Application of tendulin fragment in preparation of medicine for treating Tau proteopathy

Abstract

The invention discloses an application of a tendulin fragment in preparation of a medicine for treating Tau proteopathy, and belongs to the technical field of biology. The amino acid sequence of the tenascin fragment is shown as SEQ ID NO.1. The tenascin fragment can inhibit Tau protein aggregation, inhibit cell oxidative stress induced by K18PFFs and improve neuronal apoptosis and synaptic dysfunction induced by K18PFFs, and can be used for preparing a Tau protein aggregation inhibitor and a medicine for treating Tau proteopathy.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to the application of tenomodulin (TNMD) fragments in the preparation of drugs for treating Tau protein diseases. Background Art

[0002] Tauopathies are a type of neurodegenerative disease characterized by abnormal Tau protein deposition in the brain (Kovacs GG (2017) Tauopathies. Handb Clin Neurol 145: 355-368. https: / / 10.1016 / B978-0-12-802395-2.00025-0). Under physiological conditions, Tau protein exists in neurons and interacts with microtubules to maintain the normal morphology of neurons (Avila J, Lucas JJ, Perez M, Hernandez F (2004) Role of tauprotein in both physiological and pathological conditions. Physiol Rev 84 (2): 361-384. https: / / 10.1152 / physrev.00024.2003). However, under pathological conditions, Tau protein undergoes abnormal phosphorylation, acetylation and other modifications to increase aggregation tendency, resulting in loss of its normal function and formation of toxic pathological aggregates, leading to the occurrence of Tauopathy. Tauopathy is divided into different types according to the morphology of aggregates, etiology and other conditions, such as Alzheimer's disease, frontotemporal dementia, progressive supranuclear palsy, etc. (Arakhamia T, Lee CE, Carlomagno Y, Duong DM, Kundinger SR, Wang K, Williams D, Deture M et al (2020) Posttranslational Modifications Mediate the Structural Diversity of Tauopathy Strains. Cell 180 (4): 633-644. https: / / 10.1016 / j.cell.2020.01.027). At present, there is no effective treatment for Tauopathy.

[0003] Tau pathology in the blood and cerebrospinal fluid of patients with tauopathy can characterize the severity of the patient's clinical symptoms (Zhang Y, Wu KM, Yang L, Dong Q, Yu JT (2022) Tauopathies: new perspectives and challenges. Mol Neurodegener 17(1): 28. https: / / 10.1186 / s13024-022-00533-z). Injecting abnormal Tau protein into the brain of healthy mice can induce disease in healthy mice (Iba M, Mcbride JD, Guo JL, Zhang B, Trojanowski JQ, Lee VM (2015) Tau pathology spread in PS19 tau transgenic mice following locus coeruleus (LC) injections of synthetic tau fibrils is determined by the LC's afferent and efferent connections. Acta Neuropathol 130 (3): 349-362. https: / / 10.1007 / s00401-015-1458-4). These research results indicate that clearing or inhibiting Tau protein aggregation in the patient's brain is expected to be an effective method for treating Tau protein disease. Summary of the invention

[0004] The purpose of the present invention is to provide a TNMD protein fragment that inhibits Tau protein aggregation and a preparation method thereof, as well as the use of the TNMD protein fragment in the preparation of a drug for treating Tau disease. The TNMD protein fragment of the present invention can inhibit Tau aggregation and alleviate cell and neuronal damage induced by Tau repeat domain preformed fibrils (K18 PFFs), and has great application prospects for the treatment of Tau disease.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] The present invention provides a TNMD protein fragment for inhibiting Tau protein aggregation, and its amino acid sequence is shown in SEQ ID NO.1.

[0007] The present invention also provides a biological material related to the TNMD protein fragment, which is any one of the following A1) to A4):

[0008] A1) a nucleic acid molecule encoding the TNMD protein fragment, the sequence of which is preferably as shown in SEQ ID NO.2;

[0009] A2) an expression cassette containing the nucleic acid molecule described in A1);

[0010] A3) a recombinant expression vector containing the nucleic acid molecule described in A1), or a recombinant expression vector containing the expression cassette described in A2); the recombinant expression vector is preferably pET28c;

[0011] A4) a recombinant cell containing the nucleic acid molecule described in A1), or a recombinant cell containing the expression cassette described in A2), or a recombinant cell containing the recombinant expression vector described in A3); the recombinant cell preferably uses Escherichia coli BL21 as a host cell.

