Application of FAM134B gene in preparation of medicine for treating Alzheimer disease

Abstract

The invention provides application of an FAM134B gene in preparation of a medicine for treating Alzheimer's disease, and belongs to the technical field of gene therapy. The preparation method comprises the following steps: inserting an FAM134B gene into a plasmid to construct a recombinant plasmid vector for overexpressing the FAM134B gene, packaging the recombinant plasmid vector by using an adeno-associated virus to obtain an adeno-associated virus for overexpressing the FAM134B gene, and injecting the adeno-associated virus for overexpressing the FAM134B gene into a human body or an animal body to realize treatment of the Alzheimer's disease. On the basis of an epigenetic regulation mechanism of the FAM134B and a key effect of the FAM134B in APP removal, APP degradation in an Alzheimer's disease model is promoted and A beta deposition is relieved by targeting an FAM134B-mediated endoplasmic reticulum autophagy pathway, and a brand-new gene therapy strategy is provided for treatment of the Alzheimer's disease.

Description

Technical Field

[0001] The present invention relates to the technical field of gene therapy, and in particular to the application of the FAM134B gene in preparing a drug for treating Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is an irreversible degenerative disorder of the central nervous system with an insidious onset and slow progression, primarily occurring in the elderly or pre-elderly. Key clinical features include memory impairment, impaired visual-spatial abilities, impairment of abstract thinking and computational skills, and personality and behavioral changes. It is the most common type of dementia in the elderly, and the risk of developing the disease increases with age. According to the 2021 World Alzheimer Report, over 50 million people worldwide are living with AD, a number projected to reach 150 million by 2050, making it a significant public health burden.

[0003] The pathological characteristics of AD mainly include the formation of neurofibrillary tangles due to hyperphosphorylated tau protein in neurons and the formation of amyloid plaques due to the deposition of Aβ peptide outside neurons. Among them, the abnormal accumulation of Aβ is considered to be the key event that triggers a series of neurotoxic reactions and cell death. Aβ is generated by the cleavage of amyloid precursor protein (APP) during processing in the endoplasmic reticulum and Golgi apparatus. An increasing number of studies have shown that the occurrence of AD involves not only increased production of Aβ but also impaired clearance of Aβ. In particular, dysfunction of the autophagy-lysosome system is considered to be one of the key factors in the pathogenesis of AD (Nixon RA. The role of autophagy in neurodegenerative disease. Nat Med. 2013 Aug;19(8):983-97).

[0004] Currently, the drugs that improve the cognitive function of AD patients include: 1) Acetylcholinesterase inhibitors (AChEI): including donepezil, rivastigmine, and huperzine A, which mainly increase the level of acetylcholine in the brain and enhance synaptic transmission; 2) N-methyl-D-aspartate (NMDA) receptor antagonists: memantine, which can antagonize NMDA receptors and has the effect of regulating glutamate activity. It is now used to treat patients with moderate to severe AD; 3) Monoclonal antibodies targeting Aβ: Aduhelm is the first new therapy approved for AD since 2003; lecanemab is the second drug ever approved for the treatment of AD by the U.S. Food and Drug Administration (FDA) in January 2023; On March 31, 2025, the new drug for AD treatment-donezumab injection, was prescribed for the first time in the country at Xuanwu Hospital, and 5 patients received treatment at the same time. There are some AD-specific testing reagents, such as the Elecsys β-Amyloid (1-42) and Elecsys Phospho-Tau (181P) CSF test kits, as well as gene therapy research. However, there is no specific drug that can cure AD or effectively reverse the disease process. The combination of drug therapy, non-drug treatment, and careful nursing care can only alleviate symptoms and delay disease progression. The exact pathogenesis remains unclear. Therefore, the development of novel therapeutic strategies that can precisely intervene in the pathological process of AD is urgent.

[0005] Endoplasmic reticulum autophagy (ER-phagy) is a selective autophagy mechanism that recognizes and degrades dysfunctional or overloaded ER structures through specific receptors to maintain ER homeostasis. In highly differentiated cells such as neurons, ER-phagy plays an important role in clearing abnormally aggregated proteins. Since 2015, several ER-phagy receptors have been discovered, including the FAM134 family (FAM134A / B / C), RTN3L, ATL3, TEX264, and CCPG1 (He L. et al. Advances in ER-Phagy and Its Diseases Relevance. Cells. 2021 Sep 6;10(9):2328). Among them, FAM134B was the first receptor discovered to mediate the entry of ER fragments into autophagosomes (Khaminets A. et al. Regulation of endoplasmic reticulum turnover by selective autophagy. Nature. 2015 Jun 18;522(7556):354-8).

[0006] Based on this, the present invention provides the use of the FAM134B gene in the preparation of a drug for treating Alzheimer's disease, providing a new idea for the treatment of Alzheimer's disease. Summary of the Invention

[0007] In view of the above deficiencies in the prior art, the present invention provides the use of the FAM134B gene in the preparation of a drug for treating Alzheimer's disease.

[0008] To achieve the above purpose, the specific technical solutions of the present invention are as follows:

[0009] The present invention provides use of the FAM134B gene in preparing a medicine for treating Alzheimer's disease.

[0010] Furthermore, the nucleotide sequence of the FAM134B gene is shown as SEQ ID No. 1, and the amino acid sequence of the protein encoded by the FAM134B gene is shown as SEQ ID No. 2.

