Application of tesc as a target for prevention and treatment of alzheimer's disease

Abstract

The application discloses application of TESC as an Alzheimer's disease prevention and treatment target and relates to the field of biological medicines. A TESC overexpression (TESC-OE) hippocampal neuron cell line is constructed through lentivirus transfection, and the result shows that TESC-OE has the ability to resist A beta-induced neuron apoptosis and synapse damage. A TESC overexpression model in the hippocampus of a mouse is constructed through stereotactic injection of an adeno-associated virus, and the result shows that the hippocampal gray matter volume in the brain of the TESC-OE mouse is significantly larger than that of a wild type mouse. A beta stereotactic modeling is performed on the basis of TESC overexpression in the hippocampus, and the result shows that the TESC-OE mouse can significantly resist A beta-induced hippocampal atrophy, learning and memory dysfunction and impaired synaptic plasticity. Immunoblotting analysis reveals the molecular mechanism that TESC can increase the expression of anti-apoptotic genes in the hippocampus and reduce the expression of pro-apoptotic genes to exert a neuroprotective effect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of TESC as an Alzheimer's disease prevention and treatment target. BACKGROUND

[0002] Thousands of single nucleotide polymorphisms (SNPs) associated with human brain imaging phenotypes have been discovered by genome-wide association studies (GWAS). These variants can be matched to target genes by methods such as expression quantitative trait locus (eQTL) analysis, but their preparation and accuracy are affected by linkage disequilibrium patterns, minimum allele frequency, tissue-specific sample size and many other problems. More importantly, most GWAS studies only stop at finding SNPs associated with phenotypes, neither explore the biological causal relationship between the two, nor provide specific biological molecular mechanisms, which largely limits the significance of such association to the prevention, diagnosis and treatment of brain diseases characterized by hippocampal damage.

[0003] The hippocampus is a brain structure that is essential for learning, memory and other cognitive functions, and its structural and functional damage exists in many brain diseases such as Alzheimer's disease (AD). As one of the well-known neuroimaging markers of AD, hippocampal volume calculated by structural magnetic resonance imaging (sMRI) is highly heritable, and some GWAS studies have reported dozens of genetic variants associated with hippocampal volume. Among these variants, eQTL analysis shows that rs7294919 or rs4767492 regulates the expression of calcineurin B homologous protein 3 (CHP3) in temporal lobe tissue, including hippocampus, temporal lobe cortex and frontal lobe cortex.

[0004] CHP3, also known as TESCalcin (TESC), is a Ca 2+ / Mg 2+ binding protein that is also an essential cofactor in Na + / H + exchange activity, involved in regulating intracellular pH, cell volume and cytoskeletal organization. However, it is not yet known whether TESC can prevent hippocampal volume shrinkage to prevent and stop the progression of AD and its potential molecular mechanisms. SUMMARY

[0005] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application proposes the use of a substance that specifically up-regulates the expression or activity of TESC protein in the preparation of a product for preventing and treating Alzheimer's disease. By specifically up-regulating the expression or activity of TESC protein, the volume of hippocampal gray matter can be significantly increased, Aβ-induced hippocampal atrophy, neuronal apoptosis and impaired synaptic plasticity can be effectively alleviated, and the learning and memory dysfunction of the Alzheimer's disease mouse model can be improved. The present application reveals a neuroprotective molecular mechanism of increasing the expression of anti-apoptotic genes and reducing the expression of pro-apoptotic genes, and has broad application prospects in the prevention and treatment of Alzheimer's disease.

[0006] The present application also provides a product for preventing and treating Alzheimer's disease.

[0007] The present application also provides the use of TESC gene or TESC protein as a target in the screening or development of drugs for preventing and treating Alzheimer's disease.

[0008] The present application also provides the use of hippocampus-specific overexpression TESC mice in the screening or development of drugs for preventing and treating Alzheimer's disease.

[0009] The use of a substance that specifically up-regulates the expression or activity of TESC protein according to the first aspect of the present application in the preparation of a product for preventing and treating Alzheimer's disease.

[0010] According to some embodiments of the present application, the product is used for treating Alzheimer's disease, and specifically includes at least one of reducing neuronal apoptosis, hippocampal atrophy, impaired synaptic plasticity, increasing the expression of anti-apoptotic proteins, and improving the cognitive behavior of Alzheimer's disease patients.

