontaneous firing interval in Purkinje cells at Prmt5 fl / fl The significant increase in spontaneous discharge activity in mice indicates that the regularity of spontaneous discharge activity is disrupted. Figure 3 (Middle F).

[0111] The data above indicate that the absence of Prmt5 in endothelial cells disrupts the excitation / inhibition (E / I) balance of the cerebellar cortex neural circuits and impairs the firing activity of Purkinje cells, which may be the cause of the motor deficits in mice.

[0112] 4. Single-cell RNA sequencing of mouse cerebellar endothelial cells For Prmt5 20 days after birth fl / fl Single-cell RNA sequencing (scRNA-seq) was performed on cerebellar endothelial cells of control mice.

[0113] The specific steps are as follows: I. FACS sorting of endothelial cells 1) From P20's Prmt5 fl / fl ;ROSA26 LSL-tdTomato Mice and Prmt5 fl / + ;ROSA26 LSL-tdTomato Cells were obtained from the cerebellum of littermates.

[0114] 2) Stained with APC anti-mouse CD31 (eBiosciences, 17-0311-82) and PE anti-mouse CD45 (eBiosciences, 12-0453-82) for 1 hour at 4°C.

[0115] 3) Cell viability was assessed with 7-AAD (eBiosciences, 00-6993-50) to exclude dead cells.

[0116] 4) Live ECs (RFP + CD31 + CD45 - 7-AAD - ).

[0117] II. Single-cell RNA sequencing processing 1) Freshly sorted cells were immediately processed with droplet-based single-cell RNA sequencing (10x Genomics Chromium platform).

[0118] 2) Single-cell libraries were prepared with Chromium Single Cell 3' Reagent Kits v3 (10x Genomics) following the manufacturer's protocol: FACS-sorted cells were washed three times with Dulbecco's Phosphate-Buffered Saline (DPBS) containing 0.04% BSA, resuspended at 700-1200 cells per pL with cell viability > 85%. Cell encapsulation was performed with Chromium Controller (10x Genomics), monitoring cell capture efficiency in real time, and reverse transcription to generate unique barcoded cDNA within droplets. After reverse transcription, emulsion was broken, and barcoded cDNA was purified with Dynabeads. Purified cDNA was amplified by PCR. For 3' gene expression library construction, 50 ng of amplified cDNA was fragmented, end-repaired, and double-size selected with SPRIselect. Final libraries were sequenced on the Illumina NovaSeq X platform, generating 150 bp paired-end reads.

[0119] III. 10x Genomic data processing 1) Raw reads were demultiplexed and mapped to the reference genome with 10X Genomics Cell Ranger (v1.1.0) with default parameters.

[0120] 2) Cells were strictly filtered, retaining cells meeting the following criteria: Cell-level filtering (300-5000 genes detected; total UMI count >1000; exclude top 3% high UMI outliers). Hemoglobin gene filtering (Hb[ ] <0.5). Mitochondrial content threshold (MT[ ] <10).

[0121] 3) Data normalization and clustering were performed in Seurat (v5.2.1) with SCTransform normalization: 2000 highly variable genes (HVGs) were selected based on the graph-based clustering, principal component analysis (PCA) was performed on the scaled data. SNN graph was constructed with the top 30 principal components, and clustering was performed with the Leiden algorithm (resolution=0.5). Cell population clusters were annotated to known cell types by assessing the expression of specific marker genes determined in previous literature.

[0122] 4) Differential expression analysis was performed with Wilcoxon rank-sum test, only genes with FDR <0.05 and |log2FC| >0.5 were considered as differentially expressed genes (DEGs).

[0123] 5) Functional enrichment analysis of DEGs was performed with ClusterProfiler (v4.14.6), biological process terms were from Gene Ontology (GO) database.

[0124] After quality control, 24,209 Prmt5 fl / fl cells and 31,911 control cells were subjected to uniform manifold approximation and projection (UMAP) analysis, which resulted in 20 different cell clusters, including 11 endothelial cell subtypes and 9 non-endothelial cell populations.