[0011] Furthermore, the application is to insert the FAM134B gene into a plasmid to construct a recombinant plasmid vector that overexpresses the FAM134B gene, then package the recombinant plasmid vector with an adeno-associated virus to obtain an adeno-associated virus that overexpresses the FAM134B gene, and finally inject the adeno-associated virus that overexpresses the FAM134B gene into a human or animal.

[0012] Furthermore, the structural elements of the recombinant plasmid vector include: hSyn, EGFP, P2A, FAM134B, 3xFLAG, and WPRE. Specifically, the sequence of these structural elements is: hSyn-EGFP-P2A-FAM134B-3xFLAG-WPRE. Among the structural elements of the plasmid containing the FAM134B gene, hSyn is a neuron-specific promoter, ensuring that expression is restricted to the central nervous system; EGFP is enhanced green fluorescent protein, used for observing infection and protein expression efficiency in cells or in vivo; P2A is a self-cleaving peptide sequence (linker) that enables co-expression of EGFP and FAM134B proteins; 3xFLAG is a triple FLAG tag consisting of three tandem FLAG peptides, used for subsequent protein tracking and purification, facilitating detection and in vivo tracing of the target protein; and WPRE is a post-transcriptional regulatory element that significantly enhances target gene expression by stabilizing mRNA and improving nucleocytoplasmic transport efficiency. A primer for titration is also designed at this position.

[0013] Furthermore, the adeno-associated virus is of the AAV2 / 9 serotype. The present invention utilizes the AAV2 / 9 serotype adeno-associated virus and utilizes a hybrid viral vector (rAAV2 / 9) produced by combining the AAV2 genome with different capsid proteins. The recombinant virus possesses the stable expression and gene integration capabilities of the AAV2 serotype, while also possessing the tissue infection tropism and good nervous system penetration of other serotypes.

[0014] Furthermore, the method for preparing the adeno-associated virus overexpressing the FAM134B gene comprises the following steps:

[0015] (1) Construction of recombinant plasmid vector: hSyn-GFP-P2A-FAM134B-3 x FLAG-WPRE;

[0016] (2) hSyn-GFP-P2A-FAM134B-3 x FLAG-WPRE, AAV2 / 9 serotype adeno-associated virus, and auxiliary packaging plasmid were co-transfected with cells. After amplification and purification, adeno-associated virus overexpressing the FAM134B gene was obtained.

[0017] Furthermore, the cells are HEK293T cells.

[0018] Furthermore, the injection site is the hippocampus of the brain.

[0019] The present invention also provides a drug for treating Alzheimer's disease, the active ingredient of which includes a recombinant plasmid vector overexpressing the FAM134B gene or an adeno-associated virus overexpressing the FAM134B gene, the nucleotide sequence of the FAM134B gene is shown in SEQ ID No. 1, and the amino acid sequence of the protein encoded by the FAM134B gene is shown in SEQ ID No. 2.

[0020] Furthermore, the structure of the recombinant plasmid vector for overexpressing the FAM134B gene includes hSyn-GFP-P2A-FAM134B-3 x FLAG-WPRE.

[0021] Compared with the prior art, the present invention is beneficial in that:

[0022] Current treatments for Alzheimer's disease (AD) primarily focus on inhibiting Aβ production or promoting its clearance. However, these treatments often rely on antibody drugs or enzyme inhibitors, which can lead to inconsistent efficacy, significant side effects, and imprecise targeting. Furthermore, existing treatment strategies generally overlook the source of Aβ production—an imbalance in the APP metabolic pathway—and its key regulatory mechanism: autophagy. This invention, leveraging the central role of FAM134B in endoplasmic reticulum autophagy, provides the use of the FAM134B gene in the preparation of a drug for treating Alzheimer's disease, as well as a drug for treating Alzheimer's disease, with the following advantages:

[0023] (1) Strong targeting and clear mechanism of action: The present invention uses adeno-associated virus (AAV) to mediate the specific expression of FAM134B in the hippocampus of the brain, restores damaged endoplasmic reticulum autophagy, promotes the degradation of APP, and inhibits the generation of Aβ from the source.

[0024] (2) Significant therapeutic effect: Animal experiments have shown that overexpression of the FAM134B gene significantly improved the learning and memory abilities of 5XFAD mice, reduced Aβ plaque deposition, restored neuronal and synaptic structures, and exhibited a good neuroprotective effect.

[0025] (3) Dependence on the LIR domain and structural controllability: By comparing FAM134B with FAM134B mutLIR The present invention clearly characterizes the functional core region through experiments, which is beneficial for the subsequent development of small drug molecules or optimization of gene construction.

[0026] (4) Easy operation and controllable cost: AAV virus has the characteristics of low immunogenicity and stable expression, which is suitable for clinical transformation; and the virus preparation and injection technology is mature, suitable for large-scale application. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of mouse propagation and virus injection;

[0028] Figure 2 developing periodograms for mouse behavior;

[0029] Figure 3 HE staining results;

[0030] Figure 4 This is an electron micrograph of the morphology of neurons and endoplasmic reticulum in the hippocampus;

[0031] Figure 5 This is an electron micrograph of the myelin sheath in the hippocampus;

[0032] Figure 6 This is an electron micrograph of synapses in the hippocampus;

[0033] Figure 7Statistical graphs of synapse number, membrane thickness, and synaptic cleft;

[0034] Figure 8 The results are for the detection of endoplasmic reticulum marker proteins Calnexin, Climp63, and Reep5;

[0035] Figure 9 Fluorescence image of virus detection in mouse hippocampus;

[0036] Figure 10 This is an image of Aβ immunofluorescence staining in mouse brain;

[0037] Figure 11 This is a statistical graph for Aβ quantification;

[0038] Figure 12 Dot Blot detection of Aβ deposition, Aβ 42 Quantitative statistical chart. DETAILED DESCRIPTION

[0039] The technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0040] The present invention provides the use of the FAM134B gene in preparing a drug for treating Alzheimer's disease. The nucleotide sequence of the FAM134B gene is shown in SEQ ID No. 1, and the amino acid sequence of the protein encoded by the FAM134B gene is shown in SEQ ID No. 2.

