Application of rab5 as a target in promoting central nervous axon regeneration and motor function recovery

Abstract

The application discloses a method for regulating central nervous axon regeneration and motor function recovery by using Rab5 protein, and provides a new target of Rab5 protein for promoting central nervous axon regeneration and function recovery. When the Rab5 protein in the application is overexpressed in vivo, the axon regeneration and function recovery of the central nervous can be significantly promoted, and vice versa, the axon regeneration and function recovery of the central nervous can be inhibited.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical technology, and particularly relates to a protein Rab5 and application thereof in promoting central nervous axon regeneration and motor function recovery. BACKGROUND

[0002] Axon injury of central neurons is a common disease in life, which is characterized by nerve axon rupture and is commonly seen in various accidents induced by external force, such as car accidents, falls, etc. Axon injury can lead to loss of motor function, bringing great inconvenience to the life of patients. At the current treatment level, there are few successful cases of axon repair in clinic. The main reason is that the axon of central neurons of higher animals almost cannot regenerate after injury, leading to difficulty in recovery of motor function. Therefore, exploring the molecular mechanism of regeneration of central neuron axon after injury can help us understand the principle of regeneration and promote the repair of central neuron axon injury.

[0003] The Rab family is one of the largest Ras subfamilies, which exists in all eukaryotes, and Rab5 is extremely conserved in all eukaryotes. The main role of Rab5 in vivo is to mediate the generation and transport of endosomes. However, there is no research on the relationship between Rab5 and central nervous axon regeneration and motor function recovery. SUMMARY

[0004] The problem to be solved by the present application is to provide the application of Rab5 as a target in promoting nerve axon regeneration and motor function recovery. Overexpression of Rab5 in vivo can significantly promote the regeneration of zebrafish Muller neuron axon, providing a new target for axon regeneration of central neurons.

[0005] Specifically, the present application provides the following technical solutions:

[0006] In one aspect, the present application provides the use of an agent for promoting the expression of Rab5 in the preparation of a medicament for promoting the regeneration of central nervous axon and the recovery of motor function.

[0007] In some embodiments, the agent comprises a system for overexpressing Rab5 protein.

[0008] In some embodiments, the GAL4 / UAS expression system is used to overexpress the Rab5 protein, the GAL4 gene and the UAS gene are present in two expression vectors respectively, the nucleotide sequence of the Rab5 protein is connected with the UAS gene, the promoter is connected with the GAL4 gene, and the expression of the Rab5 protein is started after the GAL4 gene is combined with the UAS gene.

[0009] In some embodiments, the promoter is a CMV promoter.

[0010] In some embodiments, the nucleotide sequence of the CMV promoter is set forth in SEQ ID NO: 4.

[0011] In some embodiments, the nucleotide sequence of the GAL4 gene is set forth in SEQ ID NO: 5.

[0012] In some embodiments, the nucleotide sequence of the UAS gene is set forth in SEQ ID NO: 6.

[0013] In another aspect, the present application provides a pharmaceutical composition for promoting central nervous axon regeneration and motor function recovery, characterized in that the composition comprises a Rab5 protein.

[0014] In some embodiments, the Rab5 protein comprises or consists of a sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3.

[0015] In another aspect, the present application provides a method for screening a drug for promoting central nervous axon regeneration and motor function recovery, the method comprising detecting the expression level of Rab5.

[0016] In another aspect, the present application provides a pharmaceutical composition for promoting central nervous axon regeneration and motor function recovery, characterized in that the pharmaceutical composition comprises a Rab5 protein and a pharmaceutically acceptable carrier.

[0017] In another aspect, the present application provides the use of an agent or a nucleic acid construct for promoting the expression of Rab5 in the preparation of a drug for promoting central nervous axon regeneration and motor function recovery in a subject.

[0018] In another aspect, the present application provides a method for promoting central nervous axon regeneration and motor function recovery, characterized in that the method comprises administering to a subject an effective amount of a Rab5 protein or overexpressing Rab5 in a subject.

[0019] In some embodiments, the subject can be a zebrafish or a human or other mammal, including but not limited to monkeys, cats, dogs, horses, rabbits, rodents (e.g., mice, rats, hamsters, and guinea pigs), cattle, sheep, and goats.

[0020] Embodiments of the present application provide a protein Rab5, which can be used as a protein target for repairing central nervous axon injury.

[0021] The present application also provides the use of Rab5 for the preparation of a drug for promoting neural axon regeneration and motor function recovery.

[0022] wherein the neural injury is a two-photon lesion of the unilateral axon of a zebrafish hair cell neuron.

[0023] The application also provides the use of Rab5 in promoting nerve axon regeneration and motor function recovery, comprising the following verification steps:

[0024] S1, obtain green fluorescent labeled Mauthner neurons of zebrafish, and overexpress Rab5 in single cells by using single cell electroporation technology, and observe the overexpression of Rab5 in promoting axon regeneration after axon damage by using two-photon laser;

[0025] S2, use drugs to treat green fluorescent labeled Mauthner neurons of zebrafish, and observe the inhibition of axon regeneration by inhibiting the normal expression of Rab5 after axon damage by using two-photon laser;

[0026] S3, collect the zebrafish obtained in the previous two steps, and take a picture of the axon regeneration length after 48h of axon damage, and place it under a behavior device to observe the relationship between the axon regeneration length and the motor function recovery.

