CN115032400A

CN115032400A - Use of alpha-synuclein in auxiliary diagnosis of neurodegenerative diseases

Info

Publication number: CN115032400A
Application number: CN202210317186.8A
Authority: CN
Inventors: 冯涛; 郑元初; 于震维
Original assignee: Beijing Tiantan Hospital
Current assignee: Beijing Tiantan Hospital
Priority date: 2022-03-28
Filing date: 2022-03-28
Publication date: 2022-09-09
Also published as: WO2023185871A1

Abstract

The utility model provides an application of a product for detecting oral mucosa protein in preparing a reagent or a kit for auxiliary diagnosis of neurodegenerative diseases, the oral mucosa can be collected noninvasively, the updating period is short, and repeated sampling in a short time is easy.

Description

Use of alpha-synuclein in auxiliary diagnosis of neurodegenerative diseases

Technical Field

The disclosure relates to the technical field of biomedicine, in particular to application of a product for detecting oral mucosa alpha-Syn protein in preparing a reagent or a kit for auxiliary diagnosis of neurodegenerative diseases.

Background

Alpha-synuclein is a small protein encoded by the 4q21-22SNCA gene, has a molecular weight of 19kDa and consists of 140 amino acids. There is a dynamic equilibrium between normal, misfolded and oligomerized α -synuclein, when this equilibrium is broken fibrils rapidly aggregate into large, insoluble fibrils; α -synuclein exhibits a variety of morphologies under various influences, including an extended state, a pre-globular solubilized state, an α -helical state (membrane-bound), a β -lamellar state, a dimeric state, an oligomeric state, and insoluble amorphous and fibrous states; structural changes caused by point mutations of alpha-synuclein, increase of intracellular content, accumulation of a large number of protein molecules, truncation of structural sequences, concentration of intracellular anions and salts (change of pH), neurotoxic molecules (heavy metals, organic solvents, carbon monoxide, MPTP, pesticides and herbicides), post-translational modifications (oxidation, phosphorylation and nitration), and the like can promote aggregation of alpha-synuclein to form insoluble fibers.

Parkinson's Disease (PD) is a common degenerative disease of the central nervous system, and PD diagnosis is mainly based on medical history and physical examination, and the accuracy of early diagnosis of the disease is limited. The neuropathological features of PD are loss of dopaminergic neurons in the substantia nigra pars compacta and abnormal deposition of alpha-synuclein (alpha-Syn). Studies have shown that abnormal alpha-Syn deposition occurs not only in the central nervous system, but also in body fluids and peripheral tissues, including blood, cerebrospinal fluid, olfactory mucosa, skin, salivary glands, retina, adrenal medulla, heart, and gastrointestinal tract. Pathological diagnosis of the Parkinson disease can be realized through body fluid or peripheral tissue alpha-Syn detection. However, most of the body fluid and peripheral tissue sampling at present are invasive operations, and the sampling difficulty is high and the compliance of patients is poor. At present, a noninvasive and convenient detection means is urgently needed.

Disclosure of Invention

In order to solve the defects of the prior art, the present disclosure aims to provide a biological specimen for diagnosing neurodegenerative diseases, which can be collected noninvasively and has a short renewal period, and can intuitively display the intracellular expression condition of the parkinson-related protein through immunofluorescence staining.

Specifically, the present disclosure proposes the following technical solutions:

in one aspect of the disclosure, an application of a product for detecting oral mucosa protein in preparing a reagent or a kit for auxiliary diagnosis of neurodegenerative diseases is provided. The collection process of the oral mucosa is non-invasive and is easily accepted by patients and normal people. In addition, oral mucosal epithelial cells are renewed every 7-21 days, facilitating repeated sampling in a short period of time.

In one embodiment, the neurodegenerative disease includes Cerebral Ischemia (CI), Brain Injury (BI), epilepsy; the brain injury includes Alzheimer's Disease (AD), Parkinson's Disease (PD), Huntington's Disease (HD), Amyotrophic Lateral Sclerosis (ALS), different types of spinocerebellar ataxia (SCA), Pick's disease, etc.

