Application of TBCD inhibitor in preparation of medicine for treating and / or preventing Alzheimer's disease

Abstract

The invention finds that the expression quantity of TBCD in high-expression tau protein cells is up-regulated, and experiments show that the high expression of TBCD obviously increases the formation of tau aggregates, which prompts that the inhibition of TBCD can reduce the aggregation of tau protein, so that the TBCD has the potential of treating Alzheimer's disease. The present invention provides a novel therapeutic method for reducing fibrillary tangles of tau protein by inhibition of TBCD. The research result of the invention not only provides a theoretical basis for reducing aggregation of tau protein, but also can provide a new drug target for clinical treatment of Alzheimer's disease.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology and prevention and treatment of neurodegenerative diseases, and more specifically, the present invention relates to the field of tau protein neurofibrillary tangles for treating Alzheimer's disease. The present invention provides a new tau protein aggregation inhibitor with the potential to treat Alzheimer's disease. Background Art

[0002] Alzheimer's disease, one of the most common neurodegenerative diseases in my country, is characterized by older patients, gradual memory loss, and cognitive decline. At present, there is still no way to completely cure Alzheimer's disease. In clinical practice, it is mainly to slow down the further aggravation of the disease by inhibiting the amyloid deposition protein produced by Aβ and the neurofibrillary tangles produced by tau protein. Therefore, studying the pathogenic mechanism of Alzheimer's disease and finding key targets for reducing amyloid protein and neurofibrillary tangles are key research directions in this field.

[0003] The TBCD gene is located on chromosome 17q25.3, has a total length of 1192 amino acids, contains 58 exons, and encodes tubulin folding factor D. In addition, high expression of TBCD is significantly correlated with the folding, stability, phosphorylation, and disordered aggregation of other tubulins. However, the function and mechanism of action of TBCD in Alzheimer's disease have not been reported. Therefore, TBCD can be used as a therapeutic target for Alzheimer's disease associated with tau protein aggregation, providing a new method for the clinical treatment of Alzheimer's disease. Summary of the invention

[0004] The purpose of the present invention is to provide a method for reducing protein aggregation of tau protein in the cytoplasm by inhibiting TBCD, thereby having a potential effect of treating Alzheimer's disease.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides the use of TBCD inhibitors in the preparation of drugs for treating and / or preventing Alzheimer's disease in humans or animals;

[0007] A new approach to reduce pathological protein aggregation in cells by inhibiting TBCD.

[0008] The present invention found through mass spectrometry that the expression of TBCD in cells with high expression of tau protein treated with copper ions was upregulated, and Western blotting showed that the expression of TBCD in cells with high expression of tau protein was upregulated, indicating that the high expression of TBCD was related to the pathological protein aggregation in Alzheimer's disease, and the immunofluorescence detection method showed that inhibiting the expression of TBCD could significantly reduce the protein aggregation in cells. Therefore, in the present invention, we propose an application and detection method of TBCD inhibitor, which provides technical support for the treatment and mechanism research of Alzheimer's disease by reducing the aggregation of tau.

[0009] Furthermore, the inhibitor includes: anti-TBCD antibodies, small molecule inhibitors, RNA interference molecules or antisense oligonucleotides targeting TBCD coding sequences for TBCD protein inhibition.

[0010] Furthermore, the anti-TBCD antibodies include: monoclonal antibodies, polyclonal antibodies and antibody fragments with immunological activity against TBCD or its active fragments, which can specifically recognize and bind to the amino acid sequence or spatial structure of TBCD.

[0011] The antibody of the present invention can be detected by immunoprecipitation, immunohistochemistry or immunofluorescence.

[0012] The small molecule inhibitors of the present invention can be prepared into various dosage forms as required, and can be administered alone or in various combinations.

[0013] The RNA interference molecule targeting the TBCD coding sequence of the present invention is selected from one or more of shRNA, siRNA, miRNA and dsRNA.

[0014] Further, the preferred sequence of the RNA interference molecule siRNA is siRNA1, sense strand: 5′-GGGUGUCAUUACCAUGGAUTT-3′ and antisense strand: 5′-AUCCAUGGUAAUGACACCCTT-3′;

[0015] siRNA2, sense strand: 5′-GGAUGAGAAGGCAGUGCAATT′ and antisense strand: 5′-UUGCACUGCCUUCUCAUCCTT-3′;

[0016] siRNA3, sense strand: 5′-GCAAGCAAGUGAGAAGAUUTT-3′ and antisense strand: 5′-AAUCUUCUCACUUGCUUGCTT-3′.

