WO2019134481A1

WO2019134481A1 - Uses of protein in preparing drug for preventing and treating alzheimer's disease

Info

Publication number: WO2019134481A1
Application number: PCT/CN2018/120172
Authority: WO
Inventors: 张英豪; 付晶鹏
Original assignee: 上海清流生物医药科技有限公司
Priority date: 2018-01-02
Filing date: 2018-12-11
Publication date: 2019-07-11
Also published as: CN109985231A

Abstract

Uses of a protein in preparing a drug for preventing and treating Alzheimer's disease, said protein being an sDSS1 protein used in preparing a drug for preventing neurodegenerative and vascular dementias, or a drug for treating said diseases in the prophase, early, middle and later stages. Said sDSS1 protein interacts with an amyloid β-protein (Aβ) to contribute to Aβ removal while protecting against Aβ toxicity. Moreover, the Aβ deposition is effectively reduced in animals, and the concentration of a soluble Aβ is reduced, leading to disease symptom relief.

Description

Application of a protein in preparing medicine for preventing and treating dementia

Technical field

The present invention relates to the use of the sDSS1 protein drug for the preparation of a medicament for preventing and treating dementia, comprising a prophylactic medicament for the preparation of degenerative dementia and vascular dementia, or for preparing a pre-, early, intermediate or late stage of these diseases. Therapeutic drugs.

Background technique

Dementia is a type of brain disease that causes long-term and progressive deterioration of patient thinking and learning memory. The most common form of dementia is Alzheimer's Disease (AD), which accounts for dementia. 50-70% of patients, others include vascular dementia (

25%), Louise-type dementia (

15%) and so on. According to data released by Alzheimer's Disease International in 2015, the number of people with dementia worldwide is about 46.8 million, and the number of patients worldwide is expected to reach 75 million in 2030 [1]. AD is a typical neurodegenerative disease with clinical manifestations of progressive learning and memory impairment and neurological damage. A typical pathological feature of AD patients is the presence of β-amyloid (Aβ) plaques and tau deposition in nerve tissue. The β amyloid hypothesis suggests that the progressive accumulation of toxic proteins caused by the imbalance of Aβ protein production and clearance in nerve tissue and the resulting synaptic dysfunction and neuronal death are key mechanisms leading to disease [2] [3]. Based on the beta amyloid hypothesis, the researchers hope to prevent Aβ protein production and aggregation, or promote Aβ clearance, in order to prevent or treat diseases [4]. The results of AD studies based on animal experiments show that antibodies [5] [6], peptide drugs [7], small molecule drugs [8] can block Aβ aggregation or production, reduce plaque formation, and reduce animal disease indications. Therefore, the drug inhibits and clears the pathological deposition of Aβ or can be an effective way to treat AD.

In the past ten years, dozens of pharmaceutical companies, including Lilly, AstraZeneca, Johnson & Johnson, Pfizer, and Roche, have invested tens of billions of dollars in the development of AD treatments. However, the progress has not been smooth, and the difficulty in developing AD treatment drugs can be seen. With complexity. Lily uses the Aβ protein as a target monoclonal antibody, solanezumab, which has been shown to slow down cognitive decline in 34% of patients with mid-term AD and decline in behavioral capacity in 18% of patients in phase III clinical trials, but is subject to extensive side effects. The comprehensive effect evaluation was not affected by statistical differences, and the drug was declared a failure in December 2016. Biogen's Phase Ib clinical trial published in July 2018 showed that the aducanumab, an experimental monoclonal antibody against Aβ protein, can reduce beta amyloid in the brain of patients and significantly improve the cognitive level of patients. Phase II clinical trials of BAN2401, another monoclonal antibody against Aβ protein, released by Baishen and Eisai in November 2018, also achieved significant clinical results. In addition, in 2017-2018, international pharmaceutical giants such as Roche, Eli Lilly, AstraZeneca, and Merck have announced new drug development plans for AD diseases, which shows that the pharmaceutical industry is focusing on AD diseases.

Shfm1 (split hand/split foot malformation type 1) gene is one of the key genes in human crab claw disease. It is highly conserved in evolution and its encoded protein DSS1 is involved in stable genome, homologous gene recombination, DNA damage repair and cell proliferation. Etc. [9-12]. The results of the inventors of the present invention show that the DSS1 protein can be added to the oxidized protein by a energy-consuming enzymatic reaction to help the cells clear the oxidized protein [13]. These results show the important role of DSS1 protein in biological activities.

The citations for the above are as follows:

1. Alzheimer’s Disease International (2016) World Alzheimer Report 2015: the global impact of dementia, an analysis of prevalence,incidence, cost and trends.

2. Gupta A, Iadecola C (2015) Impaired Aβclearance: a potential link between atherosclerosis and Alzheimer's disease. Front Aging Neurosci 7:115.

3.Bu XL, Xiang Y, Jin WS, Wang J, Shen LL, Huang ZL, Zhang K, Liu YH, Zeng F, Liu JH, Sun HL, Zhuang ZQ, Chen SH, Yao XQ, Giunta B, Shan YC, Tan J, Chen XW, Dong ZF, Zhou HD, Zhou XF, Song W, Wang YJ (2017) Blood-derived amyloid-β protein induces Alzheimer's disease pathologies. Mol Psychiatry. [Epub ahead of print]

4. Citron M. (2010) Alzheimer's disease: strategies for disease modification. Nat Rev Drug Discov 9(5): 387-98.

5.Winblad B, Andreasen N, Minthon L, Floesser A, Imbert G, Dumortier T, Maguire RP, Blennow K, Lundmark J, Staufenbiel M, Orgogozo JM, Graf A (2012) Safety, tolerability, and antibody response of active Aβ Immunotherapy with CAD106 in patients with Alzheimer's disease: randomised, double-blind, placebo-controlled, first-in-human study. Lancet Neurol 11(7): 597-604.

6. Sevigny J, Chiao P, Bussière T, Weinreb PH, Williams L, Maier M, Dunstan R, Salloway S, Chen T, Ling Y, O'Gorman J, Qian F, Arastu M, Li M1, Chollate S, Brennan MS, Quintero-Monzon O, Scannevin RH, Arnold HM, Engber T, Rhodes K, Ferrero J, Hang Y, Mikulskis A, Grimm J, Hock C, Nitsch RM, Sandrock A. The antibody aducanumab reduces Aβ plaques in Alzheimer's disease. Nature 537: 50–56.

7.Chang L, Cui W, Yang Y, Xu S, Zhou W, Fu H, Hu S, Mak S, Hu J, Wang Q, Ma VP, Choi TC, Ma ED, Tao L, Pang Y, Rowan MJ, Anwyl R, Han Y, Wang Q (2015) Protection against β-amyloid-induced synaptic and memory impairments via altering β-amyloid assembly by bis(heptyl)-cognitin.Sci Rep 5:10256.

8.Kim HY, Kim HV, Jo S, Lee CJ, Choi SY, Kim DJ, Kim Y (2015) EPPS rescues hippocampus-dependent cognitive deficits in APP/PS1 mice by disaggregation of amyloid-β oligomers and plaques.Nat Commun 6 :8997.

9.Van Silfhout AT, van den Akker PC, Dijkhuizen T, Verheij JB, Olderode-Berends MJ, Kok K, Sikkema-Raddatz B, van Ravenswaaij-Arts CM (2009) Split hand/foot malformation due to chromosome 7q aberrations (SHFM1 ): additional support for functional haploinsufficiency as the causative mechanism. Eur J Hum Genet 17(11): 1432-8.

10.Li J,Zou C,Bai Y,Wazer DE,Band V,Gao Q(2006)DSS1 is required for the stability of BRCA2.Oncogene 25:1186–1194.

11. Liu J, Doty T, Gibson B, Heyer WD (2010) Human BRCA2 protein promotes RAD51 filament formation on RPA-covered single stranded DNA. Nat Struct Mol Biol 17: 1260–1262.

12.Zhou Q, Kojic M, Cao Z, Lisby M, Mazloum NA, Holloman WK (2007) Dss1 interaction with Brh2 as a regulatory mechanism for recombinational repair. Mol Cell Biol 2:2512–2526.

13.Zhang Y, Chang FM, Huang J, Junco JJ, Maffi SK, Pridgen HI, Catano G, Dang H, Ding X, Yang F, Kim DJ, Slaga TJ, He R, Wei SJ (2014) DSSylation, a novel Protein modification targets proteins induced by oxidative stress, and facilitates their degradation in cells. Protein Cell 5(2): 124-40.

Summary of the invention

The transient accumulation of Aβ protein is one of the key factors in the pathogenesis of AD. It is one of the main ways to treat AD diseases by removing Aβ or inhibiting Aβ aggregation. The sDSS1 protein provided by the invention can bind to Aβ protein to accelerate the clearance of Aβ protein, reduce the accumulation of Aβ protein, improve the symptoms of the disease, and has the potential for preparing clinical AD and other dementia prevention drugs and therapeutic drugs.

The specific technical solutions are as follows:

The use of a protein for the preparation of a medicament for the prevention and treatment of dementia, which is the use of the sDSS1 protein for the preparation of a medicament for the prevention and treatment of dementia.

Preferably, the dementia refers to degenerative dementia, including Alzheimer's disease, Alzheimer's disease, Lewy body dementia, and frontotemporal dementia.

Preferably, the dementia is vascular dementia.

Preferably, the dementia is pre-dementia dementia, mild dementia dementia, moderate dementia dementia, and severe dementia dementia.