In one embodiment, the oral mucosal protein is an α -Syn protein and the neurodegenerative disease is parkinson's disease.

In one embodiment, the product for detecting α -Syn protein comprises a reagent for detecting the α -Syn protein.

In one embodiment, the product for detecting alpha-Syn protein comprises a reagent for detecting alpha-Syn oligomeric protein and phosphorylating alpha-Syn protein.

In a preferred embodiment, the phosphorylated α -Syn protein is phosphorylated α -Syn protein 129 (pS 129).

In one embodiment, the product for detecting oral mucosa protein comprises an alpha-Syn protein antibody.

In one embodiment, the product for detecting oral mucosa proteins is selected from one or more of alpha-Syn protein antibodies, alpha-Syn protein oligomer antibodies or anti-phosphorylation alpha-Syn protein antibodies.

In one embodiment, the detection is based on immunofluorescence analysis.

In one embodiment, the detection is based on an electrochemiluminescence immunoassay, which allows the binding of the α -Syn antibody to the analyte in an electron-rich well followed by the generation of an electrochemiluminescence signal, which can be detected to the accuracy of 5-10 pg/ml.

In one embodiment, the product comprises a capture antibody comprising one or more selected from an a-Syn protein antibody, an a-Syn oligomer antibody or an anti-phosphorylated a-Syn protein antibody, and an observation antibody, which is a Sulfo-TAG labeled a-Syn antibody.

In still another aspect of the present disclosure, there is provided a reagent or kit for assisting in the diagnosis of parkinson's disease, the reagent or kit comprising a reagent for detecting the oral mucosal protein.

In one embodiment, the oral mucosal protein is an α -Syn protein.

In one embodiment, in the above reagent or kit, the reagent for detecting α -Syn protein comprises one or more of α -Syn antibody, α -Syn oligomer antibody and phosphorylated α -Syn antibody.

In one embodiment, the reagent or kit for detecting α -Syn protein comprises recombinant anti- α -Syn MJFR-1(ab138501, Abcam, Cambridge, MA, USA), recombinant anti- α -Syn oligomer MJFR-14(ab209538, Abcam, Cambridge, MA, USA) and anti-phosphorylated α -Syn (ps129,825701, BioLegend, San Diego, CA, USA) antibodies.

Drawings

Figure 1 shows the alpha-Syn immune response signals in oral mucosal cells of PD patients and controls.

FIG. 2 shows the subcellular distribution of α -Syn in the oral mucosal cells of PD patients.

FIG. 3 shows the results of quantitative analysis of α -Syn in oral mucosal cells.

Figure 4 shows the results of ROC analysis of oral mucosal cells.

FIG. 5 shows a standard curve for an electrochemiluminescence immunoassay.

Detailed Description

For better understanding of the present disclosure, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below with reference to the accompanying drawings of the present disclosure, and it is obvious that the described embodiments are only some embodiments of the present disclosure, not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments disclosed herein without making any creative effort, shall fall within the protection scope of the present disclosure.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described. For the purposes of this disclosure, the following terms are defined below.

The term "oligomer" refers to a short polymer of a relatively small number of monomers, repeated by covalent bonds, and includes short polymers of amino acids, sugars, nucleotides, the number of monomers being below 20.

The term "phosphorylated a-Syn" refers to a modified a-Syn protein that is phosphorylated at serine residue 129.

The term "immunofluorescence assay" refers to an assay in which a fluorochrome that does not affect the activity of an antigen-antibody is labeled on an antibody (or antigen), and after binding to its corresponding antigen (or antibody), exhibits specific fluorescence under a fluorescence microscope for detection.

The term "electrochemiluminescence immunoassay" refers to an immunoassay technique using the principle of chemiluminescence generated electrochemically using a terpyridyl ruthenium-labeled antigen or antibody. The basic principle is that tripropylamine and terpyridyl ruthenium in the immunoreaction compound lose electrons around the electrode to form trivalent free radicals, photons with the wavelength of 620nm are emitted when the excited terpyridyl ruthenium is de-excited, more photons are generated on the surface of the electrode repeatedly, and the photons are measured to strengthen the signal of a detected object.

