Application of FCN2 as target spot in screening or treating Alzheimer's disease

Abstract

The invention relates to the technical field of medical biological detection, provides a new application of FCN2, particularly relates to an application of FCN2 as a target spot in screening or treating Alzheimer's disease, and further provides a kit for performing early screening or curative effect evaluation on Alzheimer's disease by utilizing quantitative detection aiming at FCN2 and a medicine taking the kit as the target spot. The kit and the detection method disclosed by the invention are simple, convenient, reliable, short in period, high in specificity and easy to clinically popularize. The liver-derived plasma protein Ficolin-2 (FCN2) can be used as an early screening target and a drug treatment target of the Alzheimer's disease (AD), is particularly used as a kit and other medical equipment products to be applied, and has important significance on early discovery, precise treatment, curative effect monitoring and the like of high-risk patients.

Description

Technical Field

[0001] The present invention relates to the field of medical biological detection technology, and relates to the application of FCN2 as a molecular marker in early screening or efficacy monitoring or evaluation of Alzheimer's disease, and also provides its application as a therapeutic target for Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is one of the most common age-related brain diseases and the leading cause of health burden for people over 65 years old worldwide. Currently, clinical trials for AD face major challenges, and more mechanisms, therapeutic targets and effective treatments remain to be explored. Increasing evidence shows that circulating proteins actively secreted or released into the blood by organs have indirect or direct regulatory effects on the brain, mediating the interaction between peripheral organs and the brain, and their imbalance can promote cognitive impairment and neurological diseases including AD.

[0003] Currently, drugs used to treat AD can only delay symptoms for a limited time but cannot reverse disease progression. Therefore, there is an urgent need to explore more detailed mechanisms, identify promising therapeutic targets, and develop effective treatments to improve the quality of life and reduce the medical burden on society.

[0004] Ficolin belongs to complement lectins and is an important natural immune molecule. As a soluble pattern recognition receptor (PRR), ficolin can recognize pathogen-associated molecular patterns (PAMPs) on the surface of pathogenic microorganisms, bind to mannose-binding lectin (MBL)-associated serine proteases (MASPs) to activate the complement lectin pathway, and can also act as an opsonin to bind to phagocyte surface receptors to mediate opsonization and phagocytosis.

[0005] Ficolin-2 (FCN-2) is a type of fibrin protein ficolin, also known as P35, L-ficolin, and EBP-37hucolin. FCN-2 protein is mainly synthesized and secreted by hepatocytes. It has similar structure and function to MBL, has lectin activity, can recognize oligosaccharide structures on the surface of pathogens, and then activate complement through the lectin pathway. Ficolin-2 in normal human serum is an important complement lectin protein. Its concentration increases during acute emergency response, inflammatory response or infection, and can be used as an early warning indicator for inflammatory diseases. Its concentration returns to normal after the disease recovers, and it is also positively correlated with CRP, so it can be used as a monitoring indicator for inflammation. Based on this, many application studies of FCN2 have been conducted in the prior art, such as patent document US11940450B2, which explores FCN2 as a biomarker for non-invasive diagnosis of congenital renal insufficiency; patent document US20230117596A1, which studies the diagnosis of non-alcoholic fatty liver disease with blood proteins including FCN2; patent document US20220326258A1, which studies the application of blood proteins including FCN2 as targets for amyotrophic lateral sclerosis (ALS) and motor neuron disease; Patent documents EP3955001A1, WO / 2020 / 179954, KR1020200112728A, US20220146531A1, etc. have studied the application of blood proteins including FCN2 in the treatment of eye diseases; patent document CN102712687A has studied ficolin-related polypeptides, and polypeptides derived from these ficolin-related polypeptides, which are used to treat diseases related to inflammation, apoptosis, autoimmunity, coagulation, thrombosis or coagulopathy. Summary of the invention

[0006] The present invention provides a protein marker for assessing high-risk AD and treating AD and its application. The protein marker is a hepatic plasma protein FCN2, which is applied as an early screening target, a target for AD treatment, and a target for efficacy monitoring.