Preferably, the sDSS1 protein comprises human, chimpanzee, bonobo, gorilla, orangutan, white-cheeked gibbons, golden monkey, rhesus monkey, golden monkey, East African pheasant, Angora simian, white-tailed white-browed monkey, ghost A basic protein formed by any sDSS1 protein sequence in scorpionfish, porpoise monkey, wherein the amino acid sequence of human sDSS1 is SEQ ID NO: 1, the amino acid sequence of chimpanzee sDSS1 is SEQ ID NO: 2, and the amino acid sequence of porcine chimpanzee sDSS1 is SEQ. ID NO: 3, the amino acid sequence of gorilla sDSS1 is SEQ ID NO: 4, the amino acid sequence of orangutan sDSS1 is SEQ ID NO: 5, the amino acid sequence of white buccal gibbon sDSS1 is SEQ ID NO: 6, and the golden monkey sDSS1 The amino acid sequence of SEQ ID NO: 7, the amino acid sequence of rhesus sDSS1 is SEQ ID NO: 8, the amino acid sequence of gilt monkey sDSS1 is SEQ ID NO: 9, and the amino acid sequence of ssDSS1 is SEQ ID NO: 10, Angola. The amino acid sequence of simian sDSS1 is SEQ ID NO: 11, the amino acid sequence of leucocephalus sDSS1 is SEQ ID NO: 12, and the amino acid sequence of scorpion sDSS1 is SEQ ID NO: 13. M.nemestrina sDSS1 amino acid sequence as SEQ ID NO: 14.

Preferably, the sDSS1 protein is the first protein having a similarity to the basic protein of 70% or more.

Preferably, the sDSS1 protein is based on 58 amino acids of the nitrogen terminus of the basic protein, and fuses other polypeptide fragments at the nitrogen terminal or the carbon terminal, and the structural features or amino acid sequence characteristics of the polypeptide fragment for fusion are The second protein of the same or similar sequence of the carbon end of the basic protein.

Preferably, the sDSS1 protein is based on the basic amino acid of the basic protein of 58 amino acids, and the other amino acid fragments are fused at the nitrogen or carbon end, and the fused protein can realize the transmembrane transport function. .

Preferably, the sDSS1 protein is a fusion formed by using the basic protein, the first protein, the second protein or the third protein to form a protein, a carrier protein, an antibody or other amino acid fragments of any length. protein.

Preferably, the sDSS1 protein is a polypeptide/protein modification produced by modification of the basic protein, the first protein, the second protein, the third protein or the fusion protein.

Preferably, the polypeptide/protein modification is directed to an amino group on an amino acid side chain, a carbonyl group on an amino acid side chain, a nitrogen terminal amino group, a carbon terminal carbonyl group, a cysteine, a tyrosine, a serine, a tryptophan. Specific or non-specific chemical modification of 1-20 sites.

Preferably, the method for modifying the polypeptide/protein modification comprises glycosylation modification, fatty acid modification, acylation modification, Fc fragment fusion, albumin fusion, polyethylene glycol modification, dextran modification, heparin modification, polyethylene Pyrrolidone modification, polyamino acid modification, polysialic acid modification, chitosan and its derivative modification, lectin modification, sodium alginate modification, carbomer modification, polyvinylpyrrolidone modification, hydroxypropyl methylcellulose modification, One or more of hydroxypropylcellulose modification, acetylation modification, formylation modification, phosphorylation modification, methylation modification, sulfonation modification, and other pharmaceutically acceptable polypeptide/protein drug modification methods.

Preferably, the sDSS1 protein is an amino acid other than the 20 basic amino acids based on the amino acid sequence of the basic protein, the first protein, the second protein, the third protein or the fusion protein. - 31 non-natural amino acid replacement proteins substituted with any amino acid site.

Preferably, the amino acid substitution of the non-natural amino acid replacement protein comprises replacement with hydroxyproline, hydroxylysine, selenocysteine, D-form amino acid or synthetic non-natural amino acid and derivatives thereof.

Preferably, the sDSS1 protein is pharmaceutically applicable to the basic protein, the first protein, the second protein, the third protein, the fusion protein, the polypeptide/protein modification or the non-natural amino acid replacement protein. Part or all of the complex formed by the drug carrier.

Preferably, the pharmaceutical carrier of the complex comprises one or one of an enteric coating preparation, a capsule, a microsphere/capsule, a liposome, a microemulsion, a double emulsion, a nanoparticle, a magnetic particle, a gelatin, and a gel. the above.

Preferably, the base of the protein, the first protein, second protein and third protein, fusion protein, polypeptide / protein or complex modifications play operation starting concentration of toxic proteins toxic shield is not less than 0.2μg /mL .

Preferably, the basic protein, the first protein, the second protein, the third protein, the fusion protein, the polypeptide/protein modification or the complex exhibits a molar concentration ratio of shielding toxic protein toxicity of not less than 0.010; The molar concentration ratio refers to the ratio of the molar concentration of the protein, polypeptide/protein modification or complex in the reaction system to the molar concentration of the toxic protein.

Preferably, the sDSS1 protein targets the individual's own sDSS1 protein, and affects the level of the individual's own sDSS1 protein by the exogenous drug.

Preferably, the drug is a drug target of a sDSS1 protein, a gene of sDSS1 protein, a regulatory element of a gene of sDSS1 or a gene of sDSS1.

Preferably, the drug modulates the amount of sDSS1 protein in the blood or cerebrospinal fluid by affecting protease/peptidase activity in blood or cerebrospinal fluid.

Preferably, the drug is the first drug formed by a chemical small molecule drug, an antibody, a polypeptide/protein drug, a nucleic acid drug or a nano drug.

Preferably, the sDSS1 protein is any one of the basic protein, the first protein, the second protein, the third protein, the fusion protein, the polypeptide/protein modification, the complex, and the first drug. A second drug formed by a combination of two or more of one component.

Preferably, the sDSS1 protein is a basic protein, a first protein, a second protein, a third protein, a fusion protein, a polypeptide/protein modification, a non-natural amino acid replacement protein, a complex, A third drug formed by the combination of one, two or more of any one of the first drugs with a pharmaceutically acceptable excipient.

Preferably, the sDSS1 protein is a fourth obtained by introducing a nucleotide sequence encoding the basic protein, the first protein, the second protein, the third protein or the fusion protein into the body and expressing the same by an expression system. Protein.

Preferably, the expression system is eukaryotic expression plasmid vector, adenovirus, adeno-associated virus, lentivirus, retrovirus, baculovirus, herpes virus, pseudorabies virus, ZFN gene editing technology, TALEN gene editing technology, CRISPR/Cas gene editing technology or other medically available gene editing technology or viral vector.

Preferably, the sDSS1 protein is a fifth protein formed by the basic protein, the first protein, the second protein, the third protein or the fusion protein obtained by the transplanted cell in the individual.

Preferably, the cell is a stem cell, a precursor cell or an adult cell of any one of humans.

Preferably, the stem cells are embryonic stem cells, induced pluripotent stem cells, cells obtained by transdifferentiation, or stem cells derived from primary culture, pluripotent or pluripotent stem cells differentiated from mother cells.

Preferably, the sDSS1 protein is a sixth protein formed by the basic protein, the first protein, the second protein, the third protein or the fusion protein obtained by transplanting tissues or organs in an individual.

Preferably, the tissue is a whole organ or part of a tissue block of the brain, liver, kidney, spleen, islet, or blood, fat, muscle, bone marrow, skin.

Preferably, the sDSS1 protein is a seventh protein introduced into the body by serum, cerebrospinal fluid, and interstitial fluid infusion.

Preferably, the prophylactic agent comprises a basic protein, any of the first to seventh proteins, a fusion protein, a polypeptide/protein modification, a non-natural amino acid replacement protein, a complex, a first drug, and a second Drugs, third drugs, protein drugs in expression systems, cells, tissues, organs, body fluids, tissue fluids, peptide drugs, nucleic acid drugs, chemical small molecule drugs, cell products, commercial transplant tissues, injections, lyophilized powder, One or more of health care products and food additives.

Preferably, the therapeutic agent comprises a basic protein, any of the first to seventh proteins, a fusion protein, a polypeptide/protein modification, a non-natural amino acid replacement protein, a complex, a first drug, and a second Drugs, third drugs, protein drugs in expression systems, cells, tissues, organs, body fluids, tissue fluids, peptide drugs, nucleic acid drugs, chemical small molecule drugs, cell products, commercial transplant tissues, injections, lyophilized powder, One or more of health care products and food additives.

Features and/or benefits of the present invention are:

1. The sDSS1 protein provided by the invention binds to Aβ protein, inhibits the aggregation of Aβ protein and reduces the binding ability of Aβ protein to its receptor protein, and effectively alleviates the cytotoxicity caused by Aβ protein.

2. The sDSS1 protein provided by the present invention can accelerate the clearance of Aβ protein by microglia, and shields Aβ oligomer toxicity and reduces microglia activation induced by Aβ oligomer.

3. The sDSS1 protein provided by the present invention significantly improves the disease symptoms of the AD model nematode on the CL4176 nematode and reduces the accumulation of Aβ protein in the nematode tissue.

4. The sDSS1 protein provided by the present invention helps mice clear Aβ and reduce Aβ levels in the blood. Long-term injection of sDSS1 protein into AD model APP/PS1 mice can effectively reduce Aβ plaque deposition in brain tissue.