The term "capture antibody" refers to a substance that specifically binds to a target protein to be detected in an electrochemiluminescence immunoassay, causing it to become immobilized and form a molecule to be detected.

The term "viewing antibody" refers to a antibody with a Sulfo-TAG label ((S))Why Electrochemiluminescence Meso Scale Discovery) The antibody capable of forming a complex with the capture antibody and the antigen to be detected is used for displaying the concentration of the antigen to be detected.

The terms "patient" and "subject" are used interchangeably to refer to both human or other mammalian patients and subjects, and include any individual being examined or treated using the methods of the present disclosure. However, it should be understood that "patient" does not mean that symptoms are present.

The term "kit" refers to any delivery system used to deliver a substance. In reaction assays, such delivery systems include systems that store, transport or deliver reaction reagents (e.g., oligonucleotides, enzymes, etc. in appropriate containers) and/or support materials (e.g., buffers, instructions for performing the assay, etc.) from one location to another. For example, a kit comprises one or more housings (e.g., cassettes) containing the relevant reaction reagents and/or support materials.

Examples

Hereinafter, the present disclosure is described in more detail with reference to specific examples, which, however, are for illustrative purposes only and do not have a limiting effect on the present disclosure. The reagents and biomaterials described in the following examples are commercially available, unless otherwise specified.

Example 1

1. Materials and methods

1.1 study object

During the period of 3 months at 2021 to 10 months at 2021, 57 primary PD patients and 51 age-matched healthy controls were recruited from the dyskinetic disease department of the beijing tiantan hospital affiliated with the university of capital medicine. All PD patients met the international association for dyskinesia 2015 edition PD clinical diagnostic criteria. Exclusion criteria for the PD group included those diagnosed with atypical or secondary parkinsonism, severe head trauma, history of stroke, severe psychiatric disease, severe systemic disease, and oral mucosal disease. Exclusion criteria for the control group included those diagnosed with PD or other dyskinetic disorders, family history of dyskinetic disorders, severe head trauma, stroke history, severe psychiatric disorders, severe systemic disorders, and oral mucosal disorders. Demographic and clinical data including age, gender, education, mini-mental state examination scale (MMSE) and montreal cognitive assessment scale (MOCA) were collected for all participants. For PD patients, the course of the disease, the off-stage H-Y staging scale, and the third part of the society for dyskinesias unified parkinson's disease rating scale (MDS-UPDRS iii) were used to assess the severity of the disease. The protocol of the cross-sectional study was reviewed and approved by the ethical committee of the beijing tiantan hospital affiliated with the university of capital medicine and informed written consent was obtained from all participants.

1.2 oral mucosal cell sample Collection and preparation

Participants rinsed their mouths thoroughly with saline prior to sampling to avoid food residue in the mouth and contamination of saliva. Immediately after rinsing, samples of oral mucosa cells were collected from both cheek parts using two tissue cell brushes with 2 cm long brush heads. The inner side wall of the buccal cavity on one side is scraped by rotation for 30 times.

After sampling was completed, a tissue cell was brushed on a silane-coated microscope slide for a thin smear for immunofluorescence staining. Slides were stored at-20 ℃. The other cell brush was immersed in a 1.5ml centrifuge tube containing 200. mu.l RIPA buffer (Applygen, cat. No. C1053+) for an Electrochemiluminescence (ECL) immunoassay. After mixing for 1 minute with Vortex mixer, the cell brush was discarded. And (3) carrying out ultrasonic treatment on the residual lysate in the centrifugal tube for 1min by using an ultrasonic cell disruption instrument, and putting the centrifugal tube on ice in the process. After completion of sonication, centrifugation was carried out at 12000 Xg centrifugal force at 4 ℃ for 10 minutes. Transferring the oral mucosa cell extract protein containing the supernatant into a new centrifuge tube, storing at-80 deg.C, and thawing the sample during analysis.

Protein concentrations in oral mucosal samples were assessed at 562nm absorbance using a bicinchoninic acid (BCA) protein assay kit (Pierce/Thermo Fisher Scientific, Rockford, IL, USA) as a protein standard.