[0012] The present invention also provides a method for preparing the above-mentioned TNMD protein fragment, which comprises the following steps: inoculating the above-mentioned recombinant cells into a culture medium, obtaining the above-mentioned TNMD protein fragment by culture, or obtaining the above-mentioned TNMD protein fragment by culture and induced expression.

[0013] The present invention also provides the use of the above TNMD protein fragment or biological materials related thereto in the preparation of Tau protein aggregation inhibitors.

[0014] The present invention also provides the use of the above TNMD protein fragment or related biomaterials in the preparation of drugs for treating Tau diseases, which include Alzheimer's disease, frontotemporal dementia, progressive supranuclear palsy, cortical basal degeneration, chronic traumatic encephalopathy, etc.

[0015] Advantages and beneficial effects of the present invention: The TNMD protein fragment of the present invention can inhibit Tau protein aggregation, inhibit K18 PFFs-induced cell oxidative stress, and improve K18 PFFs-induced neuronal apoptosis and synaptic dysfunction. The present invention provides a new drug for the treatment of Tau protein disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The results of the TNMD protein fragment inhibiting Tau aggregation in Example 1. K18 monomers were incubated in the presence or absence of the TNMD protein fragment, and the aggregation kinetics were monitored using the thioflavin T (ThT) assay. The results showed that the TNMD protein fragment significantly inhibited K18 aggregation ( Figure 1 A), n = 3 independent repeated experiments. K18 PFFs were added to Tau RD-YFP HEK293 cells in the presence or absence of TNMD protein fragments. The results showed that K18 PFFs successfully promoted the formation of Tau inclusion bodies, which was inhibited by TNMD protein fragments ( Figure 1B, C), n = 5 independent repeated experiments, scale bar: 20 μm. ANOVA, **P < 0.01, ****P < 0.0001.

[0017] Figure 2 The results of Example 2 that TNMD protein fragment inhibited oxidative stress induced by K18 PFFs. K18 PFFs were added to SH-SY5Y cells in the presence or absence of TNMD protein fragments, and oxidative stress indicators were detected. The results showed that TNMD protein fragments significantly reduced the intracellular reactive oxygen species ( Figure 2 AD), MDA ( Figure 2 E) and hydrogen peroxide ( Figure 2 F) levels, indicating that TNMD protein fragments significantly inhibited K18 PFF-induced oxidative stress. n = 5 independent repeated experiments, scale bar: 50 μm. ANOVA, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0018] Figure 3 This is the result of Example 3 that TNMD protein fragment improves K18 PFFs-induced neuronal apoptosis and synaptic dysfunction. K18 PFFs were added to neurons in the presence or absence of TNMD protein fragments. Tunel results showed that TNMD protein fragments significantly reduced K18 PFFs-induced neuronal apoptosis ( Figure 3 A, B), n = 6 independent repeated experiments, scale bar: 20 μm. DiI staining results showed that TNMD protein fragments significantly improved the decrease in dendritic spine density induced by K18 PFFs ( Figure 3 C, D), n = 8 independent repeated experiments, scale bar: 10 μm. ANOVA, ***P < 0.001, ****P < 0.0001. DETAILED DESCRIPTION

[0019] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0020] [Example 1] TNMD protein fragment inhibits Tau aggregation