[0011] According to some embodiments of the present application, the improvement of the behavior of Alzheimer's disease patients includes at least one of alleviating the impairment of cognitive function and alleviating the impairment of learning and memory ability.

[0012] According to some embodiments of the present application, the anti-apoptotic protein includes a Bcl-2 family protein.

[0013] According to some embodiments of the present application, the substance includes at least one of a nucleic acid molecule, an antibody drug, and a recombinant virus.

[0014] The product for preventing and treating Alzheimer's disease according to the second aspect of the present application includes a substance that specifically up-regulates the expression or activity of TESC protein.

[0015] According to some embodiments of the present application, the substance includes at least one of a nucleic acid molecule, an antibody drug, and a recombinant virus.

[0016] According to some embodiments of the present application, the nucleic acid molecule comprises at least one of an antisense oligonucleotide, a dsRNA, a micro RNA, a siRNA and a shRNA.

[0017] According to some embodiments of the present application, the recombinant virus is an adeno-associated virus vector, an adenovirus vector or a lentivirus vector.

[0018] The skilled in the art can choose a variety of known viral vectors to be modified for use in the present application.

[0019] According to some embodiments of the present application, the recombinant virus can express a nucleic acid molecule that specifically up-regulates the expression or activity of a TESC protein.

[0020] According to some embodiments of the present application, the product further comprises a pharmaceutically acceptable carrier or excipient.

[0021] The TESC gene or TESC protein according to the third aspect of the embodiments of the present application is used as a target in screening or developing drugs for preventing or treating Alzheimer's disease.

[0022] The hippocampus-specific TESC-overexpressing mouse according to the fourth aspect of the embodiments of the present application is used in screening or developing drugs for preventing or treating Alzheimer's disease.

[0023] According to some embodiments of the present application, the hippocampus-specific TESC-overexpressing mouse has increased hippocampal gray matter volume, increased number of neurons, improved synaptic plasticity, increased expression of anti-apoptotic proteins and improved cognitive function.

[0024] According to some embodiments of the present application, the AD model is an Aβ 1-42 and IBO mixture stereotaxic injection into the CA1 region of the hippocampus of C57BJ / 6L mice.

[0025] According to some embodiments of the present application, the AD model mouse has significantly impaired cognitive function, increased neuron apoptosis, decreased synaptic plasticity, decreased hippocampal gray matter volume and inhibited expression of anti-apoptotic proteins.

[0026] According to some embodiments of the present application, the AD model mouse has significantly impaired cognitive function, increased neuron apoptosis, decreased synaptic plasticity, decreased hippocampal gray matter volume and inhibited expression of anti-apoptotic proteins.

[0027] The present application has at least the following beneficial effects:

[0028] The TESC-OE mouse constructed based on hippocampal stereotactic injection of the embodiment of the application has improved hippocampal gray matter volume, neuron survival, synaptic plasticity, anti-apoptotic protein expression and cognitive function performance compared with wild-type mice. After continuing Aβ-induced AD modeling, TESC-OE can significantly resist Aβ-induced hippocampal atrophy, neuron apoptosis, impaired synaptic plasticity, inhibited anti-apoptotic protein expression and learning and memory dysfunction. The application explores the regulatory role of TESC in AD at the animal model and cell experiment level, reveals the molecular mechanism of TESC in regulating hippocampal gray matter volume, neuron apoptosis and synaptic plasticity in AD. It is also proved that TESC can be used as a target for preventing and treating AD, which helps to prevent the impairment of cognitive function and learning and memory ability of Alzheimer's disease patients in the early stage, and can be used for guiding gene therapy of Alzheimer's disease, and developing and screening effective anti-Alzheimer's disease drugs, and has broad application prospects in preventing and treating Alzheimer's disease.

[0029] Other features and advantages of the present application will be set forth in the following description, and in part will become apparent to those skilled in the art from the description, or can be learned by practice of the application. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 : TESC expression detection of TESC-OE hippocampal neurons.

[0031] Figure 2 : Synaptic network morphology analysis of TESC-OE hippocampal neurons.

[0032] Figure 3 : Cell activity detection of TESC-OE hippocampal neurons under Aβ induction.

[0033] Figure 4 : TESC expression detection in hippocampus of TESC-OE mice.

[0034] Figure 5 : Hippocampal gray matter volume detection of TESC-OE mice.

[0035] Figure 6 : Hippocampal gray matter volume detection of TESC-OE+Aβ mice.

[0036] Figure 7 : Cognitive behavior detection of TESC-OE, TESC-OE+Aβ mice.