[0007] The purpose of the present invention is to screen a protein marker that can assess the risk of AD and its application, so as to provide a reference for the auxiliary diagnosis or treatment of clinical AD.

[0008] To achieve the above purpose, the present invention firstly analyzes the deCODE database to find that FCN2 protein has a protective effect on AD, and analyzes that plasma FCN2 originates from the liver; then compares the serum FCN2 protein levels in clinical young, elderly, and old AD patients, showing that serum FCN2 is negatively correlated with age, positively correlated with MoCA cognitive function scores, and positively correlated with MMSE cognitive function scores. Finally, an AD mouse model was constructed, and by transfecting a recombinant virus that overexpresses FCN2, it was shown that FCN2 can improve the patient's AD motor coordination and cognitive function, and has a protective effect on AD liver, and related mechanism analysis was performed, showing that FCN2 overexpression significantly inhibits the downregulation of cognitive / AD-related genes and the activity of pathways.

[0009] Based on the above analysis process, the specific technical solutions of the present invention are as follows:

[0010] In a first aspect, the present invention provides the use of a reagent for detecting the expression level of FCN2 in the preparation of a kit for early screening or efficacy evaluation of Alzheimer's disease.

[0011] Preferably, the reagent for detecting the expression level of FCN2 is a reagent for detecting the expression level of FCN2 protein in a biological sample, and the kit comprises a reagent for detecting the expression level of FCN2 protein in a biological sample.

[0012] Further preferably, the reagent for detecting the expression level of FCN2 protein in the biological sample is selected from the reagents required for detection using protein chip and microfluidic chip, ELISA, mass spectrometry, immunoturbidimetry, immunohistochemistry, immunofluorescence or Western blot technology, and preferably ELISA is used for detection.

[0013] Preferably, the biological sample is selected from peripheral blood.

[0014] Preferably, the risk assessment standard for Alzheimer's disease is an FCN2 protein concentration of 2000-8000 ng / ml, with a preferred concentration threshold of 5000 ng / ml.

[0015] In a second aspect, the present invention provides a kit for early screening or efficacy evaluation of Alzheimer's disease, comprising a reagent for detecting the expression level of FCN2 in a biological sample.

[0016] Preferably, the reagent is selected from the reagents required for detection using protein chips and microfluidic chips, ELISA, mass spectrometry, immunoturbidimetry, immunohistochemistry, immunofluorescence or Western blot technology. Most preferably, the ELISA method is used to detect the concentration of FCN2 protein in blood samples of high-risk patients to assess the risk of AD, and the level of FCN2 protein expression is negatively correlated with the incidence of AD.

[0017] Therefore, the detection product provided by the present invention includes the above-mentioned kit, iTRAQ labeling, liquid chromatography, Raman spectroscopy, etc. for detecting FCN2 protein in a sample.

[0018] In a third aspect, the present invention provides the use of FCN2 in the preparation of an Alzheimer's disease drug, wherein the drug is selected from a liposome or nanomaterial encapsulating exogenous FCN2, an FCN2 recombinant expression vector, or a substance that promotes the in vivo expression of FCN2 or inhibits the degradation of FCN2.

[0019] Specifically, the drugs of the present invention are products that are targeted for AD treatment, including monoclonal antibody drugs, small molecule compounds, nucleic acids, recombinant viruses, etc. They achieve the purpose of maintaining a high concentration level of FCN2 protein by increasing FCN2 protein expression and inhibiting FCN2 degradation signal pathways.

[0020] After transfecting the overexpression FCN2 virus in the liver of AD mouse animal model, the FCN2 protein is significantly upregulated, and the coordination, action ability and cognitive function of AD mice are significantly improved; the freezing time of AD mice in the FCN2 overexpression group is longer than that of the control group in the conditioned fear memory test; the mechanism study shows that the area of ​​6E10 and MXO4 positive plaques in the hippocampus of FCN2 group mice is reduced, and the activation of microglia and astrocytes is reduced. FCN2 has a protective effect on the liver of AD mice.

[0021] In a fourth aspect, the present invention provides a pharmaceutical composition for treating Alzheimer's disease, comprising an active ingredient and a medically acceptable excipient, wherein the active ingredient comprises FCN2 protein or FCN2 recombinant expression vector.