[0021] 1. Preparation of recombinant TNMD protein fragments

[0022] The amino acid sequence of the TNMD protein fragment is shown in SEQ ID NO.1, and its coding sequence is shown in SEQ ID NO.2. The NotⅠ and SalⅠ double enzyme digestion method obtains the TNMD protein fragment coding sequence with sticky ends and the pET28c expression vector, and T4 DNA ligase connects the two to insert the TNMD protein fragment coding sequence into the pET28c expression vector to obtain a recombinant plasmid. Subsequently, the recombinant plasmid is transformed into Escherichia coli BL21 to obtain protein expression bacteria. Take an appropriate amount of protein expression bacteria and add it to LB liquid culture medium, place it in a shaker, shake at 37°C and 160rpm for 6 hours, then reduce the shaker temperature to 20°C, and after the bacterial solution is adapted to 20°C for 1 hour, add isopropyl-beta-D-thiogalactopyranoside (IPTG) to make its final concentration 1mM, and shake the bacterial solution overnight (culture 14h). On the second day, the bacteria were collected by centrifugation at 10000 rpm for 10 minutes, and the obtained precipitate was placed in a solvent (20 mM Tris-HCl mixture, pH = 8), followed by ultrasonic treatment at a power of 450 W for 20 minutes. The precipitate was collected by centrifugation at 10000 rpm for 15 minutes, and the precipitate was repeatedly washed with a washing solution (20 mM Tris-HCl, 1 mM EDTA, 2 M urea, 1 M sodium chloride, 1% Triton X-100, pH = 8.0), and the precipitate was collected by centrifugation at 10000 rpm for 15 minutes, and repeated three times. The obtained precipitate was dissolved in a dissolution buffer (20 mM Tris-HCl, 5 mM DTT, 8 M urea, pH = 8.0) and allowed to stand overnight at 4°C. On the third day, centrifuge at 10,000 rpm for 15 minutes to discard the precipitate, slowly add diluent (20 mM Tris-HCl, 0.15 M sodium chloride, pH = 8.0) to the supernatant to gradually renature the protein, stop dilution when the solution becomes slightly turbid, put the obtained solution into a dialysis bag, and dialyze it with double distilled water at 4°C for 24 hours. On the last day, measure the concentration and purity of the obtained soluble protein, freeze the protein solution at -80°C, and then freeze-dry it in a freeze dryer to obtain the target protein dry powder.

[0023] 2. Preparation of K18 Monomer Protein

[0024] First, the pRK172-His-K18 expression plasmid was constructed, specifically, the truncated human Tau coding sequence (SEQ ID NO.3) containing only the microtubule binding domain (K18) was fused with the N-terminal His tag and inserted into the Kpn1 site of the pRK172 bacterial expression vector. Then the plasmid was transformed into Escherichia coli BL21 (DE3). The expression conditions of pRK172-His-K18 were 1mM isopropyl β-D-1-thiogalactopyranoside (IPTG) induced expression at 37°C for 6-8 hours; the induced bacteria were sonicated in a buffer containing 20mM Tris (pH8.0), 500mM NaCl and 5mM imidazole, and the solution was clear and centrifuged at 12000rpm for 30min at 4 degrees; the bacterial supernatant collected after centrifugation was added to the Ni-NTAHis·Bind Resin (MERCK) pre-equilibrated column. The column was washed with 10 column volumes of a buffer containing 20 mM Tris (pH = 8.0), 500 mM NaCl and 15 mM imidazole; the protein was eluted in the same buffer containing 100 mM imidazole; the purified protein was dialyzed against double distilled water for 48 hours, freeze-dried in a freezer, and placed in a -80 degree refrigerator for use.

[0025] 3. K18 PFFs Preparation and ThT Assay

[0026] Take the above-mentioned freeze-dried TNMD protein fragment and K18 monomer protein, dissolve them in PBS, and centrifuge them at 15000rpm for 20 minutes to remove insoluble components. Three groups of solutions were prepared, namely PBS, K18 solution (final concentration 1mg / mL) and K18 (final concentration 1mg / mL) + TNMD protein fragment (final concentration 0.05mg / mL) solution, and DTT (final concentration 2mM) and low molecular weight heparin (final concentration 12.5μM) were added to the three groups of solutions. Then, the solution was incubated on a shaker at 37°C and 1000rpm for 13h. During the incubation period, the aggregation process of K18 in the presence or absence of TNMD protein fragments was evaluated by thioflavin T (ThT) experiment. Specifically: take out a 10μL aliquot from the above incubation mixture, add PBS and ThT to make the total liquid volume 100μL, and the final concentration of ThT is 2mM. After incubation at room temperature for 10 minutes, the fluorescence of the mixture was recorded using a SpectraMax microplate reader at an excitation wavelength of 450 nm and an emission wavelength of 510 nm. The above operation was repeated at different time points, and the results are shown in Figure 1 A, indicating that TNMD protein fragments significantly inhibited K18 aggregation. Among them, the final product of the K18 solution group was K18 PFFs.