5. The sDSS1 protein provided by the present invention is a protein possessed by humans and other primates, has a relatively small molecular weight, low immunogenicity, and has a natural protein degradation mechanism in vivo, and therefore, clinical application does not cause significant immunity. The reaction or other toxic side effects are safe and reliable.

In summary, the present invention provides an sDSS1 protein drug for the treatment of dementia. The sDSS1 protein can bind to Aβ protein, inhibit Aβ aggregation, and reduce Aβ protein and receptor by molecular, cell and animal level experiments. The binding efficiency promotes microglia clearance of Aβ and reduces microglia activation. In animal experiments, sDSS1 protein promotes Aβ clearance in the blood, reduces the accumulation of Aβ protein in tissues, and alleviates disease symptoms. sDSS1 protein has low immunogenicity and remarkable efficacy, and has the potential to be used for clinical preparation of dementia prevention drugs or for preparing pre-treatment, early, intermediate, and advanced dementia drugs.

DRAWINGS

The invention is further described in detail below with reference to the accompanying drawings, in which

Figure 1A-1B. Molecular experiments show that A[beta] protein binds to sDSSl protein.

1A, Aβ 1-42 protein and Aβ 1-40 protein were incubated with sDSS1 protein, respectively, and a complex of Aβ 1-42 protein and sDSS1 protein was detected by Aβ protein antibody (molecular weight 10KD-37KD, Lane 3 and lane 4). The Aβ 1-40 protein is incubated alone to form a partial dimer, and incubation with the sDSS1 protein reduces dimer formation.

Figure 1B, sDSS1 protein and Aβ 1-42 protein (lane 3 and lane 4) or sDSS1 protein and Aβ 1-40 protein (lane 6 and lane 7) can also be detected by sDSS1 protein antibody ( The molecular weight is between 37 and 150 KD), and the complex formation increases as the concentration of sDSS1 protein in the reaction system increases.

Figure 1C. ThT assay detects A[beta] 1-42 protein aggregation. The Aβ1-42 protein aggregates at 12 hours and shows an ascending curve. Addition of 5% or 10% sDSS1 protein according to the molecular weight ratio inhibited the ascending curve and prolonged the detection time without observing the rising curve, suggesting that no Aβ 1-42 protein aggregation occurred.

Figure 1 D. ThT assay detects Aβ1-40 protein aggregation. The Aβ 1-40 protein began to show a rising curve at 24 hours, indicating aggregate formation. Addition of 5% or 10% sDSS1 protein according to the molecular weight ratio inhibited the ascending curve, and the prolongation of the detection time did not observe a rising curve, suggesting that no Aβ 1-40 protein aggregation occurred.

Figure 1E. ThT assay detects sDSS1 protein mutant 4 inhibits Aβ1-40 protein aggregation. The Aβ1-40 protein aggregates at 20-24 hours and shows an ascending curve. When 0.667%, 0, 3.33%, or 16.67% sDSS1(M4) protein was added according to the molecular weight ratio, the rise curve was inhibited, and the rise time was not observed, indicating that no Aβ 1-40 protein aggregation occurred.

Figure 1F. ThT assay detects sDSS1 protein mutant 11 inhibiting Aβ1-40 protein aggregation. The Aβ1-40 protein aggregates at 20-24 hours and shows an ascending curve. When 0.667%, 0, 3.33%, or 16.67% sDSS1(M11) protein was added according to the molecular weight ratio, the rising curve was inhibited, and the rise time was not observed, indicating that no Aβ 1-40 protein aggregation occurred.

2A-2B. Molecular experiments show that Aβ protein oligomers bind to sDSS1 protein.

2A, the Aβ 1-42 protein oligomer and the Aβ 1-40 protein oligomer were incubated with the sDSS1 protein, respectively, and the complex formed by the Aβ 1-42 protein and the sDSS1 protein was detected by the Aβ protein antibody (molecular weight 10KD). Between -37KD, lane 3 and lane 4). No significant difference was detected between the Aβ 1-42 protein and the sDSS1 protein.

2B, a complex of sDSS1 protein and Aβ 1-42 oligomer (lane 3) or sDSS1 protein and Aβ 1-40 oligomer (lane 5) can be detected by using sDSS1 protein antibody (molecular weight 37-150KD) between).

Figure 2C. ThT assay detects the reaction of sDSS1 protein with Aβ1-40 oligomer. The addition of 50% and 100% sDSS1 protein in a molecular weight ratio of 30 μM Aβ1-40 oligomer showed an increase in the fluorescence value of the Aβ 1-40 oligomer, and the fluorescence value remained substantially unchanged during the 12-hour detection period.

Figure 2D. ThT assay detects the reaction of the sDSS1 protein with the Aβ 1-42 oligomer. The addition of 50% and 100% sDSS1 protein in a molecular weight ratio of 30 μM Aβ 1-42 oligomer showed an increase in the fluorescence value of the Aβ 1-42 oligomer. Within the 12-hour detection limit, the fluorescence value of the Aβ1-42 oligomer also increased, and the fluorescence value increased after the addition of the sDSS1 protein.

Figure 3A. The sDSS1 protein is unable to interact with the RAGE receptor protein. The results of molecular interaction test showed that on the binding curve, it can be seen that after the injection of sDSS1 protein, no obvious ascending binding curve was observed, and no obvious dissociation curve was observed.

Figure 3B. sDSS1 protein inhibits the interaction of Aβ protein with RAGE receptor protein. Aβ 1-42 protein can bind to RAGE receptor protein, and the addition of sDSS1 protein can inhibit the binding efficiency of Aβ protein to RAGE, the binding amount of Aβ protein decreases, and the effect shows a typical dose-dependent effect. The greater the concentration of sDSS1 protein, the lower the binding efficiency of Aβ protein.

Figures 4A-4C. Cell experiments demonstrate that sDSS1 protein shields Aβ 1-42 oligomer toxicity and protects N2a cell viability.

1A, Aβ1-42 oligomers cause N2a cytotoxicity, and the addition of Aβ 1-42 oligomers causes abnormal aggregation and rounding of cells, and sDSS1 protein can alleviate these phenomena.

Figure 2B, detection of cell viability revealed that sDSS1 protein can reduce cell viability by Aβ 1-42 oligomer. After the addition of the same amount of sDSS1 protein, cell viability is almost unaffected by the toxicity of Aβ 1-42 oligomer.

In Fig. 2C, Aβ 1-42 caused cytotoxicity, which showed an increase in the LDH content of the solution. After the addition of sDSS1 protein, the LDH content of the solution was significantly decreased, indicating a decrease in cytotoxicity. N=5 per group. Data were analyzed by ANOVE, *, p-value < 0.05; **, p-value < 0.01.

Figures 5A-5B. Cell experiments demonstrate that sDSS1 protein shields Aβ 1-42 oligomer toxicity and protects primary neuronal cell viability.

Figure 5A, 10 μM Aβ 1-42 oligomers can cause a decrease in neuronal cell viability, and cell viability can be recovered after the addition of 5 μM and 10 μM sDSS1 protein.

Figure 5B, sDSS1 protein can reduce the cytotoxicity level caused by Aβ 1-42 protein, and 10 μM sDSS1 protein can protect cells from the toxicity of Aβ 1-42 oligomer. N=10 per group. Data were analyzed by ANOVE, *, p-value < 0.05; **, p-value < 0.01.

Figures 6A-6B. The sDSS1 protein promotes microglia clearance of Aβ protein.

Figure 6A, Aβ 1-42 protein and sDSS1 protein were added to the medium. The concentration of Aβ protein in microglia was significantly higher than that of the control with only Aβ protein added after 24 hours, with the increase of the concentration of sDSS1 protein added. The cytosolic Aβ protein concentration is increased.

Fig. 6B, correspondingly, the content of A? 1-42 protein in the culture medium to which sDSS1 protein was added was significantly lower than that of the control sample to which only A? was added, and showed a dose-dependent effect. N=3 per group. Data were analyzed by ANOVE, *, p-value < 0.05; **, p-value < 0.01.

Figures 6C-6D. The sDSS1 protein reduces microglial activation by Aβ 1-42 protein oligomers.

In Fig. 6C, Aβ 1-42 protein oligomers caused microglia activation, which showed an increase in ROS levels in BV2 cells. After adding different concentrations of sDSS1 protein, ROS levels in BV2 cells gradually decreased, which was close to the control group.

Figure 6D, Aβ 1-42 protein oligomer stimulates microglia to release inflammatory factor TNF-α, and sDSS1 protein reduces TNF-α release. N=3 per group. Data were analyzed by ANOVE, *, p-value < 0.05; **, p-value < 0.01.

Figure 7A. The sDSS1 protein ameliorate the disease symptoms of CL4176 nematodes. As the culture time prolonged, the sputum rate of the AD model CL4176 nematode gradually increased, and the addition of sDSS1 protein to the culture medium significantly slowed down the rate of sputum. And the mitigation efficiency showed drug dose dependence, and the mitigation effect increased with the increase of sDSS1 protein concentration. At 26 hours, the ratio of the control group to the nematode was only 15.09%, the 500 μg/mL sDSS1 protein administration group was 39.47%, and the 1000 μg/mL sDSS1 protein administration group was 65.71%. At 43 hours, the nematode in the control group was almost all sputum (the ratio of the nematode was less than 1%), while in the 500 μg/mL sDSS1 protein-administered group, the proportion of the untreated nematode was 18.42%, and the 1000 μg/mL sDSS1 protein-administered group was 48.57. %. The number of nematodes per group is 90. The experiment was repeated 3 times and the data was analyzed by Log-rank (Mantel-Cox) test, ***, p-value < 0.001.