[0022] Furthermore, the pharmaceutical composition is used in combination with other drugs for treating Alzheimer's disease.

[0023] The beneficial protection and effects of the present invention are as follows:

[0024] The present invention first discovered that liver-derived FCN2 protein has a protective effect on the occurrence of AD, and that AD can be alleviated after increasing FCN2 protein. It proves that FCN2 protein can be used as an early screening target for AD, a drug treatment target, and especially as a medical device product such as a kit, which is of great significance for early detection of high-risk patients, early intervention treatment, and prognosis monitoring.

[0025] In terms of technology, the detection of FCN2 is essentially a quantitative detection of proteins in peripheral blood. There are many ways to implement it in the existing technology. It has the characteristics of simple operation, sensitive detection, good specificity and high repeatability. It has been increasingly used in clinical testing technology and is an effective supplement to the current detection methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Mendelian randomization showed that FCN2 protein in human blood was a protective factor for AD: a. Mendelian randomization analysis results showed that FCN2 protein in human plasma could reduce the risk of AD; b. Mendelian randomization analysis results showed that FCN2 protein in human plasma had a protective effect against AD; c. Colocalization analysis showed that the effect of FCN2 on AD was robust and reliable, and its PPshared value was close to 1 (PPshared greater than 0.85 indicated that the association between the two was reliable).

[0027] Figure 2 The figure shows the results of human organ proteomics and GEO database analysis: a. Based on human organ proteomics data, it was found that plasma FCN2 originated from the liver; b. Based on human organ transcriptomics data, analysis showed that FCN2 was mainly expressed in human liver regardless of male or female; c. Based on human liver single-cell sequencing data in the GEO database (GSE115469), analysis showed that liver FCN2 was mainly expressed in endothelial cells; d. Based on human brain single-cell sequencing data in the GEO database (GSE138852), analysis showed that FCN2 was not expressed in any brain region or brain cells.

[0028] Figure 3 The correlation analysis of serum FCN2 protein levels in young, old and elderly AD patients with age and cognitive function is shown: a. Clinical information of young, old and elderly AD patients; b. Comparison of serum FCN2 in AD patients with that in young and elderly patients; c. Serum FCN2 is negatively correlated with age; d. Serum FCN2 is positively correlated with MoCA cognitive function score; e. Serum FCN2 is positively correlated with MMSE cognitive function score.

[0029] Figure 4 The figure shows the alleviating effect of FCN2 protein on Alzheimer's disease: a. Experimental flow chart of tail vein injection of human FCN2 liver overexpression virus in AD mice; b. FCN2 overexpression virus successfully transfected the liver; c. After liver transfection with FCN2 virus, FCN2 protein was significantly upregulated; de. In the water maze experiment, the number of times the AD mice in the FCN2 overexpression group crossed the target platform increased significantly; f. In the new object recognition, the cognitive index of the AD mice in the FCN2 overexpression group was significantly increased; g. In the open field test, the number of times the FCN2 overexpression AD mice entered the central area was significantly higher than that of the control group; h. In the conditioned fear memory test, the freezing time of the AD mice in the FCN2 overexpression group was longer than that of the control group; i. In the rotarod test, it was observed that the duration of the AD mice in the FCN2 overexpression group was significantly longer; jk. The area of ​​6E10 and MXO4 positive plaques in the hippocampus of the FCN2 group mice was reduced, and the activation of microglia and astrocytes was reduced.

[0030] Figure 5 The results of HE staining of the main organs of the mouse model are shown: a. HE staining of the lungs, liver, spleen, heart, kidneys and small intestine of AD control mice and AD mice with liver-specific human FCN2 overexpression showed no significant differences; b. Elisa test was performed on the plasma of AD control mice and AD mice with liver-specific human FCN2 overexpression, and no significant differences were found in the routine blood tests between the two groups; c. Liver function test found that FCN2 overexpression significantly reduced the concentrations of ALT and AST, indicating that FCN2 has a protective effect on the liver of AD mice.