[0017] SiRNA4, sense strand: 5′-GCCUACCUAUAGAUACCAUTT-3′ and antisense strand: 5′-AUGGUAUCUAUAGGUAGGCTT-3′.

[0018] The RNA interference molecule siRNA described in the present invention uses a transient transfection method.

[0019] The antisense oligonucleotide of the present invention specifically binds to TBCD gene DNA or mRNA to inhibit TBCD gene expression, and is a molecular drug regulated at the gene level, including antisense DNA and antisense RNA that are artificially synthesized or expressed in vivo.

[0020] The present invention provides a method for detecting TBCD, including: immunoblotting detection based on anti-TBCD antibodies, immunofluorescence detection based on anti-TBCD antibodies, immunohistochemistry detection based on anti-TBCD antibodies, targeted mass spectrometry detection and non-targeted mass spectrometry detection.

[0021] The Alzheimer's disease described in the present invention includes: familial Alzheimer's disease, sporadic Alzheimer's disease, and early-onset Alzheimer's disease. The Alzheimer's disease described in the present invention is characterized by one or more of the global dementia manifestations of memory impairment, aphasia, apraxia, agnosia, visual-spatial skill impairment, executive dysfunction, and personality and behavioral changes, including one or more of the early-onset, sporadic, or familial types.

[0022] Currently, in the treatment of Alzheimer's disease, a large number of clinical experiments have shown that the use of drugs to prevent or delay the fibrillary tangles of Aβ or tau protein is an effective method. Therefore, finding drug inhibition targets for Aβ or tau protein and developing inhibitors thereof are the key to treating Alzheimer's disease. The present invention found that the expression of TBCD in cells with high expression of tau protein was upregulated, and experiments showed that high expression of TBCD significantly increased the formation of tau aggregates, suggesting that inhibiting TBCD can reduce the aggregation of tau protein, thereby having the potential to treat Alzheimer's disease. The present invention provides a new treatment method for reducing the fibrillary tangles of tau protein by inhibiting TBCD. The research results of the present invention not only provide a theoretical basis for reducing the aggregation of tau protein, but also provide new drug targets for the clinical treatment of Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The expression of TBCD protein was upregulated in HT22-tau cells with high expression of tau protein treated with copper ions;

[0024] Figure 2 The TBCD protein was successfully knocked down in HT22-Tau cells;

[0025] Figure 3 After knocking down TBCD protein in HT22-Tau cells, the cell survival rate was higher than that of the group with normal TBCD expression. DETAILED DESCRIPTION

[0026] The present invention is described in detail below with reference to specific examples. In the following examples, the experimental methods without specific conditions are usually carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. The percentages in the solutions used are by volume unless otherwise specified.

[0027] Example 1. TBCD protein expression is upregulated in HT22-tau neurons with high tau expression treated with copper ions

[0028] To investigate the expression level of TBCD in Alzheimer's disease cells and Alzheimer's disease cells treated with copper ions, label-free quantitative proteomics analysis of mouse neuronal cells HT22-tau (Nanjing Jingmai Biotechnology Co., Ltd.) that highly express tau before and after copper ion treatment revealed that the abundance of TBCD in HT22-tau treated with copper ions was significantly higher than that in HT22-tau. Western blotting was used to further detect the expression level of TBCD protein in the two cells.

[0029] HT22-tau cells were cultured in a 10 cm dish until the cell density was about 60%. The cells in the control group were subjected to the same operation. 100 μmol / L copper ions (copper sulfate pentahydrate was dissolved in DMEM to prepare a 500 μmol / L stock solution and then diluted) were added to the experimental group cells, and copper ions were not added to the control group cells. When the cell density reached 80%-90% (about 5×10 6Cells were first washed three times with 6 ml PBS (pH 7.4) (purchased from Gibco, USA), and then 300 μl RIPA lysis buffer (purchased from Shanghai Biyuntian Biotechnology Co., Ltd., the main components of the lysis buffer are 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and sodium orthovanadate, sodium fluoride, EDTA, leupeptin and other inhibitors) were added. The cells were lysed on ice for 30 min, centrifuged at 15000g for 30 min at 4 °C, and the precipitate was removed to obtain the protein supernatant. The protein concentration was determined using the BCA kit (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.). The expression level of TBCD protein was detected by Western blotting, and the protein sample loading amount for each lane was 50 μg. Bio rad electrophoresis instrument and Bio rad membrane transfer instrument were purchased from Biorad, USA. TBCD antibody was purchased from Proteitech, catalog number 14867-1-AP. β-actin antibody was used as internal reference and was purchased from Sigma, USA, catalog number A5441.

