Pathological tau protein obtained by means of multi-round amplification, preparation method therefor and use thereof

Abstract

Disclosed is a preparation technical method capable of preparing a large number of pathogenic tau proteins having a pathological conformation similar to that in the brain of a patient with neurodegenerative diseases by means of in vitro preparation and continuous multi-round amplification. The method can achieve continuous multi-round amplification of a patient brain-derived pathological tau protein by using only a small amount of the patient brain-derived pathological protein as a template, a recombinant human-derived tau protein monomer, and a negatively charged polyanion compound as an inducing cofactor, thereby achieving the in vitro preparation of a large amount of the pathological tau protein. In addition, the pathological tau protein prepared by the method can maintain the special pathological conformation of the original tau protein of the disease, has the same pathological activity as the original tau protein, and can be used for probe screening, antibody preparation, and verification model development.

Description

A pathological tau protein obtained through multigenerational amplification, its preparation method, and its application. Technical Field

[0001] This disclosure pertains to the biomedical field and relates to the preparation and application of in vitro multigenerational amplification of pathological tau protein in a class of neurodegenerative diseases characterized by tau protein deposition. Background Technology

[0002] Several neurodegenerative diseases, including Alzheimer's disease (AD) and frontotemporal dementia (FTD), are major causes of dementia, and currently there are no effective methods for early and accurate diagnosis and intervention. A common feature of AD and FTD is the deposition of tau protein in the brain, and the degree of tau protein deposition is closely related to the disease progression. Therefore, pathological tau protein has become a recognized effective target for the detection and intervention of these diseases.

[0003] Currently, both the detection and intervention of pathological tau proteins in AD / FTD suffer from imprecise targeting. Pathological tau proteins in the patient's brain have a specific conformation that cannot be perfectly replicated using existing animal models or through conventional in vitro protein synthesis. The only way to obtain proteins with a completely identical pathological conformation is through extraction from patient brain tissue. This yields a very limited number of pathological tau proteins, largely dependent on precious, high-quality human brain samples. This severely restricts the development of probes, antibody preparation, and validation models that precisely target disease-specific tau proteins, resulting in a continued lack of effective early diagnosis and intervention methods for AD / FTD. Currently, the detection probes, antibodies, and inhibitors for tau pathology in AD / FTD patients being developed by major pharmaceutical companies are designed and prepared using tau proteins without disease-specific pathological conformations, limiting their potential for clinical translation.

[0004] Virginia Lee from the University of Pennsylvania and her collaborators at Mei Hong's lab at MIT reported that by using pathological tau protein extracted from patient brain tissue as a template, they were able to amplify the pathological tau protein ADP1 (PMID: 33385254, 39214304) with a core fold region identical to that found in the brains of people with Alzheimer's disease (AD). However, this method is limited to one round of in vitro amplification and cannot be used as a template for further amplification, thus restricting the large-scale in vitro preparation of pathological tau protein with the same conformation as that found in AD / FTD.

[0005] Therefore, there is an urgent need in the field for a method, its products, and its uses for preparing pathological tau protein monomers with similar pathological conformations to human pathological tau proteins through multi-generation amplification in vitro. Summary of the Invention

[0006] The purpose of this disclosure is to provide a method for preparing pathological tau protein monomers with similar pathological conformation and activity to human pathological tau protein through continuous multi-generation amplification in vitro, and the resulting products thereof.

[0007] In one aspect, this disclosure provides a method for in vitro amplification of pathological tau protein through two or more generations, the method comprising the steps of:

[0008] (1) Provides a non-pathological state of tau protein;

[0009] (2) In the presence of a template pathological tau protein and an optional inducing cofactor, the non-pathological tau protein is incubated to form a pathological tau protein with a pathological conformation.

[0010] Wherein, the concentration A1 of the template pathological tau protein and the concentration A0 of the non-pathological tau protein satisfy 1% ≤ A1 / A0 ≤ 20%; the inducing cofactor is selected from the following group: heparan sulfate, heparan sulfate-derived oligosaccharide molecules, heparin, polyphosphate, phosphatidylinositol triphosphate, nucleic acid, or combinations thereof; the concentration of the inducing cofactor satisfies 2-1000 μg / mL;

[0011] The pathological tau protein obtained in step (2) is used as the template pathological tau protein, and steps (1)-(2) are repeated.

[0012] In some embodiments, the concentration A1 of the template pathological tau protein and the concentration A0 of the non-pathological tau protein satisfy 0.2≤A1 / A0≤20%, such as 5%≤A1 / A0≤20% or 5%≤A1 / A0≤10%.

[0013] In some embodiments, the template pathological tau protein includes: primary pathological tau protein, ADP. n .

[0014] In some implementations, the ADP n It is obtained by n-generation amplification using the primary pathological tau protein and the non-pathological tau protein.

[0015] In some implementations, the ADP n Including: ADP1 and ADP2.

[0016] In some implementations, the primary pathological tau protein includes: human pathological tau protein and non-human mammalian pathological tau protein.

[0017] In some implementations, the human pathological tau protein is a pathological tau protein derived from patients with neurodegenerative diseases.

[0018] In some implementations, the neurodegenerative disease-derived pathological tau proteins include: Alzheimer's disease pathological tau protein (AD-tau) and frontotemporal dementia pathological tau protein (FTD-tau).

[0019] In some implementations, the human pathological tau protein is AD-tau.

[0020] In some implementations, the AD-tau is selected from the group consisting of AD-PHF, AD-SF, or combinations thereof.

[0021] In some implementations, the non-human mammals include rodents and non-human primates.

[0022] In some implementations, the rodents include mice and rats.

[0023] In some embodiments, the non-pathological tau protein includes: human tau protein or fragments thereof, recombinant tau protein or fragments thereof, and chemically synthesized tau protein or fragments thereof.

[0024] In some embodiments, the human nonpathological tau protein is selected from the group consisting of: ON3R(T44), 1N3R(T37), 2N3R(T39), ON4R(T43), 1N4R(T34), 2N4R(T40), or combinations thereof.

[0025] In some embodiments, the recombinant tau protein includes: microbial tau protein, non-human mammalian cell-derived tau protein, and insect cell-derived tau protein.

[0026] In some embodiments, the recombinant tau protein is a microbial tau protein.

[0027] In some implementations, the microorganisms include Escherichia coli.

[0028] In some implementations, the microorganism expresses the human tau protein.

[0029] In some embodiments, the recombinant tau protein or chemically synthesized tau protein is selected from the group consisting of full-length tau protein, mutant tau protein, truncated tau protein, or combinations thereof.

[0030] In some embodiments, the chemically synthesized tau protein includes phosphorylated tau protein.

[0031] In some embodiments, the mutant tau protein has at least 70%, such as 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% amino acid sequence identity with the full-length tau protein.

[0032] In some embodiments, the mutant tau protein has at least 70%, such as 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% amino acid sequence identity with a fragment of the full-length tau protein.

[0033] In some embodiments, the truncated tau protein has at least 70%, such as 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% amino acid sequence identity with the full-length tau protein.

[0034] In some embodiments, the truncated tau protein has at least 70%, such as 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% amino acid sequence identity with a fragment of the full-length tau protein.

[0035] In some embodiments, the mutant Tau protein includes T40. P301L .

[0036] In some embodiments, the truncated Tau protein includes T40. 297-391 .

[0037] In some implementations, the non-pathological tau protein is T40.

[0038] In some embodiments, the phosphorylated tau protein is PAD12.

[0039] In some implementations, the PAD12 is ON3R PAD12.

[0040] In some embodiments, the heparin sulfate-derived oligosaccharide molecule includes sulfonated heparin oligosaccharides and oligosaccharides.

[0041] In some embodiments, the sulfonated heparin oligosaccharide is selected from the group consisting of sulfonated heparin tetrasaccharide, sulfonated heparin trisaccharide, sulfonated heparin disaccharide, or combinations thereof.

[0042] In some embodiments, the sulfonated heparin tetrasaccharide comprises the compound shown in Figure 2A.

[0043] In some embodiments, the sulfonated heparin tetrasaccharide preferably has 3-6 sulfonation groups, more preferably 4-6.

[0044] In some embodiments, the sulfonation sites of the sulfonated heparin tetrasaccharide include Y1 and Y3 sites.

[0045] In some embodiments, the sulfonated heparin trisaccharide comprises the compound shown in Figure 2B.

[0046] In some embodiments, the sulfonated heparin trisaccharide preferably has a sulfonation degree of 5.

[0047] In some embodiments, the sulfonation substitution site of the sulfonated heparin trisaccharide includes the Y1 site.

[0048] In some embodiments, the sulfonated heparin disaccharide comprises the compound shown in Figure 6A.

[0049] In some embodiments, the sulfonated heparin disaccharide preferably has a sulfonation degree of 2-3.

[0050] In some embodiments, the sulfonation substitution site of the sulfonated heparin disaccharide includes the X2 site.

[0051] In some embodiments, the oligosaccharide comprises the compound shown in Figure 2C.

[0052] In some implementations, the heparin includes low molecular weight heparin.

[0053] In some embodiments, the low molecular weight heparin comprises the compound shown in Figure 5A.

[0054] In some embodiments, the low molecular weight heparin is selected from the group consisting of HO-24, HO-10, HO-04, enoxaparin, or combinations thereof.

[0055] In some implementations, the nucleic acid includes whole-cell RNA, DNA, or its chiral isoforms.

[0056] In some implementations, the inducing cofactor is heparan sulfate.

[0057] In some embodiments, the concentration of the inducing cofactor is 20-160 μg / mL, preferably 20-80 μg / mL, and more preferably 40 μg / mL.

[0058] In some embodiments, the concentration of the inducing cofactor is 40 μg / mL.

[0059] In some implementations, in step (1), the non-pathological tau protein is present in the reaction system.

[0060] In some implementations, the reaction system further includes a reducing agent and a buffer solution.

[0061] In some implementations, the pH of the reaction system is 3-10.

[0062] In some implementations, the pH of the reaction system is 6.5-7.5.

[0063] In some implementations, the pH of the reaction system is 7.0-7.4.

[0064] In some implementations, the reducing agent includes: DTT, BME, TCEP, GSH.

[0065] In some embodiments, the buffer includes: Tris-HCl buffer, sodium acetate (NaOAc) buffer, bis(hydroxyethyl) phosphate buffer (PBS), 4-hydroxyethylpiperazine ethanesulfonic acid buffer (HEPES), 3-morpholinopropanesulfonic acid buffer (MOPS), and piperazine-1,4-diethanesulfonic acid buffer (PIPES).

[0066] In some implementations, step (1) further includes the step of:

[0067] (1.1) Heat shock the reaction system at the heat shock temperature for 10 ± 1 minute;

[0068] (1.2) Cool the reaction system for 10 ± 1 minutes;

[0069] In some implementations, in step (1.1), the heat shock temperature is 56±2°C, preferably 56°C.

[0070] In some implementations, in step (1.1), the reaction system is thermally stimulated for 10 minutes.

[0071] In some implementations, in step (1.2), the reaction system is cooled for 10 minutes.

[0072] In some implementations, in step (2), the non-pathological tau protein, the template pathological tau protein, and the optional inducing cofactor are present in the induction system.

[0073] In some implementations, the concentration of the non-pathological tau protein is 15-50 μM.

[0074] In some embodiments, the concentration of the template pathological tau protein is 0.05-5 μM, such as 0.1-5 μM, 0.15-5 μM, 0.75-5 μM, 0.75-2.5 μM, such as 0.09 μM, 0.1 μM, 0.15 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.75 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM.