[0037] Figure 8 : Synaptic plasticity analysis of TESC-OE, TESC-OE+Aβ mice.

[0038] Figure 9 : Apoptosis-related protein analysis of TESC-OE, TESC-OE+Aβ mice. DETAILED DESCRIPTION

[0039] The concept and the technical effects of the present application will be described clearly and completely in combination with the embodiments, so that the purpose, features and effects of the present application can be fully understood. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0040] The specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments not mentioned by the manufacturer are all conventional products that can be obtained by market purchase.

[0041] In the description of the present application, the terms "comprising" and "having" and any variations thereof are intended to cover the non-exclusive inclusion, for example, the process, method, system, product or equipment comprising a series of steps or units do not have to be limited to the clearly listed steps or units, but can include other steps or units that are not clearly listed or inherent to these processes, methods, products or equipment.

[0042] 1. TESC expression detection of TESC-OE hippocampal neurons

[0043] (1) TESC-OE plasmid construction and virus packaging

[0044] The process of constructing TESC overexpression (TESC-OE) plasmid is roughly as follows: after obtaining the TESC gene coding sequence from the mouse genome template, it is inserted into the enzyme-digested PCDH-CMV-MCS-EF1-T2A-Puro plasmid by homologous recombination. At the same time, the PCDH-CMV-MCS-EF1-T2A-Puro plasmid without containing TESC coding sequence is used as a control plasmid. Then, the TESC-OE plasmid and packaging plasmid (PAX8, VSVG) are transfected into 293T cells to produce TESC-OE lentivirus; the control plasmid and packaging plasmid (PAX8, VSVG) are transferred into 293T cells to produce control lentivirus.

[0045] (2) HT22 cell infection and positive cell screening

[0046] The HT22 cells with good growth state and density reaching 60-80% are centrifuged at 1000 rpm for 5 min. The cell precipitate is washed with an appropriate amount of PBS and centrifuged at 1000 rpm for 5 min. The cell precipitate is resuspended with the virus-containing culture supernatant, and is uniformly spread in the culture dish, and an appropriate amount of HI solution is supplemented to provide nutrition and balance pH. After 12 h of infection, the solution in the culture dish is replaced with complete culture medium.

[0047] After 48h of continuous culture, fresh medium was added and puromycin solution was added to a final concentration of 2 μg / mL. Meanwhile, a set of HT22 cells without virus infection was prepared as a puromycin efficacy detection control. The screening was completed after 48h of continuous culture when the cells in the control were all dead. If the control was not all dead, the drug was continuously added until the control was all dead. The cells were continuously cultured, and the RNA of the positive cells after drug screening was extracted. Real-time quantitative PCR (RT-qPCR) was used to detect the overexpression efficiency at the mRNA level, and the protein of the positive cells was collected to detect the overexpression efficiency at the protein level by Western blotting.

[0048] The results of TESC expression detection of TESC-OE hippocampal neurons are shown in FIG. 2. The expression level of TESC protein and the relative expression amount of mRNA in TESC-OE hippocampal neurons were significantly increased, which proved the successful construction of the hippocampal neuron model with specific up-regulation of TESC. Figure 1

[0049] 2. Synaptic network morphology analysis of TESC-OE hippocampal neurons

[0050] The TESC-OE cells and the control cells were seeded on the coverslips in a 24-well plate at a density of 5 x 10 4 When the cell density reached 60-80%, cell immunofluorescence imaging was performed. The specific steps are as follows: the culture medium was discarded, and the coverslips with cells were washed with PBS for 2 times, 3 min each time; the cells were fixed with 4% paraformaldehyde for 15 min, and the coverslips were washed for 3 times, 5 min each time; the coverslips were permeabilized with 0.5% Triton X-100 at room temperature for 20 min, and washed for 3 times, 3 min each time; the residual PBS on the coverslips was absorbed with absorbent paper, and 5% BSA solution was added to the coverslips, which were incubated at room temperature for 30 min; the coverslips were washed with PBST for 3 times, 5 min each time; the primary antibody (MAP2, 1:500 dilution) was added to the coverslips, which were incubated in a wet box at 4°C overnight; the primary antibody was recovered, and the coverslips were washed with PBST for 3 times, 5 min each time; the secondary antibody (goat anti-rabbit IgG, 1:1000 dilution) was added, and the coverslips were incubated at room temperature for 1 hour in the dark; the coverslips were washed with PBST for 3 times, 5 min each time; DAPI solution (1:2000 dilution) was added to the coverslips for nuclear staining, and the coverslips were incubated in the dark for 5 min; the coverslips were washed with PBST for 4 times, 5 min each time; the liquid on the coverslips was absorbed, and the coverslips were sealed with an anti-fluorescence quenching mounting medium; finally, the coverslips were observed and images were collected under an inverted fluorescence microscope equipped with NIS imaging software. As shown in FIG. 3, the TESC-OE hippocampal neurons showed thicker neurites and more complex connection networks than the control (Control) neurons. Figure 2 ​​

