Application of USP46 inhibitor in preparation of medicine for treating and / or preventing neurodegenerative diseases

Abstract

The invention relates to a method and a reagent for inhibiting aggregation of microtubule-associated protein Tau, in particular to application of a USP46 inhibitor in preparation of a medicine for treating and / or preventing neurodegenerative diseases. The invention finds that the expression quantity of the deubiquitinating enzyme USP46 is up-regulated in a Tau pathology mouse model PS19 (P301S) for the first time, and a cellular level aggregate formation experiment shows that the expression up-regulation of the USP46 obviously increases the formation of Tau aggregates, reveals that the USP46 is closely related to the occurrence and development of Tau pathology, and prompts that the USP46 inhibitor can be used for inhibiting Tau protein aggregation and further relieving the Tau pathology. The research result of the invention not only provides a theoretical basis for the molecular mechanism research of Tau protein aggregation in the occurrence process of neurodegenerative diseases, but also provides a new drug target for slowing down Tau pathology.

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 inhibiting Tau pathology. The present invention provides a new USP46 inhibitor for inhibiting Tau pathology, and its application in neurodegenerative diseases. Background Art

[0002] Misfolding and accumulation of proteins in the brain are common features of a range of neurodegenerative diseases, including those characterized by pathological Tau accumulation. Several neurodegenerative diseases, including Alzheimer's disease (AD), dementia with Lewy bodies (DLB), Parkinson's disease (PD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and related diseases such as Down syndrome (DS), have abnormal Tau protein aggregation in the brain. Understanding the aggregation mechanism of Tau protein remains a major challenge in preclinical research.

[0003] During cell growth and development, ubiquitination plays an important regulatory role in maintaining protein stability and cell function. Deubiquitinating enzymes are often closely related to the abnormal aggregation of some proteins. USP11, as an X-chromosome-associated deubiquitinating enzyme, plays an important role in increasing the susceptibility of Alzheimer's disease in women (Yan Y, Wang X, Chaput D, Shin MK, Koh Y, Gan L, Pieper AA, Woo JA, Kang DE. X-linkedubiquitin-specific peptidase 11 increases tauopathy vulnerability in women. Cell. 2022 Oct 13; 185(21): 3913-3930.). USP11 directly interacts with Tau protein, deubiquitinates Tau protein, and prevents it from being degraded through the ubiquitin proteasome pathway. It is further acetylated under the action of acetylase, causing Tau protein to aggregate. Studies have shown that knocking down USP7 in AD mice significantly slowed down Tau pathology (Kim J, deHaro M, Al-Ramahi I, Garaicoechea LL, Jeong HH, Sonn JY, Tadros B, Liu Z, Botas J, Zoghbi HY. Evolutionarily conserved regulators of tau identify targets for new therapies. Neuron. 2023 Mar 15; 111 (6): 824-838. e7.). USP46 is a member of the deubiquitinase family and plays an important role in the development of the nervous system and the occurrence and development of cancer. However, the role of USP46 in Tau protein aggregation has not been reported.The present invention utilizes mass spectrometry-based quantitative proteomics technology screening and biological function research (such as using protein blotting (refer to Sambrook et al., Molecular Cloning: A Laboratory Guide (New York: Cold Spring Harbor Laboratory Press, 1989) and fluorescence experiments) to find that: 1) compared with wild-type mice (C57BL / 6), USP46 expression is significantly upregulated in PS19 mice (C57BL / 6 transgenic Tau P301S); 2) using protein blotting, real-time quantitative PCR detection found that USP46 expression is significantly upregulated in the Tau pathological model; 3) fluorescence confocal qualitative analysis showed that USP46 significantly increased the formation of Tau aggregates; 4) protein blotting quantitative analysis showed that USP46 caused a significant increase in insoluble Tau protein; 5) siRNA interference experiments found that knocking down USP46 reduced the production of Tau aggregates. The present invention discovered for the first time the molecular function of USP46 in Tau pathology; it was found that inhibiting USP46 can significantly reduce the formation of Tau protein aggregates; therefore, USP46 can be used as a therapeutic target for neurodegenerative diseases associated with Tau pathology, providing a new method for the clinical treatment of typical neurodegenerative diseases such as Alzheimer's disease. Summary of the invention

[0004] The purpose of the present invention is to provide an inhibitor of USP46 for use in reducing or inhibiting Tau aggregation. In order to achieve the above purpose, the present invention adopts the following technical solution:

[0005] The present invention provides a method for inhibiting USP46 by an inhibitor.