[0125] Based on known endothelial cell markers Sema3g, Vegfc, Fbln2, Tfrc, Slclal, Meoxl, Hs3stl, Flrt2, Slc38a5, these endothelial cell clusters were annotated as Arterial 0 (A0), Arterial 1 (Al), Capillary 0 (C0), Capillary 1 (Cl), Capillary 2 (C2), Capillary 3 (C3), Capillary 4 (C4), Venous (V), Mitotic Endothelial (M), Capillary / Pericyte (P / C), and Capillary / Astrocyte (Ast / C) (Fig. 2A and B). Figure 4 The P / C cluster showed features of both capillary endothelial cells and pericytes, while the Ast / C cluster represented a mixed population containing capillary endothelial cells and astrocytes (Fig. 2A and B). Figure 4 The non-endothelial cell clusters included pericytes, fibroblasts, smooth muscle cells, microglia, monocytes, ependymal cells, marginal zone macrophages, neurons, and an undefined population (Fig. 2A and B). Figure 4 The non-endothelial cell clusters included pericytes, fibroblasts, smooth muscle cells, microglia, monocytes, ependymal cells, marginal zone macrophages, neurons, and an undefined population (Fig. 2A and B).

[0126] To assess the impact of Prmt5 gene deletion, differentially expressed genes (DEGs) between mouse endothelial cell clusters were examined using thresholds of |log2FC| > 0.5 and adjusted p-value < 0.05.

[0127] Results show that collagen-containing extracellular matrix (ECM) components secreted by endothelium were significantly dysregulated in A0, A1, C1, C2 and V subgroups Figure 4 C. Given the critical roles of ECM secretion and assembly in structural scaffolding and microenvironment regulation, as well as intercellular signal communication, it is hypothesized that PRMT5-mediated changes in endothelial secreted ECM can affect innervation of neurons. Among the top 5 DEGs with the most significant changes in each subgroup by differential analysis of genes encoding ECM components, Angptl4 was significantly elevated in each subgroup Figure 4 D and E). ANGPTL4 is a multifunctional secreted protein with important effects on lipid metabolism, tumorigenesis, angiogenesis, and vascular permeability of endothelial cells, but its potential role in neurodegenerative diseases is not well understood.

[0128] The expression changes of ANGPTL4 were verified by qPCR, Western blot and ELISA analysis of brain endothelium of Prmt5 fl / fl and control mice. The specific analysis steps are as follows: A. Isolation of mouse primary brain vascular endothelial cells 1) The mouse was decapitated and the brain tissue was quickly isolated and surface residual blood was removed by continuous shaking in pre-cooled PBS.

[0129] 2) The brain tissue was crushed using sharp forceps and placed in 37°C digestion for 50 min, with 5 pipette blows every 10 min.

[0130] 3) After the digestion was liquid, it was collected in a centrifuge tube and centrifuged at 4°C, 310 g for 7 min, and the supernatant was discarded.

[0131] 4) The pellet was resuspended with 20% BSA and filtered to a new tube using a 100 μm filter.

[0132] 5) Centrifugation at 4°C, 310 g for 5 min.

[0133] 6) After centrifugation, the bottom of the tube was the cell population containing ECs, while the supernatant was a milky fat layer. The pellet was retained and all the supernatant was transferred to a new tube and centrifuged at 4°C, 350 g for 5 min, and the cells were collected 3 times. 7) Collect all cell pellets into one centrifuge tube, add red blood cell lysing solution, incubate on ice for 5-10 min, then centrifuge at 1000 r / min for 10 min at 4°C.

[0134] 8) Discard the supernatant, resuspend the cells with 1 mL 0.1% BSA per tube, then collect the cells with 1 mL and pass through a 70 μm filter.

[0135] 9) Centrifuge at 1200 rpm for 4 min.

[0136] 10) Resuspend with 500 uL 0.1% BSA.

[0137] 11) Add 5-10 uL Bio-CD31 antibody, gently pipette 10-15 times, rotate and incubate at 4°C for 30 min.

[0138] 12) Centrifuge at 1200 rpm for 4 min, and wash twice with 0.1% BSA.

[0139] 13) Discard the supernatant, add magnetic beads according to the volume ratio of resuspension liquid: avidin magnetic beads = 4:1, 20 uL magnetic beads for a maximum of 10 7 cells.

[0140] 14) Wrap with tin foil, rotate and incubate for 30 min, filter with a 40 μm filter before placing on a magnetic stand.

[0141] 15) Add 1 mL 0.1% BSA, centrifuge at 1200 rpm for 4 min, and resuspend with 1 mL 0.1% BSA.

[0142] 16) Place the MS column on a magnetic stand, add 1-2 mL magnetic bead buffer per tube, and wait for the magnetic bead buffer to flow down, then add the cell suspension, and when the magnetic column does not flow, add 1 mL magnetic column buffer to the MS column, and push it out with a plunger. The resulting product is the endothelial cells.