[0051] 3. Detection of cellular viability of TESC-OE hippocampal neurons under Aβ induction

[0052] This experiment was conducted according to the instructions of the CCK-8 kit. The specific procedure included: first, TESC-OE cells and control cells were inoculated at 1×10⁻⁶ cells / mL. 4 Cells were seeded at a density of 100 cells / well in 96-well plates, and then different concentrations (0 μM, 4 μM, 6 μM, and 8 μM) of Aβ were added. 1-42 After incubating with the oligomer solution for 24 hours, washing, and adding 10 μL of CCK-8 reagent to each well, the cells were incubated at 37°C in the dark for 0.5 hours. Finally, the absorbance at 490 nm was measured using a multi-mode microplate reader. Five replicates were performed for each cell type at each concentration. The results are as follows: Figure 3 As shown, Figure 3 China and Israel Aβ 1-42 The concentration of oligomers was plotted on the x-axis, and cell viability on the y-axis to examine changes in cell viability in the absence of Aβ. 1-42 Under oligomer treatment, there was no significant difference in cell viability between TESC-overexpressing (TESC-OE) cells and control cells. With Aβ... 1-42 Increased oligomer concentration significantly reduced HT22 cell viability, indicating that Aβ... 1-42 Oligomers can lead to neuronal death; however, HT22 cells overexpressing TESC showed significantly higher viability than control cells (all significant P < 0.001), indicating that TESC overexpression can resist Aβ. 1-42 Oligomer-induced neuronal death.

[0053] 4. Detection of TESC expression in the hippocampus of TESC-OE mice

[0054] (1) Animal husbandry

[0055] This study used 8-week-old male C57BL / 6J mice, weighing 21-23g, purchased from Beijing Huafukang Biotechnology Co., Ltd. They were housed under standard experimental conditions, maintaining a 12h / 12h light / dark cycle environment, and provided with sufficient food and water. All animal experiments were conducted in accordance with the requirements of the Animal Protection and Use Ethics Committee of Tianjin Medical University (IACUC No. E2015093).

[0056] (2) Stereoscopic injection of hippocampal infection

[0057] C57BL / 6J mice were divided into the following three groups (n = 20 / group): wild type group (denoted as WT), sham operation group (injected with AAV-GFP virus, denoted as Sham), and TESC-OE group (injected with AAV-TESC-OE virus). The specific steps are as follows: after the mice were weighed, they were placed in the anesthesia induction box, and 3% isoflurane was used to induce anesthesia for about 3-5 min, and then the mice were quickly fixed on the stereotaxic instrument; the hair on the top of the mouse's head was removed, and after alcohol disinfection, the scalp was clamped with forceps, cut along the midline, and probed on both sides to expose the skull; after positioning the origin, completing the front-back and left-right adjustment, the syringe was moved back to the origin.

[0058] The coordinates of the hippocampal CA1 region are: anterior-posterior, -2.0 mm; left-right, ±1.5 mm; dorsal-ventral, -1.0 mm. Raise the syringe, move the microsyringe 2.0 mm backward along the Y axis and 1.5 mm rightward along the X axis, and lower the syringe. When the front end of the syringe reaches the surface of the skull, make a mark on the surface of the skull. Raise the syringe to a slightly higher position, drill a hole at the mark with a skull drill, expose the brain tissue, and moisten it with normal saline. The contralateral treatment requires moving the syringe leftward along the X axis to the position where the digital display is 1.5 mm, and the rest of the operations are the same as for the right side. Recalibrate the bregma, move the microsyringe containing the virus to the position above the hole in the target brain region, lower the syringe to Z axis -1.0 mm, and slowly and evenly during the needle insertion process. Use a microsyringe pump to maintain the injection speed at 100 nL / min during the injection process, inject 1 μL of virus (virus titer is 3.5 × 10 12 vp / mL) per side, and after the injection is completed, the needle stays at the injection site for 10 min, then the syringe is slowly removed, and the wound is sutured.