[0075] In some implementations, in step (2), the non-pathological tau protein is incubated at an incubation temperature for an incubation period.

[0076] In some embodiments, the incubation temperature is 37±2°C, preferably 37°C.

[0077] In some implementations, the incubation period is 3-8 days, preferably 5-7 days, and more preferably 7 days.

[0078] In some implementations, the method further includes the step of: (3) separating and washing the tau pathological protein.

[0079] In some implementations, step (3) further includes the step of:

[0080] (3.1) The reaction system was subjected to ultracentrifugation at centrifugation temperature, and the precipitate was collected;

[0081] (3.2) Resuspend the precipitate in buffer solution and soak for ≥12 hours;

[0082] (3.3) The reaction system was subjected to ultracentrifugation at the centrifugation temperature, and the precipitate was collected;

[0083] (3.4) Resuspend the precipitate with buffer solution, mix the buffer solution and the precipitate to obtain a tau pathological protein suspension.

[0084] In some implementations, in steps (3.1) and (3.3), the centrifugation temperature is 22±2℃, preferably 22℃.

[0085] In some embodiments, the ultracentrifugation speed is ≥100,000×g, such as 100,000×g.

[0086] In some implementations, the ultracentrifugation time is ≥60 minutes, such as 60 minutes.

[0087] In some implementations, in steps (3.2) and (3.4), the buffer solution comprises phosphate-buffered saline (PBS).

[0088] In some implementations, in step (3.4), the buffer solution and the precipitate are mixed using ultrasound.

[0089] In another aspect, this disclosure provides a pathological tau protein obtained by amplifying a template pathological tau protein through two or more generations, wherein the pathological tau protein is prepared by the method described in this disclosure.

[0090] In some embodiments, the template pathological tau protein includes: primary pathological tau protein, ADP. n .

[0091] In some implementations, the ADP n It was obtained by n-generation amplification using primary pathological tau protein and non-pathological tau protein.

[0092] In some implementations, the primary pathological tau protein includes: human pathological tau protein.

[0093] In some implementations, the human pathological tau protein is a pathological tau protein derived from patients with neurodegenerative diseases.

[0094] In some implementations, the human pathological tau protein is AD-tau.

[0095] In some implementations, the AD-tau is selected from the group consisting of AD-PHF, AD-SF, or combinations thereof.

[0096] In some embodiments, the non-pathological tau protein includes: human tau protein or fragments thereof, recombinant tau protein or fragments thereof, and chemically synthesized tau protein or fragments thereof.

[0097] In some embodiments, the non-pathological tau protein is selected from the group consisting of: ON3R(T44), 1N3R(T37), 2N3R(T39), ON4R(T43), 1N4R(T34), 2N4R(T40), or combinations thereof.

[0098] In some implementations, the non-pathological tau protein is T40.

[0099] In some implementations, the pathological tau protein includes ADP1, ADP2, and ADP3.

[0100] In some implementations, the ADP1 includes: T40-ADP1, T40-ADP1, T39-ADP1, and T44-ADP1.

[0101] In some embodiments, the ADP2 includes: heparan sulfate (HS)-T40-ADP2, heparin (Hep)-T40-ADP2, polyphosphate (polyP)-T40-ADP2, phosphatidylinositol triphosphate (PIP3)-T40-ADP2, and RNA-T40-ADP2.

[0102] In some embodiments, the ADP3 comprises: heparan sulfate (HS)-T40-ADP3.

[0103] In some embodiments, the pathological tau protein has structural similarity to the template pathological tau protein.

[0104] In some implementations, the structural similarity is assessed by evaluating the root mean square deviation (RMSD).

[0105] In some embodiments, the pathological tau protein structure is the same as that of the primary pathological tau protein structure.

[0106] In some embodiments, the pathological tau protein structure is similar to the AD-tau structure.

[0107] In some embodiments, the ADP1 structure and the AD-PHF structure like

[0108] In some implementations, the ADP1 structure and the AD-SF structure like

[0109] In some embodiments, the ADP2 structure and the AD-PHF structure

[0110] In some implementations, the ADP2 structure and the AD-SF structure like

[0111] In some embodiments, the pathological tau protein structure is similar to the APD1 structure.

[0112] In some implementations, the ADP2 structure and the ADP1 structure like

[0113] In another aspect, this disclosure provides the use of an inducing cofactor or a pharmaceutically acceptable salt thereof for the preparation of a formulation, composition, or kit for inducing and / or promoting the formation of pathological tau protein from non-pathological tau protein; wherein the inducing cofactor or a pharmaceutically acceptable salt thereof is selected from the group consisting of heparan sulfate, heparan sulfate-derived oligosaccharide molecules, heparin, polyphosphate, phosphatidylinositol triphosphate, nucleic acids, or combinations thereof.

[0114] In some embodiments, the formulation, composition, or kit includes one or more components selected from the group consisting of:

[0115] (A) Heparan sulfate;

[0116] (B) Compounds as shown in Formula I:

[0117] X1 is selected from the following group: acetyl or sulfonyl;

[0118] X2 is selected from the following group: acetyl or sulfonyl;

[0119] Y1 is sulfonyl;

[0120] Y2 is selected from the following group: H, or sulfonyl;

[0121] Y3 is a sulfonyl group;

[0122] Y4 is selected from the following group: H, or sulfonyl;

[0123] "-" indicates that the structure is the anionic part of a salt;

[0124] (C) Compounds as shown in Formula II:

[0125] X1 is selected from the following group: acetyl or sulfonyl;

[0126] X2 is selected from the following group: acetyl or sulfonyl;

[0127] Y1 is sulfonyl;

[0128] Y2 is selected from the following group: H, or sulfonyl;

[0129] Y3 is a sulfonyl group;

[0130] Y4 is a sulfonyl group;

[0131] "-" indicates that the structure is the anionic part of a salt;

[0132] (D) Compounds as shown in Formula III:

[0133] X1 is selected from the following group: acetyl or sulfonyl;

[0134] X2 is selected from the following group: acetyl or sulfonyl;

[0135] Y1 is sulfonyl;

[0136] Y2 is a sulfonyl group;

[0137] Y3 is a sulfonyl group;

[0138] Y4 is a sulfonyl group;

[0139] "-" indicates that the structure is the anionic part of a salt;

[0140] (E) Compounds as shown in Formula IV:

[0141] X1 is selected from the following group: acetyl or sulfonyl;

[0142] X2 is selected from the following group: acetyl or sulfonyl;

[0143] Y1 is sulfonyl;

[0144] Y2 is a sulfonyl group;

[0145] Y3 is a sulfonyl group;

[0146] Y4 is a sulfonyl group;

[0147] "-" indicates that the structure is the anionic part of a salt;

[0148] (F) Compounds as shown in Formula V:

[0149] Wherein, X1 is sulfonyl;

[0150] X2 is a sulfonyl group;

[0151] Y1 is sulfonyl;

[0152] Y2 is selected from the following group: H, or sulfonyl;

[0153] Y3 is a sulfonyl group;

[0154] Y4 is a sulfonyl group;

[0155] "-" indicates that the structure is the anionic part of a salt;

[0156] (G) Compounds as shown in Formula VI:

[0157] X1 is selected from the following group: acetyl or sulfonyl;

[0158] X2 is selected from the following group: acetyl or sulfonyl;

[0159] Y1 is sulfonyl;

[0160] Y2 is selected from the following group: H, or sulfonyl;

[0161] Y3 is a sulfonyl group;

[0162] "-" indicates that the structure is the anionic part of a salt;

[0163] (H) Compounds as shown in Formula VII:

[0164] X1 is selected from the following group: acetyl or sulfonyl;

[0165] X2 is selected from the following group: acetyl or sulfonyl;

[0166] Y1 is sulfonyl;

[0167] Y2 is a sulfonyl group;

[0168] Y3 is a sulfonyl group;

[0169] "-" indicates that the structure is the anionic part of a salt;

[0170] (I) Compounds as shown in Formula VIII:

[0171] X1 is selected from the following group: acetyl or sulfonyl;

[0172] X2 is selected from the following group: H, or sulfonyl;

[0173] X3 is selected from the following group: H, or sulfonyl;

[0174] "-" indicates that the structure is the anionic part of a salt;

[0175] (J) Compounds as shown in formula panta-1:

[0176] Wherein, "-" indicates that the structure is the anionic part of a salt;

[0177] (K) Compounds as shown in formula panta-2:

[0178] Wherein, "-" indicates that the structure is the anionic part of a salt;

[0179] (L) Compounds as shown in formula AO-3:

[0180] Wherein, "-" indicates that the structure is the anionic part of a salt;

[0181] (M) Compounds as shown in formula AO-4:

[0182] Wherein, "-" indicates that the structure is the anionic part of a salt;

[0183] (N) Enoxaparin;

[0184] (O)HO-24;

[0185] (P)HO-10;

[0186] (Q)HO-04.

[0187] In some embodiments, the formulation or composition comprises one or more components selected from the group consisting of:

[0188] (-0) Heparan sulfate;

[0189] (Tetra-1) is a compound of formula I, wherein X1 is acetyl, X2 is acetyl, Y1 is sulfonyl, Y2 is H, Y3 is sulfonyl, and Y4 is H;

[0190] (Tetra-2) is a compound of formula I, wherein X1 is acetyl, X2 is acetyl, Y1 is sulfonyl, Y2 is H, Y3 is sulfonyl, and Y4 is sulfonyl;

[0191] (Tetra-4) is a compound of formula I, wherein X1 is acetyl, X2 is acetyl, Y1 is sulfonyl, Y2 is sulfonyl, Y3 is sulfonyl, and Y4 is sulfonyl;

[0192] (Tetra-5) is a compound of formula I, wherein X1 is sulfonyl, X2 is sulfonyl, Y1 is sulfonyl, Y2 is H, Y3 is sulfonyl, and Y4 is H;

[0193] (Tetra-9) is a compound of formula I, wherein X1 is sulfonyl, X2 is sulfonyl, Y1 is sulfonyl, Y2 is H, Y3 is sulfonyl, and Y4 is sulfonyl;

[0194] (Tetra-11) is a compound of formula I, wherein X1 is sulfonyl, X2 is sulfonyl, Y1 is sulfonyl, Y2 is sulfonyl, Y3 is sulfonyl, and Y4 is sulfonyl;

[0195] (Tetra-3) is a compound as shown in Formula II, wherein X1 is acetyl, X2 is acetyl, Y1 is sulfonyl, Y2 is H, Y3 is sulfonyl, and Y4 is sulfonyl;

[0196] (Tetra-7) is a compound as shown in Formula II, wherein X1 is acetyl, X2 is acetyl, Y1 is sulfonyl, Y2 is sulfonyl, Y3 is sulfonyl, and Y4 is sulfonyl;

[0197] (Tetra-10) is a compound as shown in Formula II, wherein X1 is sulfonyl, X2 is sulfonyl, Y1 is sulfonyl, Y2 is H, Y3 is sulfonyl, and Y4 is sulfonyl;

[0198] (Tetra-13) is a compound of formula II, wherein X1 is sulfonyl, X2 is sulfonyl, Y1 is sulfonyl, Y2 is sulfonyl, Y3 is sulfonyl, and Y4 is sulfonyl;

[0199] (Tetra-6) is a compound of formula III, wherein X1 is acetyl, X2 is acetyl, Y1 is sulfonyl, Y2 is sulfonyl, Y3 is sulfonyl, and Y4 is sulfonyl;