[0006] Furthermore, the inhibitor includes: anti-USP46 antibodies for inhibiting USP46 protein, RNA interference molecules or antisense oligonucleotides for USP46 coding sequences.

[0007] Furthermore, the anti-USP46 antibodies include: monoclonal antibodies, polyclonal antibodies and antibody fragments with immunological activity that have immunological activity against USP46 or its active fragments and can specifically recognize and bind to the amino acid sequence or spatial structure of USP46.

[0008] The RNA interference molecule targeting the USP46 coding sequence of the present invention is selected from shRNA, siRNA, miRNA, and dsRNA. Further, the preferred sequence of the RNA interference molecule siRNA is siRNA1, with a sense strand: 5'-GGUCCAGAGCAGUUUCCAATT-3' and an antisense strand: 5'-UUGGAAACUGCUCUGGACCTT-3';

[0009] siRNA2, sense strand: 5′-GCCGUCCAUUCCGGGAGAATT-3′ and antisense strand: 5′-UUCUCCCGGAAUGGACGGCTT-3′;

[0010] siRNA3, sense strand: 5′-GUUGAUGUGGAGCAGAAUATT-3′ and antisense strand: 5′-UAUUCUGCUCCACAUCAACTT-3′.

[0011] The antisense oligonucleotide of the present invention specifically binds to USP46 gene DNA or mRNA to inhibit USP46 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.

[0012] The Tau pathological models described in the present invention include: neurodegenerative diseases, Down syndrome, etc.

[0013] Further, neurodegenerative diseases include: Alzheimer's disease (AD), dementia with Lewy bodies (DLB), Parkinson's disease (PD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD).

[0014] The Tau pathology detection method described in the present invention includes: fluorescence confocal detection of aggregated Tau and immunoblotting detection of insoluble Tau protein.

[0015] The present invention provides a new method for inhibiting Tau protein aggregation by inhibiting USP46. The present invention found that USP46 expression was upregulated in PS19 mice, a Tau pathology model, and overexpression of USP46 increased the formation of Tau insoluble aggregates. Inhibition of USP46 can significantly slow down the aggregation of Tau protein. The present invention provides a new drug target for delaying Tau pathology.

[0016] The present invention relates to a method and reagent for inhibiting the aggregation of microtubule-associated protein Tau. The present invention first discovered that in the Tau pathological mouse model PS19 (P301S), the expression of the deubiquitinase USP46 was upregulated, and the cell-level aggregate formation experiment showed that the upregulated expression of USP46 significantly increased the formation of Tau aggregates, revealing that USP46 is closely related to the occurrence and development of Tau pathology, suggesting that USP46 inhibitors can be used to inhibit Tau protein aggregation and further alleviate Tau pathology. The research results of the present invention not only provide a theoretical basis for the study of the molecular mechanism of Tau protein aggregation in the process of neurodegenerative diseases, but also provide new drug targets for slowing down the occurrence of Tau pathology. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1.USP46 protein overexpression accelerates the formation of Tau aggregates in SH-SY5Y cells. The fluorescence images are co-expression of USP46 and TauP301L (USP46_TauP301L), overexpression of TauP301L and treatment with TauPFF (TauP301L+TauPFF), co-expression of USP46 and Tau and treatment with TauPFF (USP46_Tau+TauPFF), and co-expression of USP46 and TauP301L and treatment with TauPFF (USP46_TauP301L+TauPFF).