[0041] After APP is synthesized and initially glycosylated in the endoplasmic reticulum, it is transported to the Golgi apparatus for further processing and finally secreted and localized to the plasma membrane. In AD models, the degradation of APP is significantly impaired, leading to its accumulation and increased Aβ production. The inventors have discovered that FAM134B can directly bind to APP localized in the endoplasmic reticulum, promoting its encapsulation into autophagosomes and transport to lysosomes for degradation through the endoplasmic reticulum autophagy pathway. This mechanism not only helps maintain APP levels but may also alleviate AD-related Aβ toxicity. More importantly, in the 5XFAD mouse model, FAM134B expression was significantly downregulated, and abnormal histone modifications were present in its promoter region, leading to TFEB / TFE3 transcriptional activator binding disorders. This epigenetic regulatory change ultimately leads to decreased endoplasmic reticulum autophagy activity, increased APP and Aβ deposition, and impaired cognitive function. The inventors further found through AAV-mediated hippocampus-specific FAM134B overexpression experiments that they could significantly restore the cognitive function of 5XFAD mice and reduce the Aβ plaque load, providing a new gene therapy strategy for the treatment of Alzheimer's disease.

[0042] Example 1 Construction and purification of recombinant AAV vector

[0043] Adenovirus is a non-enveloped, linear, double-stranded DNA virus. The linear double-stranded DNA and core protein form a core with a diameter of 60-65 nm, enclosed in a protein coat. The protein coat is a regular icosahedron with a diameter of approximately 80 nm and consists of 252 capsomers with a diameter of 8-10 nm. These capsomers are arranged on triangular faces, with 6 capsomers per side. 240 of these capsomers are hexons (non-vertex capsomers) and the remaining 12 are penton bases (vertex capsomers). Adenoviral vectors are gene therapy vectors developed based on adenoviruses and are capable of infecting both dividing and non-dividing cells. Adenoviral vectors enter cells through receptor-mediated endocytosis, transferring the adenoviral genome to the cell nucleus, where it remains extrachromosomally and does not integrate into the host cell genome. This example aims to construct and prepare adeno-associated virus (AAV2 / 9) expressing human FAM134B (WT or mutLIR).

[0044] (1) Vector construction:

[0045] Human FAM134B (WT) (reference sequence: NM 001034850.3) was subcloned into the pAAV expression vector driven by the hSyn promoter. The ligation sequence was: hSyn-EGFP-P2A-FAM134B WT -3 x FLAG-WPRE; in addition, the control construct included a LIR mutant with impaired autophagy receptor function (FAM134B mutLIRThe mutant lost the ability to bind to LC3B and could not promote endoplasmic reticulum autophagy) expression vector, that is, all 6 amino acids in the LIR region were mutated to alanine by point mutation, that is, the amino acid sequence was mutated from AspAspPheGluLeuLeu to AlaAlaAlaAlaAlaAla, and the nucleotide sequence was mutated from GATGACTTTGAACTACTT to GCTGCCGCTGCAGCAGCT, as well as an empty vector (hSyn-EGFP-P2A-MCS-3 xFLAG-WPRE).

[0046] Amplification primers are designed to include homologous recombination sequences at their 5' ends. These primers are used to amplify the target gene fragment, ensuring that the sequences at the 5' and 3' ends of the amplified product are identical to those at the ends of the linearized cloning vector. A recombination reaction is then prepared using the linearized vector and the target gene amplification product to achieve in vitro circularization of the linearized vector and target gene fragment. The recombinant product is directly transformed, and single colonies are identified by PCR. Positive clones are sequenced and analyzed. The correct clones are cultured and extracted to obtain high-purity plasmids for downstream viral packaging.

[0047] (2) Virus packaging:

[0048] 1) HEK293T (ATCC, Cat# CRL-3216) adenovirus packaging cells are anchorage-dependent epithelial-like cells grown in DMEM supplemented with 10% FBS. HEK293T cells in logarithmic growth phase were dissociated with 0.25% trypsin and seeded into cell culture flasks at a cell density of 30%–40%. Cultured in a 37°C, 5% CO2 incubator. Transfection was possible after 24 hours of culture when the cell density reached 50%–60%. (Change to serum-free medium 2 hours before transfection.)

[0049] 2) Transfect HEK293T cells by mixing 5 µg of each pAAV, pRC, and pHelper plasmid with 350 µL of Opti-MEM and incubating at room temperature for 5 minutes. Simultaneously, mix 15 µL of Neofect with 350 µL of Opti-MEM and incubate at room temperature for 5 minutes. Gently mix the diluted DNA solution with Neofect and incubate at room temperature for 15 minutes to form the DNA / Neofect transfection complex.