[0200] (Tetra-14) is a compound of formula III, wherein X1 is sulfonyl, X2 is sulfonyl, Y1 is sulfonyl, Y2 is sulfonyl, Y3 is sulfonyl, and Y4 is sulfonyl;

[0201] (Tetra-8) is a compound as shown in Formula IV, wherein X1 is acetyl, X2 is acetyl, Y1 is sulfonyl, Y2 is sulfonyl, Y3 is sulfonyl, and Y4 is sulfonyl;

[0202] (Tetra-12) is a compound as shown in Formula IV, wherein X1 is sulfonyl, X2 is sulfonyl, Y1 is sulfonyl, Y2 is sulfonyl, Y3 is sulfonyl, and Y4 is sulfonyl;

[0203] (Tetra-15) is a compound of formula V, wherein X1 is sulfonyl, X2 is sulfonyl, Y1 is sulfonyl, Y2 is H, Y3 is sulfonyl, and Y4 is sulfonyl;

[0204] (Tetra-16) is a compound of formula V, wherein X1 is sulfonyl, X2 is sulfonyl, Y1 is sulfonyl, Y2 is sulfonyl, Y3 is sulfonyl, and Y4 is sulfonyl;

[0205] (Tri-1) is a compound as shown in Formula VI, wherein X1 is acetyl, X2 is acetyl, Y1 is sulfonyl, Y2 is H, and Y3 is sulfonyl;

[0206] (Tri-2) is a compound as shown in Formula VI, wherein X1 is acetyl, X2 is acetyl, Y1 is sulfonyl, Y2 is sulfonyl, and Y3 is sulfonyl;

[0207] (Tri-4) is a compound of formula VI, wherein X1 is sulfonyl, X2 is sulfonyl, Y1 is sulfonyl, Y2 is H, and Y3 is sulfonyl;

[0208] (Tri-5) is a compound as shown in Formula VI, wherein X1 is sulfonyl, X2 is sulfonyl, Y1 is sulfonyl, Y2 is sulfonyl, and Y3 is sulfonyl;

[0209] (Tri-3) is a compound as shown in Formula VII, wherein X1 is acetyl, X2 is acetyl, Y1 is sulfonyl, Y2 is sulfonyl, and Y3 is sulfonyl;

[0210] (Tri-6) is a compound as shown in Formula VII, wherein X1 is sulfonyl, X2 is sulfonyl, Y1 is sulfonyl, Y2 is sulfonyl, and Y3 is sulfonyl;

[0211] (HD001) The compound shown in Formula VIII, wherein X1 is sulfonyl, X1 is sulfonyl, X1 is sulfonyl;

[0212] (HD002) The compound shown in Formula VIII, wherein X1 is sulfonyl, X1 is H, and X1 is sulfonyl;

[0213] (HD003) The compound shown in Formula VIII, wherein X1 is acetyl, X1 is sulfonyl, and X1 is sulfonyl;

[0214] (HD004) The compound shown in Formula VIII, wherein X1 is sulfonyl, X1 is sulfonyl, and X1 is H;

[0215] (HD005) The compound shown in Formula VIII, wherein X1 is sulfonyl, X1 is H, and X1 is H;

[0216] (HD006) The compound shown in Formula VIII, wherein X1 is an acetyl group, X1 is H, and X1 is H;

[0217] (HD007) A compound as shown in Formula VIII, wherein X1 is an acetyl group, X1 is H, and X1 is a sulfonyl group; or

[0218] (HD008) The compound shown in Formula VIII, wherein X1 is an acetyl group, X1 is a sulfonyl group, and X1 is H;

[0219] (-1) Enoxaparin;

[0220] (-2)HO-24;

[0221] (-3)HO-10;

[0222] (-4)HO-04.

[0223] In some embodiments, the formulation or composition comprises one or more components selected from the group consisting of: Tetra-1, Tetra-2, Tetra-3, Tetra-4, Tetra-5, Tetra-6, Tetra-7, Tetra-8, Tetra-9, Tetra-10, Tetra-11, Tetra-12, Tetra-13, Tetra-14, Tri-1, Tri-2, Tri-3, Tri-5, Penta-1, Penta-2, AO-3, AO-4, HD001, HD004, HO-24, HO-10, HO-04, heparan sulfate, and enoxaparin.

[0224] In some embodiments, the formulation or composition further includes a pharmaceutically acceptable carrier, diluent, or excipient.

[0225] In another aspect, this disclosure provides a formulation or composition comprising:

[0226] (i) the pathological tau protein obtained by amplification of the template pathological tau protein through two or more generations as described in this disclosure; and

[0227] (ii) Inducing cofactors or their pharmaceutically acceptable salts;

[0228] The inducing cofactor or a pharmaceutically acceptable salt thereof is selected from the group consisting of heparan sulfate, heparan sulfate-derived oligosaccharide molecules, heparin, polyphosphate, phosphatidylinositol triphosphate, nucleic acids, or combinations thereof.

[0229] In some embodiments, the formulation, composition, or kit may further include a pharmaceutically acceptable carrier, diluent, or excipient.

[0230] In another aspect, this disclosure provides a kit comprising:

[0231] (c1) the formulations or compositions described in this disclosure; and

[0232] (c2) Optional non-pathological state tau protein.

[0233] In some embodiments, the formulation or composition and the non-pathological tau protein are located in different containers.

[0234] In some implementations, the kit also includes a label or instructions.

[0235] In some implementations, the label or instructions specify that the kit is used to prepare pathological tau protein obtained by amplifying template pathological tau protein through two or more generations.

[0236] In some embodiments, the label or specification also indicates the methods described in this disclosure.

[0237] In another aspect, this disclosure provides the use of the pathological tau protein obtained by amplifying the template pathological tau protein through two or more generations, or the formulations or compositions described in this disclosure, comprising:

[0238] (a) Inducing and / or promoting the deposition of pathological proteins in cells / tissues in vitro / in vivo;

[0239] (b) Establishing animal models of neurodegenerative diseases;

[0240] (c) Preparation of biomacromolecule probes / antibodies;

[0241] (d) Large-scale compound screening.

[0242] In some implementations, the cells include nerve cells.

[0243] In some implementations, the nerve cells are derived from human or non-human mammals.

[0244] In some implementations, the non-human mammals include rodents.

[0245] In some implementations, the rodents include mice and rats.

[0246] In some embodiments, the nerve cells are primary wild-type nerve cells.

[0247] In some implementations, the organization includes the hippocampus.

[0248] In some implementations, the animal includes mammals.

[0249] In some implementations, the mammals include: rodents, non-human primates, rabbits, cats, dogs, pigs, sheep, cattle, horses, and donkeys.

[0250] In another aspect, this disclosure provides a method for inducing and / or promoting the production of pathological deposits in cells in vitro, the method comprising the steps of:

[0251] (S1) Provide a cell containing tau protein in a non-pathological state;

[0252] (S2) The pathological tau protein obtained by amplifying the template pathological tau protein of the present disclosure through two or more generations, or the preparation or composition of the present disclosure, is incubated with the cells to induce and / or promote the deposition of pathological proteins in the cells.

[0253] The inducing cofactor is selected from the group consisting of heparan sulfate, heparan sulfate-derived oligosaccharide molecules, heparin, polyphosphate, phosphatidylinositol triphosphate, nucleic acid, or combinations thereof.

[0254] In some implementations, the cells include: neuronal cells and non-neuronal cells.

[0255] In some embodiments, step (S2) further includes: incubating the cells with the pathological tau protein obtained by amplifying the template pathological tau protein of the present disclosure through two or more generations in the presence of a protein transfection reagent.

[0256] In some embodiments, the protein transfection reagent includes: cell-penetrating peptides and lipid mixtures.

[0257] In some implementations, the method includes the steps of:

[0258] (S1) provides a neuron cell;

[0259] (S2) The pathological tau protein obtained by amplifying the template pathological tau protein of the present disclosure through two or more generations is incubated with the neuronal cells to induce and / or promote the deposition of pathological proteins in the neuronal cells.

[0260] In some implementations, the method includes the steps of:

[0261] (S1) provides a non-neuronal cell;

[0262] (S2) The pathological tau protein obtained by amplifying the template pathological tau protein through two or more generations, as described in this disclosure, and the protein transfection reagent are incubated with the non-neuronal cells to induce and / or promote the deposition of pathological proteins in the non-neuronal cells.

[0263] In some implementations, the incubation time is 1-12 hours, preferably 4-8 hours.

[0264] It should be understood that, within the scope of this disclosure, the above-described technical features and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0265] Figure 1 shows the preparation and characterization results of the HS-induced recombinant full-length tau protein T40 filament system (HS-T40).

[0266] Figure 2 shows the structures of sulfonated heparin tetrasaccharide, sulfonated heparin trisaccharide, and oligosaccharide that can induce tau protein aggregation to form filaments.

[0267] Figure 3 shows the results of the oligosaccharide-induced recombinant tau protein system. NC: blank control group.

[0268] Figure 4 shows the induction of neuronal tau pathological deposition by oligosaccharide-T40 PFF in primary cultured neurons in vitro and in wild-type mice. AD: Results in Alzheimer's disease patients as a positive control.

[0269] Figure 5 shows the preparation and characterization results of the heparin and low molecular weight heparin glycan-induced recombinant full-length tau protein T40 fiber system (heparin glycan-T40).

[0270] Figure 6 shows the preparation and characterization results of the unsaturated standard heparin disaccharide-induced recombinant full-length tau protein T40 fiber system (heparinoid-T40). NC: blank control group.

[0271] Figure 7 shows the amplification of AD-tau into ADP1 and ADP2. (a) Characterization results of AD-derived tau pathological proteins; scale bar: large field of view 100 nm; small field of view 200 nm. (b) Schematic diagram of amplification methods. (c) Characterization results of the ultrafiltration sedimentation experiment of ADP1 preparation, stained with Coomassie brilliant blue (CBB). (d) Characterization results of NS-TEM of ADP1 preparation; scale bar, 200 nm. (e) Quantitative statistics of AD-tau amplification and pathological fiber formation by different tau monomers. (f) NS-TEM characterization results of ADP2 preparation; scale bar, 200 nm. (g) Characterization results of ultrafiltration sedimentation experiment of ADP2 preparation, stained with CBB. (h) Quantitative statistics of AD-tau amplification and pathological fiber formation assisted by different cofactors. (i) Dosage effect of cofactor HS inducing recombinant T40 fibrosis.

[0272] Figure 8 shows the cryo-electron microscopy structures of ADP1 and ADP2. (a) Cryo-electron microscopy results of AD-tau derived from brain tissue of AD patients; resolution marker in the lower left corner; scale bar, 5 nm. (b) Cryo-EM structures of ADP1 obtained from the amplification of four monomers; resolution marker in the lower left corner; scale bar, 5 nm. (c) Cryo-EM structures of three types of ADP2; resolution marker in the lower left corner; scale bar, 10 nm. (d) Comparison of similarity between different pathological fibers.

[0273] Figure 9 shows the results of pathological activity tests for ADP1 and ADP2. (a) Pathological activity of ADP1 obtained from the amplification of the four monomers; scale bar, 20 μm. (b) Statistical results of a. (c) Pathological activity of HS-T40-ADP2 in neurons; scale bar, 20 μm. (d) Statistical results of c. (e) Pathological activity of HS-T40-ADP2 in vivo; scale bar, 100 μm. (f) Statistical results of e.