1.3 immunofluorescence staining analysis and three-dimensional reconstruction of oral mucosal cells

The slides were fixed in 4% paraformaldehyde for 10 minutes and then washed 3 times with Phosphate Buffered Saline (PBS). After washing, the slides were permeabilized in 1% Triton X-100 for 10 minutes. After 3 washes, slides were blocked in 5% Bovine Serum Albumin (BSA) blocking solution for 1 h. Recombinant anti- α -Syn MJFR-1(ab138501, Abcam, Cambridge, MA, USA), recombinant anti- α -Syn oligomer MJFR-14(ab209538, Abcam, Cambridge, MA, USA) and anti-phosphorylated α -Syn (pS129,825701, BioLegend, San Diego, Calif., USA) antibodies were diluted 1:1000 with blocking solution and slides were incubated overnight at 4 ℃. Using PBST buffer (PBS + 0.05%

20) After 3 washes, goat anti-murine Alexa Fluor 488(1:1000 dilution) and goat anti-rabbit Alexa Fluor 488(1:1000 dilution) were incubated for 1h at room temperature. Nuclei were stained with DAPI (0.2. mu.g/mL) for 5 min. Marking phalloidin (1:200 dilution) with rhodamine to show oral mucosa finenessCytoskeletal extent of the cells. After mounting with fluorescence quenching mounting medium, the slides were observed using a 40 × eyepiece of a zeiss LSM 700 confocal microscope. Three-dimensional reconstruction of oral mucosa cells is performed by a Z-stacks image mode with a magnification of 40 x and is used for visualizing intracellular localization of alpha-Syn.

1.4 electrochemiluminescence immunoassay

Standard protein: recombinant α -Syn monomers (RP-001, Proteos, Inc.), recombinant phosphorylated α -Syn (RP-004, Proteos, Inc.), and α -Syn oligomers (RP-002, Proteos, Inc.). Standard proteins were assayed for concentration using a NanoDrop OneC spectrophotometer, diluted to 1. mu.g/ml in Diluent 35(D35, MSD, Rockville, Md., USA), followed by a 1:3 gradient dilution to prepare a standard curve.

Observation of the antibody: Sulfo-TAG-labeled anti- α -Synclone 42(624096, BD Bioscience, San Jose, Calif., USA).

Capture antibody: biotinylated recombinant anti- α -Syn-MJFR-1(ab138501, Abcam, Cambridge, MA, USA), biotinylated recombinant anti- α -Syn oligomer MJFR-14(ab209538, Abcam, Cambridge, MA, USA), biotinylated anti-pS 129 antibody (825701, BioLegend, San Diego, Calif., USA).

The three capture antibody solutions were added to a Meso Scale Discovery (MSD, Rockville, MD, USA) U-Plex plate, respectively, at room temperature and incubated for 1 hour with shaking at 600rpm to attach the capture antibodies to the electrochemiluminescence wells. After washing 3 times with 150. mu.l of washing buffer (MSD, Rockville, Md., USA), blocking was performed at 600rpm with 150. mu.l of D35 for 1 hour at room temperature, followed by 3 additional washes. Samples containing oral mucosal cell protein extracts were diluted 1:2.5 in D35 and incubated for 1 hour at 600 rpm. After 3 washes, the diluted observation antibody solution (1. mu.g/ml) of D35 was added and incubated at 600rpm for 1 hour. After the incubation was completed, the plate was washed 3 times with washing buffer, and then 150. mu.l of 2 Xreading buffer (MSD, Rockville, Md., USA) was added thereto, and the detection results of α -Syn, α -Syn oligomer, and anti-phosphorylated α -Syn were analyzed on a Sector Imager 6000 detection platform reading plate, respectively.