[0027] In the step S1, the specific steps are as follows:

[0028] S1.1, place three female T056 strain zebrafish and two WT male zebrafish in the same spawning tank, and treat them under dark conditions for 12h overnight.

[0029] S1.2, place all the zebrafish under light in the morning, and mix the male and female zebrafish.

[0030] S1.3, after the zebrafish finish spawning, collect the zebrafish, and collect the spawned zebrafish eggs.

[0031] S1.4, transfer the zebrafish eggs to a glass dish, and pick out unfertilized and dead fish eggs, and place them in a light incubator for 24h.

[0032] S1.5, take out the zebrafish eggs, change the water and pick out dead eggs, and add an appropriate amount of PTU to inhibit the generation of pigment. Place in the incubator for 24h.

[0033] S1.6, take out the zebrafish eggs, change the water and pick out dead fish and dead eggs, and add an appropriate amount of PTU to inhibit the generation of pigment. Place in the incubator for 48h.

[0034] S1.7, take out the zebrafish larvae four days (4dpf) after fertilization, anesthetize them with MS222, and fix them on a special electroporation plate with 1% melted agarose.

[0035] S1.8, mix CMV-GAL4 and UAS-mCherry-Rab5 plasmids according to the proportion and add a certain amount of red fluorescent dye.

[0036] S1.9, the mixed plasmid is injected into the electrode wire which is pre-drawn, and is installed on the micro manipulator.

[0037] S1.10, the fixed zebrafish larvae are placed under the confocal microscope, and the electrode wire is touched to the cell body of the Mauthner neuron under the microscope, and an electric stimulus is applied, so that the cell body transfection successfully shows red fluorescence.

[0038] S1.11, the fixed zebrafish larvae are released and placed in an incubator for 24 hours.

[0039] S1.12, the zebrafish is taken out, the water is changed, and the dead fish is picked out and placed in an incubator for 24 hours.

[0040] S1.13, the zebrafish larvae six days after fertilization (6dpf) are taken out, anesthetized with MS222, and fixed on a glass slide with 1% melted agarose.

[0041] S1.14, the glass slide with the fixed zebrafish larvae is placed under the confocal microscope equipped with a two-photon laser, and the axon site near the cloaca is found, and the axon is damaged by using a high-energy two-photon laser.

[0042] S1.15, the zebrafish larvae after axon injury are released and placed in an incubator for 24 hours.

[0043] S1.16, the zebrafish is taken out, the water is changed, and the dead fish is picked out and placed in an incubator for 24 hours.

[0044] S1.17, the zebrafish is taken out, anesthetized with MS222, and fixed on a glass slide with 1% melted agarose.

[0045] S1.18, the fixed zebrafish larvae are placed under the confocal microscope, the axon regeneration is imaged, and the length of each group is counted.

[0046] The specific steps of step S2 are:

[0047] S2.1, three female T056 strain zebrafish and two WT male zebrafish are placed in the same spawning tank, and are treated in dark conditions for 12 hours overnight.

[0048] S2.2, in the morning, all the zebrafish are placed under bright light, and male and female zebrafish are mixed.

[0049] S2.3, after the zebrafish finish spawning, the zebrafish are recovered, and the spawned zebrafish eggs are collected.

[0050] S2.4, the zebrafish eggs are transferred to a glass dish, and the unfertilized and dead fish eggs are picked out and placed in a bright incubator for 24 hours.

[0051] S2.5, Take out the zebrafish eggs, change the water and pick up the dead eggs, add appropriate amount of PTU to inhibit the generation of pigment. Place in incubator for 24h.

[0052] S2.6, Take out the zebrafish eggs, change the water and pick up the dead fish and dead eggs, add appropriate amount of PTU to inhibit the generation of pigment. Place in incubator for 48h.

[0053] S2.7, Take out the zebrafish, change the water and pick up the dead fish, add appropriate amount of PTU to inhibit the generation of pigment. Place in incubator for 48h.

[0054] S2.8, Take out the zebrafish larvae six days after fertilization (6dpf), anesthetize them with MS222 and fix them on a glass slide with 1% melted agarose.

[0055] S2.9, Place the glass slide with the zebrafish larvae fixed on it under a confocal microscope equipped with a two-photon laser, and find the axon site near the cloaca, and use the high energy of the two-photon laser to damage the axon.

[0056] S2.10, Release the zebrafish larvae after axon damage, add 10 μM, 20 μM, 50 μM and 100 μM of Rab5 inhibitor CID-1067700 respectively, and put them in the incubator for 24h.

[0057] S2.11, Take out the zebrafish, change the water and pick up the dead fish, add water containing Rab5 inhibitor CID-1067700, and place in the incubator for 24h.