[0018] Figure 2 Overexpression of USP46 significantly increased Tau aggregation in 293T cells. The fluorescence images were overexpression of TauP301L and treatment with TauPFF (TauP301L+TauPFF), co-expression of USP46 and TauP301L and treatment with TauPFF (USP46_TauP301L+TauPFF).

[0019] Figure 3 Overexpression of USP46 reduced soluble Tau and significantly increased Tau aggregation. Figure A is the result of immunoblotting, and Figures BD are the quantitative results of total protein (Total), soluble Tau protein (Soluble), and insoluble Tau protein (Insoluble) in Figure A. DETAILED DESCRIPTION

[0020] 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.

[0021] Example 1. Inhibition of USP46 expression reduces Tau aggregation

[0022] 1. siRNA design and synthesis based on USP46 gene (Gene ID: 64854 ) sequences were designed and synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd. siRNA1, siRNA2, and siRNA3; and a negative control siRNA (siRNA NC) with no sequence homology to the USP46 gene was also provided.

[0023] siRNA NC:

[0024] Sense strand: 5'-UUCUCCGAACGUGUCACGUTT-3'

[0025] Antisense strand: 5'-ACGUGACACGUUCGGAGAATT-3'

[0026] siRNA USP46:

[0027] siRNA1, sense strand: 5′-GGUCCAGAGCAGUUUCCAATT-3′ and antisense strand: 5′-UUGGAAACUGCUCUGGACCTT-3′;

[0028] siRNA2, sense strand: 5′-GCCGUCCAUUCCGGGAGAATT-3′ and antisense strand: 5′-UUCUCCCGGAAUGGACGGCTT-3′;

[0029] siRNA3, sense strand: 5′-GUUGAUGUGGAGCAGAAUATT-3′ and antisense strand: 5′-UAUUCUGCUCCACAUCAACTT-3′.

[0030] 2. Cell Transfection

[0031] Take 293T cells (ATCC, CRL-3216) that are growing well and in the logarithmic growth phase for 10 5 The cells 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 group was transfected with siRNA USP46. The experimental operation and reagent dosage were referred to the instructions of the transfection kit. After 48 hours of culture, the protein expression level was detected by Western blotting (refer to "Molecular Cloning"). USP46 antibody was purchased from Wuhan Sanying Biotechnology Co., Ltd., item number 13502-1-AP. HSP90 antibody was used as an internal reference and was purchased from Santacruz Biotochnotogy, USA, item number SC-13119. The results of Western blotting showed that after the experimental group was transfected with siRNA USP46, the expression level of USP46 was lower than that of the control group, indicating that the transfection was successful and siRNA USP46 could achieve the knockdown of USP46 expression. The distribution of Tau protein in soluble and insoluble components was detected using EGFP antibody (recognizing EGFP fused with TauP301L protein). Knockdown of USP46 significantly reduced the production of insoluble Tau, indicating that USP46 plays a key role in Tau protein aggregation.

[0032] Example 2. Overexpression of USP46 protein accelerates the formation of Tau aggregates in SH-SY5Y cells. To study the expression level of USP46 in wild-type mice and PS19 mice, label-free quantitative proteomics analysis of wild-type mice and PS19 mice revealed that the abundance of USP46 in PS19 mice was significantly higher than that in wild-type mice. Further verification was carried out at the cellular level.

