Application of TBCD inhibitors in the preparation of drugs for the treatment and / or prevention of Alzheimer's disease

Abstract

This invention discovered that TBCD expression is upregulated in cells with high tau protein expression, and experiments showed that high TBCD expression significantly increases the formation of tau aggregates, suggesting that inhibiting TBCD can reduce tau protein aggregation, thus possessing the potential for therapeutic treatment of Alzheimer's disease. This invention provides a novel therapeutic method for reducing tau protein fibrillary tangles by inhibiting TBCD. The research findings of this invention not only provide a theoretical basis for reducing tau protein aggregation but also offer new drug targets for the clinical treatment of Alzheimer's disease.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and the prevention and treatment of neurodegenerative diseases. More specifically, this invention relates to the field of tau protein neurofibrillary tangles in the treatment of Alzheimer's disease. This invention provides a novel tau protein aggregation inhibitor with the potential to treat Alzheimer's disease. Background Technology

[0002] Alzheimer's disease, one of the most common neurodegenerative diseases in my country, is characterized by advanced age of onset, gradual memory loss, and cognitive decline. Currently, there is no cure for Alzheimer's disease. Clinically, treatment primarily focuses on slowing the disease's progression by inhibiting the production of amyloid deposits by Aβ and the formation of neurofibrillary tangles by tau protein. Therefore, researching the pathogenesis of Alzheimer's disease and identifying key targets for reducing amyloid protein and neurofibrillary tangles is a key research direction in this field.

[0003] The TBCD gene, located on chromosome 17q25.3, is 1192 amino acids long and contains 58 exons, encoding tubulin folding factor D. Furthermore, high expression of TBCD is significantly associated 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 could serve as a therapeutic target for tau protein aggregation-related Alzheimer's disease, providing a novel approach for its clinical treatment. Summary of the Invention

[0004] The purpose of this invention is to provide a protein aggregation method that reduces the production of tau protein in the cytoplasm by inhibiting TBCD, thereby having a potential therapeutic effect on Alzheimer's disease.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] This invention provides the use of TBCD inhibitors in the preparation of drugs for the treatment and / or prevention of Alzheimer's disease in humans or animals;

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

[0008] This invention utilizes mass spectrometry analysis to reveal that copper ion treatment upregulates TBCD expression in cells with high tau protein expression. Western blotting further confirms this upregulation, indicating that high TBCD expression is associated with pathological protein aggregation in Alzheimer's disease. Furthermore, immunofluorescence assays show that inhibiting TBCD expression significantly reduces protein aggregation in cells. Therefore, this invention proposes an application and detection method for a TBCD inhibitor, providing technical support for the treatment and mechanistic research of Alzheimer's disease by reducing tau aggregation.

[0009] Furthermore, the inhibitors include: anti-TBCD antibodies that inhibit TBCD protein, small molecule inhibitors, RNA interference molecules or antisense oligonucleotides that target the TBCD coding sequence.

[0010] Furthermore, the anti-TBCD antibody includes: monoclonal antibodies, polyclonal antibodies, and immunologically active antibody fragments that are immunologically active against TBCD or its active fragments and are capable of specifically recognizing and binding to the amino acid sequence or spatial structure of TBCD.

[0011] The antibodies described in this invention can be detected by immunoprecipitation, immunohistochemistry, or immunofluorescence.

[0012] The small molecule inhibitors described in this invention can be prepared into various dosage forms as needed, and can be administered alone or in various combinations.

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

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

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

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

[0017] siRNA4, positive strand: 5′-GCCUACCUAUAGAUACCAUTT-3′ and negative strand: 5′-AUGGUAUCUAUAGGUAGGCTT-3′.

[0018] The RNA interference molecule siRNA described in this invention is generated using a transient transfection method.

[0019] The antisense oligonucleotides described in this invention specifically bind to TBCD gene DNA or mRNA to inhibit TBCD gene expression, and are molecular drugs that regulate at the gene level, including artificially synthesized or in vivo expressed antisense DNA and antisense RNA.