1.5 statistical analysis

Statistical analysis was performed using SPSS 22.0 software and GraphPad Prism 8. Prior to analysis, the concentrations of α -Syn, pS129, and α -Syn oligomers were normalized based on the total protein concentration of the oral mucosal cell protein extract. The nonparametric Mann-Whitney U test was used to compare alpha-Syn levels between PD and healthy control groups, and the Spanish-Scale correlation coefficient was used to analyze the correlation between alpha-Syn levels and disease severity. Binary logistic regression was used to build multivariate logistic regression models containing oral mucosal cells alpha-Syn, pS129, and alpha-Syn oligomers for PD diagnosis. The area under the receiver operating characteristic curve (ROC) curve (AUC) was used and the john index (sensitivity + specificity-1) was calculated to give the optimal cut-off values for PD and control groups.

2. Results of the study

2.1 demographic and clinical characteristics

A total of 57 PD patients and 51 age-matched Healthy (HC) controls were included in this study. Table 1 is the demographic and clinical data for all subjects. There was no difference in the mean gender, age, MMSE scale, MOCA scale distribution between PD patients and controls. The median disease course of PD patients was 5 years (ranging from 7 months to 16 years), the median off-stage H-Y score was 3 stages (ranging from 1 to 5 stages), and the median off-stage MDS-UPDRS III score was 41.5 points (ranging from 5 to 74 points).

TABLE 1 demographic and clinical data

Grouping	PD,n＝57	HC,n＝51	p
				Female/male	25/32	27/24	0.346
Age (age)	62.11±10.30	62.94±6.35	0.732
				Course of disease	6.18±3.69	NA	NA
H-Y staging	2.90±0.80	NA	NA
				MDS-UPDRSⅢ	42.50±16.00	NA	NA
MMSE	26.91±3.32	28.06±1.77	0.163
				MOCA	23.55±4.73	24.84±2.34	0.394

2.2 expression of alpha-Syn in oral mucosal cells of PD and healthy control groups

Immunofluorescent staining results showed that immunoreactive signals of α -Syn, pS129 and α -Syn oligomers (FIG. 1) were detectable in oral mucosal cells in both PD patients and controls. Immunoreaction signals of alpha-Syn, pS129, and alpha-Syn oligomers in oral mucosal cells of PD patients were significantly increased compared to the control group.

Three-dimensional reconstruction of Z-stack confocal images showed that the alpha-Syn, pS129 and alpha-Syn oligomers had different subcellular distribution characteristics within the oral mucosal cells. alpha-Syn was detected predominantly in the nuclei and cytoplasm of oral mucosal cells from PD patients (fig. 2A), pS129 was predominantly distributed in the cytoplasm of oral mucosal cells (fig. 2B), and alpha-Syn oligomers were expressed at higher levels in the nuclei and perinuclear cytoplasm of oral mucosal cells (fig. 2C). However, there was no difference in intracellular distribution of α -Syn between PD patients and controls.

There was differential expression of α -Syn, pS129, and α -Syn oligomers in oral mucosal cells from PD patients and controls. Immunofluorescence results show that the expression levels of alpha-Syn, pS129 and alpha-Syn oligomers in oral mucosa cells of PD patients are higher than those of a control group, and abnormal alpha-Syn deposition exists in the oral mucosa cells of the PD patients. Levels of alpha-Syn, pS129, and alpha-Syn oligomers were significantly elevated in samples of the oral mucosa from PD patients compared to controls. The differential expression characteristic shows that the alpha-Syn in the oral mucosa cells can be used as a biomarker of PD.

2.3 quantitative analysis of alpha-Syn in oral mucosal cells

Before the quantitative analysis of oral mucosal cell alpha-Syn, pS129 and alpha-Syn oligomer concentrations were normalized based on total protein concentration in oral mucosal samples.

Compared with HC control group, PD patients have significantly increased levels of alpha-Syn, pS129 and alpha-Syn oligomers in oral mucosal cells (Table 2, FIG. 3). In the PD group, the α -Syn and α -Syn oligomers in oral mucosal cells had a mean that was significantly correlated with the off-phase H-Y score (α -Syn r ═ 0.495, p ═ 0.001; α -Syn oligomers r ═ 0.324, p ═ 0.03). However, levels of oral mucosal cells pS129 did not significantly correlate with off-stage H-Y scores. In addition, the levels of alpha-Syn, pS129 and alpha-Syn oligomers of oral mucosal cells have no significant correlation with disease severity indexes such as disease course and off-stage MDS-UPDRS III scores.