[0030] The results showed that the protein expression level of TBCD in HT22-tau treated with copper ions was significantly higher than that in control cells HT22-tau ( Figure 1 ), indicating that the expression of TBCD is associated with nerve damage caused by copper ions.

[0031] Example 2. TBCD protein expression is upregulated in HT22-tau neurons with high tau expression treated with copper ions

[0032] HT22-tau cells were cultured in a 10 cm dish until the cell density was about 60%. The cells in the control group were subjected to the same operation. 100 μmol / L copper ions (copper sulfate pentahydrate was dissolved in DMEM to prepare a 500 μmol / L stock solution and then diluted) were added to the experimental group cells, and no copper ions were added to the control group cells. The i-FASP method of ionic liquid was used to pretreat the samples, and the TBCD expression of the cells was detected by targeted proteomics. The pretreatment steps refer to the article Fang F, Zhao Q, Chu H, Liu M, Zhao B, Liang Z, Zhang L, Li G, Wang L, Qin J, Zhang Y. Molecular dynamics simulation-assisted ionic liquid screening for deep coverage proteome analysis. Mol Cell Proteom. 2020; 19(10): 1724–37. The TBCD protein expression level of the experimental group treated with copper ions was higher than that of the control group without copper ion treatment.

[0033] Example 3. Inhibition of TBCD expression increases the survival rate of HT22-tau cells

[0034] 1. siRNA design and synthesis

[0035] Shanghai Jima Pharmaceutical Technology Co., Ltd. designed and synthesized siRNA1 based on the TBCD gene sequence, and also provided a negative control siRNA (siRNA NC) with no sequence homology to the TBCD gene.

[0036] siRNA NC

[0037] Sense strand: 5′-UUCUCCGAACGUGUCACGUTT-3′

[0038] Antisense strand: 5′-ACGUGACACGUUCGGAGAATT-3′

[0039] SiRNA TBCD1 (SiRNA1), sense strand: 5′-GGGUGUCAUUACCAUGGAUTT-3′ and antisense strand: 5′-AUCCAUGGUAAUGACACCCTT-3′;

[0040] SiRNATBCD 2 (SiRNA2), sense strand: 5′-GGAUGAGAAGGCAGUGCAATT′ and antisense strand: 5′-UUGCACUGCCUUCUCAUCCTT-3′;

[0041] SiRNATBCD 3 (SiRNA3), sense strand: 5′-GCAAGCAAGUGAGAAGAUUTT-3′ and antisense strand: 5′-AAUCUUCUCACUUGCUUGCTT-3′.

[0042] SiRNA TBCD 4 (SiRNA4), sense strand: 5′-GCCUACCUAUAGAUACCAUTT-3′ and antisense strand: 5′-AUGGUAUCUAUAGGUAGGCTT-3′.

[0043] 2. Cell Transfection

[0044] HT22-tau cells (10 5 ) were spread onto six-well plates and transfected using the transfection kit Lipofectamine RNAiMAX (purchased from Invitrogen). The control group was transfected with siRNA NC, and the experimental groups were transfected with siRNA TBCD1-3. The company did not synthesize siRNA TBCD4. The experimental operations and reagent dosages can be referred to the instructions of the transfection kit. Protein expression levels were detected using Western blotting (refer to "Molecular Cloning"). TBCD antibody (diluted 1000 times with TBST) was purchased from Proteitech, catalog number 14867-1-AP. β-actin antibody (diluted 1000 times with TBST) was used as an internal reference and was purchased from Sigma, USA, catalog number A5441. The results of Western blotting showed that after the experimental group was transfected with siRNA1 TBCD, the expression level of TBCD was lower than that of the control group ( Figure 2 ), indicating that the transfection was successful and siRNA1 and siRNA2 could knock down the expression of TBCD.