Figure 7B. Immunohistochemical staining for the detection of Aβ 1-42 deposition in C14176 nematode tissue. AD model 4176 elegans tissues were fixed and immunofluorescent stained, Aβ 1-42 specific antibodies were used to display Aβ protein (green fluorescence), Hoechst dye was used to stain nuclei (blue fluorescence), and white arrows indicate Aβ plaque deposition. Typical Aβ 1-42 plaques and diffuse Aβ precipitates were seen in the control nematode tissues. After treatment with 500 μg/mL sDSS1 protein, Aβ plaques and diffuse deposition were significantly reduced. The 1000 μg/mL sDSS1 protein administration group showed almost no plaque and less diffuse deposition.

Figure 7C - Figure 7D. 4176 Reduction of A[beta] 1-42 aggregates and total A[beta] protein content in nematode tissue mills.

Figure 7C, Western blotting was used to detect Aβ 1-42 aggregates in nematode tissue mills. It can be seen that after treatment with sDSS1 protein, most Aβ aggregate bands decreased or disappeared completely (black arrow indicates position).

In Fig. 7D, the total Aβ protein in the tissue slurry was detected by ELISA, and it was found that the total Aβ protein of the nematode tissue of the sDSS1 protein administration group was significantly decreased, and the content of the 1000 μg/mL sDSS1 protein administration group was only 59.07% of the control group. The number of nematodes used in each group was 400 in the experiment.

Figures 8A-8B. sDSS1 protein promotes Aβ protein clearance in mouse blood.

Figure 8A shows a model of acute Aβ rise by intravenous injection of Aβ protein into mice. The results show that sDSS1 protein administration accelerates Aβ protein clearance. As the amount of sDSS1 protein administered increased, the Aβ content decreased significantly.

Figure 8B. Injecting sDSS1 protein into AD model APP/PS1 mice, a significant decrease in Aβ levels was also detected in the blood. Acute model 5 mice per group, APP/PS1 mice 3 per group. Data were analyzed by ANOVE, **, p-value < 0.01; ***, p-value < 0.001.

Figures 9A-9C. sDSS1 protein reduces Aβ plaque deposition in brain tissue of APP/PS1 mice.

Fig. 9A, after staining with FSB dye, a large amount of Aβ plaque precipitate (blue fluorescence) was observed in the brain tissue of APP/PS1 mice at 11 months of age. After administration of sDSS1 protein, plaque deposition in the hippocampus of brain tissue decreased. The number of plaques is reduced and the brightness is reduced.

Fig. 9B, after the photo was processed by Phototshop CS software, the plaque area was counted. The results showed that the plaque area of brain tissue after sDSS1 protein administration was significantly lower than that of the control group.

Figure 9C, the number of small plaques (marked as 1 in Figure 9A), medium-sized plaques (marked as 2 in Figure 9A), and large plaques (marked as 3 in Figure 9A) were counted according to plaque size. In contrast, the number of medium-sized plaques and large plaques in tissues was significantly decreased after administration of sDSS1 protein, and there was no significant difference in small plaques. APP/PS1 mice were 3 in each group. The data was analyzed by ANOVE, n.s., p-value>0.5; **, p-value<0.01.

Detailed ways

The preferred embodiments of the present invention are described and illustrated in the following examples, which are not intended to limit the scope of the invention. All ranges of the invention are defined by the claims.

The experimental methods used in the following examples are routine experimental methods unless otherwise specified.

The sDSS1 protein used in the following examples is the human sDSS1 protein produced by the company itself, and the protein sequence is shown in SEQ ID NO: 1. The company controls the quality of protein. The purity of the tested protein is greater than 95%. The endotoxin (less than 3EU/mg protein) and other impurities are in compliance with the standard and can be used in animal experiments without causing significant animal toxicity.

The materials and reagents in the following examples, except for the sDSS1 protein, are commercially available.

Example 1. The sDSS1 protein inhibits Aβ protein aggregation.

experimental method

1.ThT experiment Aβ1-42 and Aβ1-40 protein were entrusted to Suzhou Qiang Yao Biotechnology Co., Ltd. to synthesize and make lyophilized powder, and the protein purity was detected to be greater than 95%. The thioflavin T dye (ThT, Sigam-Aldrich, T3516) was first dissolved in methanol to 1 mg/mL (3.1 mM) and continued to be diluted to 1 mM with PBS. The Aβ protein was dissolved to 2 mg/mL with 20 mM NaOH and diluted to 100 μM with PBS. sDSS1 protein, sDSS1 mutants 4 (sDSS1 (M4)) (amino acid sequence is shown in SEQ ID 15), sDSS1 mutants 11 (amino acid sequence see SEQ ID 16) (sDSS1 (M11 )) were diluted to 1mg / mL in PBS (100 μM). The experiment was carried out in a PBS environment. First, a PBS was added in a 1.5 mL centrifuge tube, and then sDSS1 protein or mutant protein, Aβ protein and ThT dye were sequentially added, and the sDSS1 protein molar concentration was added according to a preset concentration gradient. For the Aβ 1-42 protein, the molar concentrations of the Aβ protein and the ThT dye were 30 μM and 5 μM, respectively; for the Aβ 1-40 protein, the molar concentrations of the Aβ protein and the ThT dye were 30 μM and 10 μM, respectively. After completion, the solution was shaken and mixed on the suspension, and then 200 μL was added to a black fluorescence detection plate (Costar, 3792) for two duplicate wells per group. The black fluorescence detection plate was placed on a multi-function microplate reader (Molecular Devices, SpectraMax i3x) to detect the fluorescence value, and the excitation light was set to 450 nm, the emission light was 485 nm, the detection interval was 30 minutes, and the detection was continued for 48 hours.

2. Protein Interactions Aβ 1-42 and Aβ 1-40 protein monomers were diluted to 20 μM with PBS and mixed with 10 μM or 20 μM sDSS1 protein, and 20 μM Aβ protein was used as a positive control. The protein solution was mixed and incubated overnight at 4 °C. 5x loading buffer was added to the incubated protein solution, mixed and heated at 100 ° C for 10 minutes, and the prepared samples were used for Western Blotting analysis.

3. Western Blotting 15 μL of the prepared sample was added to the well, and 10% of the pre-formed gel (Life Technology C#NP0321BOX) was used to separate the protein and transferred to the PVDF membrane. The membrane was blocked by 5% skim milk for 1 hour, and the first-stage antibody Rabbit-anti-Aβ (Cell Signaling Technology, #8243) or Rabbit-anti-sDSS1 (invented by Shanghai Ruizhi Chemical Research Co., Ltd.) was incubated at 4 °C. Overnight, the TBST solution was washed three times; the second antibody (Goat-anti-rabbit HRP antibody) was incubated for 2 hours at room temperature. It was washed three times with TBST, and after completion, it was developed with a luminescent liquid and the strip was displayed with an X-ray film.

Experimental result

In the buffer, Aβ 1-42 or Aβ 1-40 protein was co-incubated with sDSS1 protein, and the mixture after incubation was detected. It was found that Aβ 1-42 protein forms a complex with sDSS1 protein (molecular weight 10KD-37KD). Co-incubation of the Aβ 1-40 protein with the sDSS1 protein significantly reduced the formation of dimers (Fig. 1A). The Aβ protein and sDSS1 protein complex signals were detected by the same technique using the sDSS1 protein antibody, and the complex signal was enhanced as the sDSS1 protein concentration was increased (Fig. 1B). Aβ 1-42 or Aβ 1-40 monomeric proteins aggregate to form an amyloid deposit, which is enhanced by binding to ThT dye. In the control group, it can be seen that as the incubation time is prolonged, the fluorescence brightness gradually increases, indicating that Aβ 1-40 and Aβ 1-42 proteins are aggregated. However, after adding different concentrations of sDSS1 protein or sDSS1 mutant protein in the reaction system, the fluorescence brightness did not increase and remained near the initial value, indicating that sDSS1 protein significantly inhibited Aβ protein aggregation. Moreover, it is only necessary to add 5% of sDSS1 protein to the reaction system to inhibit the Aβ protein aggregation reaction (Fig. 1C, Fig. 1D); 0.667% or 3.33% of sDSS1(M4) protein or sDSS1 (M11) is added to the reaction system. The protein can inhibit the Aβ protein aggregation reaction (Fig. 1E, Fig. 1F) . These results indicate that the sDSS1 protein can interact with the Aβ protein to form a complex and inhibit the formation of Aβ protein aggregates.

Example 2. The sDSS1 protein binds to an Aβ protein oligomer.

experimental method

1. Aβ 1-42 protein solubilization and oligomer preparation 1 mg Aβ1-42 or Aβ1-40 protein was added to 500 μL of 20 mM NaOH solution, shaken and mixed for 10 minutes, and then sonicated for 10 minutes to help dissolve the protein. Dilute to 100 μM with PBS solution, incubate at 4 ° C for 24 hours, centrifuge at 12000 g for 10 minutes to remove possible precipitates, and obtain Aβ 1-42 or Aβ 1-40 oligomers. Aβ oligomers are labeled according to the concentration of Aβ monomer protein prior to incubation.

