Application of Caspr4 inhibitor in preparation of axonal injury medicine

Abstract

The invention belongs to the field of biological medicine, and particularly relates to application of a Caspr4 inhibitor in preparation of axonal injury repair medicine. It is found that after the sciatic nerve axon of a mouse is damaged, the expression quantity of Caspr4 on a corresponding DRG neuron is remarkably reduced, and regeneration of the damaged axon is promoted by inhibiting the activity of Caspr4 in DRG and RGC neuron, which indicates that Caspr4 plays an important role in regulation and control of regeneration of central and peripheral axons, regeneration of the damaged axon can be effectively promoted by adopting an effective Caspr4 inhibitor, and the activity of Caspr4 is inhibited. The compound is expected to become a medicine for treating axonal injury.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to the application of a Caspr4 inhibitor in the preparation of axonal damage repair drugs. Background Art

[0002] In my country, about 30 million people are suffering from sensory dysfunction such as limb numbness and pain, and even motor dysfunction such as paralysis and disability and sympathetic nerve disorders caused by nerve axon injury, and the number is estimated to increase by 2 million people each year. The causes of axon injury are complex, mainly including traumatic injury, local compression, nerve tumors, etc. Most of them are caused by trauma. Nowadays, various treatment methods can solve the functional recovery of the affected limb to a certain extent, but they still leave sequelae of varying degrees, which brings a heavy burden to the family and society. Axon injury repair has become an important social need and one of the key problems in the medical field. Early accurate diagnosis and timely treatment are the key to a good prognosis of axon injury. Current treatment methods mainly include surgical decompression, anti-inflammatory and nerve nutrition drug treatment, but it is difficult to achieve satisfactory results. The drugs currently used to treat axon injury include methylprednisolone, gangliosides, etc., but the clinical efficacy is uncertain, there are strict application restrictions and may bring many adverse reactions. Therefore, starting from the molecular mechanism of axon regeneration is the key to fundamentally overcome the problem of axon injury. Axon regeneration is a series of complex and coordinated processes initiated by the body after nerve injury. Compared with the central nervous system, the peripheral nervous system of adult mammals has a stronger endogenous regeneration ability and can achieve a certain degree of tissue structure remodeling and functional reconstruction after injury. Therefore, further exploring the biological mechanism of axon regeneration and finding new targets that can more effectively promote axon regeneration will lay a theoretical foundation for the development of new treatment methods, which is of great significance for overcoming the problem of axon injury repair.

[0003] Contactin-related proteins (Casprs) are members of the neuropilin superfamily, mainly including 5 members, namely Casprs1, Casprs2, Casprs3, Casprs4 and Casprs5, which are specifically expressed on neurons. All of their members are composed of a large extracellular domain, a single transmembrane region and a short cytoplasmic region. The formation and stability of myelinated axons of neurons are partly dependent on members of the Casprs family. Caspr4 is abundantly expressed in neurons of the cortex, dentate gyrus of the hippocampus, brainstem, cerebellum and substantia nigra. It can be seen that Caspr4 may play an important role in the development of the nervous system, and is also highly expressed in sensory neurons such as the peripheral trigeminal ganglion and dorsal root ganglion (DRG). Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide an application of a Caspr4 inhibitor in the preparation of axonal damage repair drugs.

[0005] In a first aspect, the present invention provides use of a Caspr4 inhibitor in the preparation of axonal damage repair drugs.

[0006] The Caspr4 inhibitor is a virus that interferes with Caspr4.

[0007] The Caspr4 interference virus is AAV2-shCaspr4.

[0008] A second aspect of the present invention is a drug for treating axonal damage and repair, wherein the active ingredient is a small molecule compound inhibitor of Caspr4.

[0009] The Caspr4 inhibitor is a small molecule compound inhibitor of Caspr4.

[0010] The present invention found that after the sciatic nerve axon of mice was injured, the expression of Caspr4 on the corresponding DRG neurons was significantly reduced, and reducing the expression of Caspr4 in DRG and RGC neurons promoted the regeneration of damaged axons, which means that Caspr4 plays an important role in the regulation of central and peripheral axon regeneration. The use of effective Caspr4 inhibitors can effectively promote the regeneration of damaged axons and is expected to become a drug for treating axon injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying creative labor, other drawings obtained based on these drawings still belong to the scope of the present invention.

[0012] Figure 1 The expression changes of Caspr4 in damaged DRG sensory neurons 3 and 7 days after sciatic nerve injury in mice; (A) Caspr4 mRNA expression detected by qPCR; (B) Caspr4 expression detected by western blot; (C) Quantitative analysis of the relative expression of Caspr4 protein detected by western blot; (D) Immunofluorescence staining technique was used to detect the localization and expression of Caspr4 in DRG tissue neurons.