[0045] 3. MTT cell proliferation and toxicity assay

[0046] The initial density of the culture in a six-well plate was 5*10 4 HT22-tau cells were grown on the wall for 24 hours before cell transfection. The transfection kit Lipofectamine RNAiMAX was used for transfection. The control group was transfected with siRNA NC, and the experimental groups were transfected with siRNA TBCD1, siRNA TBCD2, and siRNA TBCD3. The experimental operation and reagent dosage can be referred to the instructions of the transfection kit. When the cell density is about 80%, digestion is performed. 0.1 ml of trypsin is used for digestion for 2 minutes, and 2 ml of DMEM medium is added to blow the cell fluid. After counting the cells, dilute with DMEM medium, take 100 microliters of cell fluid (containing 5*10 3Cells) were added to each well of a 96-well plate. HT22-tau cells were cultured in a 96-well culture dish. After 24 hours of culture, copper ions dissolved in DMEM were added at a final concentration of 100 μmol / L. After 24 hours of culture, 10 μl of MTT (3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide) solution (China Bio-Tech Company, catalog number C0009S) was added to each well. After the cells were cultured for another 4 hours, all the liquid in the wells was discarded, 100 μl of DMSO (dimethyl sulfoxide) was added to each well, and after shaking on a shaker for 3 minutes, the absorbance at 490 nm was detected using an enzyme marker.

[0047] The results showed that when copper ions were added, the survival rate of cells that successfully knocked down TBCD was significantly higher than that of the siRNA NC group ( Figure 3 ), the results showed that inhibition of TBCD expression improved the survival rate of HT22-tau, indicating that inhibition of TBCD expression has potential relevance to the treatment of Alzheimer's disease.

[0048] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of TBCD inhibitors in the preparation of drugs for treating and / or preventing Alzheimer's disease.

2. The use according to claim 1, characterized in that: TBCD inhibitors inhibit tau aggregation in neuronal cells.

3. The use according to claim 1 or 2, characterized in that: The TBCD inhibitor is selected from one or more of the following: one or more of anti-TBCD antibodies for TBCD protein inhibition, RNA interference molecules for TBCD coding sequences, and antisense oligonucleotides.

4. The use according to claim 3, characterized in that: Anti-TBCD antibodies include: one or more of monoclonal antibodies, polyclonal antibodies and antibody fragments with immunological activity that have immunological activity against TBCD or its active fragments and can specifically recognize and bind to the amino acid sequence or spatial structure of TBCD; The RNA interference molecule targeting the TBCD coding sequence is selected from: at least one or more than two of shRNA, siRNA, miRNA, and dsRNA; The antisense oligonucleotide is a molecular drug that specifically binds to TBCD gene DNA or mRNA to inhibit TBCD gene expression and regulates at the gene level, including one or more of artificially synthesized or in vivo expressed antisense DNA and antisense RNA.

5. The use according to claim 4, characterized in that: The sequence of the RNA interference molecule siRNA is one or more of the following: siRNA1, sense strand: 5′-GGGUGUCAUUACCAUGGAUTT-3′ and antisense strand: 5′-AUCCAUGGUAAUGACACCCTT-3′; siRNA2, sense strand: 5′-GGAUGAGAAGGCAGUGCAATT′ and antisense strand: 5′-UUGCACUGCCUUCUCAUCCTT-3′; siRNA3, sense strand: 5′-GCAAGCAAGUGAGAAGAUUTT-3′ and antisense strand: 5′-AAUCUUCUCACUUGCUUGCTT-3′. SiRNA4, sense strand: 5′-GCCUACCUAUAGAUACCAUTT-3′ and antisense strand: 5′-AUGGUAUCUAUAGGUAGGCTT-3′.

6. The use according to claim 2, characterized in that The tau protein includes 6 tau subtypes encoded by the human MAPT gene, which are six isoforms produced by alternative splicing of exons 2, 3 and 10, including one or more of 2N4R, 1N4R, 0N4R, 2N3R, 1N3R and 0N3R.

7. The use according to claim 1, characterized in that The TBCD detection method includes one or more sample processing methods using ionic liquid i-FASP, SPA, SP3, free solution, etc., a method of using a mass spectrometer to detect the expression level of TBCD in the whole proteome or targeted proteome, including one or two of the immunoblotting detection method or the immunofluorescence detection method, including one or two of the MTT method to detect the cell survival rate after high expression or inhibition of TBCD, etc.

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