[0027] 4. Cell Immunofluorescence

[0028] HEK293 cell lines stably expressing Tau repeat domains (Tau RD-YFP HEK293 cells) produce Tau inclusions when exposed to exogenous K18 PFFs. The morphology of Tau aggregates can be observed under fluorescence microscopy and are used as a cell model for detecting Tau aggregation (Sanders DW, Kaufman SK, Devos SL, Sharma AM, Mirbaha H, Li A, Barker SJ, Foley AC et al (2014) Distinct tau prion strains propagate in cells and mice and define different tauopathies. Neuron 82(6): 1271-1288. https: / / 10.1016 / j.neuron.2014.04.047).

[0029] Tau RD-YFP HEK293 cells were plated in a six-well plate. When the cells adhered and the cell density reached about 50%, TNMD protein fragments (final concentration 0.25 μg / mL) and K18 PFFs (final concentration 5 μg / mL) were added, or equal amounts of PBS and K18 PFFs were added. Only PBS was added as the control group, and the cells were cultured for 36 hours. The culture medium in the six-well plate was removed, and PBS was slowly added along the wall of the well. The six-well plate was gently shaken to wash the cells, and the PBS was removed. Then 4% paraformaldehyde containing 1% Triton X-100 was added to the wells for fixation and permeabilization for 30 minutes. Blocked with 3% bovine serum albumin (BSA) for 30 minutes, stained with DAPI for 5 minutes, and images were taken using a fluorescence microscope. The results are shown in Figure 1 B, C, K18 PFFs successfully promoted the formation of Tau inclusion bodies, and this promotion was inhibited by TNMD protein fragments.

[0030] [Example 2] TNMD protein fragment inhibits K18 PFFs-induced cellular oxidative stress

[0031] SH-SY5Y cells were plated into the dish. When the cells were attached and the density reached about 50%, TNMD protein fragments (final concentration 0.25 μg / mL) and K18 PFFs (final concentration 5 μg / mL) were added, or equal amounts of PBS and K18 PFFs were added. Only PBS was added as the control group. After 36 hours, the culture medium was removed, PBS was slowly added along the well wall, and the dish was gently shaken to wash the cells and remove the PBS. 1 mL of PBS was added to the dish again, and the cells in the dish were resuspended and placed in a centrifuge tube. Centrifuged at 3700 rpm for 5 minutes at room temperature, the supernatant was removed, and the cell pellet was obtained for subsequent operations.

[0032] DCFH-DA (Nanjing Jianshe Bioengineering Institute, E004-1-1) and DHE detection kit (Beyotime, S0063) were used to measure the level of intracellular reactive oxygen species. Malondialdehyde (MDA) detection kit (Nanjing Jianshe Bioengineering Institute, A003-1-2) was used to assess lipid peroxidation in cells. Hydrogen peroxide detection kit (Nanjing Jianshe Bioengineering Institute, A064-1-1) was used to measure the concentration of hydrogen peroxide in cells. All test procedures were performed according to the manufacturer's instructions. All results were normalized to the corresponding total protein content.

[0033] Results Figure 2 TNMD protein fragment significantly reduced the intracellular reactive oxygen species induced by K18 PFF ( Figure 2 AD), MDA ( Figure 2 E) and hydrogen peroxide ( Figure 2 F) levels, indicating that the TNMD protein fragment significantly inhibited the oxidative stress induced by K18 PFF.

[0034] [Example 3] TNMD protein fragment improves K18 PFFs-induced neuronal apoptosis and synaptic dysfunction

[0035] 1. Isolation and culture of neurons: 18-day pregnant C57BL / 6 female mice were killed, and after alcohol disinfection, mouse embryos were removed from their uteri. After washing the embryos with 75% ethanol and sterile PBS, the brains were removed from the head one by one using a dissecting microscope and then transferred to a fresh culture dish containing PBS. The fetal meninges were peeled off, the fetal cerebral cortex was removed, and the cortical tissue was carefully cut with sterile scissors. DMEM (DMEM-HS-FS) containing horse serum (final concentration 5%) and fetal bovine serum (final concentration 5%) was added, and the sterile Pasteur pipette was used to blow gently repeatedly until no large lumps of tissue were visible to the naked eye. The mixture was centrifuged at 1000 rpm for 5 minutes at 4°C, the supernatant was discarded, the precipitate was resuspended in 3 mL of DMEM-HS-FS, blown again, centrifuged, and a neuronal cell suspension was obtained after resuspension. The neurons were placed in a six-well plate with round glass slides according to the ratio of one suckling mouse to a whole six-well plate.

