Application of phosphorylated Parkin protein and PINK1 kinase expression promoter in preparation of drugs

Abstract

The invention belongs to the field of biological medicine, and particularly relates to application of phosphorylated Parkin protein in preparation of a medicine for treating neurodegenerative diseases carrying pathological alpha-synuclein aggregate characteristics. According to the application, research finds that the Parkin protein and the PINK1 protein have obviously different expression modes in a monkey brain in a physiological state, but the phosphorylation level of the Parkin depends on the expression of the PINK1 protein. Subsequently, it is further found that Parkin phosphorylation expression reduction and obvious aggregation of toxic pS129-alpha-syn occur in both the normal senescent monkey brain and the brain of a sporadic PD patient, and further, it is found through overexpression of wild Parkin that the S65 site phosphorylated Parkin can reduce accumulation of pS129-alpha-syn in the monkey brain, so that the accumulation of pS129-alpha-syn in the monkey brain can be reduced, and the accumulation of pS129-alpha-syn in the monkey brain can be reduced. And the mutant Parkin, which cannot be phosphorylated by PINK1, cannot reduce the aggregation of toxic pS129-alpha-syn. Therefore, the phosphorylated Parkin protein provided by the invention can be used for preparing medicines for treating neurodegenerative diseases carrying pathological alpha-synuclein aggregate characteristics.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine, and specifically relates to the use of phosphorylated Parkin protein and PINK1 kinase expression promoter in the preparation of drugs for treating neurodegenerative diseases characterized by pathological α-synuclein aggregates. Background Art

[0002] Neurodegenerative diseases are a class of diseases that affect the brain and nervous system and are characterized by the gradual loss of nerve cells over time, leading to cognitive and motor dysfunction. Such diseases include Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, etc.

[0003] Take Parkinson's disease (PD) as an example. It is the second largest neurodegenerative disease after Alzheimer's disease. Its clinical manifestations are bradykinesia, muscle rigidity, static tremor and gait disorder. The current treatment methods for Parkinson's disease can alleviate the condition to a certain extent, but they cannot prevent the degeneration and death of brain nerve cells and therefore cannot completely cure it.

[0004] At present, clinical treatment focuses on alleviating symptoms and is helpless in completely curing degenerative diseases. One of the important reasons is that there is no effective animal model that can simulate the important pathological characteristics of the progressive degeneration and death of brain nerve cells in Parkinson's patients, which greatly limits clinical drug development.

[0005] Alpha-synuclein is one of the most abundant proteins in the brain and is found primarily at the ends of nerve cells called presynaptic terminals. Alpha-synuclein is known to interact with phosphates and proteins to help regulate the release of the neurotransmitter dopamine. Human alpha-synuclein consists of more than 140 amino acids and is encoded by the SNCA gene. The main pathological features of Parkinson's disease (PD) are the progressive neurodegeneration of substantia nigra dopamine neurons with age and the formation of Lewy bodies, which are formed by the accumulation of toxic phosphorylated alpha-synuclein (pS129-α-syn). Lewy bodies are intracellular protein aggregates that are often associated with neurodegenerative diseases. They are most commonly seen in Parkinson's disease and Alzheimer's disease, but are also present in neurological diseases such as Huntington's disease and amyotrophic lateral sclerosis (ALS). In these diseases, the formation of Lewy bodies is associated with neuronal damage and degeneration. However, there are currently no drugs available clinically to reduce the formation of Lewy bodies.

[0006] PD can also be caused by genetic gene mutations. Among them, Parkin gene mutation is the number one pathogenic gene for recessive Parkinson's disease, with a high incidence rate. Parkin protein is an E3 ubiquitin-protein ligase. Its mutation will cause early-onset Parkinson's disease due to gene function loss, and the condition gradually worsens with age. In vitro and small animal experiments have found that Parkin is usually in an inactive state under physiological conditions, and is only activated and functions after being phosphorylated by PINK1 kinase.

[0007] In the treatment mechanism, post-translational modifications such as phosphorylation and ubiquitination often play a key role in physiological and pathological processes mediated by cell signaling pathways, but how different modifications work is unknown. Therefore, it is necessary to explore new drug targets related to phosphorylation. Summary of the invention

[0008] Based on this, one embodiment of the present application provides the use of phosphorylated Parkin protein in the preparation of a drug for treating neurodegenerative diseases characterized by pathological α-synuclein aggregates.

[0009] On the one hand, the present application provides the use of phosphorylated Parkin protein in the preparation of a drug for treating neurodegenerative diseases characterized by pathological α-synuclein aggregates;

[0010] Compared with the wild-type Parkin protein, the serine at position 65 of the phosphorylated Parkin protein is phosphorylated.

[0011] On the other hand, the present application provides the use of a PINK1 kinase expression promoter in the preparation of a drug for treating a neurodegenerative disease characterized by pathological α-synuclein aggregates;

[0012] In one embodiment, the α-synuclein aggregates include pS129-α-synuclein aggregates.