[0143] B. Extraction of cell RNA 1) Discard the cell culture medium, wash once with PBS, and discard the PBS.

[0144] 2) Add Trizol according to the cell amount (1 mL Trizol for every 1 x 10 7 cells), gently pipette the cells to dissolve them into the Trizol, and transfer into a 1.5 mL EP tube.

[0145] 3) Add 1 / 5 volume of chloroform to the Trizol, use a vortex shaker to shake thoroughly, and stand at room temperature for 3 min.

[0146] 4) 13000 r / min centrifugation for 15 min.

[0147] 5) The water sample supernatant was sucked into a new EP tube, and the protein precipitate on the tube wall was not touched.

[0148] 6) 500 μL of isopropyl alcohol was added, and after gentle mixing, it was incubated at room temperature for 10 min.

[0149] 7) Put into the 4℃ centrifuge, 13000 r / min centrifugation for 10 min.

[0150] 8) The supernatant was sucked off, and the RNA was precipitated at the bottom of the tube. 1 mL of 75% ethanol was added to wash off the organic matter.

[0151] 9) Put into the 4℃ centrifuge, 13000 r / min centrifugation for 2 min.

[0152] 10) Suck off the ethanol, dry at room temperature for 5-10 min, and add 30 μL of RNase-free H2O to dissolve the RNA.

[0153] C, RT-qPCR 1) The RNA sample was quantified using a NanoDrop spectrophotometer.

[0154] 2) The reverse transcription system was prepared in a 0.2 mL tube according to the following proportion, 2000 ng of RNA per 10 μL of system reverse transcription. RNA, 2000 ng; 5×RT Master Mix, 2 μL; RNase-free H2O, the system was supplemented to 10 μL.

[0155] 3) The prepared sample was reverse transcribed according to the following program to obtain cDNA. 37℃, 15 min, 1 cycle; 50℃, 5 min, 1 cycle; 98℃, 5 min, 1 cycle; 4℃, 30 min, 1 cycle.

[0156] 4) The qPCR reaction system was prepared according to the following system: cDNA template, 1 μL; ddH2O, 3.8 uL; qPCR MasterMix, 5 μL; upstream and downstream mixed primers, 0.2 μL.

[0157] 5) Start qPCR according to the following procedure: pre-denaturation, 1 cycle (95 °C, 10 min); denaturation, annealing and extension, 40 cycles (95 °C, 15 s; 1.6 °C / s, 21.8 s; 60 °C, 34 s); re-annealing, 1 cycle (1.6 °C / s, 21.8 s); annealing, 1 cycle (1.6 °C / s, 21.8 s); extension, 1 cycle (60 °C, 1 min); re-annealing (1.5 °C / s, 23.3 s).

[0158] 6) Analyze qPCR data.

[0159] D. Western Blot I. Isolation and quantification of cellular proteins 1) Add appropriate amount of RIPA lysis buffer to the cells and break them using a tissue homogenizer.

[0160] 2) Place the RIPA sample of the above cells on ice for 10 min lysis, and then break them using ultrasonic, with a program of 60% power ultrasonic for 6 s and interval for 9 s, for a total of 3.5 min, with low temperature throughout.

[0161] 3) Centrifuge the protein sample at 4 °C, 13000 g for 15 min, collect the supernatant into a new tube.

[0162] 4) Quantify using a protein quantification kit and a microplate reader.

[0163] 5) After quantification, dilute the sample to no less than 2 μg / μL using RIPA and 5x Loading buffer, and incubate in boiling water for 4 min to denature it.

[0164] II. Electrophoresis 1) Load 20 μg of the quantified protein into each lane, and electrophorese using Running Buffer at 25 mA for 45 min.

[0165] 2) Transfer the SDS-PAGE into PVDF membrane in the environment of Transferring Buffer, with a condition of 100 V for 1.5 h.

[0166] III. Protein hybridization and color development 1) According to the molecular weight of the target protein, cut the PVDF membrane to an appropriate size, and block using 3% milk powder for 1 h.

[0167] 2) Incubate the PVDF membrane with an appropriate amount of primary antibody at 4 °C for 12 h.

[0168] 3) Recover the primary antibody, and rinse the PVDF membrane with PBST for 3 times, each for 5 min.

[0169] 4) Based on the species origin of the primary antibody, add the secondary antibody diluted 1:1000 and incubate at room temperature for 1 h.

[0170] 5) Discard the secondary antibody and wash the PVDF membrane three times with PBST, 5 min each time.

[0171] 6) Develop the bands using a colorimetric reagent and take pictures using a Western blotting imager.