TABLE 2 results of quantitative analysis of α -Syn in oral mucosal cells

Grouping	PD	HC	p
				α-Syn,pg/mg	30.13[11.29-64.20]	9.60[2.73-24.35]	0.001*
pS129α-Syn,pg/mg	82.06[42.13-138.21]	43.06[33.37-84.59]	0.002*
				alpha-Syn oligomer, pg/mg	63.31[31.75-166.32]	38.04[20.09-104.08]	0.013*

The recombinant alpha-Syn monomer is used as standard protein, detection is carried out according to the steps of the electrochemical luminescence immunoassay method of 1.4 to obtain a standard curve shown in figure 5, and as can be seen from figure 5, when the electrochemical luminescence immunoassay is used for detecting the alpha-Syn protein, the detection range can be accurate to 5-10 pg/ml.

2.4ROC Curve analysis

ROC analysis was used to analyze the efficacy of a-Syn, pS129 and a-Syn oligomers in oral mucosal cells in the diagnosis of PD. As shown in FIG. 4, in the electrochemiluminescence immunoassay, the sensitivity of PD was 60.8%, the specificity was 77.6%, AUC was 0.684, and the Cut off value was 25.09pg/mg when using oral mucosal cell α -Syn. pS129 differentiated PD from the control group by AUC of 0.674, sensitivity of 45.3%, specificity of 88.0%, Cut off value of 99.98 pg/mg. The α -Syn oligomer distinguished PD from the control group by an AUC of 0.641, a sensitivity of 74.1%, a specificity of 50.0%, and a Cut off value of 35.94 pg/mg.

Based on the quantitative results of alpha-Syn, pS129 and alpha-Syn oligomers in the oral mucosa sample, a multivariate logistic regression PD diagnosis model of the alpha-Syn, pS129 and alpha-Syn oligomer levels in the oral mucosa sample of the multivariate PD diagnosis model is established by adopting binary logistic regression. The diagnostic model has an AUC of 0.749, a sensitivity of 66.7%, a specificity of 72.5%, and a moderate overall sensitivity. TABLE 3

Claims

1. The application of the product for detecting the oral mucosa protein in preparing a reagent or a kit for assisting in diagnosing the neurodegenerative disease.

2. The use according to claim 1, wherein the oral mucosa protein is alpha-Syn protein, and the neurodegenerative disease is Parkinson's disease;

preferably, the product for detecting alpha-Syn protein comprises a reagent for detecting the alpha-Syn protein.

3. The use according to claim 1 or 2, the product for detecting oral mucosa proteins comprises alpha-Syn protein antibodies.

4. The use according to any one of claims 1 to 3, wherein the product for detecting oral mucosa proteins is selected from one or more of alpha-Syn protein antibodies, alpha-Syn protein oligomer antibodies or anti-phosphorylation alpha-Syn protein antibodies.

5. The use according to any one of claims 1 to 4, wherein the detection is based on an immunofluorescence assay.

6. The use according to any one of claims 1 to 4, wherein the detection is based on an electrochemiluminescence immunoassay.

7. Use according to claim 6, the product comprising a capture antibody comprising one or more selected from the group consisting of an a-Syn protein antibody, an a-Syn oligomer antibody or an anti-phosphorylated a-Syn protein antibody, and an observation antibody being a Sulfo-TAG labeled a-Syn antibody.

8. A reagent or a kit for assisting in diagnosing Parkinson's disease, wherein the reagent or the kit comprises a reagent for detecting the oral mucosa protein; wherein the oral mucosa protein is alpha-Syn protein.

9. The reagent or kit according to claim 7, wherein the reagent for detecting α -Syn protein comprises one or more of α -Syn antibodies, α -Syn oligomeric antibodies, and phosphorylated α -Syn antibodies.

10. The reagent or kit according to claim 8 or 9, wherein the detection is an immunofluorescence or electrochemiluminescence immunoassay based detection.