[0013] In one embodiment, the neurodegenerative disease with pathological α-synuclein aggregates includes one or both of Parkinson's disease and dementia with Lewy bodies.

[0014] In one embodiment, the Parkinson's disease is sporadic Parkinson's disease and familial Parkinson's disease characterized by pathological α-synuclein aggregates.

[0015] In one embodiment, the drug includes the phosphorylated Parkin protein or its expression vector.

[0016] In one embodiment, the gene expression vector comprises one or more of an adenoviral vector, a lentiviral vector and a retroviral vector.

[0017] In one embodiment, the serotype of the adenovirus vector is any one of AAV2, AAV9, AAV Mac and php.eb.

[0018] On the other hand, the present application provides a drug for treating neurodegenerative diseases characterized by pathological α-synuclein aggregates, wherein the active ingredient of the drug includes phosphorylated Parkin protein or PINK1 kinase expression promoter.

[0019] In one embodiment, the drug further comprises a pharmaceutically acceptable excipient;

[0020] In one embodiment, the auxiliary material is selected from one or more of a diluent, a binder, a disintegrant, a lubricant and a wetting agent.

[0021] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0023] Figure 1 Two plasmid expression vectors constructed in one embodiment of the present application;

[0024] Figure 2 Phosphorylates Parkin for PINK1;

[0025] Figure 3 For dephosphorylation experiments;

[0026] Figure 4 PINK1 phosphorylates Parkin in primate brain;

[0027] Figure 5 To phosphorylate Parkin to clear the Lewy bodies formed by syn aggregation;

[0028] Figure 6 PINK1 can also clear Lewy bodies formed by pS129-a-synuclein aggregation;

[0029] Figure 7Knockout of the upstream kinase PINK1 in the monkey brain can lead to reduced Parkin S65 phosphorylation and significant accumulation of pS129-a-synuclein;

[0030] Figure 8 Overexpression of Parkin Ser 65 effectively eliminated pS129-a-synuclein aggregates in the brains of PD monkeys;

[0031] Fig. 9 The brains of sporadic PD patients are accompanied by pS129-a-synuclein aggregation and reduced Parkin S65 expression;

[0032] Fig.10 In the normally aged monkey brain, Parkin Ser65 phosphorylation is reduced, while endogenous pS129-a-synuclein aggregation is increased. DETAILED DESCRIPTION

[0033] Below in conjunction with embodiment and example, the application is further described in detail.It should be understood that these embodiments and examples are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiments and examples is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0034] 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 application belongs.

[0035] the term

[0036] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0037] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0038] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0039] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0040] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0041] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0042] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0043] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0044] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the integer group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all sub-ranges included therein.

[0045] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0046] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0047] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the invention purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0048] The term "α-synuclein aggregation" in this specification refers to the aggregation of two or more α-synuclein proteins. In one embodiment of the present invention, the α-synuclein aggregates may have a larger molecular weight and / or size than non-aggregated α-synuclein.

[0049] The term "gene vector" in this specification refers to a method of delivering a nucleic acid sequence or a composition containing a nucleic acid sequence to a cell or tissue. For example, the gene vector may include: a viral vector or a non-viral vector (such as retrovirus, adenovirus, adeno-associated virus and other nucleic acid-based delivery vectors), naked nucleic acid injection or microinjection, and a polymer-based delivery system (such as liposomes and metallic particle systems), biolistic injection of lipid nanoparticles (Lipid Nano Particle; LNP), etc., but is not limited thereto.

[0050] This application found that PINK1 kinase is specifically expressed in the primate brain and can phosphorylate and activate Parkin, which is essential for maintaining primate brain function (Yang et al., Cell Res 2019; Yang et al., ProteinCell 2021; Han et al., J Clin Invest 2024). In mouse and pig models, the expression of PINK1 kinase and phosphorylated Parkin (Ser65) protein is low and undetectable (Chen et al., Zool Res 2024). This may explain why PINK1 / Parkin knockout mouse and pig models cannot reproduce the important pathological characteristics of neurodegeneration and toxic α-synuclein aggregation in the brain of PD patients.

[0051] Since PINK1 and Parkin mutations can both cause early-onset Parkinson's disease with similar clinical phenotypes due to loss of gene function, and PINK1 is specifically expressed in the primate brain and can phosphorylate and activate Parkin, it suggests that Parkin phosphorylation also plays a specific function in the non-human primate brain.

[0052] Therefore, it is irreplaceable and of great significance to use non-human primate models that are closer to humans to study the functions of PD pathogenic genes PINK1 and Parkin.