[0050] 3) Slowly add the transfection complex dropwise to the HEK293T culture medium, gently shake to mix, and incubate in a 37°C, 5% CO2 incubator. After 6 hours of incubation, replace with fresh medium and continue culturing. Observe the cells daily. If the medium turns yellow, replenish with fresh medium as appropriate. Continue culturing for 10 days. When 50% of the cells begin to detach from the cell wall and most cells show typical cytopathic effect (CPE) under a microscope, collect the cells by low-speed centrifugation and resuspend in 2 mL of DMEM medium. Repeat the freeze-thaw cycle at -80°C / 37°C with shaking three times. Centrifuge at 7000 g for 5 minutes at 4°C. Collect the viral supernatant and store at -80°C until further use.

[0051] 4) Adenovirus Amplification: For the first round of amplification, discard the cell culture medium in the T25 flask (at this point, the HEK293T cell density is approximately 60%) and immediately add 2 mL of the adenovirus supernatant obtained in step 3). Incubate in a cell culture incubator for 90 minutes, then add 3 mL of complete culture medium and continue culturing. When most cells show typical CPE and 50% of the cells have detached from the cell wall, harvest the cells by low-speed centrifugation and resuspend in 2 mL of DMEM medium. Repeat the freeze-thaw cycle at -80°C / 37°C with shaking three times. Centrifuge at 7000 g for 5 minutes at 4°C to collect the viral supernatant. For the second round of amplification, discard the cell culture medium in the T25 flask (at this point, the HEK293T cell density is approximately 90%) and add 2 mL of the viral supernatant obtained in the first round of amplification. Incubate in a cell culture incubator for 90 minutes, then add 10 mL of complete culture medium and continue culturing. When most cells showed typical CPE and 50% of the cells were detached from the cell wall, the cells were collected by low-speed centrifugation and resuspended in 2 mL of DMEM medium. The cells were repeatedly frozen and thawed at -80°C / 37°C and shaken three times. The cells were centrifuged at 7000 g for 5 minutes at 4°C. The viral supernatant was collected and stored at -80°C for later use.

[0052] (3) Virus purification and quality testing:

[0053] 1) Preliminary filtration of 10 mL of the viral supernatant obtained in step (2) was performed through a 0.45 µm filter membrane, followed by further purification through a filtration device, and viral concentration was performed using a high-speed refrigerated centrifuge.

[0054] 2) Determination of viscosity: Use a 20-200 μL pipette to slowly aspirate 50 μL of adenovirus storage solution. No noticeable viscosity or aspiration lag should be observed.

[0055] Sterility test: The virus was added to HEK293T cells for verification. After 24 hours of normal culture, microscopic examination showed no bacterial or fungal contamination. At the same time, referring to the empty cell group, there were no obvious particles in the intercellular space and the culture medium was clear and transparent.

[0056] 3) Adenovirus titer determination - endpoint dilution method: plate a 96-well plate and transfer 1 x 10 3 HEK293T cells; prepare 12 sterile EP tubes, add 990 μL complete medium to the first EP tube, and add 900 μL complete medium to each of the remaining 11 EP tubes; take 10 μL of adenovirus stock solution and add it to the first EP tube for a 1:100 dilution; then take 100 μL of virus dilution solution and add it to the next EP tube for a 1:10 dilution, until the dilution reaches 10 -13 ; 96-well plate discarded old culture medium, added dilution of 10 -13 to 10 -6 Each dilution of the virus solution occupied a row, and 90 μL of the virus dilution solution was added to each of the first 10 wells in each row. 90 μL of complete culture medium without virus was added to wells 11-12 as a control. The 96-well plate was placed in a 37°C, 5% CO2 cell culture incubator and continued to be cultured. After 10 days, the cytopathic effect was observed and the cell wells with CPE were counted. The CPE positive rate after treatment with each dilution of the virus solution was calculated, and the virus titer was calculated. The results are as follows:

[0057] The titer of pcAAV-hSyn-EGFP-P2A-MCS-3 x FLAG-WPRE is 3.57 x 10 13 vg / mL, 100 µL;

[0058] pcAAV-hSyn-EGFP-P2A-FAM134B WT -3 x FLAG-WPRE titer was 2.49 x 10 12 vg / mL, 200µL;

[0059] pcAAV-hSyn-EGFP-P2A-FAM134B mutLIR -3 x FLAG-WPRE titer was 5.49 x 10 12 vg / mL, 200 μL;

[0060] The virus solution was diluted to a final concentration of 1 x 10 12 vg / mL, set aside.

[0061] Example 2 Mouse model injection and behavioral studies

[0062] In this example, normal (WT) and 5XFAD transgenic mice were used for virus injection and subsequent behavioral testing.

[0063] (1) Five-month-old female mice were randomly divided into groups, with 10 mice in each group. The mice were treated as follows:

[0064] Group 1: WT+pcAAV-hSyn-EGFP-P2A-MCS-3 x FLAG-WPRE (referred to as WT+AAV);

[0065] Group 2: 5XFAD+pcAAV-hSyn-EGFP-P2A-MCS-3 x FLAG-WPRE (abbreviated as 5XFAD+AAV);

[0066] Group 3: 5XFAD+pcAAV-hSyn-EGFP-P2A-FAM134B WT -3 x FLAG-WPRE (abbreviated as 5XFAD+FAM134B WT );

[0067] Group 4: 5XFAD+pcAAV-hSyn-EGFP-P2A-FAM134B mutLIR -3 x FLAG-WPRE (abbreviated as 5XFAD+FAM134B mutLIR ).

