Detection of AD (Alzheimer's disease), FTLD (frontotemporal lobar degeneration), ALS (amyotrophic lateral sclerosis), PD (Parkinson's disease) and DLB (Lewy body type dementia) by MARCKS phosp
阅读说明：本技术 以marcks磷酸化为指标的ad(阿尔茨海默病)、ftld(额颞叶变性症)、als(肌萎缩侧索硬化症)、pd(帕金森病)和dlb(路易小体型痴呆)的检测 (Detection of AD (Alzheimer's disease), FTLD (frontotemporal lobar degeneration), ALS (amyotrophic lateral sclerosis), PD (Parkinson's disease) and DLB (Lewy body type dementia) by MARCKS phosp) 是由 冈泽均 于 2019-03-08 设计创作，主要内容包括：本发明提供一种高灵敏度且高特异性地检测神经系统变性疾病的方法,所述神经系统变性疾病选自人AD(阿尔茨海默病)、FTLD(额颞叶变性症)和ALS(肌萎缩侧索硬化症)。一种检测神经系统变性疾病的方法,所述神经系统变性疾病选自人AD(阿尔茨海默病)、FTLD(额颞叶变性症)和ALS(肌萎缩侧索硬化症),所述方法包括以下步骤：(i)对从受试者采集的样本试样中的46位磷酸化的MARCKS蛋白和非磷酸化MARCKS蛋白进行测定；(ii)由(i)中得到的测定值计算下式表示的DO值；[式1]<Image he="79" wi="700" file="DDA0002674620930000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>[式中,“pSer46-MARCKS”表示46位磷酸化的MARCKS蛋白的量,“非磷酸化MARCKS”表示非磷酸化MARCKS蛋白的量]；以及,(iii)以DO值为指标检测神经系统变性疾病。(The present invention provides a method for detecting a neurodegenerative disease selected from the group consisting of human AD (alzheimer's disease), FTLD (frontotemporal lobar degeneration) and ALS (amyotrophic lateral sclerosis) with high sensitivity and high specificity. A method for detecting a neurodegenerative disease selected from the group consisting of human AD (Alzheimer's disease),FTLD (frontotemporal lobar degeneration) and ALS (amyotrophic lateral sclerosis), the method comprising the steps of: (i) determining the 46 phosphorylated MARCKS protein and the non-phosphorylated MARCKS protein in a sample taken from the subject; (ii) (ii) calculating a DO value represented by the following formula from the measurement value obtained in (i); [ formula 1] [ in the formula, "pSer 46-MARCKS" represents the amount of MARCKS protein phosphorylated at position 46, "non-phosphorylated MARCKS" represents the amount of non-phosphorylated MARCKS protein](ii) a And, (iii) detecting a neurodegenerative disease using the DO value as an indicator.)
1. A method of detecting a neurodegenerative disease selected from the group consisting of human AD (alzheimer's disease), FTLD (frontotemporal lobar degeneration) and ALS (amyotrophic lateral sclerosis), comprising the steps of:
(i) determining the 46 phosphorylated MARCKS protein and the non-phosphorylated MARCKS protein in a sample taken from the subject;
(ii) (ii) calculating a DO value represented by the following formula from the measurement value obtained in (i);
[ formula 1]
Wherein "pSer 46-MARCKS" represents the amount of phosphorylated MARCKS protein at position 46 and "non-phosphorylated MARCKS" represents the amount of non-phosphorylated MARCKS protein; and
(iii) detecting the degenerative disease of the nervous system by using the DO value as an index.
2. The method of claim 1, wherein the sample specimen is cerebrospinal fluid.
3. The method of claim 1 or 2, wherein "pSer 46-MARCKS" and "non-phosphorylated MARCKS" are determined by mass spectrometry.
4. The method of any one of claims 1 to 3, wherein "pSer 46-MARCKS" and "non-phosphorylated MARCKS" in the formula are values normalized to the total protein amount in the sample.
5. The method according to any one of claims 1 to 4, wherein in step (iii), the DO value calculated for the subject is compared with a predetermined cut-off value, and when the DO value is higher than the cut-off value, it is judged that the neurodegenerative disease is detected.
6. The method of claim 5, wherein the threshold value of DO is 50-55.
7. A method of detecting a neurodegenerative disease selected from the group consisting of FTLD (frontotemporal lobar degeneration), ALS (amyotrophic lateral sclerosis), PD (parkinson's disease) and DLB (dementia of lewy body type) in a human, comprising the steps of:
(i) determining the 46 phosphorylated MARCKS protein in a sample taken from the subject;
(ii) comparing the amount of 46 phosphorylated MARCKS protein in a sample taken from the subject with the amount of 46 phosphorylated MARCKS protein in a sample taken from a healthy human; and
(iii) when the amount of 46 phosphorylated MARCKS protein in a sample specimen collected from the subject is more than the amount of 46 phosphorylated MARCKS protein in a sample specimen collected from a healthy person, it is judged that the neurodegenerative disease is detected in the subject.