2. ThT assay The sDSS1 protein was diluted to 1 mg/mL (100 μM) with PBS. The experiment was carried out under PBS conditions. First, PBS was added in a 1.5 mL centrifuge tube, and then sDSS1 protein, Aβ protein oligomer and ThT dye were sequentially added, and the molar concentration of sDSS1 protein was added according to a preset concentration gradient. For the Aβ 1-42 protein, the molar concentrations of the Aβ protein and the ThT dye were 30 μM and 5 mΜ, respectively; for the Aβ 1-40 protein, the molar concentrations of the Aβ protein and the ThT dye were 30 μM and 10 mΜ, respectively. After completion, the solution was shaken and mixed on the suspension, and then 200 μL was added to a black fluorescence detection plate (Costar, 3792) for two duplicate wells per group. The black fluorescent detection plate was placed on a multi-function microplate reader to detect the fluorescence value, and the excitation light was set to 450 nm, the emission light was 485 nm, the detection interval was 30 minutes, and the detection was continued for 12 hours.

3. Protein Interaction The Aβ 1-42 or Aβ 1-40 protein oligomer was diluted to 20 μM with PBS and mixed with 10 μM or 20 μM sDSS1 protein, and 20 μM Aβ oligomer was used as a positive control. The protein solution was mixed and incubated overnight at 4 °C. 5x loading buffer was added to the incubated protein solution, mixed and heated at 100 ° C for 10 minutes, and the prepared samples were used for Western Blotting analysis.

4. Western Blotting 15 μL of the prepared sample was added to the well, and 4-12% pre-formed (Life Technology C#NP0321BOX) was used to separate the protein and transferred to the PVDF membrane. The membrane was blocked by 5% skim milk for 1 hour, and the first-stage antibody Rabbit-anti-Aβ (Cell Signaling Technology, #8243) or Rabbit-anti-sDSS1 (invented by Shanghai Ruizhi Chemical Research Co., Ltd.) was incubated at 4 °C. Overnight, the TBST solution was washed three times; the second antibody (Goat-anti-rabbit HRP antibody) was incubated for 2 hours at room temperature. It was washed three times with TBST, and after completion, it was developed with a luminescent liquid and the strip was displayed with an X-ray film.

Experimental result

The oligomer of Aβ 1-40 or Aβ 1-42 is the major form of Aβ protein producing cytotoxicity. In order to detect the reaction of sDSS1 protein with Aβ protein oligomer, the Aβ protein was first incubated to form an oligomer, and then incubated with sDSS1 protein for western blotting and ThT detection. The results showed that the reaction of sDSS1 protein with Aβ 1-42 oligomer form was detected by Aβ protein antibody, and the complex content increased with the increase of sDSS1 protein concentration in the reaction system (Fig. 2A). A complex formed by the sDSS1 protein and the Aβ protein can also be detected using the sDSS1 protein antibody (Fig. 2B). In the ThT fluorescence experiment, it can be seen that the sDSS1 protein can react with the amyloid precipitation of Aβ 1-40 or Aβ 1-42, and the fluorescence intensity increases after the sDSS1 protein reacts with the Aβ 1-40 amyloid precipitate (Fig. 2C); After the reaction of the sDSS1 protein with the Aβ 1-42 amyloid precipitate, the fluorescence intensity was significantly reduced (Fig. 2D). The above results indicate that the sDSS1 protein can also form a complex with the Aβ protein oligomer form, and these interactions may inhibit the cytotoxicity of the Aβ protein.

Example 3, sDSS1 protein reduces the binding efficiency of Aβ protein to receptor protein.

experimental method

1. Molecular interaction experiments The biomacromolecular interaction instrument (GE, Biacore 3000) was used to detect the binding efficiency of RAGE protein to sDSS1 protein. The chip was first rinsed with PBS for 20 minutes until the baseline was stable, and the protein was coupled to the sensor chip NTA chip (GE) with a His tag on 1 μg/mL RAGE protein with a coupling amount of approximately 50 RU. The sDSS1 protein was diluted to 10 μg/mL, the loading amount was 20 μL, and the loading time was 200 seconds. After completion, rinse with PBS solution and measure the elution curve.

2. Ligand receptor binding assay In this experiment, the ELISA method was used to detect the change of binding efficiency of Aβ protein monomer and receptor glycoprotein (receptor of advanced glycation end product (RAGE). RAGE protein was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd. (11629-H02H). The blank plate was washed once with PBS, and 100 μL of 0.5 μg/mL RAGE protein diluted with PBS was added to each well overnight at 4 ° C, and washed once with PBS. The PBS diluted 2% BSA in PBST (0.05% Tween 20) was added and blocked at 37 ° C for 1 hour, and then washed once with PBS. Add 100 μL of Aβ1-42 protein or Aβ1-42 protein and sDSS1 protein mixture, set Aβ1-42 protein concentration to 0.625μg/mL and 0.3125μg/mL, respectively, and sDSS1 protein concentration of 0.2μg/mL and 1μg/mL, respectively, incubate at room temperature. 2 hours. After washing once with PBST (0.05% Tween 20), 100 μL of Rabbit-anti-Aβ (1:10000 diluted in PBST solution containing 0.5% BSA) was added, and the mixture was incubated at room temperature for 1 hour, and then washed three times with PBST. Continue to add 100 μL of horseradish peroxidase (HRP)-coupled goat-derived anti-rabbit antibody (1:10000 diluted in PBST solution containing 0.5% BSA), incubate for 1 hour at room temperature, and wash 6 times with PBST after completion. Add 200 μL of TMB color solution and incubate for 30 minutes at room temperature. After completion, add 50 μL of stop solution. The 96-well plate reads the 450 nm absorbance value on the microplate reader, reflecting the ligand binding amount of the ligand.

Experimental result

Using the BIAcore device, it was found that the sDSS1 protein could not bind to the RAGE protein and showed no significant binding peak on BIAcore (Fig. 3A). ELISA experiments showed that Aβ1-42 protein as a ligand can bind to RAGE protein, showing a higher binding amount. The addition of 0.2 μg/mL and 1 μg/mL sDSS1 protein reduced the binding amount of Aβ 1-42 to RAGE, and this effect exhibited a significant concentration-dependent effect ( FIG. 3B ). These results indicate that the sDSS1 protein reacts with the Aβ1-42 protein to reduce the binding efficiency of Aβ protein binding to the receptor.

Example 4, sDSS1 protein shields cytotoxicity caused by Aβ oligomers.

experimental method

1. Cell Culture Mouse-derived neuroblastoma cells (N2a) were purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences (Cell No.: TCM29). N2a cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific, 11995065) complete medium containing 10% fetal calf serum (FBS, Gibco, 10091148) and subcultured every two days.

2. Primary neuron culture The mice that were pregnant for 16 days were deeply anesthetized with ether and then sacrificed by cervical dislocation. The E16 stage fetus was dissected. After the fetal rats were anesthetized with ice, the cervical vertebrae were sacrificed, the rat brain was dissected, and immersed in a pre-cooled DMEM solution (containing 2% double-antibody). The pia mater was removed by ophthalmology and repeatedly washed to remove blood. The frontal cortex was dissected and the tissue was cut with an ophthalmic scissors. All tissues were collected into a centrifuge tube and digested with 5 mL of 0.05% trypsin for 15 minutes at room temperature. After completion, the digestion was terminated by adding an equal volume of DMEM complete medium, and the tissue was repeatedly beaten and filtered with a 40 μm cell strainer to obtain a cell suspension. After the cells were counted, they were added to a 96-well plate at a rate of 3.0 x 10 ⁴ cells per well, 200 μL of DMEM complete medium, and the plate was incubated at 37 °C. After 24 hours, switch to 200 μL of neuronal medium containing 98% Neurobasal (Gibco, 21103049), 2% B27 supplement (Gibco, 17504044), 1% GlutaMAX-1supplement (Gibco, 35050061). Fresh neuronal medium was changed every two days thereafter. Cultured neurons matured after 8 days and can be used in subsequent experiments.

2. Aβ1-42 protein solubilization and oligomer preparation 1 mg of Aβ1-42 was diluted to 100 μM with serum-free DMEM-/- medium, and the dilution was incubated at 4 ° C for 24 hours to obtain Aβ1-42 oligomer protein. The Aβ oligomer protein is labeled according to the concentration of Aβ monomer protein before incubation.

3. Cytotoxicity assay N2a cells were seeded into 96-well plates at 2x10 ⁴ cells per well. After the cells were attached for 12 hours, they were replaced with serum-free DMEM medium, and the cells were starved for 24 hours. The Aβ 1-42 oligomer was diluted to a 20 μM solution in serum-free DMEM. The negative control group was treated with serum-free DMEM, and the positive control group was added with 20 μM Aβ protein solution. The experimental group contained 10 μM and 20 μM sDSS1 protein in addition to 20 μM Aβ protein. For neuronal cells, after cell maturation, Aβ was diluted with serum-free DMEM according to the N2a cell experiment and the same sDSS1 protein gradient was set. Neurons were processed for 48 hours. The supernatant of the cells in the 96-well plate was collected, centrifuged at 100 g for 10 minutes, and the supernatant was used for cytotoxicity detection; the cells were used for cell viability assay.

4. Detection of cytotoxicity The lactate dehydrogenase cytotoxicity test kit was purchased from Biyuntian Biotechnology Co., Ltd. (C0016). When detecting the enzyme activity, first configure a sufficient amount of the working fluid according to the kit instructions. The supernatant collected in a 120 μL cytotoxicity experiment was added to each well of a 96-well plate, then 60 μL of the test working solution was added, mixed slightly, and incubated at 37 ° C for 30 minutes. The absorbance at 490 nm was measured on a microplate reader, and the absorbance was directly proportional to the cytotoxicity level.