[0068] (2) Virus injection:

[0069] The anterior fontanelle, also known as the Bregma point, is located at the intersection of the coronal and sagittal sutures. Taking it as the origin O of the three-dimensional coordinate system of the skull, the positions of the bilateral hippocampi are X = ± 1.5 mm, Y = -2.0 mm, and Z = -2.0 mm.

[0070] 1) Weigh the mouse and anesthetize it with an injection of 1% sodium pentobarbital. Remove the top fur of the mouse. Then, insert the mouse's upper incisors into the horizontal bar and adjust the knob to tighten the nose bar. Insert the ear bar into the mouse's ear canal and balance the left and right ear bars so that the line connecting the mouse's ears and the ear bars are aligned. Ensure the scales on the left and right ear bars are aligned, then adjust the knob to tighten the ear bars. Ensure the mouse's nose is facing the center, the head is still, and the tail does not fall when lifted. Visually check that the brain is level (the skull is level).

[0071] 2) The Bregma point location procedure is as follows: Select a central location in the brain and disinfect the area where the top hair was removed with iodine or alcohol. Make a longitudinal incision in the scalp and separate the skin with hemostats. Use a blade to scrape off the periosteum, or use hydrogen peroxide to corrode the periosteum. Completely remove the periosteum to reveal the Bregma point. Use a brain locator to read the Bregma point value as the three-dimensional coordinate origin O. After precisely determining the left and right hippocampal regions, mark and drill holes.

[0072] 3) Using a 10 µL microsyringe, 500 nL of a 1 x 10 12vg / mL virus dilution. Set the injection rate to 50 nL / min and the injection time to 10 min. After the injection is completed, stop the needle for 5 minutes, slowly withdraw the needle, and inject the remaining virus liquid. Inject the other hippocampus in the same way. After the injection is completed, suture it and place it in an incubator to wait for the mouse to recover. Schematic diagram of mouse propagation and virus injection is shown in the figure. Figure 1 shown.

[0073] (3) Postoperative observation: The mice recovered well after surgery and showed no abnormal neurological behavior after one week of observation.

[0074] (4) Behavioral testing time: 3 weeks after surgery, mice were transferred to the behavioral testing room and behavioral testing was performed after one week of adaptation.

[0075] (5) Behavioral testing methods:

[0076] 1) Novel object recognition (NOR) test: Hippocampal-dependent memory was assessed by performing a novel object recognition test in an open-field apparatus (40 × 40 × 40 cm). Before testing, mice were allowed to freely explore the testing apparatus for 10 minutes. On the first day, mice were presented with two identical objects (familiar objects) placed in the left and right corners of the open-field apparatus and allowed to freely explore the objects for 5 minutes. On the second day, one of the two familiar objects was replaced with the other (novel object), and mice were again allowed to freely explore the objects for 5 minutes. The time spent exploring each object (familiar and novel) was recorded with a digital video camera and recorded using the ANY maze software. "Exploration" was defined as touching the object (except with the tail) or sniffing the object (at a distance < 2 cm). Any time the mouse remained on the object without actively scanning or sniffing was not counted as exploration time. To analyze recognition ability, the inventors defined a discrimination index as the ratio of time spent exploring the novel object to the total time spent exploring the familiar and novel objects. Preference for the novel object indicates intact spatial recognition memory.

[0077] 2) Morris Water Maze (MWM): This test exploits the natural swimming and water-phobic nature of mice, forcing them to swim and search for a way out. Climbing onto a submerged platform is the only way for mice to escape the water. Using spatial reference landmarks to locate the platform allows them to escape the water more quickly. By observing and recording the time it takes for animals to learn to swim and find the submerged platform, as well as their swimming trajectories and search strategies, we can analyze and infer their learning, memory, and spatial cognition abilities. A conspicuous marker (e.g., a circle, square, star, triangle, etc.) (of a different color than the water and the maze) is placed above the water surface in each of the four perpendicular directions where the arms of the maze intersect. The water surface is divided into four quadrants according to the location of the markers, and the platform is placed in the center of each quadrant. The platform is placed 1-2 cm underwater (the platform should not be exposed to the surface after adding white dye, as the water surface fluctuates during the mouse's swimming). The platform should not be too deep, as this may cause the mouse to continue swimming after reaching the platform. From Days 1 to 5, each mouse was placed in the midpoint of each of the three quadrants (excluding the platform) once daily. The mouse was allowed to move freely for 1 minute to search for the platform. If the platform was found within 1 minute, the time was recorded, and the animal was allowed to remain on the platform for 3 seconds (less than 3 seconds was not considered to have found the platform) and observe its surroundings. If the animal did not find the platform within 1 minute, it was guided to the platform and remained there for 15 seconds, allowing it to observe its surroundings. On Day 6, the platform was removed from the water, while all other aspects remained unchanged. The mouse was placed into the water maze from the quadrant opposite the platform and allowed to move freely in the water for 1 minute. (At least 30 minutes should be allowed between immersions for the same mouse to allow the animal to recharge.) From Days 1 to 5, the time to first platform arrival (latency), total movement path, distance, and swimming speed were recorded. On Day 6, the time to first platform arrival (latency), number of platform crossings, total time spent swimming in the platform quadrant, total movement path, distance, and swimming speed were recorded within 1 minute. The shorter the latency period, the more likely the mouse has learned to find the platform and the stronger its learning ability.