[0033] Human neuroblastoma SH-SY5Y cells (ATCC, CRL-2266) were cultured in 12-well plates. When the cell density reached 40%-60% (about 2×10 5 Cells) were transfected. The empty vector group pcDNA3.1-myc-His (PPL plasmid and protein shared library, V855-20) was set as the control group, and the experimental groups were pcDNA3.1-USP46-myc-His plasmid (PPL plasmid and protein shared library, PPL01472-2a, hereinafter referred to as USP46)_TauP301L plasmid (Addgene, 46908), TauP301L+Tau protein precursor fiber (StressMarq, SPR-329, TauPFF), USP46_Tau+TauPFF, and USP46_TauP301L+TauPFF. The plasmid and NEOFECT were added to 100 μl Opti-MEM (Gibco, 11058021) at a ratio of 1 μg of the above plasmid and 1 μl of NEOFECT transfection reagent (Beijing Codein Technology Co., Ltd., TF201201 (TF20121201)) per well, and incubated at room temperature for 20 minutes to obtain a plasmid-NEOFECT complex. The plasmid-NEOFECT complex was added dropwise to the well plate and continued to be cultured in a 37°C, 5% CO2 incubator. After 24 hours, 3 μg of TauPFF (StressMarq, SPR-329) was added to each experimental group and cultured for another 24 hours. After 24 hours, the fluorescence was observed and it was found that only the USP46_TauP301L+TauPFF group produced obvious aggregates ( Figure 1 ), while the P301L+Tau PFF group without USP46 co-transfection did not produce aggregates. This suggests that USP46 plays a key role in the abnormal aggregation of Tau protein.

[0034] Example 3. Overexpression of USP46 significantly increased Tau aggregation in 293T cells

[0035] 293T cells (ATCC, CRL-3216) were cultured in 12-well plates. When the cell density reached 40%-60% (about 2×10 5Cells) were transfected. TauP301L plasmid (Addgene, 46908) + TauPFF (StressMarq, SPR-329) was set as the control group, and USP46 plasmid (PPL plasmid and protein shared library, PPL01472-2a)_TauP301L + TauPFF was set as the experimental group. Plasmids and NEOFECT were added to 100 μl Opti-MEM ( Thermo Fisher Scientific , 11058021), incubated at room temperature for 20 minutes to obtain plasmid-NEOFECT complex. The plasmid-NEOFECT complex was added dropwise to the well plate and continued to be cultured in a 37°C, 5% CO2 incubator. After 24 hours, 3 μg TauPFF (StressMarq, SPR-329) was added to the experimental group and cultured for another 24 hours. After 24 hours, fluorescence was observed and it was found that the USP46_TauP301L+TauPFF group produced more obvious aggregates ( Figure 2 ), while the P301L+TauPFF group without USP46 co-transfection produced fewer aggregates. This further suggests that USP46 plays a key role in the abnormal aggregation of Tau protein.

[0036] Example 4. Overexpression of USP46 reduces soluble Tau and significantly increases Tau aggregation

[0037] 293T (American Cell Culture Collection Center ATCC, CRL-3216) cells were cultured in 12-well plates. When the cell density reached 40%-60% (about 2×10 5 Cells) were transfected. The empty vector group pcDNA3.1-myc-His plasmid (PPL plasmid and protein shared library, V855-20) was set as the control group, and the experimental groups were USP46 plasmid (PPL plasmid and protein shared library, PPL01472-2a), TauP301L plasmid (Addgene, 46908), USP46_TauP301L, TauP301L+TauPFF, and USP46_TauP301L+TauPFF. Plasmids and NEOFECT were added to 100μl Opti-MEM (1μg plasmid and 1μl NEOFECT transfection reagent (Beijing Code Technology Co., Ltd., TF201201 (TF20121201)) in the ratio of 1μg plasmid per well. Thermo Fisher Scientific, 11058021), incubate at room temperature for 20 minutes to obtain the plasmid-NEOFECT complex. The plasmid-NEOFECT complex was added dropwise to the well plate and continued to be cultured in a 37°C, 5% CO2 incubator. After 24 hours, 3 μg Tau PFF (StressMarq, SPR-329) was added to the experimental group and cultured for 48 hours. After 48 hours, the culture supernatant was discarded, the cells were rinsed twice with PBS, the cells were digested with trypsin and collected, and 100 μl Western and IP cell lysis buffer (Shanghai Biyuntian Biotechnology Co., Ltd., P0013) was added to each tube of cell pellet. The main components of the lysis buffer were 20mM Tris (pH7.5), 150mM NaCl, 1% Triton X-100, and sodium pyrophosphate, β-glycerophosphate, EDTA, Na3VO4, leupeptin and other inhibitors. Use a 200μl pipette to blow and beat repeatedly 100 times, and lyse on ice for 30 minutes. Centrifuge at 30000g, 4℃ for 30 minutes, and transfer the supernatant to a new EP tube, which is recorded as the soluble component. The precipitate was washed twice with lysis buffer, and then centrifuged at 30000g, 4℃ for 30 minutes to obtain the precipitate, which was recorded as the insoluble component. Add 100μl 1% SDS TBS solution to each insoluble component, resuspend, and sonicate at 80W, on for 5s, off for 5s for 1min. Use BCA kit (Shanghai Biyuntian Biotechnology Co., Ltd., P0009) to measure the protein concentration of soluble and insoluble components. Use Loading Buffer (Takara, 9156) for protein denaturation. Use immunoblotting to detect Tau protein. The electrophoresis equipment was purchased from BioRad, and 30μg of soluble components and 15μg of insoluble components were loaded. The results showed that the soluble Tau protein in the USP46_TauP301L+TauPFF group was significantly reduced, and the insoluble Tau protein was significantly increased, further suggesting that USP46 plays a regulatory role in Tau aggregation ( Figure 3 ).