[0059] (3) Western blot analysis of hippocampal tissue

[0060] After the mice were killed, the bilateral hippocampal tissues were quickly separated on ice and placed in a 1.5 mL centrifuge tube containing RIPA lysis buffer. The lysis buffer needs to be added with PMSF protease inhibitor in advance before use. The tissue was quickly cut with an ophthalmic scissors, vortexed at the maximum speed for 10 s, and then placed in an ice bath and transferred to a tissue homogenizer. The sample was ultrasonicated for 10 min. Finally, the supernatant was transferred to a new 1.5 mL centrifuge tube, and an appropriate amount of protein loading buffer was added. The sample was placed in a 95°C metal bath and heated for 30 min to denature the protein. The sample was added to a polyacrylamide gel for separation. The protein bands separated from the gel were transferred to a PVDF membrane by transfer electrophoresis. The transfer membrane was removed and placed in blocking solution, and blocked at room temperature on a shaker for 1 h. The blocked PVDF membrane was placed in TESC primary antibody working solution (1:1000 dilution) and reacted at 4°C overnight. The free primary antibody was removed by TBST washing three times. The reaction membrane was placed in secondary antibody working solution (horseradish peroxidase-labeled goat anti-rabbit IgG, 1:2000 dilution) and reacted for 2 h. The free secondary antibody was removed by TBST washing three times. Finally, the band was detected by enhanced chemiluminescence (ECL), and the fluorescent signal was obtained by Image Lab gel imaging analysis system. Image J software was used for quantitative analysis of band gray value. The ratio of the gray value of the target protein to the gray value of the internal reference protein GAPDH represents the relative expression amount of the target protein. Figure 4 As shown in FIG. 5, the GFP fluorescence signal and the expression level of TESC protein in the hippocampus were significantly increased, indicating that the mouse model with specific up-regulation of TESC in the hippocampus was successfully constructed.

[0061] 5. Detection of hippocampal gray matter volume of Tes-OE and TESC-OE + Ab mice

[0062] (1) High-resolution structural image acquisition

[0063] The high-resolution T2 image data of mouse brain were collected by a 9.4T nuclear magnetic resonance scanner (Bruker BioSpec 94 / 30USR, Germany) in Tianjin Medical University General Hospital. During the scanning process, the mice were in a prone position throughout the process, and the respiration was monitored and the animal body temperature was maintained. During the scanning process, the mice inhaled 1% concentration of isoflurane to maintain anesthesia, while maintaining a breathing rate of 70-100 times per minute. High-resolution 3D-T2 structural images were collected using the coronal fast spin echo relaxation-enhanced TurboRARE sequence, and the specific scanning parameters were as follows: repetition time (TR) = 5500 ms, echo time (TE) = 45 ms, field of view (FOV) = 19.2 x 19.2 mm, matrix size = 192 x 192, slice thickness = 0.3 mm, image resolution = 0.1 mm x 0.1 mm x 0.3 mm, acceleration factor = 8.

[0064] (2) High-resolution structural image processing

[0065] In this study, the magnetic resonance imaging data were preprocessed based on the dcm2nii software, the software package DPABIV4.3 in Matlab (2016b), SPM12 and its plug-in VBM. The specific steps were as follows: using dcm2nii software to convert the collected 3D-T2 structural images into SPM8 (3D NIFTI nii) format; using DPABI V4.3 to magnify the 3D-T2 structural images and TMBTA template voxels; referring to the TMBTA template, using SPM 12 to adjust the coordinate origin of the 3D-T2 structural image. The VBM plug-in in SPM 12 was used to register the 3D-T2 structural image to the stereoscopic space of the TMBTA template, and the images were segmented into gray matter, white matter and cerebrospinal fluid by referring to the TMBTA-gray matter, TMBTA-white matter and TMBTA-cerebrospinal fluid three templates. After regression of the total intracranial volume, the average volume of the bilateral hippocampus and each hippocampal subregion (CA1, CA2, CA3 and DG region) was defined as the relative volume of the hippocampus and its subregion.