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

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

[0022] Currently, numerous clinical trials have demonstrated that using drugs to block or delay the formation of fibrillary tangles of Aβ or tau proteins is an effective method in the treatment of Alzheimer's disease. Therefore, identifying drug targets for Aβ or tau protein inhibition and developing their inhibitors is crucial for treating Alzheimer's disease. This invention discovered that TBCD expression is upregulated in cells with high tau protein expression, and experiments showed that high TBCD expression significantly increases the formation of tau aggregates, suggesting that inhibiting TBCD can reduce tau protein aggregation, thus possessing the potential to treat Alzheimer's disease. This invention provides a novel therapeutic method for reducing tau protein fibrillary tangles by inhibiting TBCD. The research results of this invention not only provide a theoretical basis for reducing tau protein aggregation but also provide new drug targets for the clinical treatment of Alzheimer's disease. Attached Figure Description

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

[0024] Figure 2 Successful knockdown of TBCD protein 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 expressing TBCD normally. Detailed Implementation

[0026] The present invention will be specifically described below with reference to specific embodiments. In the following embodiments, experimental methods without specific conditions are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Guide (New York: ColdSpring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise specified, all percentages in the solutions used are volume ratios.

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

[0028] To investigate the expression levels of TBCD in Alzheimer's disease cells and copper-treated Alzheimer's disease cells, label-free quantitative proteomics analysis was performed on HT22-tau mouse neuronal cells (Nanjing Jingmai Biotechnology Co., Ltd.) that highly expressed tau before and after copper ion treatment. The results showed that the abundance of TBCD in copper-treated HT22-tau was significantly higher than that in copper-treated HT22-tau. Western blotting was used to further detect the expression levels of TBCD protein in both cell types.

[0029] HT22-tau cells were cultured in 10 cm culture dishes until the cell density reached approximately 60%. The same procedure was performed on the control group cells. 100 μmol / L copper ions were added to the experimental group cells (copper sulfate pentahydrate was dissolved in DMEM to prepare a 500 μmol / L stock solution, which was then diluted), while no copper ions were added to the control group cells. After 24 hours of culture, when the cell density reached 80%-90% (approximately 5 × 10⁻⁶ cells / day), the cells were cultured... 6Cells were first washed three times with 6 ml of PBS (pH 7.4) (purchased from Gibco, USA), then 300 μl of RIPA lysis buffer (purchased from Beyotime Biotechnology Co., Ltd., Shanghai; 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 various inhibitors such as sodium orthovanadate, sodium fluoride, EDTA, and leupeptin) were added. Cells were lysed on ice for 30 min, centrifuged at 15000 g for 30 min at 4°C, and the precipitate was removed to obtain the protein supernatant. Protein concentration was determined using a BCA kit (purchased from Beyotime Biotechnology Co., Ltd., Shanghai). TBCD protein expression levels were detected by Western blotting, with 50 μg of protein sample loaded into each lane. The Bio-Rad electrophoresis apparatus and Bio-Rad transfer apparatus were purchased from Bio-Rad Laboratories, USA. The TBCD antibody was purchased from Proteitech, catalog number 14867-1-AP. The β-actin antibody, used as an internal control, was purchased from Sigma-Aldrich, USA, catalog number A5441.

[0030] The results showed that the protein expression level of TBCD in HT22-tau cells after copper ion treatment was significantly higher than that in control cells HT22-tau. Figure 1 This indicates that TBCD expression is associated with copper ion-induced nerve damage.

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

[0032] HT22-tau cells were cultured in 10cm culture dishes until the cell density reached approximately 60%. The same procedure was performed on the control group cells. 100 μmol / L copper ions were added to the experimental group cells (copper sulfate pentahydrate was dissolved in DMEM to prepare a 500 μmol / L stock solution, which was then diluted), while no copper ions were added to the control group cells. The samples were pretreated using the i-FASP method with ionic liquids, and targeted proteomics detection was performed on the TBCD expression of the cells. The pretreatment steps were referenced in 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. Analysis showed that the TBCD protein expression level in the copper-treated experimental group was higher than that in the untreated control group.