[0274] Figure 10 shows the pathological structure and activity test results of ADP3 (HS-ADP3) that can be continuously amplified from ADP2 to ADP3 using HS as a cofactor.

[0275] Figure 11 shows the induction results using tau protein monomers with simulated phosphorylation mutations (AD-PAD-P1). (A) Negative staining electron microscopy image of AD-PAD-P1 fibers; (B) Detection of seeding activity (T49) of primary mouse neurons using AD-PAD-P1 fibers; (C) THT curves of AD-PAD12-P1 systems with different potassium phosphate concentrations; (D) Negative staining electron microscopy and Coomassie brilliant blue staining quantification of different AD-PAD12-P1 fibers; (E) Display of endogenous phosphorylated pathological tau protein induced by different doses of AD-PAD12-P1 fibers injected into the hippocampus of wild-type mice 6 weeks later. DG, CA3, and Fin represent different local regions of the hippocampus. Detailed Implementation

[0276] Through extensive and in-depth research, the inventors have developed for the first time a method for the continuous, multi-generation amplification of pathogenic tau proteins with a pathological conformation similar to that found in the brains of AD / FTD patients. This method uses only a small amount of pathological protein derived from human brain as a template, utilizes recombinant human tau protein monomers, and employs negatively charged polyanionic compounds as inducing cofactors to continuously amplify pathological tau proteins from human brains across multiple generations, achieving large-scale in vitro preparation of pathological tau proteins. Furthermore, a pathological tau protein prepared using this method is provided, which retains the specific pathological conformation of the original disease tau protein and possesses the same pathological activity as the original tau protein, making it suitable for the preparation of biomolecular antibodies, large-scale compound screening, etc. Based on this, the present invention was completed.

[0277] It should be understood that the specific methods and experimental conditions described below with varying degrees of detail are intended to provide a substantive understanding of this disclosure. Definitions of certain terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0278] the term

[0279] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed-ended. In other words, the terms also include “consistently made of” or “made of”.

[0280] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0281] As used in this article, the term "significant" means that, in a hypothesis test, the observed effect (such as the difference between the experimental and control groups) is unlikely to be caused solely by random error. A hypothesis test includes: the null hypothesis (H0), which assumes that the observed effect does not exist (such as no difference between the experimental and control groups); the p-value, which is the probability of observing the current or more extreme effect when H0 is true; and the significance threshold (α). The significance threshold is typically used to determine whether a hypothesis test is significant. Generally, the significance threshold is 0.05. If the p-value ≤ α, then H0 is rejected, meaning the observed effect exists, and the result is called "significant."

[0282] As used herein, the terms “pre-fabricated fiber” and “pff” are used interchangeably and refer to the formation of pathological tau proteins, pathological protein deposition, or fibrotic aggregation of tau proteins in vitro by inducing / promoting the formation of non-pathological tau proteins using inducing cofactors.

[0283] As used herein, the terms "Thioflavin T (ThT)" and "ThT" are used interchangeably, referring to a small molecule compound that can bind to the β-sheet structure of proteins and whose energy level changes before and after binding, thus emitting fluorescence upon excitation by light of a specific wavelength. In this disclosure, ThT is used to characterize the presence of the β-sheet structure; the fluorescence intensity of ThT in the system reflects the inducing effect of the inducing cofactor.

[0284] As used herein, the terms "6htau mouse" and "6htau" are used interchangeably, referring to a transgenic mouse expressing six human tau subtypes. In this disclosure, injection of HS-T40 pff into this mouse induces a variety of endogenous pathologies.

[0285] As used herein, the terms "unsaturated standard heparin disaccharide" and "sulfonated heparin disaccharide" are used interchangeably and are a class of inducing cofactors disclosed herein. Sulfonation substitution at different positions in heparin disaccharide can give it different abilities to induce and / or promote the formation of pathological tau proteins from non-pathological tau proteins.

[0286] As used in this article, the term "multi-generation" refers to the preparation of the next generation of products using the products obtained from the previous generation, where the products obtained from each generation are stable and highly reproducible.

[0287] As used herein, the term "inducing cofactor" refers to a bioactive molecule capable of inducing non-pathological tau protein into pathological tau protein in the presence of a template pathological tau protein. In the absence of an inducing cofactor, it is impossible or difficult to amplify pathological tau protein through passage. The inducing cofactor is selected from the group consisting of: heparan sulfate (HS), heparan sulfate-derived oligosaccharide molecules, heparin (Hep), polyphosphate (polyP), phosphatidylinositol triphosphate (PIP3), nucleic acids, or combinations thereof. The nucleic acid includes whole-cell RNA, DNA, or its chiral isomers, such as ZNA.

[0288] As used herein, the term "sequence identity" refers to the percentage of nucleotide / amino acid residues in the host sequence that are identical to those in the reference sequence after sequence alignment, with gaps introduced where necessary to achieve the maximum percentage of sequence identity between sequences. This is used to determine the percentage of sequence identity between two or more nucleic acid or amino acid sequences.

[0289] Non-pathological state tau protein

[0290] As used herein, the terms "tau protein," "non-pathological tau protein," and "tau protein monomer" are used interchangeably to refer to the state in which tau protein performs its normal biological functions under normal physiological conditions. This protein has the function of binding to microtubules and regulating their assembly into bundles and stability. It is mainly found in axons, but can also be found in dendrites and other neuronal parts. Full-length tau protein includes an N-terminal domain, a proline-rich domain, a microtubule-binding domain, and a C-terminal domain.

[0291] The non-pathological tau proteins disclosed herein include human tau protein or fragments thereof, recombinant tau protein or fragments thereof, and chemically synthesized tau protein or fragments thereof. The human tau protein includes different subtypes of tau: ON3R, 1N3R, 2N3R, ON4R, 1N4R, and 2N4R, abbreviated as T44, T37, T39, T43, T34, and T40, respectively.

[0292] The recombinant tau protein is a tau protein expressed on non-human cells. The non-human cells include non-human mammalian cells and insect cells. Preferably, it is a tau protein monomer expressed and purified from *E. coli*. More preferably, it is a human tau protein expressed and purified from *E. coli*.

[0293] Recombinant tau protein or chemically synthesized tau protein can be full-length tau protein, mutant tau protein, truncated tau protein, or chemically modified tau protein. The full-length tau protein is the original sequence of the tau protein. The mutant tau protein is formed by substituting one or more amino acids into the full-length tau protein. The mutant tau protein retains the biological properties and / or structure of the full-length tau protein. The truncated tau protein is formed by deleting one or more consecutive amino acids from the full-length tau protein. The truncated tau protein retains the biological properties and / or structure of the full-length tau protein.

[0294] Specifically, the full-length Tau protein used in this disclosure is the recombinant Tau protein monomer T40, expressed and purified from *E. coli*. The mutant Tau protein used in this disclosure includes a mutation at amino acid position 301 of the T40 protein, where P is mutated to L(T40). P301L This includes specific sites, including but not limited to simulated phosphorylation modifications at sites such as T181, S202, T205, T212, S214, T217, T231, S235, S396, S400, T403, and S404, such as PAD12. For PAD12, the naming convention is "human tau protein PAD12," referring to PAD12 obtained by simulated phosphorylation modifications at 12 specific sites on the basis of the human tau protein, such as ON3R PAD12. The truncated tau protein used in this disclosure is the T40 protein with amino acids truncated from position 297 to 391 (T40...). 297-391 The mutant and truncated Tau proteins disclosed herein still retain the function and activity of the wild-type Tau protein.

[0295] In some cases, mutant tau proteins are formed by substituting one or more amino acids from fragments or homologs of the full-length tau protein. Mutant tau proteins retain some of the biological properties and / or structure of the full-length tau protein. In other cases, truncated tau proteins are formed by deleting one or more consecutive amino acids from fragments or homologs of the full-length tau protein. Truncated tau proteins also retain some of the biological properties and / or structure of the full-length tau protein.

[0296] The term "fragment" generally refers to a portion of a reference protein. A "fragment" can be a functional segment, such as a functional domain of the reference protein, such as the functional domain of a full-length tau protein, or the microtubule-binding domain of a full-length tau protein. The term "homolog" generally refers to a substance that has the same or very similar origin and similar genetic information as the reference protein, including orthologs and paralogs. An "ortholog" is a variant of the reference protein produced by a different species compared to the reference protein species. A "paralog" is a variant of the reference protein expressed by a species of the same species as the reference protein. In some embodiments, fragments and homologs of the reference protein can be characterized by their ability to perform the functions performed by the reference protein.

[0297] A “fragment” can be of any length (in terms of amino acid count), optionally at least 20% of the length of a reference protein (i.e., the protein from which the fragment originates), and can have a maximum length of any one of 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the reference protein. A tau protein fragment can have a minimum length of any one of 80, 100, 120, 140, 200, 250, 300, or 400 amino acids, and a maximum length of any one of 90, 100, 120, 140, 200, 250, 300, 400, or 430 amino acids.

[0298] Preferably, the truncated tau protein is a fragment of the full-length tau protein. Preferably, the truncated tau protein is a functional fragment of the full-length tau protein. Preferably, the truncated tau protein is a microtubule-binding domain of the full-length tau protein.

[0299] Preferably, the mutant tau protein has at least 70%, such as 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% amino acid sequence identity with the full-length tau protein.

[0300] Preferably, the mutant tau protein has at least 70%, such as 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% amino acid sequence identity with the fragment of the full-length tau protein.

[0301] Preferably, the truncated tau protein has at least 70%, such as 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% amino acid sequence identity with the full-length tau protein.

[0302] Preferably, the truncated tau protein fragment has at least 70%, such as 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% amino acid sequence identity with the full-length tau protein fragment.

[0303] Template pathology tau protein

[0304] As used herein, the terms “template pathological tau protein,” “template seed,” and “seed” are used interchangeably, including pathological tau proteins obtained directly from the brains of patients with neurodegenerative diseases, i.e., primary pathological tau proteins, or pathological tau proteins obtained through the amplification methods described in this disclosure, i.e., ADP. n .

[0305] The primary pathological tau protein can be derived from humans or non-human mammals. Preferably, the primary pathological tau protein is a human-derived pathological tau protein. The human-derived pathological tau protein is a pathological tau protein derived from patients with neurodegenerative diseases. The pathological tau proteins derived from patients with neurodegenerative diseases include Alzheimer's disease pathological tau protein (AD-tau) and frontotemporal dementia pathological tau protein (FTD-tau). AD-tau is further classified according to conformation into helical filament tau protein (AD-PHF) and straight filament tau protein (AD-SF). Methods for extracting primary pathological tau proteins from patients with neurodegenerative diseases are well known to those skilled in the art; the concentration of the extracted primary pathological tau protein can be measured using methods well known to those skilled in the art, such as sandwich ELISA, Western blotting, etc. Generally, the concentration of pathological tau protein extracted from AD patients is >1000 μg / mL, accounting for >10% of the total extracted protein.

[0306] The ADP n It is obtained by amplifying pathological tau protein and non-pathological tau protein through n generations. For example, the first generation amplification is to obtain the first generation product (ADP1) by amplifying primary pathological tau protein and non-pathological tau protein; the second generation amplification is to obtain the second generation product (ADP2) by amplifying ADP1 and non-pathological tau protein, and so on.