[0031] Figure 6 The cognitive function evaluation of the mouse model and the single-cell sequencing results of the mouse hippocampus are shown: a. The distribution of hippocampal cells in the two groups of mice; b. Comparison of the proportion of hippocampal cells in the two groups of mice; c. Comparison of the number of hippocampal cells in the two groups of mice; d. FCN2 overexpression significantly caused changes in the transcriptional profiles of 10 types of cells in the mouse hippocampus; ef. FCN2 overexpression significantly activated the upregulation of cognitive / AD protection-related genes and the activation of pathways; hj. FCN2 overexpression significantly inhibited the downregulation of cognitive / AD-harmful related genes and the activity of pathways; k. AD protection or harmful pathways significantly affected by FCN2 overexpression; l. FCN2 overexpression significantly inhibited AD-harmful metabolic pathways, such as oxidative phosphorylation, steroid biosynthesis, and pentose phosphate pathway. DETAILED DESCRIPTION

[0032] The present invention will now be described in detail in conjunction with the embodiments and drawings, but the implementation of the present invention is not limited thereto.

[0033] The reagents and raw materials used in the present invention are all commercially available or can be prepared according to literature methods. The experimental methods in the following examples where specific conditions are not specified are generally carried out according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by volume.

[0034] Example 1: deCODE database and proteomics analysis revealed that FCN2 protein has a protective effect on AD. The specific screening steps are as follows:

[0035] Step 1: Data download: Download GWAS genetic association data related to human blood FCN2 protein expression level from the deCODE database (https: / / www.decode.com / summarydata / ) (corresponding data public article number PMID: 34857953); download GWAS genetic association data of AD occurrence from the OpenGWAS project database (https: / / gwas.mrcieu.ac.uk / ) (number ieu-b-2).

[0036] Step 2: Instrumental variable screening: Based on the Mendelian randomization analysis package TwoSampleMR, instrumental variables (IVs) / SNPs were screened for FCN2. The screening criteria were as follows: the instrumental variable correlation P value associated with FCN2 protein level was less than 5×10 -8 ; The instrumental variable is not related to AD, and its P value is greater than 0.05; The instrumental variable can only affect the risk of AD by affecting the level of FCN2.

[0037] Step 3: Mendelian randomization analysis: Based on the screened instrumental variables, TwoSampleMR was used to perform Mendelian randomization analysis on the FCN2 protein expression level and the risk of AD. The main method was the inverse variance weighted (IVW) method. A P value less than 0.0001 was considered to be associated with the risk of AD, and a Beta value less than 0 indicated that FCN2 had a protective effect on AD.

[0038] Step 4: Robustness assessment of Mendelian randomization analysis results: Based on the Cochran Q test, MR-PRESSO test, leave-one-out sensitive detection and reverse causality analysis in TwoSampleMR, it was determined that the protective effect of FCN2 protein levels on the risk of AD is completely credible.

[0039] Step 5: Colocalization analysis: Based on the GWAS data of FCN2 protein and the most significantly associated lead SNP and GWAS data of AD, the coloc R package was used to perform genetic association analysis on the two. The analysis results further confirmed the protective effect of FCN2 protein against AD.

[0040] The results are as follows Figure 1 As shown in the results of Mendelian randomization analysis, FCN2 protein in human plasma can reduce the risk of AD ( Figure 1 a); Mendelian randomization analysis results showed that FCN2 protein in human plasma has a protective effect against AD ( Figure 1 b); c. Co-localization analysis showed that the effect of FCN2 on AD was robust and reliable, and its PPshared value was close to 1, indicating that the association between the two was reliable ( Figure 1 C).

[0041] Human organ proteomics, human organ transcriptomics and GEO database were used for further analysis. Based on human organ proteomics data, it was found that plasma FCN2 originated from the liver ( Figure 2 a); Based on human organ transcriptomic data, analysis showed that FCN2 is mainly expressed in human liver, regardless of male or female ( Figure 2 b); Based on human liver single-cell sequencing data in the GEO database (GSE115469), analysis showed that liver FCN2 is mainly expressed in endothelial cells ( Figure 2 c); Based on the human brain single-cell sequencing data in the GEO database (GSE138852), analysis showed that FCN2 was not expressed in any brain region or brain cell ( Figure 2 d).