[0058] S2.12, Collect part of the zebrafish (about 40), add appropriate amount of cell lysis solution containing 1% protease inhibitor, and ultrasonic lysis on ice for 5-10 min until the fish body is completely lysed; centrifuge at 4℃, 13000 rpm, 10 min, collect the supernatant;

[0059] S2.13, BCA method for protein quantification;

[0060] S2.14, Perform SDS-PAGE electrophoresis, and block with 5% BSA at room temperature for 1h after transfer;

[0061] S2.15, Incubate the primary antibody, dilute r-Rab5 with primary antibody diluent (1:1000), and incubate on a shaking table at 4℃ overnight;

[0062] S2.16, Wash with 1XTBST three times, 3min each time;

[0063] S2.17, Dilute r-HRP with secondary antibody diluent (1:5000), and incubate at room temperature for 1h;

[0064] S2.18, wash three times with 1 x TBST, 3 min each time;

[0065] S2.19, incubate ECL developing solution on the membrane for 1-3 min, develop and observe the band results, and save on the computer.

[0066] S2.20, treat with antibody removal solution for 30 min to remove the antibody;

[0067] S2.21, incubate the primary antibody, dilute r-Tubulin (1:1000) with primary antibody diluent, and incubate on a shaking table at 4°C overnight;

[0068] S2.22, wash three times with 1 x TBST, 3 min each time;

[0069] S2.23, dilute r-HRP (15000) with secondary antibody diluent, and incubate at room temperature for 1 h;

[0070] S2.24, wash three times with 1 x TBST, 3 min each time;

[0071] S2.25, incubate ECL developing solution on the membrane for 1-3 min, develop and observe the band results, and save on the computer.

[0072] S2.26, take out the zebrafish, anesthetize them with MS222, and fix them on a glass slide with 1% melted agarose.

[0073] S2.27, place the fixed zebrafish larvae under a confocal microscope, image their axon regeneration, and take pictures to count the lengths of each group.

[0074] The specific steps of step S3 are:

[0075] S3.1, take out the zebrafish obtained in steps S1 and S2, and place them under the completed high-speed photography device.

[0076] S3.2, stand still for about 5 minutes until the zebrafish adapt to the environment and stop swimming.

[0077] S3.3, play the stimulating audio to cause the escape response of the zebrafish, and use a high-speed camera to shoot the video of the zebrafish turning around.

[0078] S3.4, repeat the above steps to ensure that each zebrafish is shot with one to two complete videos.

[0079] S3.5, process the high-speed photography video using software, and calculate the maximum turning angle and average angular velocity of the zebrafish in each group.

[0080] Definitions

[0081] GAL4 / UAS: GAL4 / UAS is a gene expression regulation system existing in yeast. UAS is the abbreviation of upstream activating sequence. GAL4 is a transcription regulator, and after its binding domain (BD) binds with the UAS sequence, its activation domain (AD) binds with the promoter region, thereby inducing the expression of the gene. The key point of the GAL4 / UAS system is that GAL4 and UAS genes exist in two transgenic lines respectively. There is a transcription activator in the GAL4 transgenic line, but there is no target gene, and the establishment of the system needs an organ-specific promoter or enhancer. In the UAS-target gene line, the target gene needed is inserted downstream of the UAS, and the UAS-transgenic line can be established. Because the transcription activator does not exist in the UAS-target gene line, the target gene is in a silent state, and only after the GAL4 transgenic line is crossed with the UAS-target gene line, offspring with specific expression of the target gene can be produced, which can be used for gene overexpression, gene knockout and cell-specific deletion.

[0082] mCherry: a red fluorescent protein, widely used in biotechnology as a tracer. It has low cytotoxicity and is more excellent than other fluorescent protein tags.

[0083] CMV promoter: from cytomegalovirus, it is a recognized strong promoter for initiating the expression of eukaryotic genes, and is widely used to construct efficient eukaryotic expression vectors.

[0084] Reagent: the reagent of the application should be understood in a broad sense, which includes expression vectors, plasmids, functional gene fragments and the like capable of promoting the expression of Rab5. The expression can be the expression of a gene or the expression of a protein.

[0085] The beneficial effects of the above technical solutions of the application are as follows:

[0086] 1. The application takes Rab5 as a protein intervention target, and overexpression of Rab5 promotes the axon regeneration of zebrafish hair motoneurons.

[0087] 2. The application takes Rab5 as a protein intervention target, and inhibition of Rab5 expression will lead to weakened axon regeneration of zebrafish hair motoneurons.

[0088] 3. The Rab5 provided by the application can promote the recovery of the movement function of zebrafish by promoting the axon regeneration of zebrafish hair motoneurons. It is helpful to better understand the important role of Rab5 in the process of nerve axon injury repair, and provides a new target for the treatment after axon injury. BRIEF DESCRIPTION OF DRAWINGS

[0089] Figure 1Figure A is a schematic diagram showing that overexpression of Rab5 in vivo can significantly promote axon regeneration of zebrafish Muller neuron in Example 1 of the present application. Figure A is a comparison of axon regeneration of Muller neuron in vivo between empty UAS-mCherry (negative control) and UAS-mCherry-Rab5, Bar = 50 μm. Figure B is a histogram of axon length of axon regeneration of Muller neuron in vivo between empty UAS-mCherry (negative control) and UAS-mCherry-Rab5. ****P < 0.0001.