[0038] 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 USP46 inhibitors in the preparation of drugs for treating and / or preventing neurodegenerative diseases.

2. The use according to claim 1, characterized in that: USP46 inhibitors can inhibit Tau protein aggregation.

3. The use according to claim 1, characterized in that: The USP46 protein inhibitor is one or more of an anti-USP46 antibody for inhibiting the USP46 protein, an RNA interference molecule or an antisense oligonucleotide for the USP46 coding sequence.

4. The use according to claim 3, characterized in that: The Tau protein is of 3R type or 4R type.

5. The use according to claim 2, characterized in that: The Tau pathology includes one or more of neurodegenerative diseases.

6. The use according to claim 1, characterized in that: Neurodegenerative diseases include: one or more of Alzheimer's disease (AD), progressive supranuclear palsy (PSP), corticobasal syndrome (CBS), Parkinson's disease (PD) and dementia with Lewy bodies (DLB).

7. The use according to claim 1, characterized in that: Antibodies against USP46 include: one or more of monoclonal antibodies, polyclonal antibodies and antibody fragments having immunological activity against USP46 or its active fragments, and capable of specifically recognizing and binding to the amino acid sequence or spatial structure of USP46; The RNA interference molecule targeting the USP46 coding sequence is selected from: at least one or more of shRNA, siRNA, miRNA, and dsRNA; The antisense oligonucleotide is a molecular drug that specifically binds to USP46 gene DNA or mRNA to inhibit USP46 gene expression and regulates at the gene level, including one or more of artificially synthesized or in vivo expressed antisense DNA and antisense RNA.

8. The use according to claim 7, characterized in that: The preferred sequences of the RNA interference molecule siRNA are one or more of the following: siRNA1, sense strand: 5′-GGUCCAGAGCAGUUUCCAATT-3′ and antisense strand: 5′-UUGGAAACUGCUCUGGACCTT-3′; siRNA2, sense strand: 5′-GCCGUCCAUUCCGGGAGAATT-3′ and antisense strand: 5′-UUCUCCCGGAAUGGACGGCTT-3′; siRNA3, sense strand: 5′-GUUGAUGUGGAGCAGAAUATT-3′ and antisense strand: 5′-UAUUCUGCUCCACAUCAACTT-3′.

9. The use according to claim 2, characterized in that: The Tau pathology detection method includes changes in the phosphorylation level of Tau protein or an increase in insoluble Tau protein.

10. The use according to claim 9, characterized in that: After treating Tau pathology models with anti-USP46 antibodies, RNA interference molecules or antisense oligonucleotides, they were characterized by different Tau pathology detection methods.

Images