[0307] In this disclosure, the naming convention for ADP1 can follow "non-pathological tau protein-ADP1", such as "T40-ADP1", "T43-ADP1", "T39-ADP1", and "T44-ADP1", referring to ADP1 prepared using this non-pathological tau protein. n The naming convention can follow "inducible cofactor - non-pathological tau protein - ADP". nExamples of ADP preparations, such as "HS-T40-ADP2", "Hep-T40-ADP2", "polyP-T40-ADP2", "PIP3-T40-ADP2", "RNA-T40-ADP2", and "HS-T40-ADP3", refer to ADPs prepared using the non-pathological tau protein in the presence of the inducing cofactor. n ADP n The naming rules can also follow the "inducible cofactor-ADP" rule. n "" refers to the nth generation product obtained by induction using this inducing cofactor, and the non-pathological tau protein used can be any of the non-pathological tau proteins involved in this disclosure. The naming convention for products amplified based on phosphorylated tau protein monomers can follow "AD-PAD12-P". n "" refers to the nth generation product prepared using PAD12 tau protein, and the non-pathological PAD12 tau protein used can be a phosphorylated modified protein derived from various human tau proteins mentioned in this disclosure.

[0308] Pathological tau protein

[0309] As used in this article, the terms “tau pathological protein,” “tau pathological protein with pathological conformation,” “pathological tau fiber,” “tau pathological fiber,” and “pathological protein deposition” are used interchangeably. They all refer to tau proteins that have pathological features or conformations, are pathogenic, can induce the transformation of non-pathological tau proteins into pathological tau proteins, and are diffusive.

[0310] In this disclosure, the pathological tau protein can be a template pathological tau protein or a pathological tau protein prepared by the method described in this disclosure.

[0311] Preferably, the pathological tau protein includes ADP1 and ADP2. Preferably, ADP1 includes T40-ADP1, T40-ADP1, T39-ADP1, and T44-ADP1. Preferably, ADP2 includes HS-T40-ADP2, Hep-T40-ADP2, polyP-T40-ADP2, PIP3-T40-ADP2, and RNA-T40-ADP2. Preferably, ADP3 includes HS-T40-ADP3.

[0312] Preferably, the pathological tau protein has structural similarity to the template pathological tau protein.

[0313] The pathological tau protein disclosed herein can induce and / or promote the deposition of pathological proteins in cells / tissues in vitro / in vivo, thereby enabling the preparation of animal models of neurodegenerative diseases. Furthermore, it can also be used for the preparation of animal models of neurodegenerative diseases and large-scale compound screening.

[0314] Uses of tau protein in pathology

[0315] In this disclosure, the pathological tau protein is used to prepare formulations or compositions for: inducing and / or promoting the deposition of pathological proteins in cells in vitro; inducing and / or promoting the deposition of pathological proteins in tissues and / or cells in animals, preferably, the tissues include, but are not limited to, nuclei such as the hippocampus, striatum, corpus callosum, and lentiform nucleus; preparing animal models of neurodegenerative diseases, preferably, the animal model is a mouse model; preparing biomolecular antibodies; or large-scale compound screening.

[0316] Inducing cofactor

[0317] In this disclosure, the terms "inducing cofactor" and "cofactor" are used interchangeably, referring to negatively charged polyanionic compounds, primarily including heparan sulfate (HS) and related derivative oligosaccharide molecules, heparin, polyphosphates, phosphatidylinositol triphosphate, and nucleic acids. Specifically, the heparan sulfate-derived oligosaccharide molecules include sulfonated heparin oligosaccharides, oligosaccharides, and low molecular weight heparin. The sulfonated heparin oligosaccharides are selected from the group consisting of sulfonated heparin tetrasaccharides, sulfonated heparin trisaccharides, sulfonated heparin disaccharides, or combinations thereof; the low molecular weight heparin is selected from the group consisting of HO-24, HO-10, HO-04, enoxaparin, or combinations thereof.

[0318] Preferably, the sulfonated heparin trisaccharide comprises the compound shown in Figure 2B.

[0319] Preferably, the sulfonated heparin disaccharide comprises the compound shown in Figure 6A.

[0320] Preferably, the oligosaccharide comprises the compound shown in FIG2C.

[0321] Preferably, the low molecular weight heparin comprises the compounds shown in Figure 5A. HO-24, HO-10, and HO-04 are commercially available low molecular weight heparins prepared by partial digestion of heparin with heparinase I. HO-24 has an average molecular weight of 6850 Daltons, HO-10 has an average molecular weight of 3000 Daltons, and HO-04 has an average molecular weight of 1200 Daltons.

[0322] In this disclosure, a cofactor is induced in vitro to induce / promote the formation of Tau pathological proteins, pathological protein deposition, or Tau protein fibrotic aggregation in a non-pathological state, which is referred to as pre-formed fibrous material.

[0323] Different inducing cofactors have varying abilities to induce and / or promote the formation of preformed fibers in vitro. Preferably, the inducing cofactors for inducing and / or promoting the formation of preformed fibers in vitro are selected from the group consisting of: Tetra-1, Tetra-2, Tetra-3, Tetra-4, Tetra-5, Tetra-6, Tetra-7, Tetra-8, Tetra-9, Tetra-10, Tetra-11, Tetra-12, Tetra-13, Tetra-14, Tri-1, Tri-2, Tri-3, Tri-5, Penta-1, Penta-2, AO-3, AO-4, HD001, HD004, HO-24, HO-10, HO-04, heparan sulfate, and enoxaparin.

[0324] Different inducing cofactors induce and / or promote the formation of pre-formed fibers that can induce pathological protein deposition in vitro. Preferably, the inducing cofactors that induce and / or promote the formation of pre-formed fibers capable of inducing pathological protein deposition in vitro are selected from the group consisting of: heparan sulfate, Tri-5, Tetra-9, Tetra-3, Tetra-6, Tetra-1, Tetra-8, Tetra-10, AO-4, Tetra-12, Tetra-5, Penta-1, Tetra-7, Tri-6, Tetra-11, HD001, HD004, enoxaparin, HO-24, or combinations thereof.

[0325] Different inducing cofactors induce and / or promote the formation of prefabricated fibers that can induce pathological protein deposition in vivo, and their ability to do so varies. Preferably, the inducing cofactors that induce and / or promote the formation of prefabricated fibers capable of inducing endogenous pathological changes in vivo are selected from the group consisting of: Tetra-7, Tetra-8, AO-4, Tri-5, Tetra-6, Tetra-11, heparan sulfate, Penta-1, Tetra-10, HD001, HD004, or combinations thereof.

[0326] This disclosure describes a method for amplifying pathological tau protein using two or more generations.

[0327] This disclosure provides a method for preparing and amplifying pathological tau protein in vitro for two or more generations (hereinafter referred to as the "amplification method"), the method comprising the following steps:

[0328] (1) Provides a non-pathological state of tau protein;

[0329] (2) In the presence of a template pathological tau protein and an optional inducing cofactor, the non-pathological tau protein is incubated to form a pathological tau protein with a pathological conformation.

[0330] Wherein, the concentration A1 of the template pathological tau protein and the concentration A0 of the non-pathological tau protein satisfy 1% ≤ A1 / A0 ≤ 20%; the inducing cofactor is selected from the following group: heparan sulfate, heparin, polyphosphate, phosphatidylinositol triphosphate, nucleic acid, or a combination thereof; the concentration of the inducing cofactor satisfies 2-1000 μg / mL;

[0331] The pathological tau protein obtained in step (2) is used as the template pathological tau protein, and steps (1)-(2) are repeated.

[0332] This disclosure describes a process where a second-generation pathological tau protein product, ADP2, with a similar pathological structure is generated by adding a non-pathological tau protein and an optional inducing cofactor to a first-generation pathological tau protein product, ADP1. Similarly, adding a non-pathological tau protein and an inducing cofactor to ADP2 generates a third-generation pathological tau protein product, ADP3, with a similar pathological structure. It is understood that adding a non-pathological tau protein and an inducing cofactor to ADP3 can further generate ADP4, and so on.

[0333] Structural similarity

[0334] In this disclosure, the pathological tau protein obtained through two or more generations of amplification is similar to the primary pathological tau protein.

[0335] Methods for assessing protein structural similarity are well known to those skilled in the art. These methods primarily include: geometrically based comparisons, such as root mean square deviation (RMSD), template modeling score (TM-Score), and global distance test (GDT); topologically based comparisons, such as distance matrix alignment (DALI); and machine learning-based methods.

[0336] Preferably, this disclosure uses RMSD to assess the similarity of protein structures. RMSD calculates the root mean square value of the positional deviation of corresponding atoms (such as the α-carbon atom Cα in the amino acid sequence of a protein) in two structures. The smaller this value, the stronger the similarity between the two structures. For example, when... The two structures are considered to be almost identical; RMSD falls within... Between these two, it is considered that the two structures are highly similar; RMSD falls within... They are also considered to have similarities; Therefore, it does not have the significance of similarity assessment.

[0337] Preferably, the pathological tau protein is similar to the primary pathological tau protein. Preferably, the pathological tau protein is related to the AD-tau protein. Preferably, the pathological tau protein is related to the APD1.

[0338] Reagent test kit

[0339] This disclosure provides a kit. Preferably, the kit may have at least one container containing a predetermined amount of an inducing cofactor, a non-pathological tau protein, and a template pathological tau protein; wherein the inducing cofactor is selected from the group consisting of heparan sulfate, heparin, polyphosphate, phosphatidylinositol triphosphate, RNA, or combinations thereof.

[0340] The kit can provide an inducing cofactor, non-pathological tau protein, and template pathological tau protein for the preparation of the pathological tau protein described in this disclosure, along with a label or instruction manual.

[0341] The kit may also include a label or instructions indicating the amplification method described in this disclosure.

[0342] The main advantages of this disclosure include:

[0343] (1) This disclosure provides a method for continuous in vitro amplification of tau pathological protein, which requires only a small amount of human brain tissue extract as a template and uses negatively charged polyanionic compounds as inducing cofactors to obtain a large amount of tau pathological protein.

[0344] (2) This disclosure provides a tau pathological protein obtained through multiple generations of amplification. The pathological folding conformation of the protein is almost completely consistent with the pathological conformation in the human brain, and has the same pathological activity as the pathological protein extracted from human brain tissue.

[0345] (3) This disclosure also discovers a new use for heparan sulfate and / or its related derivative oligosaccharide molecules, which can be used as cofactors to induce tau protein monomers to form pathological fibers with pathological seeding activity, and can cause pathological changes in endogenous proteins in primary cultured neurons and animal brains, thereby mimicking the seeding function of pathological tau proteins in human brain tissue.

[0346] (4) This disclosure provides the application of pathological fibers prepared in vitro, which can be used to prepare animal models of neurodegenerative diseases and can be used as samples for large-scale compound screening and preparation of biological macromolecular antibodies.

[0347] The present disclosure is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the disclosure. Experimental methods in the following embodiments, unless otherwise specified, are generally performed 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 as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0348] Materials and methods

[0349] 1. Inducing cofactor:

[0350] Polyanionic compounds, such as heparan sulfate (HS), heparin (Hep), HS-derived oligosaccharides, polyphosphates (polyP), phosphatidylinositol triphosphate (PIP3), and RNA, etc. In mouse experiments, the nucleic acid is total RNA, DNA, or a chiral isomer, such as ZNA, extracted from mouse brain tissue.