5. Cell viability assay The cell proliferation/cytotoxicity assay kit (CCK-8) was purchased from Dongren Chemical Technology (Shanghai) Co., Ltd. (CK04). The working solution was prepared by diluting the CCK-8 solution with 1:20 (volume ratio, v/v) in serum-free DMEM. After completion of the cell treatment in the 96 wells, the old medium was discarded, and 100 μl of CCK-8 working solution was added to each well. The plate was incubated in an incubator for 1-2 hours, and then the 450 nm absorbance value was measured on a multi-function microplate reader to reflect the cell viability.

Experimental result

In order to detect the shielding effect of sDSS1 protein on the cytotoxicity of Aβ1-42 protein oligomer, the protective effect of sDSS1 protein on mouse neuroblastoma cells and primary cultured mouse neurons was examined. The results showed that Aβ 1-42 oligomer protein can cause significant cytotoxicity in N2a cells, and oligomer treatment for 48 hours leads to rounding and abnormal aggregation of cells, and addition of sDSS1 protein can reduce cytotoxicity (Fig. 4A). Using CCK-8 kit to detect cell viability, it was found that sDSS1 protein can significantly block the decrease of cell viability caused by Aβ 1-42 oligomer, and 20 μM sDSS1 protein can protect cells from Aβ 1-42 oligomer (Fig. 4B). ). The cytotoxicity level was measured by the LDH kit, and the results showed that the cytotoxicity level of the sDSS1 protein group was significantly decreased (Fig. 4C). On mouse primary neuronal cells, the sDSS1 protein also showed a shielding effect on the toxicity of Aβ oligomers, the cell viability of neurons was protected by sDSS1 protein (Fig. 5A), and the level of cytotoxicity was decreased (Fig. 5B). These results show that the sDSS1 protein can significantly block the cytotoxicity caused by Aβ oligomer proteins.

Example 5. The sDSS1 protein promotes microglia clearance of A[beta] protein and reduces microglial activation.

experimental method

1. Cell Culture Mouse microglia (BV2) was purchased from the National Experimental Cell Resource Sharing Platform (3111C0001CCC000063). BV2 cells were cultured in DMEM medium containing 10% FBS and subcultured every two days.

2. Aβ clearance assay BV2 cells were seeded into 6-well plates at 3x10 ⁵ per well and cells were adhered for 12 hours. The Aβ protein was previously diluted with DMEM complete medium to a 1 μM solution. The negative control group was not added with protein, and the positive control group was added with 1 μM Aβ protein solution. The experimental group contained 0.5 μM and 2 μM sDSS1 protein except 1 μM Aβ protein. After 24 hours of treatment, 500 μL of the supernatant was collected, centrifuged at 100 g for 10 minutes, and the supernatant was taken for ELISA assay to detect Aβ 1-42 levels. All cells were collected by centrifugation, and the lysate was centrifuged at 12000 g to collect the lysate supernatant for ELISA to detect A? 1-42 levels. The cell lysate was quantified by protein concentration using a BCA protein quantification kit (ThermoFisher, 23252).

3.BV2 cell activation experiments BV2 cells were seeded per well in accordance 1.5ⅹ10 ⁵ to 6 well plates, DMEM medium was changed to serum-free 12 hours, cells were starved for 24 hours. The Aβ 1-42 oligomer was diluted to a 20 μM solution in serum-free DMEM. The negative control group was not added with protein, and the positive control group was added with 20 μM Aβ protein solution. The experimental group contained 10 μM and 20 μM sDSS1 protein except 20 μM Aβ protein. After the cells were treated for 24 hours, the supernatant was collected, centrifuged at 100 g for 10 minutes, and the supernatant was taken for ELISA assay to detect the TNFα level of the solution. All cells were collected for ROS level detection.

4. ELISA assay The culture solution obtained in the above Aβ scavenging experiment was used in an ELISA experiment, and the culture solution sample was diluted 50 times with a standard diluent buffer provided by the kit for subsequent detection. According to the instructions of ELISA kit (Invitrogen, Human Aβ42 ELISA kit, KHB3441), the standard protein and the nematode protein to be tested were sequentially incubated and washed, and the chromogenic substrate was added for 20 minutes. The stop solution was added and detected on the microplate reader. 450 nm absorbance value. The Aβ protein concentration in the protein sample was calculated from the standard curve.

5. ROS level detection Active oxygen detection kit was purchased from Biyuntian Biotechnology Co., Ltd. (S0033). Positive control cells were pretreated with 1 μM Rosup for 20 minutes at 37 °C. 300 μL of the cell suspension was taken, centrifuged at 100 g for 5 minutes, and the cells were washed twice with PBS. 300 μL of serum-free DMEM-diluted fluorescent probe (DCFDA) (1:1000) was added and incubated at 37 ° C for 20 minutes. After centrifugation at 100 g for 5 minutes, the cells were washed three times with PBS and detected by flow cytometry (Millipore, guava Easycyte). Green fluorescence (488 nm) intensity reflects cellular ROS levels.

6. Detection of TNFα level The supernatant collected in the BV2 cell activation experiment was diluted 10-fold with Standard Dilute buffer provided in the kit for ELISA detection. According to the instructions of ELISA kit (Invitrogen, TNF alpha Mouse ELISA Kit, KMC3011C), the standard protein and the nematode protein to be tested were sequentially incubated and washed, incubated with chromogenic substrate for 20 minutes, and the stop solution was added to the microplate reader. The 450 nm absorbance value was measured. The concentration of TNFα in the supernatant was calculated from the standard curve.

Experimental result

Microglia are the main cells in nerve tissue that remove toxic proteins such as Aβ protein. In order to detect whether sDSS1 protein can help microglia clear Aβ protein, 1 μM Aβ1-42 protein and different concentrations of sDSS1 protein were added to cultured mouse microglia. The results showed that Aβ protein was detected in the cytoplasm of microglia, and the concentration of cytosolic Aβ protein was significantly increased with the addition of sDSS1 protein (Fig. 6A). Correspondingly, the Aβ protein residue in the culture solution decreased, and the Aβ protein concentration became lower and lower as the concentration of sDSS1 protein in the solution increased (Fig. 6B). These results indicate that the sDSS1 protein helps microglia clear Aβ protein. Adding high concentration of Aβ protein oligomers to the culture medium and adding different concentrations of sDSS1 protein, it can be seen that the sDSS1 protein in the culture medium can significantly reduce the number of activated microglia, and the cell ROS level is significantly reduced (Fig. 6C). The release of the inflammatory factor TNF-α gradually decreased as the concentration of sDSS1 protein increased (Fig. 6D). These results indicate that sDSS1 protein can stimulate microglia to clear Aβ protein and block the effect of Aβ oligomer on microglia.

Example 6. The sDSS1 protein reduces disease symptoms in the AD model nematode.

experimental method

1. AD model nematode detection The nematode experimental platform was provided by Shanghai Southern Model Biotechnology Co., Ltd., and the AD nematode model was provided by the nematode experimental platform. First, the adult CL4176 nematode was picked into a solid medium and placed in a 16 ° C incubator. After 2 hours of culture, all the adults were picked and the eggs were placed in a 16 ° C incubator. After 48 hours, the culture dishes were transferred to a 25 ° C incubator for further culture and treated with different concentrations of sDSS1 protein, 0 μg/ml in the control group, 500 μg/ml in the low dose group, and 1000 μg/ml in the high dose group. After 18 hours of continuous drug treatment, the number of CL4167 nematodes was counted and counted every 2 hours. After the statistics were completed, the analysis was performed. The standard for nematodes is that when the wire is lightly nematode, the nematode is stiff and incapable of moving.

2. Collection of nematode protein samples CL4176 nematodes were treated for 20 hours in different concentrations of drugs, and nematodes were collected after completion, with 400 nematodes per group. The nematodes were washed three times with PBS and placed in a 1.5 mL centrifuge tube. 150 μL of nematode lysate was added to each tube (C. elegans lysate containing 62 mM Tris-HCl, 2% SDS, 10% glycerol, 4% β-mercaptoethanol, cocktail protease inhibition) Agent), liquid nitrogen freeze-thaw 2 times, 60Hz ultrasound for 5 minutes, centrifugation at 12000rpm for 10 minutes, take the supernatant is the total protein of nematodes. The nematode protein was added to a loading buffer and heat treated at 100 ° C for 10 minutes to prepare a protein sample for Wetern blotting analysis.

3. Western Blotting The nematode protein samples were separated by SDS-PAGE electrophoresis. The electrophoresis conditions were: 10% denatured pre-formed gel (C#NP0321BOX, purchased from Life Technology), MES electrophoresis solution, 200V, 30 minutes. After the electrophoresis was completed, the film was transferred, and the film transfer conditions were: 100 V constant pressure wet rotation for 60 minutes, PVDF film. The completed membrane was first blocked with 5% skim milk powder (dissolved in PBST) for 1 hour at room temperature, then sequentially incubated with rabbit-derived Aβ antibody dilution at 4 ° C overnight (1:3000 dilution in blocking solution), washed three times with PBST, HRP coupling The conjugated anti-rabbit antibody dilution was incubated for 1 hour at room temperature (1:5000 dilution into PBST). After the ECL color is developed, the tablet is compressed and photographed and saved. The internal reference used tubulin protein (antibody was purchased from Biyuntian Biotechnology Co., Ltd., AF0001).