[0053] The present application uses CRISPR / Cas9 technology to establish a monkey model with Parkin gene deletion. The present application found that Parkin deletion can cause a decrease in Parkin phosphorylation, leading to neurodegeneration and death of substantia nigra neurons in adult monkey brains and the aggregation of toxic pS129-α-syn, and that toxic pS129-α-syn aggregation appears in the early brain development of Parkin-deficient monkeys before neurodegeneration. In addition, the present application found that Parkin protein and PINK1 protein have significantly different expression patterns in the monkey brain under physiological conditions, but the level of Parkin phosphorylation depends on the expression of PINK1 protein. Subsequently, the present application found that Parkin phosphorylation expression was reduced and toxic pS129-α-syn was significantly aggregated in the brains of normal aging monkeys and sporadic PD patients, suggesting that phosphorylated Parkin is essential for neuroprotection and reduction of toxic pS129-α-syn.

[0054] To verify this hypothesis, the present application further overexpressed wild-type Parkin and found that Parkin phosphorylated at the S65 site could reduce the accumulation of pS129-α-syn in the monkey brain, while the mutant Parkin that could not be phosphorylated by PINK1 could not reduce the aggregation of toxic pS129-α-syn. Therefore, the present application proposes that Parkin phosphorylated at the S65 site can protect the substantia nigra neurons in the midbrain by clearing toxic proteins including pS129-α-syn. Since the phosphorylation of Parkin can become a drug-activatable target, the present application provides a new therapeutic target for Parkinson's disease and other neurodegenerative diseases.

[0055] Based on this, the present application provides the use of phosphorylated Parkin protein in the preparation of drugs for treating neurodegenerative diseases characterized by pathological α-synuclein aggregates;

[0056] Compared with the wild-type Parkin protein, the serine at position 65 of the phosphorylated Parkin protein is phosphorylated.

[0057] On the other hand, the present application provides the use of a PINK1 kinase expression promoter in the preparation of a drug for treating neurodegenerative diseases characterized by pathological α-synuclein aggregates.

[0058] Optionally, the α-synuclein aggregates include pS129-α-synuclein aggregates.

[0059] In a specific example, the neurodegenerative disease carrying pathological α-synuclein aggregates includes one or both of Parkinson's disease and Lewy body dementia;

[0060] Optionally, the Parkinson's disease is sporadic Parkinson's disease and familial Parkinson's disease characterized by pathological α-synuclein aggregates.

[0061] In a specific example, the phosphorylated Parkin protein or its expression vector is included.

[0062] Optionally, the gene expression vector includes one or more of an adenoviral vector, a lentiviral vector and a retroviral vector.

[0063] For example, the serotype of the adenovirus vector is any one of AAV2, AAV9, AAV Mac and php.eb.

[0064] Further optionally, the serotype of the adenovirus vector is AAV2, and the virus vector is AAV-EFs-hParkin-WT-HA. Wherein, overexpression of WT Parkin can significantly increase the phosphorylation expression level of Parkin.

[0065] On the other hand, the present application provides the use of a PINK1 kinase expression promoter in the preparation of a neurodegenerative disease drug.

[0066] The present application also provides a drug for treating neurodegenerative diseases, wherein the active ingredient of the drug includes a Parkin serine 65 site phosphorylation expression promoter or a PINK1 kinase expression promoter.

[0067] In a specific example, the drug further includes a pharmaceutically acceptable excipient;

[0068] Optionally, the auxiliary material is selected from one or more of a diluent, a binder, a disintegrant, a lubricant and a wetting agent.

[0069] Optionally, the dosage form of the drug is one or more of tablets, capsules, granules, pills, injections and sustained-release preparations.

[0070] Further optionally, the pharmaceutically acceptable excipient is selected from one or more of a diluent, a binder, a disintegrant, a lubricant and a wetting agent.

[0071] Wherein, the diluent is selected from at least one of starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate and calcium carbonate.

[0072] The adhesive is selected from at least one of starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinyl pyrrolidone and polyethylene glycol.

[0073] The disintegrant is selected from at least one of starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, polyoxyethylene, sorbitol, fatty acid ester and sodium lauryl sulfate. The lubricant is selected from at least one of talc, silicon dioxide, stearate, tartaric acid, liquid paraffin and polyethylene glycol.

[0074] Wherein, the wetting agent is selected from at least one of water, ethanol and isopropanol. Optionally, the drug is an injection, and the pharmaceutically acceptable excipient is selected from at least one of a solubilizer, a pH regulator and an osmotic pressure regulator.

[0075] Further optionally, the solubilizing agent is selected from at least one of ethanol, isopropanol, propylene glycol, polyethylene glycol, poloxamer, lecithin and hydroxypropyl-β-cyclodextrin.

[0076] Optionally, the pH regulator is selected from at least one of citrate, phosphate, carbonate, acetate, hydrochloric acid and hydroxide. Wherein, the osmotic pressure regulator is selected from at least one of sodium chloride, mannitol, glucose, phosphate, citrate and acetate.

[0077] In another aspect, the present application provides a method for treating a neurodegenerative disease, comprising administering the drug to a subject.

[0078] Optionally, the subject is a primate, and further, the subject is a human.