[0172] E. ELISA detection of ANGPTL4 secretion 1) The isolated endothelial cells were cultured in EGM-2 medium, and the medium was changed every three days.

[0173] 2) Collect the culture medium supernatant from day 4 to day 6.

[0174] 3) Perform analysis using the mouse angiopoietin-like protein 4 (ANGPTL4) ELISA kit according to the instructions.

[0175] The above data suggest that ANGPTL4 may act as a key mediator of PRMT5 in regulating endothelial dysfunction. fl / fl The mRNA and protein levels of Angptl4 in mouse brain endothelial cells were significantly upregulated. Figure 4 (F and G), ELISA detection also confirmed the presence of the virus in cultured primary Prmt5. fl / fl The secretion of ANGPTL4 is increased in the culture supernatant of brain endothelial cells. Figure 4 (H).

[0176] Example 2: Study on the effect of secreted factors from Prmt5-deficient endothelial cells on inhibitory synapse formation. The design of siPrmt5 is achieved by annealing the following two chains: Forward primer: 5'-GCACAGUUUGAGAUGCCUU-3' (SEQ ID No:4); Reverse primer: 5'-AAGGCAUCUCAAACUGUGC-3' (SEQ ID No:5).

[0177] Compared to siNC, siNC is formed by annealing the following two chains: Forward primer: 5'-UUCUCCGAACGUGUCACGUTT-3'; Reverse primer: 5'-AACGUGACACGUUCGGAGAA-3'.

[0178] Short interfering RNA (siRNA) against mouse Prmt5 gene was synthesized by GenScript (Beijing). siPrmt5 or negative control siNC was transfected into cells using jetPRIME according to the manufacturer's instructions. Cells were harvested 48 hours after transfection for further analysis.

[0179] bEnd.3 cells (ATCC, CRL-2299) with siPrmt5 or siNC were co-cultured with primary neurons isolated from E13.5 mouse embryos. The detailed steps are as follows: A. Isolation of E13.5 mouse embryonic primary neurons 1) Poly-lysine was added to the culture plate in advance and placed at 4°C for more than 6 h.

[0180] 2) The cortex of 13.5-day-old C57 / BL6 mouse embryos was isolated and the meninges were stripped.

[0181] 3) The treated cortex was added to 1 mL Accutase and digested for 4-5 min, shaking 10 times every 2 min, to prepare a single cell suspension.

[0182] 4) After the digestion was liquid, 2 volumes of DMEM complete medium were added to terminate the digestion, and filtered to a new centrifuge tube using a 100 μm filter.

[0183] 5) Centrifugation at 300 g for 5 min at 4°C.

[0184] B. Culture of primary neurons 1) Poly-lysine in the culture plate was aspirated and washed 4 times with PBS.

[0185] 2) The isolated neurons were added to DMEM complete medium and placed in a 37°C incubator.

[0186] 3) After 12 h of culture, the culture supernatant was aspirated.

[0187] 4) Neuronal medium (50 mL Neurobasal added with 1x B27-Neu, 500 μL GluMax, 1% penicillin-streptomycin) was added for culture.

[0188] C. Co-culture experiment of neurons and endothelial cells Two days before co-culture, bEnd.3 were transfected with small interfering RNA and cultured in Neurobasal medium containing 10% FBS. Neurons were isolated before co-culture and seeded in 24-well plates after digestion. On the day of co-culture, bEnd.3 were digested and plated in Transwell chambers 4 h in advance, and then co-cultured with neurons in the dish, with Neurobasal medium containing 1% penicillin-streptomycin, 1x B27-Neu and 1% GluMax. Half of the medium was changed every 3 days, and subsequent detection was performed 96 h after co-culture.

[0189] D. Immunofluorescence staining of cell climbing sheets 1) Discard the culture medium, slowly add room temperature TBS on the cells, and wash twice, each time for 5 seconds.

[0190] 2) Cell fixation: add 4% neutral formaldehyde fixing solution (prepared in TBS buffer) to fix the cells at room temperature for 15 min.

[0191] 3) Absorb the fixing solution, and use 4°C pre-cooled TBS buffer to rinse 3 times, each time for 5 min.

[0192] 4) Use membrane permeation solution to treat the climbing sheet at room temperature for 20 min.

[0193] 5) Add blocking serum dropwise, and incubate at room temperature for 30 min to block non-specific binding sites.

[0194] 6) Add the first antibody dropwise, and incubate at 4°C for 6 h.