4. ELISA detection The total nematode protein extracted from the above Western blotting experiment was used in the ELISA experiment, and the protein sample was diluted three times with the standard diluent buffer provided by the kit for subsequent detection, and the protein dilution was added. The volume was adjusted according to the data of the internal reference in the Western blotting experiment to ensure that the total amount of protein added was consistent. According to the instructions of the ELISA kit, the standard protein and the nematode protein to be tested were sequentially incubated and washed, incubated with a chromogenic substrate for 20 minutes, and the stop solution was added to measure the absorbance at 450 nm on a microplate reader. The Aβ protein concentration in the protein sample was calculated from the standard curve.

5. C. elegans immunohistochemical staining CL4176 nematodes were treated in different concentrations of drugs for 20 hours, and after completion, nematodes were collected, with 50-100 nematodes in each group. The nematodes were washed once with PBS and placed in a 1.5 mL centrifuge tube, and the supernatant was discarded, and 4% paraformaldehyde (dissolved in PBS, pH 7.2) was added at 4 ° C overnight. The fixed nematode was first rinsed once with wash buffer (containing 10 mm Tris/HCl, 1% Triton-X100, 1 mM EDTA, pH 7.4). The nematodes were sequentially incubated with a washing solution containing 1% β-mercaptoethanol for 1 hour at room temperature, boric acid buffer (containing 25 mM H ₃ BO ₃ , 12.5 mM NaOH, 10 mM DTT, pH 9.2) was incubated for 1 minute at room temperature for 5 minutes. 1% H ₂ O ₂ wash buffer was incubated for 15 minutes at room temperature. After completion, the nematodes were washed once with wash buffer and blocked by adding blocking solution (containing 1% BSA, 0.5% Triton-X100, 1 mM EDTA, PBS) for two hours at room temperature. The treated nematodes were sequentially incubated overnight at 4 °C with a primary antibody dilution (Aβ antibody, diluted 1:100 in blocking solution), and the secondary antibody dilution (added Hoechst at a ratio of 1:1000) (Donkey-anti-Rabbit-Alex Fluor) 488 (purchased from Life Technology, Inc., A-11008) was incubated for 1 hour at room temperature with a dilution of 1:5000 in PBST. The PBST was washed three times, and after completion, it was mounted and observed with a Confocol laser confocal microscope (Nikon, Nikon C1Plus), and photographed.

Experimental result

CL4176 nematode was used as an animal model for AD to detect toxicity caused by accumulation of acute Aβ 1-42 protein and was used for drug evaluation. In the linear worm experiment, the addition of different concentrations of sDSS1 protein significantly reduced the nematode sputum level, and the effect showed a significant concentration dependence (Fig. 7A). After 43 hours, the proportion of the non-nematode in the control group was less than 1%, while the nematodes with 18.42% (500 μg/mL sDSS1 protein) and 48.57% (1000 μg/mL sDSS1 protein) in the administration group were normal. In the overall analysis, the median time of occurrence of sputum in half of the nematodes was 22 hours in the control group, 24 hours in the low concentration group, and 42 hours in the high concentration group. These results indicate that the sDSS1 protein is effective in reducing the disease symptoms of the AD model nematode. Immunohistochemistry was used to detect Aβ deposition in nematodes, and obvious granular aggregates were observed in the control nematodes. However, no aggregates were detected or few particles were detected in the sDSS1 protein administration group, indicating that sDSS1 protein reduced Aβ aggregation. Object formation (Fig. 7B). Western blotting assay was used to detect the Aβ protein content in nematode grinding solution. The results also showed that sDSS1 protein reduced the content of Aβ protein multimer (Fig. 7C). The Aβ 1-42 protein content in the tissue lysate was detected by ELISA. It can be seen that the Aβ protein in the sDSS1 protein-treated nematode was decreased, and the Aβ protein content in the 1000 μg/mL sDSS1 protein treatment group was only 59.09% of the control group (Fig. 7D). . Through the above experiments, we verified that sDSS1 protein can reduce the formation of Aβ 1-42 aggregates, reduce the accumulation of tissue Aβ, and alleviate the disease symptoms of AD model nematodes in the AD nematode model.

Example 7. The sDSS1 protein accelerates clearance of Aβ protein in mice.

experimental method

1. APP/PS1 mouse blood Aβ clearance test APP/PS1 mice (2 months old, male) were purchased from the Model Animal Center of Nanjing University. The APP/PS1 mice were injected with sDSS1 protein at a dose of 25 mg/kg body weight for 8 weeks from the age of 8 months. After completion, the mice were sacrificed, blood was taken and centrifuged to obtain plasma for ELISA to detect Aβ 1-42 levels in the blood.

2. Aβ protein acute clearance test ICR mice (6-8 weeks old, male) were purchased from Shanghai Slack Laboratory Animals Co., Ltd. The mice were divided into four groups according to their body weight, including a negative control group (injected with normal saline), a positive control group (administered with Aβ protein alone), a low-dose group (Aβ-administered and 5 mg/kg body weight injected with sDSS1 protein), and high dose. The administration group (Aβ administration and 30 mg/kg body weight injection of sDSS1 protein). At the beginning of the experiment, the mice were first injected with sDSS1 protein according to the tail vein of the group. The negative control group and the positive control group were injected with the same amount of PBS. After 1 hour, the positive control group and the drug-administered group were injected with Aβ protein at the tail vein of 10 mg/kg body weight. The control group was injected with the same amount of normal saline. After the injection was completed, the mice were normally reared, and the mice were sacrificed 2 hours later. Blood was taken from the heart, and the cells were centrifuged at 3,500 g for 20 minutes to obtain plasma for ELISA to detect A? 1-42 levels.

3. ELISA test Plasma samples obtained in the above experiment (diluted 1-3 times if necessary) were used for the ELISA experiment. According to the instructions of the ELISA kit, the standard protein and the plasma sample to be tested are sequentially incubated and washed, and the chromogenic substrate is added for 20 minutes, the stop solution is added, and the 450 nm absorbance value is detected on the microplate reader. The Aβ protein concentration in plasma was calculated from the standard curve.

Experimental result

To test whether sDSS1 protein can help clear Aβ protein, we used an acute Aβ level increase model and a chronic Aβ level increase model, respectively. In the acute model, high concentrations of Aβ 1-42 protein were injected through the tail vein while the sDSS1 protein was injected. The results of the experiment showed that different concentrations of sDSS1 protein administration significantly reduced blood Aβ protein levels, and the effect was dose-dependent (Fig. 8A). In the chronic model, AD model APP/PS1 mice were injected with sDSS1 protein for a long time in the tail vein, and it was found that sDSS1 protein also reduced blood Aβ protein levels (Fig. 8B). These two results indicate that sDSS1 protein can help clear Aβ protein in the blood of mice.

Example 8. sDSS1 protein reduces plaque deposition in brain tissue of AD model mice.

experimental method

1. AD model mice administration APP/PS1 mice (2 months old, male) were purchased from the Model Animal Center of Nanjing University. The APP/PS1 mice were injected with sDSS1 protein at a dose of 50 mg/kg body weight for 8 weeks from the age of 9 months. After completion, the mice were used for behavioral experiments. After the behavioral experiment was completed, the mice were sacrificed, and plasma was taken for blood index detection. Brain tissue was taken and fixed in 4% paraformaldehyde for immunohistochemical staining to analyze plaque deposition levels.

2. Immunohistochemistry experiment The treated mice were perfused with 50m normal saline and 50m pre-cooled 4% paraformaldehyde solution. After completion, the brain tissue was immersed in 4% paraformaldehyde for 2 days. The tissue block was prepared by dehydration with a 30% sucrose solution and embedding with an embedding agent. Brain tissue was cut into 10 um thick brain slices using a cryostat for subsequent immunohistochemical analysis. The brain piece was firstly washed with PBS to remove the embedding agent, and then incubated with FSB working solution (Dojindo, F308) (diluted to 0.05% working solution with 50% ethanol solution) for 30 minutes, and then the slice was immersed in a saturated lithium carbonate solution. Finally, it was washed once with 50% ethanol solution. The sections were mounted with a sealing tablet and observed with a fluorescence microscope, and the number and size of amyloid precipitated patches were photographed.

Experimental result

Eight weeks after administration of AD model mice, brain slices were stained to observe the level of Aβ plaque deposition in the tissues. The results showed that plaques of different sizes were detected in the brain tissue of the 11-month-old APP/PS1sDSS1 mice in the control group, but plaque deposition was significantly reduced in the tissues after 8 weeks of administration of the sDSS1 protein (Fig. 9A). The plaque area was counted using photoshop CS software, and the plaque area of the drug-administered group was only one-third of the control group (Fig. 9B). The number of plaques was counted based on the plaque size, and it was found that the number of plaques in the brain tissue of the mice in the administration group was significantly reduced, and large plaque deposits were hardly found (Fig. 9C). These results indicate that sDSS1 protein administration can significantly reduce Aβ protein deposition in brain tissue of AD model mice.