[0079] The present application has found through research that Parkin protein and PINK1 protein have significantly different expression patterns in the monkey brain under physiological conditions, but the level of Parkin phosphorylation depends on the expression of PINK1 protein. Subsequently, it was further found that Parkin phosphorylation expression decreased and toxic pS129-α-syn was significantly aggregated in the brains of normal aging monkeys and sporadic PD patients. Furthermore, the present application found that Parkin phosphorylated at the S65 site can reduce the accumulation of pS129-α-syn in the monkey brain by overexpressing wild-type Parkin, while mutant Parkin that cannot be phosphorylated by PINK1 cannot reduce the aggregation of toxic pS129-α-syn. Therefore, the present application proposes that Parkin phosphorylated at the S65 site can protect the substantia nigra neurons in the midbrain by clearing toxic proteins including pS129-α-syn. That is, phosphorylated Parkin protein can be used to prepare drugs for treating neurodegenerative diseases with characteristics of pathological α-synuclein aggregates.

[0080] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0081] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0082] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0083] Example 1

[0084] 1. Construction of recombinant expression vector

[0085] The AAV2-EFs-zsGreen vector used in this example was purchased from Pagen Biotech Co., Ltd. The vector carries the EFs promoter, and the restriction endonucleases NcoI and BamHI were purchased from NEB, USA.

[0086] Schematic diagram of constructing a viral vector for expressing the target plasmid Figure 1 , the specific process is as follows:

[0087] 1. Use the NcoI and BamHI restriction sites on the AAV2-EFs-zsGreen vector to delete the zsGreen green fluorescent protein, and connect the human-Parkin-HA sequence with the NcoI and BamHI restriction site sequences to obtain the AAV-EFs-hParkin (WT) -HA vector.

[0088] 2. Use the NcoI and BamHI restriction sites on the AAV2-EFs-zsGreen vector to delete the zsGreen green fluorescent protein, and connect the human-Parkin Ser65A-HA sequence with the NcoI and BamHI restriction site sequences to obtain the AAV-EFs-hParkin (Ser65A)-HA vector.

[0089] 3. The successful expression of the modified AAV plasmid vector was verified by sequencing.

[0090] The results showed that the AAV-EFs-hParkin (WT)-HA vector carried the normally phosphorylated Parkin Ser 65 site, while the Ser65 site in the AAV-EFs-hParkin (Ser65A)-HA vector was mutated to A and could not be phosphorylated.

[0091] 2. Verification of the effectiveness of the constructed plasmid and viral packaging in 293 cells

[0092] AAV-EFs-hParkin (WT)-HA and AAV-EFs-hParkin (Ser65A)-HA plasmids were transfected into 293 cells, and samples were collected 48 hours later for Western blot verification. The results were as follows: Figure 2 shown.

[0093] The results showed that both constructed plasmids could effectively express Parkin protein in 293 cells. The AAV-EFs-hParkin (WT)-HA plasmid could be phosphorylated at Ser65 by its upstream kinase, while the mutant AAV-EFs-hParkin (Ser65A)-HA plasmid could not be phosphorylated by the upstream kinase PINK1.

[0094] 3. Verification of the specificity of the phosphorylated Parkin S65 antibody

[0095] It is a generally accepted view in the field that under physiological conditions, it is difficult to detect endogenous expression of PINK1 protein and its substrate Parkin Ser65 in common cell lines and mouse and pig brains. This application has demonstrated for the first time that PINK1 kinase and its substrate Parkin Ser65 phosphorylation can be specifically expressed in primate brains under physiological conditions. In order to verify the effectiveness of Parkin Ser65 antibodies, this application adopted a dephosphorylation experiment and used immunoblotting experiments to detect protein expression in PINK1 knockdown monkey brain tissue, Parkin knockdown monkey brain tissue and frozen dead human brain tissue. Immunohistochemical staining and other methods verified the specificity and effectiveness of the Parkin S65 antibody used (such as Figure 3 and Figure 4 ).

[0096] 1. Dephosphorylation of endogenous proteins

[0097] Adult wild-type monkey brainstem tissue (20 mg) was homogenized in 0.5 ml of ice-cold homogenization buffer (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl 2, 1 mM dithiothreitol, 0.5% Triton X-100, pH adjusted to 7.9) with protease inhibitors (cOmplete ULTRA EDTA-free tablets from Roche, 05892791001), and with or without phosphatase inhibitors (PhosSTOP from Roche, 04906837001). Ten micrograms of protein from brainstem lysates were treated with alkaline phosphatase (1 U / μl, FastAP, thermoscientific, EF0654) for 0.5 or 1 h at 37°C to dephosphorylate endogenous proteins. Samples were then analyzed by Western blot using two antibodies specific for pS65Parkin, anti-phospho-S112-Bad, anti-Parkin, and anti-Bad. The identification results are shown in Figure 3 .