[0090] Figure 2 Figure A is a schematic diagram showing that inhibition of Rab5 expression in vivo can inhibit axon regeneration of zebrafish Muller neuron in Example 2 of the present application. Figure A is a graph showing the expression of Rab5 in zebrafish in vivo under different concentrations of CID-1067700 treatment, DMSO is the drug negative control. Figure 2 Figure B is a histogram of axon length of axon regeneration of Muller neuron in zebrafish under different concentrations of CID-1067700 treatment. **P < 0.01, ***P < 0.001.

[0091] Figure 3 Figure A is a schematic diagram showing that overexpression of Rab5 in vivo can promote the recovery of motor function of zebrafish in Example 3 of the present application. Figure A is a schematic diagram showing the escape response of zebrafish under different treatments, uninjured is the negative control. Figure B is a histogram of the maximum turning angle of the escape response of zebrafish under different treatments. Figure C is a histogram of the average angular velocity of the escape response of zebrafish under different treatments. *P < 0.05, ***P < 0.001. DETAILED DESCRIPTION

[0092] In order to make the purpose, technical solutions and advantages of the present application clearer and more apparent, the present application is further described in detail below in combination with specific examples and with reference to the accompanying drawings.

[0093] The present application provides a protein target Rab5 (SEQ ID NO: 1, sequence from zebrafish, NCBI ID: 337737) which significantly promotes axon regeneration in the process of central nervous axon injury repair.

[0094] Rab5 is used for preparing a drug for promoting nerve axon regeneration and motor function recovery, and the nerve axon injury is two-photon damage of axon of zebrafish Muller neuron.

[0095] The present application also provides the use of Rab5 in promoting nerve axon regeneration and motor function recovery, which comprises the following verification steps:

[0096] S1, obtain green fluorescent labeled Mauthner neuron zebrafish, and use single cell electroporation technology to overexpress Rab5 in single cells, and observe the overexpression of Rab5 to promote axon regeneration after axon damage by two-photon laser;

[0097] S2, use drug to treat green fluorescent labeled Mauthner neuron zebrafish, and observe the inhibition of axon regeneration after axon damage by two-photon laser, and the inhibition of axon regeneration after axon damage by two-photon laser. 18 H 18 N2O4S2, Selleck, E0135) treatment will hinder the expression and normal function of Rab5, and the inhibition of axon regeneration after axon damage by two-photon laser.

[0098] S3, collect the zebrafish obtained in the previous two steps, and take pictures of the axon regeneration length 48h after axon damage, and place them under the behavior device to observe the relationship between axon regeneration length and motor function recovery.

[0099] Example 1 Influence of overexpression of Rab5 on axon regeneration

[0100] Obtain green fluorescent labeled Mauthner neuron zebrafish, and use single cell electroporation technology to overexpress Rab5 in single cells, and observe the overexpression of Rab5 to promote axon regeneration after axon damage by two-photon laser, and the overexpression of Rab5 to promote axon regeneration after axon damage by two-photon laser.

[0101] 1.1, obtain green fluorescent labeled Mauthner neuron zebrafish

[0102] 1.1.1, place three female T056 strain zebrafish (donated by RIKEN, Saitama, Japan) and two WT male zebrafish (purchased from China Zebrafish Resource Center, CZ1) in the same spawning tank, and treat them under dark conditions for 12h to promote spawning.

[0103] 1.1.2, place all zebrafish in a light place in the morning, and mix the male and female zebrafish.

[0104] 1.1.3, after the zebrafish finish spawning, collect the zebrafish, and collect the spawned zebrafish eggs.

[0105] 1.1.4, transfer the zebrafish eggs to a glass dish, and pick out unfertilized and dead fish eggs, and place them in a light incubator for 24h.

[0106] 1.1.5, take out the zebrafish eggs, change the water and pick out dead eggs, add appropriate amount of PTU (N-phenylthiourea, Sigma, P7629) to inhibit the generation of pigment. Place in incubator for 24h.

[0107] 1.1.6, Take out the zebrafish eggs, change the water and pick up the dead fish and dead eggs, add appropriate amount of PTU to inhibit the generation of pigment. Place in incubator for 48h.

[0108] 1.2, In vivo electroporation of overexpression Rab5 in Mauthner neuron of zebrafish

[0109] 1.2.1, Take out the T056 zebrafish larvae four days after fertilization (4dpf), anesthetize them with MS222 (3-aminobenzoic acid ethyl ester, Sigma, E10521) and fix them on a 2% agarose electroporation plate wrapped with copper wire with 1% melted agarose.

[0110] 1.2.2, Mix CMV-GAL4 (Cytomegalovirus-Galactokinase 4, CMV is the promoter, GAL4 sequence is derived from yeast, GAL4 binds to UAS to start downstream protein expression) and the constructed UAS-mCherry-Rab5 (Upstream activating sequence-mCherry-Rab5, UAS sequence is derived from yeast) plasmid in a ratio of 1:1 to a final concentration of 300ng / μL and add 1 μL of alexa 488 / 594nm red fluorescent dye (Sigma, 1623-7) to indicate the entry of the plasmid.

[0111] 1.2.3, Inject the mixed plasmid into the pre-drawn electrode (electrode wire similar to a needle, capacity greater than the volume of the configured plasmid) with a fine needle and install it on a micromanipulator (an instrument that can step in microns, can realize three-axis movement, used for fine adjustment of needle position).