[0351] 2. Induction system:

[0352] (1) Induction system without template protein:

[0353] (2) Induction system containing template protein:

[0354] (3) Induction system using tau protein monomers that mimic phosphorylation mutations:

[0355] 3. Induction steps:

[0356] Induction steps without template protein:

[0357] (1) Add tau monomer, Tris-HCl buffer, reducing agent and preservative to the EP tube;

[0358] (2) Heat shock the system at 56℃ for 10 min;

[0359] (3) Transfer the system to ice and cool for 10 minutes;

[0360] (4) Add inducing cofactors;

[0361] (5) Induce for about 7 days in a heat mixer at 37°C and 1000 rpm;

[0362] (6) Centrifuge the inducer of the reaction system at 100,000 x g for 30 min at 20 °C.

[0363] (7) Remove the supernatant and retain the precipitate at the bottom of the tube;

[0364] (8) Add PBS equal to the initial reaction volume to the tube, resuspend and wash the precipitated pathological fibrin, and soak for more than 12 hours.

[0365] (9) Centrifuge the inducer of the reaction system at 20°C at 100,000 x g for 30 min and remove the supernatant;

[0366] (10) Add 10% PBS equal to the initial reaction volume to the tube and sonicate the deposited protein until it becomes a homogeneous suspension.

[0367] (11) The prepared pathological tau fibers were quantified and quality control analysis was performed.

[0368] Induction steps involving template proteins:

[0369] (1) Calculate the required amount to be added, and add recombinant tau monomer, reaction buffer (Tris-HCl buffer: 30mM Tris-HCl, pH adjusted to 7.0-7.4 with NaOH; or commercially available PBS buffer, pH 7.0-7.4; or NaOAc buffer: 100mM NaOAc, pH adjusted to 7.0-7.4 with glacial acetic acid) and reducing agent DTT to the EP tube. No inducing cofactor is required in this step.

[0370] (2) Heat shock system at 56℃ for 10 min.

[0371] (3) Transfer the system to ice and cool for 10 minutes.

[0372] (4) Add inducing cofactor.

[0373] (5) Add tau protein extract from patient brain tissue that has been treated with ultrasound, or first-generation fibers amplified using tau pathology from patient brain tissue as a template as a reaction template.

[0374] (6) Induce at 37°C and 1,000 rpm for about 7 days in a heat mixer.

[0375] (7) Centrifuge the inducing agent of the reaction system at 22°C at 100,000×g for 60 min.

[0376] (8) Remove the supernatant and retain the sediment at the bottom of the tube.

[0377] (9) Add PBS to the tube with the same volume as the initial reaction, resuspend and wash the precipitated pathological fibrin, and soak for more than 12 hours.

[0378] (10) The precipitate system resuspended in PBS was centrifuged at 100,000×g for 60 min at 22°C to remove the supernatant and retain the precipitate.

[0379] (11) Add 10% of the initial reaction volume of PBS to the tube and sonicate the deposited protein slightly until it becomes a homogeneous suspension.

[0380] (12) Quantify and perform quality control analysis on the prepared pathological tau fibers, including negative staining transmission electron microscopy observation, and / or ultrafiltration sedimentation test, and / or thioflavin T (ThT) fluorescence binding test identification.

[0381] Induction steps using tau protein monomers with simulated phosphorylation mutations:

[0382] (1) Prepare the following stock solutions: 1M potassium phosphate (pH=7.2), 100mM TCEP (pH=6.5), 1M HEPES, and 1M potassium citrate, and filter them using a 0.22μm filter membrane.

[0383] (2) Add H2O, potassium citrate, HEPES, TCEP, potassium phosphate, PAD12, and finally AD seeds. Add the mixed liquid at a volume of 50 μL / well to a 384-well plate that has been rinsed with H2O beforehand. Add a ring of water around the wells and cover with a sealing film.

[0384] (3) Then place it on an ELISA reader for incubation at 37°C and 500 rpm, with a 1-minute interval between every 2 minutes of shaking, for 4-6 days.

[0385] (4) Remove the plate and mix the liquid from all the wells together. Then perform quality control on the fibers, and take 2 μL of the original solution, dilute it 5 times, and observe the morphology through a negative staining electron microscope.

[0386] (5) The remaining stock solution can be transferred to a 1.5 mL ultrafiltration tube and centrifuged at 45000 rpm at 25℃ for 1 h to obtain a transparent precipitate.

[0387] (6) After adding an equal amount of PBS, change the orientation of the supernatant tube and centrifuge again at 45000 rpm and 25℃ for 30 min to wash the precipitate.

[0388] (7) After adding 1 / 10 volume of the original solution in PBS, sonicate at room temperature for 40X. Use the known concentration of BSA protein as a standard curve and quantify the fiber concentration by Coomassie brilliant blue staining. Identify its seeding activity in primary neurons at 0.5 μg / well, using anti-mouse tau (T49 antibody) staining results as an indicator.

[0389] When using tau protein monomers with simulated phosphorylation mutations for induction, the addition or omission of an inducing cofactor will not affect the induction results or yield significantly. When using an inducing cofactor, its concentration can be referenced from the concentrations used in the previous two induction systems, such as 40 μg / mL.

[0390] Example 1: Preparation and characterization of HS-induced recombinant full-length tau protein T40 filament system (HS-T40)

[0391] This embodiment relates to the preparation and characterization results of a recombinant full-length tau protein T40 fibrous system induced by heparan sulfate as a cofactor (HS-T40), and compares the characterization results with those of a heparin-induced fibrous system.

[0392] Figure 1A shows schematic diagrams of the basic disaccharide units of the cofactors heparan sulfate (HS) and heparin (Hep). HS primarily uses glucuronic acid (GlcA) and glucosamine acetylglucosamine (GlcNAc) linked by 1-4 glycosidic bonds as basic repeating units. On average, each disaccharide unit has one sulfate group, located at position 6 (shown by the blue wavy line). However, positions 2 and 3 (shown by the red circle), as well as the acetyl group, can be sulfated, which is relatively rare in HS. Hep primarily uses iduronic acid (IdoA) and GlcNAc linked by 1-4 glycosidic bonds as basic repeating units. On average, each disaccharide unit has 2.7-3 sulfate groups, located at positions 2 and 6 (shown by the blue wavy lines), and the acetyl group of GlcNAc. However, position 3 (shown by the red circle) can also be sulfated.

[0393] Figure 1B shows the negative staining transmission electron microscopy characterization results of the HS and Hep-induced recombinant full-length tau protein T40 filament system. The addition of both cofactors HS and Hep induced fibrillation and aggregation of T40. This is referred to as prepared fibril (pff).

[0394] Figure 1C shows the characterization of the ultracentrifugation (Sedimentation) results of the T40 fiber-inducing system with added cofactors HS or Hep. After fibrinization and aggregation, due to the exponential increase in molecular weight and poor solubility, the fibers can settle to the bottom of the centrifuge tube under ultracentrifugation (100,000 x g). After SDS-PAGE gel electrophoresis, Coomassie Blue staining of the total inducing system and the insoluble precipitate clearly showed that the addition of HS or Hep induced fibrinization and aggregation of T40, with Hep showing a stronger inducing effect than HS.

[0395] Figure 1D shows the grayscale statistics of Figure 1C, representing the relative grayscale ratio of the insoluble precipitate T40 to the total induced system T40.

[0396] Figure 1E shows the fluorescence kinetics of thioflavin T (ThT) in the HS and Hep-induced systems. Protein fibrillation and aggregation typically form extensive interlayered β-sheet structures. In this embodiment, the addition of cofactors HS or Hep resulted in an exponential increase in fluorescence intensity, characterizing the presence of β-sheet structures in T40 fibrillation aggregation, with Hep exhibiting a stronger induction effect than HS.

[0397] The above three methods are commonly used in the field to control the formation of pathological protein fibers. They characterize the success of pathological fiber induction from the perspectives of morphology, molecular weight increase, insolubility, and fine chemical structure.

[0398] Figure 1F shows the results of a "seeding" experiment where HS-T40 pff and Hep-T40 pff, purified by ultracentrifugation according to the induction step, were applied to HEK293 human kidney epithelial cells overexpressing T40. Serial extraction biochemical experiments were performed on the seeded cells to separate insoluble pathological tau protein from the cell lysates. Combined with Western blotting experiments, different antibodies were used to detect tau in different components. It was found that cells seeded with HS-T40 pff and Hep-T40 pff produced more insoluble tau components than cells seeded with T40 monoclonal pff, indicating that both pffs have pathological activity against tau-overexpressing cell lines.

[0399] Figure 1G shows the grayscale statistics of Figure 1F, representing the relative grayscale ratio of phosphorylated tau (p-tau) in the insoluble precipitate to total tau in the whole cell lysate. HS-T40 pff and Hep-T40 pff have comparable pathological activity against tau-overexpressing cells.

[0400] Figure 1H shows the seeding results of primary cultured wild-type mouse neurons using HS-T40 pff and Hep-T40 pff. Immunocytochemical staining of mouse tau in neurons revealed that only cells treated with HS-T40 pff showed significant pathological deposition in the cytoplasm, a phenomenon not observed in the T40 monomer or Hep-T40 pff treatment groups. This indicates that HS-T40 pff has pathological activity against primary cultured wild-type mouse neurons, while Hep-T40 pff has no activity.

[0401] Figure 1I shows the staining statistics of Figure 1H, representing the relative staining area of ​​antibody T49 (recognizing mouse tau) and the staining area of ​​nuclear staining agent DAPI.

[0402] Figure 1J shows representative images of endogenous pathology in the brains of wild-type mice after stereotactic injection of HS-T40 pff or Hep-T40 pff into the hippocampus, detected by immunohistochemical staining. The antibody AT8, which recognizes phosphorylated tau at amino acid positions 202 / 205, was used. Results showed that HS-T40 pff induced endogenous pathological deposition in mice, including neurons and glial cells, three months after injection, while the control group HS did not exhibit this pathological activity. Hep-T40 pff, even with an injection duration of up to 20 months, did not induce endogenous pathology in wild-type mice.

[0403] Figure 1K shows the statistical results of Figure 1J. It represents the number of AT8 signal-positive neurons and oligodendrocytes.

[0404] Figure 1L shows the pathology detected 3 months after HS-T40 pff was injected into the hippocampus of wild-type mice, indicating whether the pathology was endogenous, i.e., composed of mouse tau, and characterizing the cell types that produced the pathology. NeuN is a neuronal marker; Olig2 is an oligodendrocyte marker; R2295M is an antibody that specifically recognizes mouse tau rather than human tau; p-tau is the AT8 antibody signal that recognizes phosphorylated tau at amino acids 202 / 205. The colocalization of the pathological signals with these cell markers indicates that the cells producing the pathology are mainly neurons and oligodendrocytes. The colocalization of the pathological signals with mouse tau indicates that these pathologies are endogenous in mice.

[0405] Figure 1M shows representative images of endogenous pathology in the brains of wild-type mice 10 months after stereotactic injection of HS-T40 pff into the hippocampus and striatum, detected by immunohistochemical staining. The antibody AT8, which recognizes phosphorylated tau at amino acid positions 202 / 205, was used. The results showed that HS-T40 pff induced neuronal and glial cell pathology in multiple brain regions and demonstrated its ability to induce pathological propagation across cells and brain regions; the control group T40 monomer itself did not possess this pathological activity.

[0406] Figure 1N shows representative images of endogenous pathology in the brains of 6htau transgenic mice, detected by immunohistochemical staining 6 months after stereotactic injection of HS-T40 pff into the hippocampus. The antibody AT8, which recognizes phosphorylated tau at amino acids 202 / 205, was used. The results show that HS-T40 pff induced abundant endogenous pathology in 6htau mice.