Claims

Use of a protein for the preparation of a medicament for preventing and treating dementia, characterized in that the application is to use the sDSS1 protein for the preparation of a dementia prevention drug and a therapeutic drug.
The use according to claim 1, wherein the dementia refers to degenerative dementia, including Alzheimer's disease, Alzheimer's disease, Louis deer type dementia, and frontotemporal dementia.
The use according to claim 1, wherein the dementia is vascular dementia.
The application according to claim 1, wherein the dementia is pre-dementia dementia, mild dementia dementia, moderate dementia dementia, and severe dementia dementia.
The use according to claim 1, wherein the sDSS1 protein comprises a human, a chimpanzee, a bonobo, a gorilla, an orangutan, a white-cheeked gibbon, a Rhesus monkey, a rhesus monkey, a golden monkey, an East African ape, A basic protein formed by any of the sDSS1 protein sequences in Angora marmoset, white-tailed white-browed macaque, podophyllum, and porpoise monkey, wherein the amino acid sequence of human sDSS1 is SEQ ID NO: 1, and the amino acid sequence of chimpanzee sDSS1 is SEQ ID NO: 2. The amino acid sequence of the chimpanzee sDSS1 is SEQ ID NO: 3, the amino acid sequence of the gorilla sDSS1 is SEQ ID NO: 4, the amino acid sequence of the orangutan sDSS1 is SEQ ID NO: 5, and the amino acid sequence of the white-cheeked gibbon sDSS1 is as SEQ ID NO: 6, the amino acid sequence of Rhinopithecus sDSS1 is SEQ ID NO: 7, the amino acid sequence of rhesus sDSS1 is SEQ ID NO: 8, and the amino acid sequence of Rhinopithecus sDSS1 is SEQ ID NO: 9, East Africa 狒狒 sDSS1 The amino acid sequence of SEQ ID NO: 10, the amino acid sequence of Angora simian sDSS1 is SEQ ID NO: 11, and the amino acid sequence of leucocephalus sDSS1 is SEQ ID NO: 12, sneaky s The amino acid sequence of DSS1 is SEQ ID NO: 13, and the amino acid sequence of porpoise monkey sDSS1 is SEQ ID NO: 14.
The use according to claim 5, wherein said sDSS1 protein is the first protein having a similarity to said basic protein of 70% or more.
The use according to claim 5, wherein the sDSS1 protein is based on 58 amino acids of the nitrogen terminus of the basic protein, and the other polypeptide fragments are fused at the nitrogen terminal or the carbon terminal, and the polypeptide is used for fusion. A second protein having a structural feature or amino acid sequence characteristic of the fragment that is identical or similar to the carbon terminal 31 sequences of the basic protein.
The use according to claim 5, wherein the sDSS1 protein is based on 58 amino acids of the nitrogen terminus of the basic protein, and the other amino acid fragments are fused at the nitrogen or carbon end, and the protein can be fused. A third protein that achieves transmembrane transport function.
The use according to any one of claims 5-8, wherein the sDSS1 protein utilizes the basic protein, the first protein, the second protein or the third protein and the protein itself, A fusion protein formed by the joining of a carrier protein, antibody or other amino acid fragment of any length.
The use according to any one of claims 5-9, wherein the sDSS1 protein is modified by the basic protein, the first protein, the second protein, the third protein or the fusion protein. Polypeptide/protein modification.
The use according to claim 10, wherein the polypeptide/protein modification is directed to an amino group on an amino acid side chain, a carbonyl group on an amino acid side chain, a nitrogen terminal amino group, a carbon terminal carbonyl group, or a cysteine. Specific or non-specific chemical modification of 1-20 sites by tyrosine, serine, tryptophan.
The method according to claim 10, wherein the modification method of the polypeptide/protein modification comprises glycosylation modification, fatty acid modification, acylation modification, Fc fragment fusion, albumin fusion, polyethylene glycol modification, Dextran modification, heparin modification, polyvinylpyrrolidone modification, polyamino acid modification, polysialic acid modification, chitosan and its derivative modification, lectin modification, sodium alginate modification, carbomer modification, polyvinylpyrrolidone modification , hydroxypropyl methylcellulose modification, hydroxypropyl cellulose modification, acetylation modification, formylation modification, phosphorylation modification, methylation modification, sulfonation modification, and other pharmaceutically useful polypeptide/protein drug modification methods One or more of them.
The use according to claims 5-9, characterized in that the sDSS1 protein is based on the amino acid sequence of the basic protein, the first protein, the second protein, the third protein or the fusion protein. A non-natural amino acid replacement protein substituted with 1-31 arbitrary amino acid positions of amino acids other than the 20 essential amino acids.
The use according to claim 13, wherein the amino acid substitution of the non-natural amino acid replacement protein comprises replacement with hydroxyproline, hydroxylysine, selenocysteine, D-type amino acid or artificial Synthetic unnatural amino acids and derivatives thereof.
The use according to any one of claims 5 to 14, wherein the sDSS1 protein is the basic protein, the first protein, the second protein, the third protein, the fusion protein, the polypeptide/ A partial or total complex of a protein modified or non-natural amino acid replacement protein with a pharmaceutically acceptable pharmaceutical carrier.
The use according to claim 15, wherein the pharmaceutical carrier of the complex comprises an enteric coating preparation, a capsule, a microsphere/capsule, a liposome, a microemulsion, a double emulsion, a nanoparticle, a magnetic particle, a gelatin And one or more of the gels.
The use according to any one of claims 5-16, wherein the basic protein, the first protein, the second protein, the third protein, the fusion protein, the polypeptide/protein modification or the complex are exerted. The initial working concentration for shielding toxic protein toxicity is not less than 0.2 μg/mL.
The use according to any one of claims 5-16, wherein the basic protein, the first protein, the second protein, the third protein, the fusion protein, the polypeptide/protein modification or the complex are exerted. The molar concentration ratio of the toxic protein toxicity is not less than 0.010; the molar ratio refers to the ratio of the molar concentration of the protein, the polypeptide/protein modification or the complex in the reaction system to the molar concentration of the toxic protein.
The use according to claim 1, wherein the sDSS1 protein targets the individual's own sDSS1 protein and affects the level of the individual's own sDSS1 protein by the exogenous drug.
The use according to claim 19, wherein the drug is a drug target of a sDSS1 protein, a gene of sDSS1 protein, a regulatory element of a gene of sDSS1 or a gene of sDSS1.
The use according to claim 19, wherein the drug modulates the content of the sDSS1 protein in blood or cerebrospinal fluid by affecting protease/peptidase activity in blood or cerebrospinal fluid.
The use according to any one of claims 19-21, wherein the drug is a first drug formed by a chemical small molecule drug, an antibody, a polypeptide/protein drug, a nucleic acid drug or a nano drug.
The use according to any one of claims 5 to 22, wherein the sDSS1 protein is the basic protein, the first protein, the second protein, the third protein, the fusion protein, the polypeptide/ A second drug formed by a combination of two or more of any one of a protein modification, a complex, and a first drug.
The use according to any one of claims 5 to 22, wherein the sDSS1 protein is the basic protein, the first protein, the second protein, the third protein, the fusion protein, the polypeptide/ A third drug formed by a combination of a protein modification, a non-natural amino acid replacement protein, a complex, one of the first drugs, a combination of two or more, and a pharmaceutically acceptable excipient.
The use according to any one of claims 5-9, wherein the sDSS1 protein encodes the basic protein, the first protein, the second protein, the third protein or a fusion by an expression system. The nucleotide sequence of the protein is introduced into the body and the resulting fourth protein is expressed.
The use according to claim 25, wherein the expression system is a eukaryotic expression plasmid vector, an adenovirus, an adeno-associated virus, a lentivirus, a retrovirus, a baculovirus, a herpes virus, a pseudorabies virus, ZFN gene editing technology, TALEN gene editing technology, CRISPR/Cas gene editing technology or other medically useful gene editing techniques or viral vectors.
The use according to any one of claims 5-9, wherein the sDSS1 protein is the basic protein, the first protein, the second protein, and the third obtained in an individual by transplanting cells. The fifth protein formed by a protein or fusion protein.
The use according to claim 27, wherein said cell is a stem cell, a precursor cell or an adult cell of any one of humans.
The use according to claim 28, wherein the stem cells are embryonic stem cells, induced pluripotent stem cells, transdifferentiated cells, or derived from primary cultured stem cells, pluripotently differentiated from mother cells or Single energy stem cells.
The use according to any one of claims 5-9, wherein the sDSS1 protein is the basic protein, the first protein, the second protein, the first obtained in the individual by transplanting tissues or organs. The sixth protein formed by three proteins or fusion proteins.
The use according to claim 30, wherein the tissue is a whole organ or part of a tissue block of the brain, liver, kidney, spleen, islet, or blood, fat, muscle, bone marrow, skin.
The use according to claim 1, wherein the sDSS1 protein is a seventh protein introduced into the body by serum, cerebrospinal fluid, and interstitial fluid infusion.
The use according to any one of claims 1 to 32, wherein the prophylactic agent comprises a basic protein, any of the first to seventh proteins, a fusion protein, a polypeptide/protein modification, and an unnatural amino acid. Substitute protein, complex, first drug, second drug, third drug, protein drug, polypeptide drug, nucleic acid drug, chemical small molecule drug, cell in expression system, cell, tissue, organ, body fluid, tissue fluid One or more of products, commercial transplant tissues, injections, lyophilized powder, health care products, food additives.
The use according to any one of claims 1 to 32, wherein the therapeutic drug comprises a basic protein, any of the first to seventh proteins, a fusion protein, a polypeptide/protein modification, and an unnatural amino acid. Substitute protein, complex, first drug, second drug, third drug, protein drug, polypeptide drug, nucleic acid drug, chemical small molecule drug, cell in expression system, cell, tissue, organ, body fluid, tissue fluid One or more of products, commercial transplant tissues, injections, lyophilized powder, health care products, food additives.