[0098] Figure 3 Results show: Brainstem (BS) lysates from an 8-year-old wild-type (WT) monkey were treated with alkaline phosphatase (1U / μL) for 0.5 h and 1 h at 37°C to dephosphorylate endogenous proteins. Western blotting analysis showed that dephosphorylation abolished the immunoreactivity of pS65-Parkin to two antibodies specific for phosphorylated Parkin (AF3500 and CST). Phosphorylated expression of Bad, a known substrate protein of PINK1 (Ser112-BAD), was used as a positive control. Brainstem (BS) tissue from a PINK1 mutant (M6) monkey was included to show that endogenous pSer65-Parkin (red arrow) expression can be reduced by knocking down the PINK1 kinase. The ratio of phosphorylated protein to total protein from 4 independent experiments is presented below the blot (n=2 animals).

[0099] 2. Verification of Parkin S65 antibody specificity using PINK1 and Parkin knockdown monkey tissues

[0100] The most important method to verify the specificity of an antibody is to use the reduction or disappearance of the target protein band or staining signal when the target gene is knocked down or knocked out of the tissue. The results of immunoblotting and immunohistochemistry can effectively prove the specificity of the two Parkin S65 antibodies used in the present invention. The identification results are shown in Figure 4 .

[0101] Figure 4 The results showed that Parkin Ser65 can be specifically expressed in primate brains (human and monkey brains), and the phosphorylation of ParkinS65 in the primate brain is dependent on the expression of PINK1 kinase, while Parkin Ser65 expression cannot be detected in the mouse brain.

[0102] Figure 4 A in the figure is an analysis of the expression of Parkin and pS65-Parkin in different brain regions of 3-year-old monkey brains and 1-month-old mouse brains. In the monkey brains with reduced Parkin, the expression of pS65-Parkin also decreased, while the expression of pS65-Parkin could not be detected in the brains of mice with Parkin genotype defects.

[0103] Figure 4 B in the figure shows that the full-length human PINK1 or the truncated PINK1 (M1A) containing most of the kinase domain was transfected into HEK293 cells, and the plasmid of WT-Parkin was transfected at the same time. Both antibodies of pS65-Parkin (AF3500 and CST) can detect pS65-Parkin activated by PINK1 specific phosphorylation. However, when the Parkin plasmid with S65A mutation was transfected (the S65A mutation makes this site unable to be phosphorylated), pS65-Parkin could not be detected. This also shows the specificity of the two antibodies of pS65-Parkin.

[0104] Figure 4 C in the figure shows that the expression pattern of pS65-Parkin was consistent with that of PINK1 detected in different brain regions of 8-year-old monkeys and 68-year-old humans, both of which were highly expressed in BS (brainstem), but different from the expression pattern of Parkin, indicating that pS65-Parkin was dependent on the expression of PINK1.

[0105] Figure 4 D in the figure shows immunohistochemical staining of the cortex (ctx), substantia nigra (SN) and striatum (Str) tissues of normal and Parkin knockdown monkeys. The immunohistochemical analysis results of the pS65-Parkin antibody also show the specificity of the antibody.

[0106] 3. The specific steps for protein immunoblotting analysis are as follows:

[0107] (1) Take fresh monkey brain tissue and place it in a 1.5 ml centrifuge tube. Add RIPA cell lysis buffer (10 mg / ml, containing protease inhibitors) and grind it thoroughly 25 times using a glass homogenizer (Dounce Homogenizer Tissue Grinders). Use an ultrasonic disruptor to sonicate the cells 6 times, 5 seconds each time, and place them on a 4°C flip shaker for 30 minutes to fully lyse them.

[0108] (2) Centrifuge the tissue lysate at 14,000×g for 10 min in a 4°C centrifuge. Transfer the supernatant to a new EP tube.

[0109] (3) Use the BCA kit to determine the concentration of the protein lysate, and then adjust the protein concentration to 2 μg / μL using RIPA lysate. Add 5×SDS loading buffer to the protein sample. Treat the protein lysate in a 98°C metal bath for 10 min to fully denature the protein. Then store the protein sample in a -80°C refrigerator.

[0110] (4) Determine the concentration of polyacrylamide gel based on the size of the target protein and prepare the running buffer.

[0111] (5) Determine the amount of protein lysate to be loaded based on the expression level of the target protein, and determine the running time based on the size of the target protein. The running voltage is usually 80-100V, and the running time is 90-150min. The run can be stopped when the marker corresponding to the target band reaches the preset position.

[0112] (6) Install the membrane transfer device and transfer the protein to the nitrocellulose NC membrane at a constant voltage of 100 V for 60 min;

[0113] (7) Prepare 5% skim milk or 5% BSA for blocking and block at room temperature for 1 h. Then wash off the remaining blocking solution on the membrane with TBST, add the primary antibody prepared with 3% BSA, and incubate overnight at 4°C on a shaker.

[0114] (8) Recover the primary antibody and wash the residual antibody on the membrane with TBST. Dilute the secondary antibody conjugated with horseradish peroxidase with milk at a ratio of 1:7000 and incubate on a shaker at room temperature for 1 h.