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

[0034] 1. siRNA design and synthesis

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

[0036] siRNA NC

[0037] Chain of Justice: 5′-UUCUCCGAACGUGUCACGUTT-3′

[0038] Antonyms: 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), with the sense strand being 5′-GCAAGCAAGUGAGAAGAUUTT-3′ and the antisense strand being 5′-AAUCUUCUCACUUGCUUGCTT-3′.

[0042] siRNA TBCD 4 (siRNA4), with the sense strand being 5′-GCCUACCUAUAGAUACCAUTT-3′ and the antisense strand being 5′-AUGGUAUCUAUAGGUAGGCTT-3′.

[0043] 2. Cell transfection

[0044] Take 10 HT22-tau cells that are growing well and in the logarithmic growth phase. 5 (Numbers) were seeded onto six-well plates and transfected using the Lipofectamine RNAiMAX transfection kit (purchased from Invitrogen). The control group was transfected with siRNA NC, while the experimental groups were transfected with siRNA TBCD1-3, respectively. The company did not synthesize siRNA TBCD4; experimental procedures and reagent dosages were performed according to the transfection kit instructions. Western blotting (referencing *Molecular Cloning*) was used to detect protein expression levels. TBCD antibody (diluted 1000-fold with TBST) was purchased from Proteitech, catalog number 14867-1-AP. β-actin antibody (diluted 1000-fold with TBST) was used as an internal control, purchased from Sigma-Aldrich, catalog number A5441. Western blotting results showed that after transfection with siRNA1 TBCD in the experimental groups, the expression level of TBCD was lower than that in the control group. Figure 2 This indicates successful transfection, and that siRNA1 and siRNA2 can knock down TBCD expression.

[0045] 3. MTT cell proliferation and toxicity detection

[0046] The initial culture density in the six-well plate was 5*10. 4 HT22-tau cells were cultured and adhered to the culture medium for 24 hours before transfection. The Lipofectamine RNAiMAX transfection kit was used. The control group was transfected with siRNA NC, while the experimental groups were transfected with siRNA TBCD1, siRNA TBCD2, and siRNA TBCD3, respectively. Experimental procedures and reagent dosages were performed according to the transfection kit instructions. When the cell density reached approximately 80%, the cells were digested with 0.1 ml trypsin for 2 min, followed by addition of 2 ml DMEM medium and agitation. After cell counting, the cells were diluted with DMEM medium, and 100 μL of cell solution (containing 5 x 10⁵ cells / mL) was collected. 3HT22-tau cells were cultured in 96-well plates for 24 hours. After 24 hours of culture, copper ions dissolved in DMEM were added to a final concentration of 100 μmol / L. After another 24 hours of culture, 10 μL of MTT (3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide) solution (Beyotime Biotech, China, catalog number C0009S) was added to each well. Cells were cultured for another 4 hours. The liquid in the wells was then discarded, and 100 μL of DMSO (dimethyl sulfoxide) was added to each well. The plates were shaken for 3 minutes, and the absorbance at 490 nm was measured using a microplate reader.

[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 inhibiting TBCD expression improved the survival rate of HT22-tau, indicating that inhibiting TBCD expression has a potential association with the treatment of Alzheimer's disease.

[0048] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The use of TBCD inhibitors in the preparation of drugs for the treatment and / or prevention of Alzheimer's disease, characterized in that: The TBCD inhibitor is TBCDsiRNA; The siRNA sequence is one or two of the following: siRNA2, sense strand: 5'-GGAUGAGAAGGCAGUGCAATT' and antisense strand: 5'-UUGCACUGCCUUCUCAUCCTT-3'; siRNA3, sense strand: 5'- GCAAGCAAGUGAGAAGAUUTT -3' and antisense strand: 5'- AAUCUUCUCACUUGCUUGCTT -3'.

2. The application as described in claim 1, characterized in that: TBCD inhibitors suppress the aggregation of tau protein in neuronal cells.

3. The application as described in claim 2, characterized in that, The tau protein includes six tau isoforms encoded by the human MAPT gene. These isoforms are six homologs generated by alternative splicing of exons 2, 3, and 10, including one or more of 2N4R, 1N4R, 0N4R, 2N3R, 1N3R, and 0N3R.

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