[0078] Mouse behavioral development cycle diagram Figure 2 The behavioral test results are shown in Table 1.

[0079] Table 1: Behavioral test results

[0080]

[0081] As shown in Table 1, 5XFAD mice injected with FAM134B WT After the injection of adeno-associated virus, learning and memory abilities improved; while the FAM134B with receptor dysfunction mutLIRAdeno-associated virus could not improve the learning and memory defect phenotype of 5XFAD mice. This result indicates that overexpression of FAM134B can alleviate the symptoms of Alzheimer's disease.

[0082] Example 3 Histological and ultrastructural analysis

[0083] 1. Mouse cardiac perfusion and brain tissue dissection

[0084] Before sectioning and immunostaining the mouse brain tissue, to eliminate the interference of blood in the experiment, the mouse heart was perfused with normal saline and then perfused with 4% paraformaldehyde to fix the brain tissue. The brain tissue was then dissected and sectioned. The specific steps are as follows:

[0085] (1) Fixation of the mouse body: After weighing, inject the corresponding dose of 1% sodium pentobarbital intraperitoneally for anesthesia. Place the mouse back in the cage and wait for about 5 minutes. Squeeze the mouse's toes or tail to observe whether it loses its reaction to confirm whether it has entered a deep state of anesthesia. After confirming that the mouse has entered an anesthetized state, place it with its abdomen facing up on an operating table covered with a polystyrene foam board. Fix its limbs with pins to prevent it from moving freely during the perfusion process. Connect the intravenous needle to a 20 mL syringe filled with saline in advance. Take another 20 mL syringe and fill it with 4% paraformaldehyde solution for later use.

[0086] (2) Aortic perfusion: Use ophthalmic forceps to pull up the fur on the outside of the chest cavity, and use anatomical scissors to cut off the skin to expose the white xiphoid process. Use ophthalmic forceps to pull up the xiphoid process, and use anatomical scissors to cut the muscle layer horizontally below the xiphoid process to expose the diaphragm. Use fine scissors to carefully cut the diaphragm, cut the ribs on both sides along the outside of the sternum, turn over the anterior wall of the chest and fix it with hemostatic forceps to completely expose the heart. Use ophthalmic forceps to fix the heart, insert the injection needle from the left ventricle close to the apex at an angle roughly parallel to the left and right midline of the heart, continue to insert the needle in the initial direction, and immediately clamp the needle with a vascular clamp to fix it in the aorta. Use fine scissors to cut a small hole in the right atrium, and you will see dark red blood flowing out. Hold the syringe and push normal saline at a constant speed of about 1mL / 5s. You will see continuous blood flowing out of the right atrium and gradual blood loss from the liver. When the outflowing fluid becomes clear and the liver is completely bloodless, stop the normal saline perfusion. Connect the intravenous needle to a 20 mL syringe prefilled with 4% paraformaldehyde. Remove any bubbles. Hold the syringe and inject the paraformaldehyde at a steady rate of approximately 1 mL / 5 seconds. As the 4% paraformaldehyde flows through the mouse, muscle contractions, an arching of the chest, and a raised tail are observed. The neck may feel stiff when touched, and the liver may turn slightly yellow. After perfusion is complete, remove the needle and remove the mouse from the operating table. The mouse will be in a state of rigor mortis.

[0087] (3) Peeling off the mouse brain: Use dissecting scissors to cut the neck and remove the mouse head to expose the skull. Use dissecting scissors to remove the brain stem and use fine scissors to cut off the muscles around the skull to expose the occipital bone at the base of the skull. Insert the single-sided blade of the fine scissors parallel to the base of the mouse brain under the occipital bone and cut the skull bones on both sides outward. Use dissecting forceps to carefully remove the skull covering the cerebellum. Insert the single-sided blade of the fine scissors into the middle suture of the skull with one side of the blade facing the skull to avoid damaging the brain tissue. Cut the skull bones above the brain to above the olfactory bulb along the middle suture. Insert the scissors into the middle suture straight at the top of the mouse brain and carefully peel off the skull bones with dissecting forceps. Place the mouse brain in 4% paraformaldehyde solution and store at 4°C.

[0088] 2. Histological and Ultrastructural Analysis

[0089] (1) HE staining: Paraffin-embedded brain tissue sections were taken and routine hematoxylin-eosin staining was performed to observe the condensation and disorder of neuronal nuclei. The specific steps are as follows:

[0090] 1) Fixation: Soak the excised brain tissue in Carnoy's fixative (ethanol: chloroform: acetic acid = 6:3:1) for 30 min. Dehydrate in ethanol for 30 min x 2. Dry in ethanol (add appropriate amount of color-changing silica gel) for 1 h x 1. Soak in methyl benzoate at room temperature overnight.

[0091] 2) Embedding: Melt paraffin at a temperature ≥ 60°C. Preheat the paraffin: methyl benzoate solution (1:1) and soak the brain tissue in the preheated mixture for 1 hour. Then, immerse the brain tissue in fresh paraffin solution for 20 minutes x 8. Place the brain tissue in an aluminum embedding box, place it horizontally, add melted paraffin, and let it condense overnight.