[0115] (9) Use TBST to wash away the residual antibodies on the membrane, mix the luminol chemiluminescent reagent A solution and B solution in the same proportion, evenly cover the NC membrane, place it in a chemiluminescent imaging instrument for detection, and develop and analyze it on a developer.

[0116] 4. The specific steps for immunohistochemical analysis are as follows:

[0117] (1) Return the frozen tissue sections to room temperature, rinse with PBS, and remove the embedding agent.

[0118] (2) Use 0.3-3% H 2 O 2 Treat until there are no bubbles in the slices, and wash with PBS 3 times, 6 minutes each time. Use 0.3% Triton X-100 solution to permeabilize the slices for 40-60 minutes. Then use a hydrophobic pen to draw circles around the tissue slices, add blocking solution (3% BSA, 2% NGS, 0.3% TritonX-100) and block for 40-60 minutes. After blocking, add primary antibody to the brain slices (primary antibody is prepared with blocking solution) and incubate at 4°C overnight.

[0119] (3) Wash the sections three times with PBS, 10 min each time. Use the secondary antibody of the same species as the primary antibody in the kit, incubate at room temperature for 1 hour, then wash three times with PBS, 5 min each time. Add horseradish peroxidase for 10 min, and wash three times with PBS, 10 min each time. Add DAB colorimetric reagent, and the reaction time depends on the thickness of the section and the specificity of the antibody. When the color develops to the appropriate depth, immediately rinse off the DAB colorimetric solution with PBS, and wash three times, 10 min each time.

[0120] (4) Dehydrate with gradient ethanol: 50% ethanol, 75% ethanol, 90% ethanol, 95% ethanol, 100% ethanol, 100% ethanol, dehydrate for 10 minutes at each concentration. Immediately after dehydration, place in xylene solution for transparency. Determine the xylene treatment time based on the degree of transparency of the tissue. Finally, before the xylene dries, use neutral gum to seal the slide, and observe the image under a microscope after drying.

[0121] 4. Enhancing phosphorylated Parkin expression can effectively eliminate endogenous pathological S129-a-synuclein in the brain of aged monkeys

[0122] 1. Overexpression of Parkin can enhance the expression of phosphorylated Parkin protein, thereby effectively eliminating pathological S129-a-synuclein in the brains of elderly monkeys

[0123] Since old monkeys are not easy to obtain and are expensive, two old wild-type monkeys aged 22 and 25 were used for stereotaxic brain injection of the substantia nigra and striatum of the monkeys in this example. Since the aggregation degree of endogenous a-synuclein varies from animal to animal, in order to avoid individual differences, the brain injection virus scheme in this example is: AAV9-WT Parkin and AAV9-S65A Parkin are injected into the left and right sides of the same animal respectively. Due to the limited range of viral infection, the non-virus-infected area was taken as the control group in this example. The animals were analyzed 9 weeks after injection.

[0124] Figure 5 The results showed that overexpression of Parkin increased the expression of Parkin Ser 65, which helped to clear endogenous pS129-a-synuclein aggregates (Lewy body) in the brains of normally aged monkeys, while the phosphorylated mutant Parkin Ser65A had no such effect.

[0125] Among them, Western blotting of substantia nigra showed that overexpression of WT Parkin significantly increased the phosphorylation expression level of Parkin, while mutant Parkin (S65A) could not be phosphorylated ( Figure 5 A in the figure). Immunostaining with Parkin antibody showed that both WT Parkin injection group and S65A Parkin could be effectively expressed in the substantia nigra and striatum of the monkey brain ( Figure 5 BD in the brain), and compared with the non-injected control group (NO AAV), the overexpression of WT Parkin can effectively reduce the accumulation of pS129-α-synuclein in the substantia nigra and striatum of the aged monkey brain, while the mutant S65A Parkin that cannot be phosphorylated cannot effectively eliminate the accumulation of pS129-α-synuclein in the aged monkey brain ( Figure 5 BE in ).

[0126] 2. Overexpression of PINK1 protein (Parkin upstream kinase) can enhance the expression of phosphorylated Parkin protein, thereby effectively eliminating a-synuclein aggregation in the brains of elderly monkeys

[0127] PINK1 kinase is the upstream kinase that phosphorylates Parkin. Overexpression of PINK1 kinase can also enhance the expression of Parkin phosphorylation. Since elderly monkeys are not easy to obtain and are expensive, two 25-year-old wild-type elderly monkeys were taken in this example for stereotaxic injection of the substantia nigra and cortex of the monkeys. Since the aggregation degree of endogenous a-synuclein varies from animal to animal, in order to avoid individual differences, the brain injection virus scheme in this example is: AAV9-Control and AAV9-hPINK1-flag viruses are injected into the left and right brains of the same animal, respectively. The animals were euthanized 10 weeks after the injection and the samples were analyzed.