[0066] As shown in Figure 5 , the hippocampal volume of TESC-OE group mice was significantly larger than that of WT group and Sham group (OE vs WT: P = 0.012, OE vs Sham: P = 0.003), indicating that TESC overexpression can promote hippocampal volume increase; as shown in Figure 5 and Figure 6 , the volume of Sham + Ab group mice was significantly smaller than that of Sham group mice, indicating that the increase of Ab content in the hippocampus can cause hippocampal atrophy; as shown in Figure 6As shown, the hippocampal volume of mice in the TESC-OE+Aβ group was significantly larger than that of mice in the Sham+Aβ group (P = 0.049), indicating that TESC overexpression can resist Aβ-induced hippocampal atrophy.

[0067] 6. Cognitive and behavioral tests of TESC-OE and TESC-OE+Aβ mice

[0068] The Morris water maze experiment is an experimental method that analyzes an animal's learning, memory, and spatial cognition abilities by recording the time it takes for an animal to learn to swim in a pool and find a hidden underwater escape platform.

[0069] (1) Experimental setup

[0070] The Morris experimental system consists of a water maze apparatus, an automatic image acquisition system, and a software analysis system. The apparatus is a circular pool (100 cm in diameter and 40 cm high) filled half with opaque white water (prepared using colorless and odorless food coloring, at a temperature of 22±1℃). High-contrast geometric patterns are affixed to the pool walls at four points to provide visual cues for the mice. A black curtain surrounds the pool, with four differently shaped pictures hanging on it to prevent distant, variable reference objects from interfering with spatial localization. Four equidistant points N, E, S, and W are marked on the pool walls. These points are then diagonally connected to divide the pool into four quadrants, with the midpoints of each quadrant marked NE, SE, SW, and NW. A hidden platform (6 cm in diameter) is placed 1 cm underwater in the center of the quadrant corresponding to SW.

[0071] (2) Experimental methods

[0072] A. Orientation and Navigation Test: This test needs to be conducted continuously for 5 days, with 4 trials per day. Each trial involves placing a mouse facing the pool wall into the water sequentially from the midpoint of one of the four quadrants (NE, SE, SW, and NW). Each mouse is allowed 60 seconds to swim and search for a hidden platform. The trial ends when the mouse finds and reaches the platform, and the mouse can stay on the platform for 10 seconds. If the mouse fails to find the platform within 60 seconds, the experimenter guides the mouse to the platform and gives it a 10-second rest period.

[0073] B. Spatial exploration experiment: 24 hours after the end of the positioning and navigation experiment, the platform was removed, and the mice were placed into the pool from the midpoint NE of the quadrant farthest from the platform, allowing them to explore freely for 60 seconds. The swimming time of the mice in each quadrant and the number of times they crossed the platform location were recorded.

[0074] like Figure 7As shown in the positioning navigation test, the TESC-OE mice found the hidden platform faster and had shorter escape latency than the WT and Sham group mice; in the spatial exploration test, the TESC-OE mice spent more time in the platform quadrant and crossed the platform more times than the other two groups. The Sham + Ab group mice took longer to find the hidden platform than the Sham group mice, and the time spent in the platform quadrant and the number of times crossing the platform were greatly reduced, indicating that the AD model was successful. The TESC-OE + Ab group mice found the hidden platform faster than the Sham + Ab group mice, and the time spent in the platform quadrant and the number of times crossing the platform were also significantly improved, indicating that overexpression of TESC in the hippocampus of mice improved Aβ-induced cognitive impairment.

[0075] 7. Synaptic plasticity analysis of TESC-OE, TESC-OE + Ab mice

[0076] The long-term potentiation (LTP) of the hippocampal SC-CA1 field excitatory postsynaptic potential (fEPSP) was measured by recording the fEPSP of the hippocampal slice SC-CA1 using a multi-electrode array (MEA) to evaluate the synaptic plasticity of the hippocampus. After the mice were anesthetized, the whole brain was quickly removed and stored in ACSF at 4°C. A brain tissue slice with a thickness of 400 μm was prepared using a vibrating microtome, and after incubation in a 33°C ACSF solution for 1 h, the brain slice was transferred to the MEA perfusion chamber. A nylon mesh was placed on the brain slice to ensure close contact between the brain slice and the electrode array. The MEA consists of 60 extracellular electrodes with a spacing of 200 μm. Each electrode can act as both a stimulating electrode and a recording electrode. After recording the baseline for 20 min, a high-frequency stimulus (HFS) was applied to the electrode point in the hippocampal SC area using an external stimulator to induce LTP, and then the fEPSP of the hippocampal CA1 area was recorded for 100 min using LTP-Director 1.3.2 and LTP-Analyzer 1.3.2. The data were analyzed using Multi-Channel Systems.