[0195] 4) Wash with PBS 3 times, each time for 5 min.

[0196] 5) Add fluorescent secondary antibody corresponding to the species origin of the first antibody dropwise, and incubate at room temperature for 1 h.

[0197] 6) Perform DAPI nuclear staining according to requirements.

[0198] 7) Mount the sheet and observe.

[0199] Quantitative immunofluorescence analysis showed that the density of VGAT+ positive points on the neuron dendrites of the siPrmt5 co-culture group was significantly increased compared with the siNC control group (P < 0.05, t-test) Figure 5 A and B), indicating that Prmt5-deficient endothelial cells promote the formation of inhibitory synapses.

[0200] Example 3, Role of Angptl4 in the formation of inhibitory synapses To further verify the effect of upregulating ANGPTL4 on inhibitory synapse formation, primary neurons isolated from E13.5 mouse embryos were treated with recombinant ANGPTL4 protein (rANGPTL4). Subsequent immunofluorescence analysis and Western blot analysis were performed.

[0201] A, rANGPTL4 protein treatment of neurons In neurons cultured in vitro for 3 days, rANGPTL4 (MedChemExpress, Cat. No. HY-P7508) was added at final concentrations of 0 ng / ml, 25 ng / ml, 50 ng / mL, 100 ng / mL, and 200 ng / mL, respectively, and treated for 72 h. Subsequent immunofluorescence analysis and Western blot analysis were performed. The steps of immunofluorescence analysis and Western blot analysis were the same as above.

[0202] Immunofluorescence analysis showed that the density of VGAT+ positive dots on the dendrites of neurons significantly increased after rANGPTL4 treatment (Fig. 2A). Figure 5 Figs. 2C and 2D.

[0203] Western blot analysis showed that the expression of VGAT protein was upregulated in a dose-dependent manner with increasing concentrations of rANGPTL4 protein (Fig. 2E). Figure 5 Fig. 2E). These results indicate that upregulation of ANGPTL4 promotes the formation of inhibitory synapses.

[0204] Example 4, Prmt5 fl / fl Knocking out Angptl4 in mouse brain endothelium restores inhibitory synaptic inputs of Purkinje cells and rescues motor defects 1. Construction of Prmt5 and Angptl4 double knockout mice Angptl4 flox / flox Mice were generated by deleting exons 4 to 6 in the Angptl4 gene (Fig. 1A). Figure 6 The nucleotide sequence of the Angptl4 gene is Genebank: 57875, updated on July 30, 2025. The encoded amino acid sequence is the Angptl4 protein of UniProtKB: Q9Z1P8.

[0205] Angptl4 flox / flox Mice were purchased from SJAIE Biological. Loxp sequences were inserted at both ends of exons 4 to 6 of Angptl4, and PCR was used to verify the successful insertion of loxp sequences (Fig. 1B). Figure 6 The Cre recombinase can recognize the loxp sites flanking exons 4 to 6 in the Angptl4 gene and delete them.

[0206] Angptl4 flox / floxMice and SP-A-Cre;Prmt5 fl / + Mice mated and produced SP-A-Cre;Prmt5 fl / + ;Angptl4 fl / + (Prmt5) fl / + ;Angptl4 fl / + SP-A-Cre;Prmt5 fl / + ;Angptl4 fl / fl (Prmt5) fl / + ;Angptl4 fl / fl SP-A-Cre;Prmt5 fl / fl ;Angptl4 fl / + (Prmt5) fl / fl ;Angptl4 fl / + ) and SP-A-Cre;Prmt5 fl / fl ;Angptl4 fl / fl (Prmt5) fl / fl ;Angptl4 fl / fl Mice.

[0207] The mice were then subjected to qPCR studies, with the specific experimental steps being the same as step 4 in Example 1.

[0208] qPCR results showed that in Prmt5 fl / + ;Angptl4 fl / fl In mouse endothelial cells, the Angptl4 gene was effectively deleted ( Figure 6 (C)

[0209] 2. Prmt5 fl / fl ;Angptl4 fl / fl Inhibitory synapse density and electrophysiological properties of mouse Purkinje cells The experimental procedures for cerebellar immunofluorescence and cerebellar slice electrophysiological recording are the same as above.

[0210] The results showed that: Prmt5 fl / fl ;Angptl4 fl / fl The density of VGAT+ inhibitory synapses in the mouse molecular layer and around Purkinje cells was significantly lower than that in Prmt5. fl / fl ;Angptl4 fl / + mice ( Figure 7 (AC); Electrophysiological recording showed: Prmt5 fl / fl ;Angptl4 fl / fl The sIPSC frequency in mice recovered to the level of Prmt5. fl / + ;Angptl4 fl / + The control group had a similar level ( Figure 7(D and F).