[0112] 1.2.4, Place the fixed zebrafish larvae under a confocal microscope, and under the microscope, touch the electrode wire to the Mauthner neuron cell body and stimulate it to make the cell body transfection successfully show red fluorescence.

[0113] 1.2.5, Release the fixed zebrafish larvae and place them in an incubator for 24h.

[0114] 1.2.6, Take out the zebrafish, change the water and pick up the dead fish, and place them in an incubator for 24h.

[0115] 1.3, Two-photon damage to the axon of Mauthner neuron of zebrafish

[0116] 1.3.1, Take out the T056 zebrafish larvae six days after fertilization (6dpf), anesthetize them with MS222 and fix them on a glass slide with 1% melted agarose.

[0117] 1.3.2, Place the slide with the zebrafish larvae on the confocal microscope equipped with a two-photon laser and find the axon site near the cloaca, and destroy the axon with high energy of the two-photon laser.

[0118] 1.3.3, Release the zebrafish larvae after axon injury and put them into the incubator for 24h.

[0119] 1.3.4, Take out the zebrafish, change the water and pick out the dead fish, and put them into the incubator for 24h.

[0120] 1.4, Observe the effect of overexpression of Rab5 on the axon regeneration of zebrafish Muller neurons

[0121] 1.4.1, Take out the T056 zebrafish, anesthetize them with MS222 and fix them on the slide with 1% melted agarose.

[0122] 1.4.2, Place the fixed zebrafish larvae under the confocal microscope, image their axon regeneration and take pictures to count the length of each group.

[0123] The results show that overexpression of Rab5 can significantly promote the axon regeneration of zebrafish Muller neurons Figure 2 ), wherein, Figure 1 A is the comparison of Muller neuron axon regeneration between empty UAS-mCherry (negative control) and UAS-mCherry-Rab5 in vivo, Bar = 50 μm. Figure 1 B is the axon statistical chart of Muller neuron axon regeneration between empty UAS-mCherry (negative control) and UAS-mCherry-Rab5 in vivo, the regeneration length of the UAS-mCherry-Rab5 group increased by about 169% compared with the control group (UAS+injured: n = 10; OE Rab5+injured: n = 8, unpaired t test was used). ****P < 0.0001.

[0124] Example 2 Effect of inhibition of Rab5 expression on axon regeneration

[0125] Female T056 zebrafish were mated with WT male zebrafish to obtain green fluorescently labeled Muller zebrafish, and the expression of Rab5 was inhibited with drugs to observe the regeneration of Muller axons, the specific steps are as follows:

[0126] 2.1, Obtain green fluorescently labeled Muller neurons of zebrafish

[0127] 2.1.1, Place three female T056 strain zebrafish and two WT male zebrafish in the same spawning tank, and treat them under dark conditions for 12h overnight.

[0128] 2.1.2, Place all zebrafish under light and mix the male and female zebrafish.

[0129] 2.1.3, After the zebrafish finish spawning, collect the zebrafish and collect the spawned zebrafish eggs.

[0130] 2.1.4, Transfer the zebrafish eggs to a glass dish and pick out the unfertilized and dead fish eggs and place them in a light incubator for 24 hours.

[0131] 2.1.5, Take out the zebrafish eggs, change the water and pick out the dead eggs, add an appropriate amount of PTU to inhibit the generation of pigments. Place in the incubator for 24 hours.

[0132] 2.1.6, Take out the zebrafish eggs, change the water and pick out the dead fish and dead eggs, add an appropriate amount of PTU to inhibit the generation of pigments. Place in the incubator for 96 hours.

[0133] 2.2, Two-photon damage to the axons of zebrafish Rohon-Beard neurons

[0134] 2.2.1, Take out the T056 zebrafish larvae six days after fertilization (6dpf) and anesthetize them with MS222 and fix them on a glass slide with 1% melted agarose.

[0135] 2.2.2, Place the glass slide with the fixed zebrafish larvae under a confocal microscope equipped with a two-photon laser and find the axon site near the cloaca. Use the high-energy two-photon laser to damage the axon.

[0136] 2.2.3, Release the zebrafish larvae after axon damage and place them in the incubator for 24 hours.

[0137] 2.2.4, Take out the zebrafish, change the water and pick out the dead fish, and place them in the incubator for 24 hours.

[0138] 2.3, Inhibition of zebrafish Rab5 expression using the drug CID-1067700

[0139] 2.3.1, Release the zebrafish larvae after axon damage and add 10 μM, 20 μM, 50 μM and 100 μM of Rab5 inhibitor CID-1067700 respectively, and place them in the incubator for 24 hours.

[0140] 2.3.2, Take out the zebrafish, change the water and pick out the dead fish, and supplement the water containing the Rab5 inhibitor CID-1067700, and place them in the incubator for 24 hours.