[0077] As shown in Figure 8 Compared with the WT and Sham groups, the slope of the local field potential intensity (fEPSP) of the TESC-OE group was significantly increased; compared with the greatly reduced fEPSP slope of the Sham + Ab group, the fEPSP slope of the TESC-OE + Ab group was significantly increased, indicating that overexpression of TESC in the hippocampus of mice can counteract the Aβ-induced synaptic plasticity damage by increasing the long-term potentiation effect.

[0078] 8. Analysis of apoptosis-related proteins in TESC-OE, TESC-OE + Ab mice

[0079] After the mice were sacrificed, the bilateral hippocampal tissues were quickly separated on ice and placed in a 1.5 mL centrifuge tube containing RIPA lysis buffer. The lysis buffer needs to be added with PMSF protease inhibitor in advance before use. The tissue was quickly cut into pieces using an ophthalmic scissors, vortexed at the maximum speed for 10 s, and then placed in an ice bath and transferred to a tissue homogenizer. The tissue was ultrasonicated for 10 min. Finally, the supernatant was transferred to a new 1.5 mL centrifuge tube, and an appropriate amount of protein loading buffer was added. The sample was placed in a 95°C metal bath and heated for 30 min to denature the protein. The sample was added to an SDS-PAGE gel for separation. The protein bands separated from the gel were transferred to a PVDF membrane by transfer electrophoresis. The transfer membrane was removed and placed in blocking solution, and blocked at room temperature on a shaker for 1 h. The primary antibody was diluted with blocking solution at a corresponding ratio. The blocked PVDF membrane was placed in the primary antibody working solution and reacted at 4°C overnight. The free primary antibody was removed by TBST washing three times. The reaction membrane was placed in the secondary antibody working solution (horseradish peroxidase-labeled goat anti-rabbit IgG, 1:2000) and reacted for 2 hours. The free secondary antibody was removed by TBST washing three times. Finally, the band was detected using enhanced chemiluminescence (ECL), and the fluorescent signal was obtained using an Image Lab gel imaging analysis system. Image J software was used for quantitative analysis of the band gray value. The ratio of the gray value of the target protein to the gray value of the internal reference protein GAPDH represents the relative expression amount of the target protein.

[0080] As shown in Figure 9 The expression detection results of the pro-apoptotic protein Caspase3 and the anti-apoptotic protein Bcl2 showed that although the Caspase3 level had no significant difference among the WT, Sham, and TESC-OE groups, the Bcl2 level in the TESC-OE group was significantly higher than that in the WT group and the Sham group, indicating that TESC overexpression had a potential anti-apoptotic effect in normal mice. The Bcl2 level was significantly increased and the Caspase3 level was significantly decreased in the TESC-OE + Aβ group compared with the Sham + Aβ group, indicating that TESC overexpression resisted Aβ-induced neuronal apoptosis by simultaneously promoting anti-apoptotic effect and inhibiting pro-apoptotic effect.

[0081] The above embodiments of the present application are described in detail in combination with examples, but the present application is not limited to the above examples. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the purpose of the present application.

Claims

1. Use of a substance that specifically up-regulates expression of TESC protein in the preparation of a medicament for preventing and treating Alzheimer's disease. The substance is a recombinant virus, and the recombinant virus is an adeno-associated viral vector, an adenoviral vector or a lentiviral vector containing a nucleic acid molecule that promotes expression of TESC protein.

2. Use according to claim 1, characterized in that, The medicament for preventing and treating Alzheimer's disease specifically includes at least one of reducing neuron apoptosis, hippocampal atrophy, impaired synaptic plasticity, increasing expression of anti-apoptotic proteins, and improving cognitive behavior in Alzheimer's disease patients.

3. Use according to claim 1, characterized in that, The medicament also includes a pharmaceutically acceptable carrier or excipient.

4. Use according to claim 1, characterized in that, The specific up-regulation of expression of TESC protein includes constructing a TESC overexpression (TESC-OE) plasmid and constructing a TESC-OE hippocampal neuron cell line by lentiviral transfection; the construction of the TESC-OE plasmid includes obtaining a TESC gene coding sequence, inserting it into a PCDH-CMV-MCS-EF1-T2A-Puro plasmid after enzyme digestion by homologous recombination, and obtaining a TESC-OE plasmid.

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