[0013] Figure 2The results are compared with the control group after siRNA effectively interfered with the expression of Caspr4 in DRGs; (A) After siRNA effectively interfered with the expression of Caspr4 in DRGs, the expression of Caspr4 was detected by western blot; (B) After siRNA effectively interfered with the expression of Caspr4 in DRGs, the quantitative analysis of Caspr4 protein bands; (C) After siRNA effectively interfered with the expression of Caspr4 in DRGs, the axon regeneration was detected by immunofluorescence staining technology; (D) After siRNA effectively interfered with the expression of Caspr4 in DRGs, the change of axon length; (E) After siRNA effectively interfered with the expression of Caspr4 in living DRGs, the axon regeneration length was detected by fluorescence technology;

[0014] Figure 3 To reduce axon regeneration in the damaged optic nerve after Caspr4 expression in RGCs. DETAILED DESCRIPTION

[0015] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0016] 1. Establishment of Sciatic Nerve Axon Injury Model

[0017] The mice were weighed and anesthetized by intraperitoneal injection of 10% chloral hydrate at a dose of 0.1 mL / 20 g. After about 25 minutes, the mice were deeply anesthetized, the hair on both thighs was removed, and routine disinfection was performed with iodine tincture. The mice were placed on their sides under a dissecting microscope. The skin of the mouse thigh was cut along the long axis of the femur, with an incision of about 0.5 cm long. The muscles were gently separated along the intermuscular space with tweezers, avoiding the femoral artery and its branches, exposing the sciatic nerve as much as possible, and the sciatic nerve was cut with microscissors about 1.0 cm long. After the operation, the incision was sutured intermittently layer by layer, and the mice were placed on a heating pad until they woke up.

[0018] 2. Detect the changes in Caspr4 expression in DRG neurons after sciatic nerve axon injury.

[0019] After the sciatic nerve axon was injured, the 4th / 5th lumbar DRG tissue corresponding to the injured axon was removed. Then, the expression of Caspr4 was detected by qPCR and western blot technology; the localization and expression of Caspr4 on DRG tissue neurons was detected by immunofluorescence staining technology.

[0020] like Figure 1 As shown, 3 and 7 days after sciatic nerve axon injury in mice, the expression of Caspr4 in damaged DRG sensory neurons was significantly decreased, suggesting that Caspr4 may inhibit axon regeneration after DRG neuron injury.

[0021] 3. Effect of Caspr4 on axon regeneration in dorsal root ganglion neurons.

[0022] 1) Axon regeneration cell experiment:

[0023] First, primary DRG neurons of mice were extracted for in vitro culture. The mice were killed by cervical dislocation, and the fur and internal organs were processed. The spine was retained and rinsed with 0.01M PBS solution. After rinsing, it was fixed on the operating table, and the corresponding lumbar vertebrae (L4, L5) were cut open under a microscope. The dorsal root ganglia were found in the vertebral body, and the surrounding adhesion tissues and nerve branches were removed with microscissors. The DRG was removed as intact as possible, and the tissue was placed in MEM solution.

[0024] After sampling, discard the MEM solution, add 1.0mL collagenase A to each tube, and digest at 37°C for 90min. After that, replace collagenase A with 500μL pancreatin (life technology) and continue digestion for 15min. After complete digestion, rinse the digested DRG three times with MEM culture medium containing 10% FBS to terminate the activity of pancreatic enzymes. Then, use a 1mL pipette to blow the DRG tissue to separate the nerve cells until no obvious tissue blocks are visible. Centrifuge the evenly dispersed free cell suspension (680rpm, 7min), discard the supernatant, and resuspend the precipitate in MEM culture medium containing 5% FBS. Inoculate the cells into a 24-well cell culture plate with an adhesion slide and place it in a 37°C, 5% CO2 incubator for culture.

[0025] To explore the effect of Caspr4 on sensory nerve axon regeneration, we transfected Caspr4-targeted siRNA and eGFP plasmids into primary DRG neurons by electroporation, and set up negative siRNA and eGFP as control groups.

[0026] For electroporation transfection, we centrifuged the free DRG cell suspension and removed the supernatant, then added siRNA (2 μL times) or DNA plasmid (10 μg / time) to the precipitate and resuspended the precipitate with 100 μL Amaxa electroporation buffer (for mouse neurons, Lonza Cologne GmbH, Germany), then quickly transferred the cells to a 2.0-mm electroporation cup and electroporated with an ECM830 electroporator (35V, 15ms pulse, 950ms interval 3 times). After electroporation, the cells were immediately inoculated in a culture plate with MEM medium containing 10% FBS. After the cells were completely attached to the slide (4-6 hours), they were replaced with MEM medium containing 5% FBS.

[0027] The observation of eGFP expression in DRGs cells is a sign of successful transfection. Three days after transfection, the axon length of eGFP and Tuj1 double-positive cells was measured by staining with the neuron-specific antibody Tuj1; live mouse DRGs were infected with AAV2-Caspr4 and Caspr4 protein was overexpressed, and AAV2-eGFP infection was set as a negative control. One week after infection, the infected DRG neurons were isolated and cultured in vitro for 3 days, and the axon length of eGFP and Tuj1 double-positive cells was measured.