[0141] 2.4, Western blot test

[0142] 2.4.1, Collect part T056 zebrafish (about 40), add appropriate amount of RIPA cell lysate containing 1% protease inhibitor (Sigma, 20-188), ultrasonic lysis on ice for 5-10 min until the fish is completely lysed; centrifuge at 4°C, 13000 rpm, 10 min, collect supernatant;

[0143] 2.4.2, use ultraviolet spectrophotometer for protein quantification, and unify at about 10 μg / μL;

[0144] 2.4.3, perform SDS-PAGE electrophoresis, and block the membrane with 5% BSA (Shenguo, C500036) at room temperature for 1 h;

[0145] 2.4.4, incubate the primary antibody (rabbit-anti-Rab5; 1:1000; GeneTex, Shanghai, China; Cat#GTX13253), dilute the primary antibody with the primary antibody diluent (1×Tris-HCl buffer saline solution + 0.02% Tween20 + 0.001% sodium azide), and incubate overnight on a shaking table at 4°C;

[0146] 2.4.5, wash with 1×TBST (1×Tris-HCl buffer saline solution + 0.02% Tween20) for three times, 3 min each time;

[0147] 2.4.6, dilute the secondary antibody (goat-anti-rabbit; 1:5000; Proteintech, Chicago, IL, USA; Cat#SA00001-2) with the secondary antibody diluent (1×Tris-HCl buffer saline solution + 0.02% Tween20 + 0.01% BSA), and incubate at room temperature for 1 h;

[0148] 2.4.7, wash with 1×TBST for three times, 3 min each time;

[0149] 2.4.8, incubate the ECL (enhanced chemiluminescence reagent, MCE, HY-K1005) developing solution on the membrane, process for 1-3 min, develop and observe the band results, and save on the computer.

[0150] 2.4.9, treat with the antibody removal solution (0.1M glutamic acid and adjust to PH 3.7) for 30 min to remove the incubated antibody, so as to facilitate the incubation of the new antibody;

[0151] 2.4.10, incubate the primary antibody (rabbit-anti-β-Tubulin; 1:1000; GeneTex; Cat#GTX18587), dilute the antibody with the primary antibody diluent, and incubate overnight on a shaking table at 4°C;

[0152] 2.4.11, wash three times with 1x TBST, 3 min each time;

[0153] 2.4.12, dilute the secondary antibody (goat-anti-rabbit; 1:5000; Proteintech, Chicago, USA; Cat#SA00001-2) with secondary antibody dilution buffer, incubate at room temperature for 1 h;

[0154] 2.4.13, wash three times with 1x TBST, 3 min each time;

[0155] 2.4.14, incubate ECL developing solution on the membrane for 1-3 min, develop and observe the band results, and save them on the computer. Protein immunization results need to be done twice, once for the target protein and once for the internal reference protein. The internal reference protein can be compared with the target protein to see the protein changes.

[0156] 2.5, observe the effect of inhibiting Rab5 on the axon regeneration of zebrafish hair motoneurons

[0157] 2.5.1, take T056 zebrafish, anesthetize them with MS222, and fix them on a glass slide with 1% melted agarose.

[0158] 2.5.2, place the fixed zebrafish larvae under a confocal microscope, image their axon regeneration, and take pictures to count the lengths of each group.

[0159] The results show that inhibiting the expression of Rab5 can significantly inhibit the axon regeneration of zebrafish hair motoneurons Figure 2 ), wherein Figure 2 A is the expression of Rab5 in zebrafish treated with different concentrations of CID-1067700, and DMSO is the drug negative control. Figure 2 B is a statistical chart of the axon regeneration length of zebrafish hair motoneurons treated with different concentrations of CID-1067700. Compared with the DMSO group, the axon regeneration length of the 50 μΜ group decreased by 43.4%, and the axon regeneration length of the 100 μΜ group decreased by 65.8% (DMSO, n = 10, 10 μΜ, n = 6, 20 μΜ, n = 9, 50 μΜ, n = 10, 100 μΜ, n = 8, unpaired t test was used). **P < 0.01, ***P < 0.001.

[0160] Example 3 Evaluation of the relationship between axon regeneration length and motor function recovery

[0161] The zebrafish obtained in Example 1 and Example 2 were collected, and the axon regeneration length was photographed 48 h after axon damage, and the relationship between axon regeneration length and motor function recovery was observed under a behavioral device, the specific steps being:

[0162] 3.1 Filming the escape response of zebrafish

[0163] 3.1.1 Take out the zebrafish obtained in Example 1 and Example 2 and place them under the completed high-speed photography device.

[0164] 3.1.2 Let it sit for about 5 minutes until the zebrafish adapts to the environment and stops swimming.

[0165] 3.1.3 Play stimulating audio to elicit an escape response from the zebrafish, and use a high-speed camera to capture a video of the zebrafish turning around.

[0166] 3.1.4 Repeat the above steps to ensure that one or two complete videos are taken from each side of each zebrafish.

[0167] 3.2 Data processing of zebrafish escape response

[0168] 3.2.1. Use software to process high-speed video footage and calculate the maximum turning angle and average angular velocity of each group of zebrafish.