[0092] 2) Slicing and baking: Carefully trim the slides (make a table surface) to ensure that the two sides in contact with the card slot are parallel. Slice as continuously as possible, then pick up the slices in a 42℃ water bath, unfold them, and after they are flattened, pick them up with a slide (make sure they are in the middle of the slide to prevent incomplete staining), place them in a baking tray, and bake them at 58℃ overnight.

[0093] 4) Staining observation: Dewaxing with xylene for 15 minutes x 3; anhydrous ethanol for 5 minutes x 1, 95% alcohol for 2 minutes x 1, 75% alcohol for 2 minutes x 1, rinse with distilled water 3-4 times; stain with hematoxylin in the dark for 8 minutes, rinse with running water 3-4 times to remove excess hematoxylin; soak in 1% hydrochloric acid alcohol for 20 seconds, rinse with running water 3-4 times; stain with eosin solution for 2 minutes; 95% alcohol for 5 minutes x 2, anhydrous ethanol for 5 minutes x 1; xylene for 5 minutes x 2; place the slides in a fume hood to dry (lay on oil paper and gently cover, press the edge of the paper with tweezers). After about 20 minutes, seal with neutral resin (control the amount of neutral resin). The staining results are as follows: Figure 3 shown.

[0094] Depend on Figure 3 It can be seen that WT+AAV and 5XFAD+FAM134B WT The nuclei of the neurons in the mouse brain were neatly arranged and had normal morphology, reflecting a relatively complete neuronal morphology; while 5XFAD+AAV and 5XFAD+FAM134B mutLIR The nuclei of neurons in the mouse brain were obviously shrunk, and a large number of vacuolar structures appeared, showing a phenotype of massive neuronal cell death.

[0095] (2) Transmission electron microscopy (TEM): The left hippocampus of the mouse was removed and fixed in 2.5% glutaraldehyde solution for 2 h. Subsequently, the tissue was fixed in 1% osmium hydroxide for 2 h and then dehydrated through a series of gradient ethanol concentrations from 50% to 100%. Next, the tissue was embedded in Spur epoxy resin and cut into 60 nm thick ultrathin sections. Finally, the sections were stained with uranyl acetate and lead citrate. Images were acquired using a transmission electron microscope (JEM-1400plus, JEOL, Japan), and the results are shown in Figure 2. Figure 4-7 As shown, Figure 4 This is a morphological diagram of neurons and endoplasmic reticulum in the hippocampus. Figure 5 This is a map of the myelin sheath in the hippocampus. Figure 6 This is a synaptic map of the hippocampus. Figure 7 The following parameters were observed: endoplasmic reticulum morphology (expansion, rupture); myelin sheath structure (loose, vacuolated); and synaptic structure (number of synapses, synaptic membrane thickness, and synaptic cleft width). The results are shown in Table 2.

[0096] Table 2: Transmission electron microscopy image analysis results

[0097]

[0098] Depend on Figure 4-7 As shown in Table 2, 5XFAD mice injected with FAM134B WT After the virus was injected into the mouse brain, the nuclei of most of the neurons were no longer condensed, and the electron microscopy results showed normal endoplasmic reticulum, regular and intact myelin sheath, increased number of synapses, restored synaptic membrane thickness, and narrowed gaps, indicating that overexpression of FAM134B WT It can promote the active transmission of neurotransmitters; while the injection of FAM134B with dysfunctional receptors mutLIR The virus did not improve neuronal damage in 5XFAD mice.

[0099] Example 5 Protein expression and Aβ deposition detection

[0100] (1) Western blot (WB) detection:

[0101] The corresponding volume of NP-40 lysis buffer was added to the hippocampal tissue, and the tissue was ground and lysed. The total protein was extracted and the expression of APP, Calnexin, Climp63, Reep5 and other proteins was detected. The results are as follows: Figure 8 shown.

[0102] Depend on Figure 8 It can be seen that compared with WT+AVV mice, the degradation of endoplasmic reticulum marker proteins Calnexin, Climp63 and Reep5 in the hippocampus of 5XFAD+AVV mice was significantly inhibited, that is, endoplasmic reticulum autophagy was inhibited; when 5XFAD mice were injected with FAM134B WT After the adeno-associated virus was injected, the levels of Calnexin, Climp63 and Reep5 proteins decreased significantly, while the injection of FAM134B mutLIR There was no significant change in the levels of Calnexin, Climp63 and Reep5 proteins in the hippocampus of 5XFAD mice injected with adeno-associated virus.

[0103] (2) Aβ detection:

[0104] 1) Immunofluorescence: Frozen sections were removed from -20°C, placed at room temperature for a short time, immersed in PBS, and hydrated on a shaker for 10 minutes; blocked with 0.3% H2O2 (30% hydrogen peroxide dissolved in methanol) for 20 minutes to remove intracellular peroxidase, and washed three times with PBS for 5 minutes each; incubated with 0.1% Triton-X 100 at room temperature for 20 minutes, washed three times with PBS for 5 minutes each; blocked with 5% BSA at room temperature for 2-3 hours; stained with anti-Aβ antibody (Cell Signaling Technology, Cat#14974) at 4°C for 48 hours, washed three times with PBS for 5 minutes each; incubated with rabbit secondary antibody at room temperature in the dark for 2-4 hours, washed three times with PBS for 5 minutes each; stained with DAPI for 10 minutes at room temperature in the dark, washed three times with PBS for 5 minutes each, then mounted and photographed. Fluorescence images of virus detection in mouse hippocampus are shown below. Figure 9 As shown, the immunofluorescence staining of Aβ in mouse brain is shown in Figure 10 As shown; Aβ was quantified and the results were as follows Figure 11 shown.