[0407] The above seeding experiment results indicate that HS-T40 pff has seeding activity that induces endogenous pathological deposition in overexpressing cell lines, primary neurons, and animals in vivo. Although Hep can induce fibrosis of recombinant tau protein in vitro, the fibers it produces cannot induce endogenous pathogenesis in physiological models, such as primary neurons and animals in vivo.

[0408] Example 2: Structures of heparin tetrasaccharide, heparin trisaccharide, and oligosaccharides that induce tau protein aggregation to form fibrous structures

[0409] This embodiment relates to the structures of sulfonated heparin tetrasaccharide, sulfonated heparin trisaccharide, and oligosaccharide.

[0410] Table 2A shows that sulfonated heparin tetrasaccharides can induce tau protein aggregation to form filaments using the system developed in this disclosure. All synthesized heparin oligosaccharide molecules were treated with a sodium ion exchange column, and the cations were all Na+, the same below.

[0411] Table 2B shows that sulfonated heparin trisaccharides can induce tau protein aggregation to form fibrous structures through the system developed in this disclosure.

[0412] Figure 2C shows that oligosaccharides with the illustrated structure can induce tau protein aggregation to form fibers through the system developed in this disclosure.

[0413] Example 3: Oligosaccharides induce recombinant tau protein to aggregate into obvious fibrillary aggregates.

[0414] This embodiment involves using oligosaccharides to induce recombinant tau protein, thereby forming fibrous aggregates.

[0415] Figure 3A shows the results of fluorescence kinetics analysis using thiosulfate T to induce oligosaccharides and recombinant tau proteins (T40, T40P301L, and T40297-391) in the system disclosed herein. The figure shows the fluorescence value at the reaction endpoint. Oligosaccharides can induce the aggregation of recombinant full-length tau protein T40 to form cross-β-sheet fibers, and can promote the assembly of mutant tau protein T40P301L and truncated tau protein T40297-391 to form tau fibers. Tau fibers can be recognized and bound by thiosulfate T, thereby producing fluorescence.

[0416] Figure 3B shows the negative staining transmission electron microscopy characterization results of the oligosaccharide-induced recombinant full-length tau protein T40 system. The results show that oligosaccharides can induce T40 aggregation to form obvious fibrillary aggregates.

[0417] Example 4: Oligosaccharide-T40 PFF can cause pathological deposits in neurons.

[0418] In this embodiment, oligosaccharide-T40 PFF was seeded into mouse neurons, and immunocytochemical staining demonstrated that the pathological protein formed in vitro could cause pathological deposition in neurons.

[0419] Figure 4A shows the seeding results of oligosaccharide-T40 PFF on primary cultured wild-type mouse neurons. Immunocytochemical staining of mouse tau in neurons revealed significant cytoplasmic pathological deposition in some neurons treated with oligosaccharide-T40 PFF, indicating that some oligosaccharide-T40 PFF possesses pathological activity against primary cultured wild-type mouse neurons.

[0420] Figure 4B shows the core region structure of the fibrous sheets formed by human T40 induced by cryo-electron microscopy, as resolved by heparin trisaccharide Tri-5. Different domains of the tau protein are labeled with different colors.

[0421] Figure 4C shows representative images of endogenous pathology in the brain of wild-type mice three months after injection of oligosaccharide-T40 PFF into the hippocampus, detected by immunohistochemical staining. Statistical results show that some oligosaccharide-T40 PFF can induce significant neuronal tau pathological deposition in the hippocampus of wild-type mice.

[0422] Example 5: Preparation and characterization of heparin and low molecular weight heparin glycan-induced recombinant full-length tau protein T40 fiber system (heparin glycan-T40)

[0423] The table shown in Figure 5A lists heparin and low molecular weight heparin (LMWH) glycans that can induce tau protein aggregation to form fibers through the system developed in this disclosure.

[0424] Figure 5B shows the results of fluorescence kinetic experiments using thiosulfate T to induce recombinant tau protein T40 in the system disclosed in this paper. The figure shows the kinetic curves. Heparin-like glycans can induce the aggregation of recombinant full-length tau protein T40 to form fibers with cross-beta sheets, which can be recognized and bound by thiosulfate T, thereby producing fluorescence.

[0425] Figure 5C shows the time it takes for heparin-like glycans to induce the aggregation of recombinant tau protein T40 to reach the half-peak value of the fluorescence kinetic curve. The results show that the higher the degree of aggregation, the shorter the time to reach the half-peak value.

[0426] Figure 5D shows the negative staining transmission electron microscopy characterization results of the heparinoid-induced recombinant full-length tau protein T40 system. The results show that heparinoids can induce T40 aggregation to form obvious fibrillary aggregates.

[0427] Figure 5E shows the seeding results of heparin glycan-T40 PFF on primary cultured wild-type mouse neurons. Immunocytochemical staining of mouse tau in neurons revealed significant cytoplasmic pathological deposition in some neurons treated with heparin glycan-T40 PFF, indicating that heparin glycan-T40 PFF has pathological activity against primary cultured wild-type mouse neurons.

[0428] Example 6: Preparation and characterization of the unsaturated standard heparin disaccharide-induced recombinant full-length tau protein T40 fibrous system (heparin glycan-T40)

[0429] The table shown in Figure 6A lists the unsaturated standard heparin disaccharides that can induce tau protein aggregation to form fibers through the system developed in this patent.

[0430] Figure 6B shows the fluorescence kinetics results of the induction of recombinant tau protein T40 by unsaturated standard heparin disaccharide and thiosulfate T using the system described in this patent. The figure shows the kinetic curve. Unsaturated standard heparin disaccharide can induce the aggregation of recombinant full-length tau protein T40 to form fibers with cross-beta sheets, which can be recognized and bound by thiosulfate T, thereby producing fluorescence.

[0431] Figure 6C shows the negative staining transmission electron microscopy characterization results of the recombinant full-length tau protein T40 system induced by unsaturated standard heparin disaccharide. The results show that unsaturated standard heparin disaccharide can induce T40 aggregation to form obvious fibrillary aggregates.

[0432] Figure 6D shows the seeding results of primary cultured wild-type mouse neurons using unsaturated standard heparin disaccharide-T40 PFF. Immunocytochemical staining of mouse tau in neurons revealed significant cytoplasmic pathological deposition in neurons treated with unsaturated standard heparin disaccharide-T40 PFF, indicating that heparin disaccharide-T40 PFF possesses pathological activity against primary cultured wild-type mouse neurons.

[0433] Figure 6E shows representative images of endogenous pathology in the brain of wild-type mice three months after injection of unsaturated standard heparin disaccharide-T40 PFF, detected by immunohistochemical staining. Statistical results show that unsaturated standard heparin disaccharide-T40 PFF can induce significant neuronal tau pathological deposition in the hippocampus of wild-type mice, and the neuronal pathology induced by HD001-T40 PFF is greater than that induced by HD004-T40 PFF.

[0434] Example 7: Results of amplification of AD-derived tau pathological protein into ADP1

[0435] This embodiment involves amplifying ADP1 from tau pathological protein derived from human brain aplasia (AD). Specifically, using AD-tau as a template seed and recombinant tau protein as a monomer, the formation of first-generation filaments (ADP1) is induced.

[0436] First, the characteristics of AD-derived tau pathological proteins were characterized, and the results are shown in Figure 7a. Figure 7a shows the negative stain transmission electro-microscopy (NS-TEM) characterization of pathological tau (named AD-tau) extracted from the gray matter of the frontal cortex of AD patients, taking tau pathological amplification in brain tissue as an example. Typical pathological tau fibers with helical structures can be seen.

[0437] ADP1 was prepared using four recombinant tau protein monomers, and the preparation process is shown in Figure 7b. The sedimentation results of the ADP1 preparation are shown in Figure 7c. Four recombinant tau protein isoforms (T40, T43, T39, and T44) at the same concentration were used as monomers. Tau fiber aggregates formed in all four ADP1 preparation systems, thus resulting in insoluble precipitates after sedimentation.

[0438] The NS-TEM characterization results of ADP1 preparations are shown in Figure 7d. As can be seen from the figure, typical pathological fibers were amplified in all four ADP1 preparation systems: T40, T43, T39, and T44.

[0439] The ThT fluorescence binding characterization results of ADP1 preparations are shown in Figure 7e. Four ADP1 preparation systems (T40, T43, T39, and T44) were diluted 10-fold and incubated with 20 μM ThT for 30 min. The emission intensity at 510 nm under 450 nm excitation light was then measured. The results showed that the ThT fluorescence signal of the four ADP1 preparation systems was several times higher than that of the tau monomer d control without AD-tau template, indicating the formation of a large number of β-sheet structures, a typical characteristic of amyloid fibers. These three characterization experiments demonstrate the successful amplification of ADP1 fibers from AD-tau.

[0440] Example 8: Results of ADP1 amplification to ADP2

[0441] This embodiment utilizes ADP1 to further amplify into ADP2. The process for preparing ADP1 is illustrated in Figure 7b.

[0442] Specifically, using AD-tau as a template seed and recombinant tau protein as a monomer, the first-generation filament (ADP1) was induced; using ADP1 as a template seed and recombinant tau protein as a monomer, and adding polyanionic cofactors such as HS, Hep, polyP, PIP3, and RNA, the second-generation filament (ADP2) was induced.

[0443] Using recombinant T40 tau protein as a monomer and ADP1 as a template seed, ADP2 was prepared using polyanionic cofactors such as HS, Hep, polyP, PIP3, and RNA. The NS-TEM characterization results of the prepared ADP2 are shown in Figure 7f. As can be seen from the figure, typical pathological fibers were amplified in all prepared systems.

[0444] The results of the ultrafiltration sedimentation experiment of ADP2 preparation are shown in Figure 7g. As can be seen from the figure, tau fiber aggregates formed in the preparation systems with added cofactors, thus resulting in insoluble precipitates after ultrafiltration; however, no precipitates were formed in the ADP2 system without added cofactors.

[0445] The ThT fluorescence binding characterization results of ADP2 preparation are shown in Figure 7h. Using T40 recombinant tau protein as a monomer, ADP1 as a template seed, and polyanions such as HS, Hep, polyP, PIP3, and RNA as cofactors, the ThT fluorescence signal of the preparation system with added cofactors increased several times compared with the control without cofactor template, indicating the formation of a large number of β-sheet structures in the system, which is a typical characteristic of amyloid fibrils. The above three characterization experiments demonstrate that adding polyanions as cofactors can successfully amplify ADP1 into ADP2 fibrils; while without adding cofactors, pathological fibrils cannot be formed.

[0446] Figure 7i shows the dose-dependent effect of cofactor-induced recombinant tau protein fibrillation, using HS as an example, characterized by ThT fluorescence kinetics. Based on these results, a moderate HS concentration of 40 μg / mL was selected, and this concentration was used for all cofactors.

[0447] Example 9: Comparison of cryo-electron microscopy structures of ADP1 and ADP2

[0448] This embodiment compares the structures of ADP1 and ADP2 with the structures of human brain-derived tau pathological proteins.

[0449] First, the cryo-electron microscopy (cryo-EM) structure of AD-tau derived from the brain tissue of AD patients was analyzed. The results, shown in Figure 8a, are identical to the previously reported AD-tau fiber structure (PMID: 28678775), exhibiting two different but similar structures: paired helical filaments (PHF) and straight filaments (SF). A C-shaped structure is observed unilaterally.