[0169] The results showed that the kinetic ability of zebrafish to escape was positively correlated with the length of axonal regeneration in Mautna neurons. Figure 3 ),in Figure 3 A is a schematic diagram of the escape response of zebrafish under different treatments. Uninjured is the negative control. Compared with the uninjured group, the maximum turning angle of the injured group decreased by 20% (Uninjured: n=7; UAS+injured: n=6; OERab5+injured: n=6; CID+injured: n=8, using unpaired t test). Figure 3 B is a statistical graph showing the maximum turning angle of zebrafish in their escape response under different treatments. Figure 3 C is a statistical graph showing the average angular velocity of zebrafish escape response under different treatments. Compared with the uninjured group, the average turning angular velocity of the injured group decreased by 60%, while overexpression of Rab5 achieved 46.4% functional recovery (Uninjured: n=7; UAS+injured: n=5; OE Rab5+injured: n=6; CID+injured: n=8, using unpaired t-test). *P<0.05,***P<0.001.

[0170] References

[0171] B. B. Hu, M. Chen, R. C. Huang, Y. B. Huang, Y. Xu, W. Yin, L. Li, B. Hu, In vivo imaging of Mauthner axon regeneration, remyelination and synapses re-establishment after laser axotomy in zebrafish larvae, Exp Neurol 300 (2018) 67-73.

[0172] R. C. Huang, M. Chen, L. Q. Yang, M. Wagle, S. Guo, B. Hu, MicroRNA-133b Negatively Regulates Zebrafish Single Mauthner-Cell Axon Regeneration through Targeting tppp3 in Vivo, Front Mol Neurosci 10 (2017).

[0173] Wang Z, Wang X, Shi L, Cai Y, Hu B. Wolfram syndrome 1b mutations suppresses Mauthner-cell axon regeneration via ER stress signal pathway. Acta Neuropathol Commun. 2022;10(1):184. Published 2022 Dec 17. doi: 10.1186 / s40478-022-01484-8

[0174] L. Q. Yang, M. Chen, J. L. Zhang, D. L. Ren, B. Hu, Hypoxia Delays Oligodendrocyte Progenitor Cell Migration and Myelin Formation by Suppressing Bmp2b Signaling in Larval Zebrafish, Front Cell Neurosci 12 (2018) 348.

[0175] Sequence

[0176] SEQ ID NO: 1 Amino acid sequence of zebrafish Rab5 protein

[0177] MANRGGATRPNGSNAGNKICQFKLVLLGESAVGKSSLVLRFVKGQFHEFQESTIGAAFLTQTLCLDDTTVKFEIWDTAGQERYHSLAPMYYRGAQAAIVVYDITNEESFARAKNWVKELQRQASPNIVIALSGNKADLANKRAVDFQDAQSYADDNSLLFMETSAKTSMNVNEIFMAIAKKLPKSEPQAAGANSGRSRGVDLTETAQPTKAPCCSN

[0178] SEQ ID NO: 2 Amino acid sequence of human Rab5 protein

[0179] MANRGGATRPNGPNTGNKICQFKLVLLGESAVGKSSLVLRFVKGQFHEFQESTIGVKFEIWDTAGQERYHSLAPMYYRGAQAAIVVYDITNEESFARAKNWVKELQRQASPNIVIALSGNKADLANKRAVDFQEAQSYADDNSLLFMETSAKTSMNVNEIFMAIAKKLPKNEPQNPGANSARGRGVDLTEPTQPTRNQCCSN

[0180] SEQ ID NO: 3 Amino acid sequence of Mus musculus Rab5 protein

[0181] MANRGGATRPNGPNTGNKICQFKLVLLGESAVGKSSLVLRFVKGQFHEFQESTIGAAFLTQTVCLDDTTVKFEIWDTAGQERYHSLAPMYYRGAQAAIVVYDITNEESFARAKNWVKELQRQASPNIVIALSGNKADLANKRAVDFQEAQSYADDNSLLFMETSAKTSMNVNEIFMAIAKKLPKNEPQNPGANSARGRGVDLTEPAQPARSQCCSN

[0182] SEQ ID NO: 4 Nucleotide sequence of CMV promoter

[0183] TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTAT

[0184] SEQ ID NO: 5 Nucleotide sequence of GAL4 (yeast)

[0185] CGTGAGAACGGATATGAATGGGCAATGAGCCATCCCATTGACGTCAATGGTGGGTGGTCCTATTGACGTCAATGGGCATTGAGCCAGGCGGGCCATTTACCGTAATTGACGTCAATGGGGGAGGCGCCATATACGTCAATAGGACCGCCCATATGACGTCAATAGGAAAGACCATATATAGAGACCATTGACGTCAATGGGGGAGTGGCTATGGGCGGTATTAGGAAGCCCCATATATGGTATATGGGACCGCCCATTGGGAGGGGCTATCTACGTCAATAGGAAAACCCATATATGGAATACTATATGGCATAGGGCCAATACATAGTATTGAACCTGGCCAATAGCCATATTGGCATAGGGCCATATTGGATATTGCCTATATATTGATCCTGGCATATAGCCAATATGGCCGCCATTATTGGCACCATGCCAATTCAATATGGCGGACCTGGCACTGTGCCAACTGGGGAGGGGTCTACTTGGCACGGTGCCAAGTTTGAGGAGGGGTCTTGGCCCTGTGCCAAGTCCGCCATATTGAATTGGCATGGTGCCAAGTCCGCCATATTGAATTGGCWSRGGTCCAGATCCACCATATTGAATTGGCAYGAGTCCATATACGCCATATTGAATTGGCACAGGTCCAGATCCACCATAT