8. The method of claim 7, wherein the sample specimen is cerebrospinal fluid.
9. The method of claim 7 or 8, wherein the phosphorylated MARCKS protein at position 46 is determined by mass spectrometry.
10. The method of any one of claims 7 to 9, wherein the amount of 46 phosphorylated MARCKS protein is a value normalized to the measurement of 46 phosphorylated MARCKS protein based on the total amount of protein in the sample or the total MARCKS protein in the sample.
11. A method for detecting a degenerative disease of the nervous system selected from PD (parkinson's disease) and DLB (dementia of the lewy body type), comprising the steps of:
(i) measuring the 46 phosphorylated MARCKS protein in samples taken from the occipital and temporal lobes, respectively, of the subject;
(ii) comparing the amount of 46 phosphorylated MARCKS protein in sample specimens taken from the occipital and temporal lobes, respectively, of the subject with the amount of 46 phosphorylated MARCKS protein in sample specimens taken from the occipital and temporal lobes, respectively, of a healthy human; and
(iii) determining that a neurological degenerative disease is detected in the subject when the amount of 46 phosphorylated MARCKS protein in a sample specimen taken from the temporal lobe of the subject is greater than the amount of 46 phosphorylated MARCKS protein in a sample specimen taken from the temporal lobe of a healthy human.
12. The method of claim 11, wherein the sample specimen is cerebrospinal fluid.
13. The method of claim 11 or 12, wherein the phosphorylated MARCKS protein at position 46 is determined by mass spectrometry.
14. The method of any one of claims 11 to 13, wherein the amount of phosphorylated MARCKS protein at position 46 is a value normalized to the measurement of phosphorylated MARCKS protein at position 46 based on the total amount of protein in the sample or the total amount of MARCKS protein in the sample.
15. The method of claim 11, wherein the 46-phosphorylated MARCKS protein is measured by PET (positron emission tomography) using a PET tracer for imaging the 46-phosphorylated MARCKS protein.
16. The method of claim 15, wherein the position 46 phosphorylated MARCKS protein imaging PET tracer is an antibody PET tracer that labels an antibody directed against a position 46 phosphorylated MARCKS protein with a positive electron nuclide.
Technical Field
The present invention relates to a method for detecting AD (alzheimer's disease), FTLD (frontotemporal lobar degeneration), ALS (amyotrophic lateral sclerosis), PD (parkinson's disease) and DLB (lewy body type dementia).
Background
The preclinical pathology of neurodegenerative diseases is of great interest and quantitative biomarkers are needed to determine such early pathology.
Alzheimer's Disease (AD) is the most common degenerative disease of the nervous system and is also a well-known cause of dementia.
One of the most supported models of AD etiology is the amyloid hypothesis premised on the cytotoxicity of amyloid fibrils resulting from the extracellular accumulation of beta-amyloid peptide (a β).
Based on this hypothesis, a β has become a major therapeutic target for AD. Therapeutic strategies including antibody therapy of bapiduzumab (Bapineuzumab) and perizumab (Solanezumab) are to reduce a β aggregation in the brain of a human AD patient in clinical examinations after onset of dementia, but recovery of dementia is insufficient among clinical symptoms so far. As a result of research interest shifted to early molecular events of AD before symptoms appeared, similar or new therapies may be more effective.
The present inventors performed a global phosphoprotein analysis of brain tissue from mouse AD models and human AD patients. 17 nucleoproteins with aberrant phosphorylation were identified in multiple AD mouse models. Interestingly, these changes occurred before symptoms, and even before a β aggregation was detected immunohistologically in the brain of model mice, abnormal phosphorylation was detected in some nucleoproteins, suggesting that abnormal phosphorylation plays an important role in the early stages of AD pathology (see non-patent document 1).
In particular, in the quantitative mass spectrum, the increase in MARCKS (substrates for myristoylated alanine-rich protein kinase C) phosphorylated at Ser46 started 1 month before the histological detection of a β aggregation in AD model mice, and remained in the brain even after the death of human AD patients (see
Summary of The Invention
Problems to be solved by the invention
The object of the present invention is to provide a method for detecting a highly sensitive and highly specific neurodegenerative disease selected from the group consisting of human AD (Alzheimer's disease), FTLD (frontotemporal lobar degeneration), ALS (amyotrophic lateral sclerosis), PD (Parkinson's disease) and DLB (Lewy body dementia or Lewy body disease).
Technical scheme for solving problems
As described above, the present inventors have previously found that: phosphorylated MARCKS increased prior to the formation of histological amyloid β (a β) aggregates in a mouse model of Alzheimer's Disease (AD), and this change continued in the post-mortem AD brain in humans. In particular, MARCKS phosphorylated at Ser46 (pSer46-MARCKS) was detected in mouse models of human patients and in dystrophic neurites around A β aggregates in senile plaques.