[0211] 3. Prmt5 fl / fl ;Angptl4 fl / fl Motor deficit assessment in mice The experimental procedure for behavioral analysis is the same as above.

[0212] The results are as follows: Prmt5 fl / fl ;Angptl4 fl / fl Mice showed significant improvement in motor behavior. Figure 8 In footprint analysis, compared with Prmt5 fl / fl ;Angptl4 fl / + Mice compared to Prmt5 fl / fl ;Angptl4 fl / fl The mice's stride length increased significantly, but was still slightly shorter than that of Prmt5. fl / + ;Angptl4 fl / + control mice ( Figure 8 Similarly, in the tests of the rotating bar and the balance beam, Prmt5 fl / fl ;Angptl4 fl / fl Mice compared to Prmt5 fl / fl ;Angptl4 fl / + Mice exhibited better motor coordination, with longer latency on the rotarod and reduced time required to cross the balance beam. Figure 8 (B and C in the middle).

[0213] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Use of a substance that reduces the expression of the ANGPTL4 gene in the manufacture of a product for the treatment and / or prevention and / or delay of cerebellar ataxia.

2. Use of a substance that reduces the expression of the ANGPTL4 gene in the manufacture of a product for the reduction of inhibitory synapse formation.

3. Use of a substance that reduces the activity of the ANGPTL4 protein in the manufacture of a product for the treatment and / or prevention and / or delay of cerebellar ataxia.

4. Use of a substance that reduces the activity of the ANGPTL4 protein in the manufacture of a product for the reduction of inhibitory synapse formation.

5. Use of a substance that reduces the content of the ANGPTL4 protein in the manufacture of a product for the treatment and / or prevention and / or delay of cerebellar ataxia.

6. Use of a substance that reduces the content of the ANGPTL4 protein in the manufacture of a product for the reduction of inhibitory synapse formation.

7. Use according to any one of claims 1 to 6, characterized in that: Said substance that reduces the expression of the ANGPTL4 gene or said substance that reduces the activity or content of the ANGPTL4 protein is any one of the following biological materials: B1) an antibody specific for the ANGPTL4 protein; B2) an inhibitor of the activity of the ANGPTL4 protein; B3) a nucleic acid molecule that targets the ANGPTL4 gene to reduce or inhibit or knock out its expression; B4) an expression cassette containing the nucleic acid molecule of B3); B5) a recombinant vector containing the nucleic acid molecule of B3) or the expression cassette of B4); B6) a recombinant microorganism containing the nucleic acid molecule of B3) or the expression cassette of B4) or the recombinant vector of B5); B7) a transgenic animal cell line in which the nucleic acid molecule of B3) or the expression cassette of B4) is integrated into the genome; B8) a transgenic animal tissue containing the nucleic acid molecule of B3) or the expression cassette of B4); B9) a transgenic animal organ containing the nucleic acid molecule of B3) or the expression cassette of B4).

8. Use of a substance that upregulates the expression of the ANGPTL4 gene, a substance that upregulates the activity or content of the ANGPTL4 protein in the manufacture of: a1) a product for promoting cerebellar ataxia; a2) a product for promoting Purkinje cell dysfunction; a3) a product for promoting inhibitory synapse formation; a4) a product for studying a disease associated with cerebellar ataxia; a5) a product for preparing an animal model with a cerebellar ataxia phenotype.

9. Use according to claim 8, characterized in that: Said substance that upregulates the expression of the ANGPTL4 gene or said substance that upregulates the activity or content of the ANGPTL4 protein is any one of the following biological materials: C1) a recombinant ANGPTL4 protein; C2) a nucleic acid molecule encoding for overexpression of the ANGPTL4 protein; C3) an expression cassette comprising the nucleic acid molecule of C2); C4) a recombinant vector comprising the nucleic acid molecule of C2) or the expression cassette of C3); C5) a recombinant microorganism comprising the nucleic acid molecule of C2), the expression cassette of C3) or the recombinant vector of C4); C6) a transgenic animal cell line in which the nucleic acid molecule of C2) or the expression cassette of C3) is integrated into the genome. C7) a transgenic animal tissue comprising the nucleic acid molecule of C2) or the expression cassette of C3); C8) a transgenic animal organ comprising the nucleic acid molecule of C2) or the expression cassette of C3).

Images