[0186] SEQ ID NO: 6 Nucleotide sequence of UAS (yeast)

[0187] ATCAGGATGGCGGAGTACTGTCCTCCGGCAAGGTCGGAGTACTGTCCTCCGACACTAGAGGTCGGAGTACTGTCCTCCGACGCAAGGCGGAGTACTGTCCTCCGGGCTGCGGAGTACTGTCCTCCGGCAAGGTCGGAGTACTGTCCTCCGACACTAGAGGTCGGAGTACTGTCCTCCGACGCAAGGTCGGAGTACTGTCCTCCGACACTAGAGGTCGGAGTACTGTCCTCCGACGCAAGGTCGGAGTACTGTCCTCCGACACTAGAGGTCGGAGTACTGTCCTCCGACGCAAGGCGGAGTACTGTCCTCCGGGCTGGCGGAGTACTGTCCTCCGGCAAGGGTCGACTCTAGAGGGTATATAATGGATCCCATCGCGTCTCAGCCTCACTTTGAGCTCCTCCACACGAATTC

[0188] SEQ ID NO: 7 Nucleotide sequence of mCherry

[0189] ATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACAAGCTGTACAAGTAG

[0190] SEQ ID NO: 8 Nucleotide sequence of zebrafish Rab5

[0191] ATGGCCAATAGGGGAGGAGCAACACGCCCCAACGGGTCCAACGCGGGCAATAAGATCTGCCAGTTTAAACTGGTCTTGCTAGGAGAGTCAGCGGTGGGGAAGTCCAGCCTCGTTCTGCGCTTCGTCAAAGGGCAGTTTCACGAGTTTCAGGAAAGCACAATAGGAGCGGCCTTTCTTACACAGACACTGTGCTTGGATGACACGACGGTAAAGTTTGAGATCTGGGACACAGCTGGACAGGAGCGCTACCACAGTCTGGCCCCCATGTACTACAGAGGTGCCCAGGCCGCCATTGTAGTTTATGACATCACCAATGAGGAGTCATTTGCAAGAGCAAAGAACTGGGTTAAAGAGCTTCAGAGGCAGGCTAGCCCAAACATTGTCATCGCTCTGTCTGGGAACAAGGCTGACCTTGCCAACAAGAGAGCTGTGGACTTCCAGGATGCTCAGTCTTATGCAGATGACAACAGTTTGCTGTTCATGGAGACGTCAGCAAAAACCTCCATGAACGTGAACGAGATCTTCATGGCCATTGCGAAAAAGTTGCCTAAGAGTGAGCCCCAAGCTGCCGGAGCCAACAGCGGGCGGAGTAGGGGTGTGGACCTCACAGAGACAGCCCAACCCACTAAAGCCCCCTGCTGCAGCAAC

[0192] The above-described specific embodiments illustrate the purposes, technical solutions, and beneficial effects of the present application. It should be understood that the above-described specific embodiments are merely specific embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent replacements, improvements, and the like made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. Use of a reagent that promotes Rab5 expression in the preparation of a medicament that promotes axonal regeneration and motor function recovery in a subject, wherein the reagent is a system that overexpresses Rab5 protein, and the loss of motor function is caused by axonal injury, and the subject is a zebrafish, human, monkey, cat, dog, horse, rabbit, rodent, cow, sheep or goat.

2. The use according to claim 1, characterized in that, The rodents are mice, rats, hamsters, or guinea pigs.

3. The use according to claim 1, characterized in that, Rab5 protein was overexpressed using the GAL4 / UAS expression system. The GAL4 gene and the UAS gene were respectively located in two expression vectors. The nucleotide sequence of the Rab5 protein was linked to the UAS gene, and the promoter was linked to the GAL4 gene. The expression of the Rab5 protein was initiated after the GAL4 gene bound to the UAS gene.

4. The use according to claim 3, characterized in that, The promoter is the CMV promoter.

5. The use according to claim 4, characterized in that, The nucleotide sequence of the CMV promoter is shown in SEQ ID NO:

4.

6. The use according to claim 3, characterized in that, The nucleotide sequence of the GAL4 gene is shown in SEQ ID NO:

5.

7. The use according to claim 3, characterized in that, The nucleotide sequence of the UAS gene is shown in SEQ ID NO:

6.

8. Use of reagents for detecting Rab5 expression levels in screening drugs that promote central nervous system axon regeneration and motor function recovery in subjects whose motor function loss is caused by axonal injury, wherein the subjects are zebrafish, humans, monkeys, cats, dogs, horses, rabbits, rodents, cattle, sheep or goats.

9. The use according to claim 8, characterized in that, The rodents are mice, rats, hamsters, or guinea pigs.

10. A method for screening drugs that promote central nervous system axon regeneration and motor function recovery in subjects, the method comprising detecting Rab5 expression levels, motor function loss caused by axonal injury, wherein the subjects are zebrafish, humans, monkeys, cats, dogs, horses, rabbits, rodents, cattle, sheep, or goats.

11. The method according to claim 10, characterized in that, The rodents are mice, rats, hamsters, or guinea pigs.

12. The use according to any one of claims 1-9 or the method according to claim 10 or 11, characterized in that, The Rab5 protein comprises, or is composed of, the sequence shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

Images