[0036] 2. Tunel staining: After 7 days of neuron culture, TNMD protein fragments (final concentration 0.25 μg / mL) and K18 PFFs (final concentration 5 μg / mL) were added, or equal amounts of PBS and K18 PFFs were added. Only PBS was added as the control group, and neurons were cultured for another 7 days. The culture medium was discarded, PBS was added to wash the neurons, and PBS was discarded. 4% paraformaldehyde was added to fix for 30 minutes to obtain cell slides, and the Tunel staining kit was used for subsequent operations. The results are shown in Figure 3 A, TNMD protein fragment significantly improved K18PFFs-induced neuronal apoptosis.

[0037] 3. DiI staining: After 7 days of neuronal culture, TNMD protein fragments (final concentration 0.25 μg / mL) and K18 PFFs (final concentration 5 μg / mL) were added, or equal amounts of PBS and K18 PFFs were added. Only PBS was added as the control group, and the neurons were cultured for another 7 days. The culture medium was discarded, PBS was added to wash the neurons, and the PBS was discarded. 4% paraformaldehyde was added to fix for 30 minutes, and the PBS was discarded after rinsing with PBS, leaving only a small amount of liquid on the round glass slide. Carefully add DiI crystals (Invitrogen, D282) to the round glass slide and incubate at room temperature for 30 minutes. Then, wash the DiI crystals with PBS. Observe the round glass slide under a confocal microscope (Lecia). To measure the density of neuronal dendritic spines, 8-10 neurons were randomly selected from each group, and the number of dendritic spines on the 50 μm long secondary dendrites of each neuron was counted, and then the dendritic spine density was calculated. See the results. Figure 3 B, TNMD protein fragment significantly improved the decrease in dendritic spine density induced by K18 PFFs.

[0038] The above embodiments are only used to help illustrate the present invention. The implementation methods 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. A TNMD protein fragment for inhibiting Tau protein aggregation, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

2. A biomaterial related to the TNMD protein fragment according to claim 1, characterized in that: Any one of the following A1) to A4): A1) a nucleic acid molecule encoding the TNMD protein fragment; A2) an expression cassette containing the nucleic acid molecule described in A1); A3) a recombinant expression vector containing the nucleic acid molecule described in A1), or a recombinant expression vector containing the expression cassette described in A2); A4) a recombinant cell containing the nucleic acid molecule described in A1), or a recombinant cell containing the expression cassette described in A2), or a recombinant cell containing the recombinant expression vector described in A3).

3. The biomaterial according to claim 2, characterized in that: The sequence of the nucleic acid molecule encoding the TNMD protein fragment is shown in SEQ ID NO.

2.

4. The biomaterial according to claim 2, characterized in that: The recombinant expression vector is pET28c.

5. The biomaterial according to claim 2, characterized in that: The recombinant cells use Escherichia coli BL21 as host cells.

6. The method for preparing the TNMD protein fragment according to claim 1, characterized in that: The method comprises the following steps: inoculating the recombinant cell described in any one of claims 2 to 5 into a culture medium, obtaining the TNMD protein fragment by culture, or obtaining the TNMD protein fragment by culture and induced expression.

7. Use of the TNMD protein fragment according to claim 1 or the biomaterial according to any one of claims 2 to 5 in the preparation of a Tau protein aggregation inhibitor.

8. Use of the TNMD protein fragment according to claim 1 or the biomaterial according to any one of claims 2 to 5 in the preparation of a drug for treating Tau disease.

9. The use according to claim 8, characterized in that: The Tau protein diseases include Alzheimer's disease, frontotemporal dementia, progressive supranuclear palsy, cortical basal degeneration, and chronic traumatic encephalopathy.

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