[0028] 2) Axon regeneration in vivo experiments:

[0029] The unilateral DRG neurons of the 4th-5th lumbar vertebrae of adult mice were selected as experimental subjects. The targeted siRNA and eGFP plasmids for Caspr4 were transfected into the target DRG neurons by in vivo electroporation technology, and negative siRNA and eGFP were set as control groups. The Caspr4 targeted siRNA used was purchased from Gene Shanghai Co., Ltd., and the sequence of siRNA is:

[0030] 5,-GGAGCACCUUUCAUUGUAATT-3,(sense strand),

[0031] 5, -UUACAAUGAAAGGUGCUCCTT-3, (antisense strand).

[0032] For electroporation, mice were first deeply anesthetized, the back hair was removed, the skin was disinfected with iodine, the skin, subcutaneous tissue and muscle were cut layer by layer, the L3-L5 lumbar vertebra on one side was exposed, and the spinal canal on one side was carefully opened to expose the DRG connected to the sciatic nerve. With a microinjection glass needle connected to the Picospritzer III (Parker Inc., USA) instrument, siRNA and plasmid solution were carefully injected into the DRG tissue until it was slightly bulged. Afterwards, electroporation was performed with homemade tweezer-shaped electrodes (diameter 12.0 mm) and BTX square wave electroporator ECM 830 (60 V, 15 ms pulse, 950 ms interval, 4 times) to allow the target siRNA and plasmid to enter the cells.

[0033] After surgery, the muscles and skin were sutured and disinfected with iodine to prevent infection. Two days later, the mice were anesthetized again, the sciatic nerve on the same side of electroporation was exposed, and 5 #The microtweezers were clamped for 2 seconds, and the clamping site was marked with a 10-0 line. Suturing and iodine disinfection were performed after surgery. Three days after the clamping, the sciatic nerve was removed from the mouse by perfusing it with 4% PFA. Afterwards, the surrounding connective tissue was carefully removed under a stereomicroscope, and finally the nerve was flattened on an adhesion slide, pressed with a weight of about 10 kg for three hours, and then sealed with a sealing agent. The axon regeneration in the sciatic nerve was then analyzed under a fluorescence microscope. The target DRG neurons were infected with AAV2-Caspr4 to overexpress the neuronal Caspr4 protein, and AAV2-eGFP infection was set as a negative control to detect the length of eGFP-labeled axons to evaluate axon regeneration.

[0034] like Figure 2 As shown, after interfering with the expression of the Caspr4 gene in DRGs, the regenerated axons became significantly longer. The results suggest that the Caspr4 protein inhibits the regeneration of axons in damaged nerves.

[0035] 4. Establishment of Optic Nerve Regeneration Model

[0036] Adult C57BL / 6J mice were intraperitoneally injected with ketamine at a dose of 100 mg / kg. After the animals were deeply anesthetized, the conjunctiva was cut on the temporal or lateral side of the eyeball, and the optic nerve was exposed in the orbit by blunt dissection. The optic nerve was clamped for 2 seconds at a distance of about 1 mm from the back of the eyeball using No. 5 microtweezers. Blood vessels were carefully avoided during the operation, and the eyes were monitored for signs of bleeding after the injury. Buprenorphine (0.05 mg / kg, Bedford, Laboratories, Bedford, MA) was given postoperatively for analgesia. Penicillin ointment was applied to the eyeball during and after the operation to prevent corneal drying. All mice with postoperative ocular vascular damage were immediately euthanized.

[0037] 5. Detect the function of Caspr4 in optic nerve axon regeneration.

[0038] Caspr4 interference virus (AAV2-shCaspr4), overexpression virus (AAV2-Caspr4) and negative control virus (AAV2-eGFP) were injected into the mouse vitreous to infect retinal ganglion cells (RGCs). Optic nerve was crushed 2 weeks after virus injection, and CTB-555 was microinjected into the vitreous 2 weeks after crush to mark the regenerated axons anterogradely. Optic nerve and retinal tissue were collected 2 days after labeling to detect optic nerve regeneration; immunofluorescence staining was used to detect the number of Tuj1-positive cells in the entire retina to analyze the survival of RGCs, and Caspase3 staining was used to detect the apoptosis of RGCs.

[0039] like Figure 3As shown, knocking down the expression of Caspr4 gene in RGCs can significantly regenerate optic nerve axons.

[0040] The present invention detects the function and mechanism of Caspr4 in axon regeneration in two models: sciatic nerve axon regeneration and optic nerve axon regeneration. In the axon regeneration process of adult mammals, Caspr4 is an inhibitory molecule. Reducing the expression of Caspr4 gene in neurons can promote axon regeneration, providing a new direction and theoretical basis for the treatment of axon regeneration in the future.

[0041] The above disclosure is only the preferred embodiment of the present invention, which cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. Application of Caspr4 inhibitors in the preparation of axonal damage repair drugs.

2. The use according to claim 1, characterized in that: The Caspr4 inhibitor is a virus that interferes with Caspr4.

3. The use according to claim 2, characterized in that: The Caspr4 interference virus is AAV2-shCaspr4.

4. A drug for treating axonal damage, wherein the active ingredient is a Caspr4 inhibitor.

5. The use according to claim 4, characterized in that: The Caspr4 inhibitor is a small molecule compound of Caspr4.

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