[0128] Immunohistochemical staining and immunoblotting experiments showed that AAV9-hPINK1-flag virus can be effectively expressed and overexpression of PINK1 can significantly increase the phosphorylation expression level of Parkin ( Figure 6 AC in the brain) and effectively cleared the accumulation of pS129-a-synuclein in the substantia nigra of the aged monkey brain, while the AAV9-Control virus injection group had no clearing effect ( Figure 6 D in.

[0129] 3. Brain stereotaxic injection method

[0130] The specific method refers to the inventor's previous publication (Yang et al., Protein Cell 2022). Specifically, the validated virus was injected into the corresponding brain area at a rate of 1 μl / min. The monkeys were euthanized by intraperitoneal injection of atropine and ketamine 2-3 months after the virus injection, and the virus injection site was dissected and taken for biochemical analysis such as immunostaining and protein blotting.

[0131] 5. Enhancing the expression of phosphorylated Parkin S65 can effectively eliminate endogenous pathological S129-a-synuclein aggregation in the brain of PD monkeys and protect the function of nerve cells

[0132] PINK1 gene mutations can cause juvenile Parkinson's disease due to loss of gene function, but unfortunately, both PINK1 knockout mouse and pig models cannot simulate the important pathological characteristics of neuronal cell death in the brains of PD patients. In the early stage of this application, it was found that the PINK1 knockdown monkey model can successfully simulate the important pathology of neuronal cell degeneration and death in the brains of PD patients (Yang et al., Cell Res 2019; Yang et al., Protein Cell, 2022), providing an effective PD animal model. This example further found that knocking down PINK1 kinase can cause a significant decrease in Parkin S65 phosphorylation and significant aggregation of pS129-a-synuclein in the monkey brain ( Figure 7 ).

[0133] The results show: Figure 7 A in the figure shows that in the brain of monkeys lacking PINK1, the expression of pS65-Parkin is reduced, and the level of pS129-a-synuclein / α-syn is increased. The relative expression ratio of pS65-Parkin and pS129-α-syn is presented as pS65-Parkin / total Parkin, pS129-a-synuclein / total a-syn or / vinculin. Figure 7 A in the figure shows that the expression of pS65-Parkin is decreased and the expression of pS129-α-syn is increased in the area where PINK1 is knocked down. Figure 7 A in the figure shows the effect of knocking down PINK1 on pS65-Parkin and pS129-a-synuclein in the substantia nigra of 10-year-old and 22-year-old monkeys.

[0134] When the expression of phosphorylated Parkin S65 was enhanced in the PINK1 knockdown PD monkey model, the endogenous pathological pS129-a-synuclein aggregation in the PD monkey brain was effectively eliminated, and the neuronal cell death caused by PINK1 knockdown was reduced, thus having the effect of protecting neurons ( Figure 8 ).

[0135] The results show: Figure 8 AB in the figure shows that brain injection of PINK1 gRNA and Cas9 virus significantly reduced the level of PINK1 through immunohistochemistry and immunoblotting. Figure 8 C shows that overexpression of WT-Parkin, but not S65A Parkin, in the brains of PINK1 knockdown monkeys significantly reduced the aggregation of pS129-a-synuclein, and the number of NeuN also increased. Figure 8 D in Figure 5 was analyzed using Two-tailed Student's t-test and is shown as mean ± SEM.

[0136] VI. pS129-a-synuclein aggregates and Parkin S65 expression is downregulated in the brains of PD patients

[0137] An important pathological feature of the brain of Parkinson's disease patients is the appearance of Lewy bodies caused by pathological pS129-a-synuclein aggregation. In order to explore the clinical significance of phosphorylated Parkin as a target for the treatment of pS129-a-synuclein aggregation, the present invention used brain tissues of non-PD patients and sporadic PD patients of similar age for immunohistochemical staining, and found that Parkin S65 phosphorylation expression was significantly downregulated and a-synuclein, Ubiquitin and other proteins aggregated in the brain of PD patients ( Fig. 9 ).

[0138] The results show: Fig. 9 A in the figure shows the distribution of pS65-Parkin in the midbrain of the human brain, and it is expressed in different regions. Fig. 9 B in the figure shows that the expression of pS65-Parkin in the substantia nigra of PD patients is lower than that in the substantia nigra of normal people. Fig. 9 C statistics were analyzed using Two-tailed Student's t-test and are shown as mean±SEM, ***P < 0.001, n=2. Fig. 9 Immunohistochemical staining in D shows significant pS129-a-synuclein accumulation in the brains of PD patients.

[0139] Since human brain tissue is not easy to obtain, the present invention further uses young and old monkey brain tissue for detection, and first found that Parkin S65 phosphorylation is also abundantly expressed in dopamine neurons in the substantia nigra of the midbrain ( Fig.10 A in the figure), and then found that Parkin phosphorylation was significantly downregulated and pathological pS129-a-synuclein was significantly aggregated in the brains of aged monkeys ( Fig.10 BC in the ), and the insolubility of Parkin protein increases with aging, indicating that its function is significantly downregulated with aging ( Fig.10 D in the above examples). The results of the above examples all show that Parkin phosphorylation is significantly downregulated in PD patients and in the normal aging process. Therefore, enhancing the expression of Parkin phosphorylation can be used as an important therapeutic drug target for clearing a-synuclein aggregation and protecting nerve cell function.