[0042] Example 2 Detection of the difference in FCN2 protein levels in the blood of healthy people and AD patients

[0043] A total of 33 young healthy adults, 42 elderly healthy people, and 32 elderly AD patients were recruited, and each volunteer was scored with the Mental State Examination (MMSE) and the Montreal Cognitive Assessment (MoCA). Figure 3 As shown in a.

[0044] The specific detection method is as follows.

[0045] 5 mL of fasting whole blood was collected from each participant, and after being placed at room temperature for 30-60 minutes, the serum was separated by centrifugation. The FCN2 protein level in the serum of the volunteers was detected by ELISA. The specific steps are as follows:

[0046] (1) Coat the sample wells of a PVC microtiter plate with capture antibody at a concentration of 1–10 μg / mL in carbonate / bicarbonate buffer (pH 9.6).

[0047] (2) Cover the microtiter plate with adhesive plastic and incubate at 4°C overnight, discard the coating solution, and wash the microtiter plate twice, adding 200 μL PBS to the microwells each time.

[0048] (3) Blocking and sample loading

[0049] Add 200 μL of blocking buffer (5% skim milk powder / PBS) to each well to block the remaining protein binding sites in the coated wells; cover the microtiter plate with adhesive plastic and incubate at room temperature for at least 1-2 hours, or incubate at 4°C overnight; wash the microtiter plate twice with 200 μL PBS, add 100 μL of diluted sample to each well, incubate at 37°C for 90 minutes, then discard the sample and wash the microtiter plate twice with 200 μL PBS.

[0050] (4) Incubate with detection antibody and secondary antibody

[0051] 100 μL of diluted detection antibody was added to each well, the microtiter plate was covered with adhesive plastic and incubated for 2 hours at room temperature. The microtiter plate was then washed four times with PBS, and 100 μL of conjugated secondary antibody was added, which was diluted in blocking buffer before use. After covering the microtiter plate with adhesive plastic and incubating for 1-2 hours at room temperature, the microtiter plate was washed four times with PBS.

[0052] (5) Detection: Measure the final product at 492 nm

[0053] Based on serum FCN2 Elisa detection and cognitive function assessment of 33 young, 42 elderly and 32 old AD patients, it was found that the serum FCN2 concentration of elderly AD patients was significantly lower than that of healthy young and elderly patients ( Figure 3 b), and FCN2 was negatively correlated with age ( Figure 3 c), and positively correlated with the Mini-Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA) scores ( Figure 3 d, 3e), indicating that blood FCN2 is affected by aging and is positively correlated with cognitive function.

[0054] Example 3 Animal model experiment verifies the protective effect of FCN2 protein on AD

[0055] 5×FAD mice were used to construct an AD animal model. Then, liver-specific human FCN2 overexpression virus was injected into the tail vein of 5×AD mice, and the control group was injected with control virus (FCN2 overexpression virus: rAAV2 / 8-TBG-FCN2-2A-EGFP-WPRE-polyA; control virus: rAAV2 / 8-TBG-EGFP-WPRE-Pa) Figure 4 a). Three months later, the two groups of mice were evaluated for cognitive function for one week, and water maze test, novel object recognition, open field test, conditioned fear memory test and rotarod test were performed. At the same time, the pathological changes of the hippocampus of AD mice were detected.

[0056] The results are as follows Figure 5 As shown, the fluorescence staining results showed that the FCN2 overexpression virus successfully transfected the liver ( Figure 4 b); After the liver was transfected with FCN2 virus, FCN2 protein was significantly upregulated ( Figure 4 c); In the water maze test, the number of times that AD mice in the FCN2 overexpression group crossed the target platform increased significantly ( Figure 4 d-4e); In the recognition of new objects, the cognitive index of AD mice in the FCN2 overexpression group was significantly increased ( Figure 4f); In the open field test, the number of times that FCN2 overexpressing AD mice entered the central area was significantly higher than that of the control group ( Figure 4 g); In the conditioned fear memory test, the freezing time of AD mice in the FCN2 overexpression group was longer than that in the control group ( Figure 4 h); In the rotarod test, it was observed that the duration of AD mice in the FCN2 overexpression group was significantly longer ( Figure 4 i); The area of ​​6E10 and MXO4 positive plaques in the hippocampus of mice in the FCN2 group was reduced, and the activation of microglia and astrocytes was reduced ( Figure 4 j-4k).