[0105] Depend on Figure 9 It can be seen that green fluorescent signals appeared only in the hippocampus of the mouse brain, indicating that the AAV virus was only injected into the hippocampus and did not spread to other areas of the brain.

[0106] Depend on Figure 10-11 It can be seen that injection of FAM134B WTIn 5XFAD mice injected with adeno-associated virus, the ER morphology was restored, ER autophagy was activated, and APP and Aβ were degraded. mutLIR There was no significant improvement in 5XFAD mice infected with adeno-associated virus. This result indicates that overexpression of FAM134B can improve the function of endoplasmic reticulum autophagy receptors that depend on this protein.

[0107] 2) Dot blot: Use a pencil to draw a grid on a nitrocellulose (NC) membrane. Use a 2.5 µL pipette tip to spot 2 µL of each sample onto the NC membrane in the center of the grid. Allow the membrane to dry at room temperature for 10 min. Block the NC membrane with 5% skim milk in TBST (TBS + 0.1% Tween-20) for 1 h and wash three times with PBS for 5 min each. Incubate with Aβ42 antibody (Thermo Fisher Scientific, Cat# 700254) overnight at 4°C. The next day, remove the primary antibody and wash three times with PBS for 5 min each. Incubate with rabbit secondary antibody for 1 h at room temperature in the dark and wash three times with PBS for 5 min each. Aβ was detected using the BIO-RAD ChemiDoc MP imaging system. 42 Sedimentation level, the results are as follows Figure 12 shown.

[0108] Depend on Figure 12 It can be seen that compared with WT+AVV mice, the Aβ in the hippocampus of 5XFAD+AVV mice 42 Deposition increased significantly; when 5XFAD mice were injected with FAM134B WT After AAV, Aβ 42 The deposition was significantly reduced, almost approaching the level of WT mice.

[0109] In summary, the present invention constructs a recombinant plasmid vector that overexpresses the FAM134B gene by inserting the FAM134B gene into a plasmid, and then packages the recombinant plasmid vector with an adeno-associated virus to obtain an adeno-associated virus that overexpresses the FAM134B gene. Injecting the adeno-associated virus that overexpresses the FAM134B gene into humans or animals can significantly promote the degradation of endoplasmic reticulum marker proteins Calnexin, Climp63, and Reep5 in the hippocampus of the brain, thereby reducing Aβ deposition and improving AD symptoms.

[0110] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details of the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. Application of the FAM134B gene in the preparation of drugs for the treatment of Alzheimer's disease.

2. The use of the FAM134B gene according to claim 1 in preparing a drug for treating Alzheimer's disease, characterized in that: The nucleotide sequence of the FAM134B gene is shown in SEQ ID No. 1, and the amino acid sequence of the protein encoded by the FAM134B gene is shown in SEQ ID No.

2.

3. Use of the FAM134B gene according to claim 1 in preparing a drug for treating Alzheimer's disease, characterized in that: The application is to insert the FAM134B gene into a plasmid to construct a recombinant plasmid vector that overexpresses the FAM134B gene, then package the recombinant plasmid vector with an adeno-associated virus to obtain an adeno-associated virus that overexpresses the FAM134B gene, and inject the adeno-associated virus that overexpresses the FAM134B gene into a human or animal.

4. The use of the FAM134B gene according to claim 3 in preparing a drug for treating Alzheimer's disease, characterized in that: The structural elements of the recombinant plasmid vector include: hSyn, EGFP, P2A, FAM134B, 3 x FLAG and WPRE.

5. Use of the FAM134B gene according to claim 3 in preparing a drug for treating Alzheimer's disease, characterized in that: The adeno-associated virus is of AAV2 / 9 serotype.

6. Use of the FAM134B gene according to claim 3 in preparing a drug for treating Alzheimer's disease, characterized in that: The method for preparing the adeno-associated virus overexpressing the FAM134B gene comprises the following steps: (1) Construction of recombinant plasmid vector: hSyn-GFP-P2A-FAM134B-3 x FLAG-WPRE; (2) hSyn-GFP-P2A-FAM134B-3 x FLAG-WPRE, AAV2 / 9 serotype adeno-associated virus, and auxiliary packaging plasmid were co-transfected with cells. After amplification and purification, adeno-associated virus overexpressing the FAM134B gene was obtained.

7. Use of the FAM134B gene according to claim 6 in preparing a drug for treating Alzheimer's disease, characterized in that: The cells are HEK293T cells.

8. The use of the FAM134B gene according to claim 3 in preparing a drug for treating Alzheimer's disease, characterized in that: The injection site is the hippocampus of the brain.

9. A drug for treating Alzheimer's disease, characterized in that: The active ingredient includes a recombinant plasmid vector overexpressing the FAM134B gene or an adeno-associated virus overexpressing the FAM134B gene. The nucleotide sequence of the FAM134B gene is shown in SEQ ID No. 1, and the amino acid sequence of the protein encoded by the FAM134B gene is shown in SEQ ID No.

2.

10. The drug for treating Alzheimer's disease according to claim 9, characterized in that The structure of the recombinant plasmid vector for overexpressing the FAM134B gene includes hSyn-GFP-P2A-FAM134B-3 x FLAG-WPRE.

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