[0140] The steps for separation and purification of soluble protein and insoluble protein in this example are as follows:

[0141] (1) Weigh approximately 150 mg of monkey cerebral cortex tissue and place it in 2 ml of Tris buffer (5 mM Tris, 140 mM NaCl, pH 7.5). Add EDTA-free protease inhibitors to the buffer and grind the sample in a glass homogenizer until no tissue clumps remain.

[0142] (2) Centrifuge at 163,202 × g for 30 min at 4°C. Transfer the soluble supernatant to a new EP tube and resuspend the precipitate containing insoluble proteins in 2 ml SDS buffer (5 mM Tris, 140 mM NaCl pH 7.5, 2% TritonX-100, 2% SDS). Incubate the resuspended sample on ice for 20 min and centrifuge at 163,202 × g for 30 min at 4°C. Transfer the supernatant to a new EP tube and discard the precipitate.

[0143] (3) Add 5× SDS loading buffer to the supernatant of the two steps, treat the protein lysate in a 98℃ metal bath for 10 min to fully denature the protein. Then store the protein sample in a -80℃ refrigerator.

[0144] Immunofluorescence staining steps in this example:

[0145] (1) After taking the slide out of the -80℃ freezer, place the slide in a humidified box and place it in PBS solution. Rewarm and remove the embedding agent. Fix with 4% PFA solution for 30 minutes. If the monkey has been perfused before dissection, this step only requires fixation for 10 minutes. Remove PFA with 1× PBS and wash three times, 5 minutes each time.

[0146] (2) Soak the slide in 0.3% Triton X-100 solution for 1-2 hours to permeabilize the membrane. Then place the slide in blocking solution (3% BSA + 2% NGS) and block at room temperature for 1 hour.

[0147] (3) Use a hydrophobic pen to draw a circle around the tissue section to reduce the amount of antibody used. Use blocking solution to prepare the primary antibody, add the primary antibody to the tissue, and incubate it in a shaker at 4°C overnight. Then recover the primary antibody. Wash with 1× PBS 3 times, 5 minutes each time. Also use blocking solution to prepare the secondary antibody. The secondary antibody has a fluorescent group, so be careful to avoid light. DAPI can be added to the secondary antibody at a ratio of 1:3000 and incubated for 1 hour. Wash with 1× PBS 3 times, 5 minutes each time.

[0148] (4) After secondary antibody treatment, Sudan black solution can be used to reduce high background, generally for 20s-2min. Use 0.3% Triton X-100 solution to remove excess Sudan black solution. Add anti-fluorescence quenching mounting medium to the tissue, gently cover with a coverslip to prevent bubbles, and fix the coverslip with transparent nail polish to prevent it from sliding. Be careful to avoid light when operating and storing.

[0149] (5) Fluorescence or confocal microscopy can be used for imaging. After neuropathological analysis, the slides can be stored in a -80℃ refrigerator.

[0150] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. Application of phosphorylated Parkin protein in the preparation of drugs for treating neurodegenerative diseases characterized by pathological α-synuclein aggregates; Compared with the wild-type Parkin protein, the serine at position 65 of the phosphorylated Parkin protein is phosphorylated.

2. Application of PINK1 kinase expression promoter in the preparation of drugs for treating neurodegenerative diseases characterized by pathological α-synuclein aggregates.

3. The use according to any one of claims 1 to 2, characterized in that: The α-synuclein aggregates include pS129-α-synuclein aggregates.

4. The use according to claim 3, characterized in that: The neurodegenerative disease carrying pathological α-synuclein aggregates includes one or both of Parkinson's disease and Lewy body dementia.

5. The use according to claim 4, characterized in that: The Parkinson's disease is sporadic Parkinson's disease and familial Parkinson's disease characterized by pathological α-synuclein aggregates.

6. The use according to any one of claims 1 to 5, characterized in that: The drug comprises the phosphorylated Parkin protein or its expression vector.

7. The use according to claim 6, characterized in that: The gene expression vector includes one or more of an adenovirus vector, a lentivirus vector and a retrovirus vector.

8. The use according to claim 7, characterized in that: The serotype of the adenovirus vector is any one of AAV2, AAV9, AAV Mac and php.eb.

9. A drug for treating neurodegenerative diseases characterized by pathological α-synuclein aggregates, characterized in that: The active ingredients of the medicine include phosphorylated Parkin protein or PINK1 kinase expression promoter.

10. The drug according to claim 9, characterized in that The drug also includes pharmaceutically acceptable excipients; Optionally, the auxiliary material is selected from one or more of a diluent, a binder, a disintegrant, a lubricant and a wetting agent.

Images