[0057] HE staining was performed on the main organs of the mouse model, and the results were as follows Figure 5 The results showed that no significant differences were found in the HE staining of the lungs, liver, spleen, heart, kidneys and small intestine of AD control mice and liver-specific human FCN2 overexpressing AD mice ( Figure 5 a); Elisa test was performed on AD control mice and liver-specific human FCN2 overexpressing AD plasma, and no significant difference was found in the blood routine test between the two groups ( Figure 5 b); Liver function tests found that FCN2 overexpression significantly reduced ALT and AST concentrations, indicating that FCN2 has a protective effect on the liver of AD mice ( Figure 5 c).

[0058] The single-cell sequencing of the hippocampus of the two groups of mice was continued, and the results were as follows Figure 6 As shown: There is no significant difference in the distribution of hippocampal cells between AD mice and AD mice overexpressing FCN2 ( Figure 6 a), but there was a difference in the proportion of hippocampal cells between the two groups of mice ( Figure 6 b), the number of hippocampal cells in mice also differed significantly ( Figure 6 c). Genetic analysis results showed that FCN2 overexpression significantly altered the transcriptional profiles of 10 types of cells in the mouse hippocampus ( Figure 6 d); FCN2 overexpression significantly activated the upregulation of cognitive / AD protection-related genes and the activation of pathways ( Figure 6 e-6f), and FCN2 overexpression significantly inhibited the downregulation of genes and pathway activity related to cognitive / AD detrimental effects ( Figure 6 h-6j); AD protective or harmful pathways significantly affected by FCN2 overexpression ( Figure 6 k), especially significantly inhibiting metabolic pathways that are harmful to AD, such as oxidative phosphorylation, steroid biosynthesis, and pentose phosphate pathway ( Figure 6 l).

[0059] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. Application of reagents for detecting FCN2 expression levels in the preparation of early screening or efficacy evaluation kits for Alzheimer's disease.

2. The use according to claim 1, characterized in that: The reagent for detecting the expression level of FCN2 is a reagent for detecting the expression level of FCN2 protein in a biological sample, and the kit comprises a reagent for detecting the expression level of FCN2 protein in a biological sample.

3. The use according to claim 2, characterized in that: The reagent for detecting the expression level of FCN2 protein in biological samples is selected from the reagents required for detection using protein chip and microfluidic chip, ELISA, mass spectrometry, immunoturbidimetry, immunohistochemistry, immunofluorescence or Western blot technology.

4. The use according to claim 2, characterized in that: The biological sample is selected from peripheral blood.

5. The use according to claim 1, characterized in that: The risk assessment standard for Alzheimer's disease is an FCN2 protein concentration of 2000-8000ng / ml.

6. A kit for early screening or efficacy evaluation of Alzheimer's disease, comprising a reagent for detecting the expression level of FCN2 in a biological sample.

7. The kit according to claim 5, characterized in that The reagents are selected from the reagents required for detection using protein chips and microfluidic chips, ELISA, mass spectrometry, immunoturbidimetry, immunohistochemistry, immunofluorescence or Western blot technology.

8. Use of FCN2 in the preparation of a drug for Alzheimer's disease, characterized in that: The drug is selected from liposomes or nanomaterials encapsulating exogenous FCN2, FCN2 recombinant expression vectors, and substances that promote the in vivo expression of FCN2 or inhibit the degradation of FCN2.

9. A pharmaceutical composition for treating Alzheimer's disease, characterized in that: The pharmaceutical composition comprises an active ingredient and a medically acceptable excipient. Wherein, the active component includes FCN2 protein or FCN2 recombinant expression vector.

10. The pharmaceutical composition for treating Alzheimer's disease according to claim 9, characterized in that: The pharmaceutical composition is used in combination with other drugs for treating Alzheimer's disease.

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