[0450] Figure 8b shows the Cryo-EM structures of four ADP1 monomers amplified by T40, T43, T39, and T44, all of which are identical to the PHF structure of the template AD-tau.

[0451] Figure 8c shows the Cryo-EM structure, taking HS-T40-ADP2, Hep-T40-ADP2 and polyP-T40-ADP2 as examples, which is the same as the structure of template T40-ADP1.

[0452] Figure 8d shows the comparison results of the PHF (AD-PHF) and SF (AD-SF) structures of AD-tau derived from brain tissue of AD patients, taking T40-ADP1 and HS-T40-ADP2 as examples. It shows that the fiber structures of ADP1 and ADP2 faithfully maintain the structure of the AD-tau template.

[0453] The quantitative results of Figure 8d are shown in Table 1. RMSD (Root Mean Square Deviation) is a commonly used parameter to measure the similarity between two structures. It calculates the root mean square value of the positional deviation of corresponding atoms (in this case, the α-carbon atom Cα in the protein amino acid sequence) in the two structures. The smaller the value, the stronger the similarity between the two structures. Pairwise comparisons were made between the structures of ADP1, HS-induced ADP2, and AD-derived tau pathological protein. The RMSD(Cα) values ​​ranged from 1.06 to 2.40, indicating a high degree of similarity in these structural frameworks, although minor differences may exist in the local orientation of the main chain or side chains, as shown in the structural diagram. Overall, the amplified ADP1 and ADP2 showed stronger similarity to AD-SF (smaller RMSD values), but also strong similarity to AD-PHF.

[0454] Table 1. Comparison of ADP1, ADP2 and tau pathological proteins derived from AD

[0455] Example 10: Detection of the pathological activity of ADP1 and ADP2 in primary wild-type mouse neurons

[0456] This embodiment relates to testing the pathological activity of ADP1 and ADP2 in primary wild-type mouse neurons.

[0457] The pathological activity of four ADP1 variants amplified using T40, T43, T39, and T44 as monomers in primary wild-type mouse neurons is shown in Figure 9a. After adding the four ADP1 variants to neuronal culture medium and culturing for 10 days, immunocytochemistry (ICC) was used to detect the pathological tau signal insoluble in the detergent HDTA (T49 is an antibody that specifically recognizes mouse tau). The results showed that all four ADP1 variants possessed neuronal pathological activity.

[0458] Figure 9b shows the pathological quantitative results of Figure a. The ratio of the relative areas of T49 signal and DAPI signal was statistically analyzed.

[0459] Taking HS-T40-ADP2 as an example, the pathological activity test results in primary wild-type mouse neurons are shown in Figure 9c. After adding HS-T40-ADP2 to neuronal culture medium and culturing for 10 days, the pathological tau signal insoluble by the detergent HDTA was detected by ICC (T49 is an antibody that specifically recognizes mouse tau). The results showed that HS-T40-ADP2 has the same neuronal pathological activity as AD-tau, while the ADP2 preparation system without added cofactors could not induce pathological activity in neurons because it did not form pathological fibers.

[0460] Figure 9d shows the pathological quantitative results of Figure c. The ratio of the relative areas of T49 signal and DAPI signal was statistically analyzed.

[0461] Taking HS-T40-ADP2 as an example, the in vivo pathological activity test results in wild-type mice are shown in Figure 9e. HS-T40-ADP2 was injected into the hippocampus of wild-type mice, and after 3 months, the antibody AT8 positive pathological signal (AT8 recognizes phosphorylated tau at positions 202 / 205) was detected by immunohistochemistry (IHC). The results showed that HS-T40-ADP2 has in vivo pathological activity in wild-type mice, while the ADP2 preparation system without added cofactors did not form pathological fibers and therefore could not be used for pathological examination in the brains of wild-type mice.

[0462] Figure 9f shows the pathological quantitative results of Figure e. The number of AT8 positive cells was counted.

[0463] Example 11: Results of ADP2 amplification to ADP3

[0464] This embodiment involves further amplifying ADP2 into ADP3.

[0465] Figure 10a shows the process of preparing HS-ADP3 from ADP2 through continuous amplification and the corresponding negative staining electron micrograph.

[0466] Figure 10b shows the negative-stain electron microscopy structure of AD-tau and HS-ADP3 tau protein filaments.

[0467] Figure 10c shows the two-dimensional classification comparison of the cryo-electron microscopy structures of AD-tau and HS-ADP3 tau protein filaments, indicating that HS-ADP3 and AD-tau have good structural similarity.

[0468] Figure 10d shows the pathological seeding activities of AD-tau and HS-ADP3 in primary cultured wild-type mouse cortical neurons; scale bar, 40 μm. Figure 10e shows the statistical results of Figure 10d. It can be seen that although the pathological activity of HS-ADP3 is lower than that of AD-tau, it is significantly higher than that of the T40 tau protein monomer.

[0469] Example 12: Products induced by tau protein monomers with simulated phosphorylation mutations and their characterization

[0470] This embodiment relates to the preparation and characterization results of the AD-PAD12-P1 system induced by tau protein monomers with simulated phosphorylation mutations.

[0471] Induction was performed according to the corresponding steps in the materials and methods. 2 μL of the stock solution was diluted 5-fold and observed morphologically using a negative staining electron microscope, as shown in Figure 11A. Seeding activity was identified on primary neurons using 0.5 μg / well anti-mouse tau (T49 antibody) staining, as shown in Figure 11B. These results demonstrate that the system can produce active AD-like pathological seeds.

[0472] Furthermore, the existing system was compared with the original 4 μM potassium phosphate system when the same amount of seeds were provided. The ON3R PAD12 system without AD seeds demonstrated that neither the 40 μM nor 4 μM potassium phosphate system caused self-aggregation of ON3R PAD12. The THT curve (Figure 11C) showed that the amplification efficiency of the 40 μM potassium phosphate system was significantly higher than that of the 4 μM potassium phosphate system.

[0473] To further verify the stability of the system, different AD seeds were amplified using a 40 μM potassium phosphate system for quality control (Figure 11D). The results showed that all seeds could be amplified well, with a conversion rate close to 50%. Adding HS as an inducing cofactor to the system also amplified AD-tau pathological fibers.

[0474] The in vivo pathogenic activity of the amplified pathological fibers was further examined. ADP1 amplified with different doses of PAD12 was injected into the hippocampus of wild-type mice. Six weeks later, the presence of pathological tau protein could be detected near the injection site using the phosphorylated tau antibody AT8, which recognizes pathological tau protein (Figure 11E).

[0475] AD-PAD12-P1 can also be used for multi-generation / multi-round pathological tau protein amplification. Compared with the amount of AD-tau seed used in the amplification systems involved in Examples 7-10, the amplification system in this example uses less seed, has a higher conversion rate, and can achieve an amplification fold of more than 150 times per round to replicate AD-tau pathology.

[0476] All documents mentioned in this disclosure are incorporated herein by reference as if each document were individually incorporated herein by reference. Furthermore, it should be understood that after reading the foregoing teachings of this disclosure, those skilled in the art can make various alterations or modifications to this disclosure, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for preparing and amplifying pathological tau protein in vitro using two or more generations, characterized in that, The method includes the following steps: (1) Provides a non-pathological state of tau protein; (2) In the presence of a template pathological tau protein and an optional inducing cofactor, the non-pathological tau protein is incubated to form a pathological tau protein with a pathological conformation. Wherein, the concentration A1 of the template pathological tau protein and the concentration A0 of the non-pathological tau protein satisfy 0.1% ≤ A1 / A0 ≤ 20%; the inducing cofactor is selected from the following group: heparan sulfate, heparan sulfate-derived oligosaccharide molecules, heparin, polyphosphate, phosphatidylinositol triphosphate, nucleic acid, or combinations thereof; the concentration of the inducing cofactor satisfies 2-1000 μg / mL; The pathological tau protein obtained in step (2) is used as the template pathological tau protein, and steps (1)-(2) are repeated.

2. The method as described in claim 1, characterized in that, The template pathological tau protein includes: primary pathological tau protein and ADP. n .

3. The method as described in claim 2, characterized in that, The ADP n It is obtained by n-generation amplification using the primary pathological tau protein and the non-pathological tau protein.

4. The method according to any one of claims 1-3, characterized in that, The non-pathological tau protein includes: human tau protein or fragments thereof, recombinant tau protein or fragments thereof, and chemically synthesized tau protein or fragments thereof.

5. The method according to any one of claims 1-4, characterized in that, The heparin sulfate-derived oligosaccharide molecules include sulfonated heparin oligosaccharides and oligosaccharides.

6. The method according to any one of claims 1-5, characterized in that, The sulfonated heparin oligosaccharide is selected from the group consisting of sulfonated heparin tetrasaccharide, sulfonated heparin trisaccharide, sulfonated heparin disaccharide, or combinations thereof.

7. The method according to any one of claims 1-6, characterized in that, The concentration of the inducing cofactor is 20-160 μg / mL.

8. A pathological tau protein obtained by amplifying a template pathological tau protein through two or more generations, characterized in that, The pathological tau protein is prepared by the method described in any one of claims 1-7.

9. The pathological tau protein as described in claim 8, characterized in that, The pathological tau protein has structural similarity to the template pathological tau protein.

10. The pathological tau protein as described in claim 9, characterized in that, The root mean square deviation between the pathological tau protein structure and the primary pathological tau protein structure 11. The use of an inducing cofactor or a pharmaceutically acceptable salt thereof, characterized in that, This preparation is intended for the formulation, composition, or kit used to induce and / or promote the formation of pathological tau protein from non-pathological tau protein; wherein the inducing cofactor or a pharmaceutically acceptable salt thereof is selected from the group consisting of: heparan sulfate, heparan sulfate-derived oligosaccharide molecules, heparin, polyphosphate, phosphatidylinositol triphosphate, nucleic acids, or combinations thereof.

12. A formulation or composition, characterized in that, The formulation or composition includes: (i) the pathological tau protein according to any one of claims 8-10; and (ii) Inducing cofactors or their pharmaceutically acceptable salts; The inducing cofactor or a pharmaceutically acceptable salt thereof is selected from the group consisting of heparan sulfate, heparan sulfate-derived oligosaccharide molecules, heparin, polyphosphate, phosphatidylinositol triphosphate, nucleic acids, or combinations thereof.

13. A reagent kit, characterized in that, The kit includes: (c1) the formulation or composition according to claim 12; and (c2) Optional non-pathological state tau protein.

14. Use of any pathological tau protein according to any one of claims 8-10, the formulation or composition according to claim 12, or the kit according to claim 13, characterized in that, include: (a) Inducing and / or promoting the deposition of pathological tau protein in cells / tissues in vitro / in vivo; (b) Establishing animal models of neurodegenerative diseases; (c) Preparation of biomacromolecule probes / antibodies; (d) Large-scale compound screening.

15. A method for inducing and / or promoting pathological deposits in cells in vitro, characterized in that, The method includes the following steps: (S1) Provide a cell containing tau protein in a non-pathological state; (S2) The pathological tau protein of any one of claims 8-10 or the preparation or composition of claim 12 is incubated with the cells to induce and / or promote the deposition of pathological proteins in the cells; The inducing cofactor is selected from the group consisting of heparan sulfate, heparan sulfate-derived oligosaccharide molecules, heparin, polyphosphate, phosphatidylinositol triphosphate, nucleic acid, or combinations thereof.

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