Based on these findings, the inventors quantified the amount of pSer46-MARCKS in the cerebrospinal fluid (CSF) of human AD (alzheimer's disease), FTLD (frontotemporal lobar degeneration) and ALS (amyotrophic lateral sclerosis). Unexpectedly, the most significant changes were detected in frontotemporal lobar degeneration (FTLD), with neural degeneration confirmed by immunohistochemistry of pSer 46-MARCKS. The specificity of pSer46-MARCKS for cerebrospinal fluid (CSF) was 1.0, which clearly distinguishes cases of neurological degeneration from those of normal.
In addition, the same change in MARCKS is known to occur in dementia caused by parkinson's disease and lewy body disease in both mouse models and human patients.
When human α -Syn-BAC-Tg/GBA-hybrid-KO mice exhibited no symptoms and before α -synuclein aggregates formed, pSer46-MARCKS levels began to rise and persisted during senescence, consistent with the post-mortem brain pattern in humans.
This result strongly suggests a common mechanism of pre-aggregate neural mutability in AD and PD/DLB lesions.
In addition, the same changes were demonstrated in both mouse models of Parkinson's Disease (PD) and dementia of the lewy body type (DLB) and human patients.
Furthermore, the inventors quantified pSer46-MARCKS and non-phosphorylated MARCKS of cerebrospinal fluid (CSF) from human patients with AD and other neurodegenerative diseases by mass spectrometry. The sensitivity and specificity of pSer46-MARCKS was sufficiently high on its own, but the inventors developed new parameters based on the integration of pSer46-MARCKS with non-phosphorylated MARCKS in order to further increase the value as a diagnostic parameter. According to this parameter, a plurality of nervous system degenerative diseases can be distinguished from healthy controls with high sensitivity and high specificity. Furthermore, based on pathological analysis, pSer46-MARCKS was shown to reflect the global neural mutational activity of neurodegenerative diseases. From these results, a new parameter called "DO (Distance from origin)" was developed as a more sensitive definitive biomarker for all neurological degenerative diseases through integration of pSer46-MARCKS with non-phosphorylated MARCKS.
Namely, the present invention is as follows:
[1] a method of detecting a neurodegenerative disease selected from the group consisting of human AD (alzheimer's disease), FTLD (frontotemporal lobar degeneration) and ALS (amyotrophic lateral sclerosis), comprising the steps of: (i) determining the 46 phosphorylated MARCKS protein and the non-phosphorylated MARCKS protein in a sample taken from the subject;
(ii) (ii) calculating a DO value represented by the following formula from the measurement value obtained in (i);
[ formula 1]
Wherein "pSer 46-MARCKS" represents the amount of phosphorylated MARCKS protein at
(iii) detecting the degenerative disease of the nervous system by using the DO value as an index.
[2] The method according to [1], wherein the sample specimen is cerebrospinal fluid.
[3] The method according to [1] or [2], wherein "pSer 46-MARCKS" and "non-phosphorylated MARCKS" are determined by mass spectrometry.
[4] The method according to any one of [1] to [3], wherein "pSer 46-MARCKS" and "non-phosphorylated MARCKS" in the formula are values normalized with respect to the total protein amount in the sample.
[5] The method according to any one of [1] to [4], wherein, in the step (iii), the DO value calculated for the subject is compared with a predetermined critical value, and when it is higher than the critical value, it is judged that the neurodegenerative disease is detected.
[6] The method according to [5], wherein the threshold value of the DO value is 50 to 55.
[7] A method of detecting a neurodegenerative disease selected from the group consisting of FTLD (frontotemporal lobar degeneration), ALS (amyotrophic lateral sclerosis), PD (parkinson's disease) and DLB (dementia of lewy body type) in a human, comprising the steps of:
(i) determining the 46 phosphorylated MARCKS protein in a sample taken from the subject;
(ii) comparing the amount of 46 phosphorylated MARCKS protein in a sample taken from the subject with the amount of 46 phosphorylated MARCKS protein in a sample taken from a healthy human; and
(iii) when the amount of 46 phosphorylated MARCKS protein in a sample specimen collected from the subject is more than the amount of 46 phosphorylated MARCKS protein in a sample specimen collected from a healthy person, it is judged that the neurodegenerative disease is detected in the subject.
[8] The method according to [7], wherein the sample specimen is cerebrospinal fluid.
[9] The method according to [7] or [8], wherein the MARCKS protein phosphorylated at
[10] The method according to any one of [7] to [9], wherein the amount of 46-position phosphorylated MARCKS protein is a value obtained by normalizing a measured value of 46-position phosphorylated MARCKS protein with respect to the total amount of protein in a sample or the total amount of MARCKS protein in a sample.
[11] A method for detecting a degenerative disease of the nervous system selected from PD (parkinson's disease) and DLB (dementia of the lewy body type), comprising the steps of:
(i) measuring the 46 phosphorylated MARCKS protein in samples taken from the occipital and temporal lobes, respectively, of the subject;
(ii) comparing the amount of 46 phosphorylated MARCKS protein in sample specimens taken from the occipital and temporal lobes, respectively, of the subject with the amount of 46 phosphorylated MARCKS protein in sample specimens taken from the occipital and temporal lobes, respectively, of a healthy human; and
(iii) determining that a neurological degenerative disease is detected in the subject when the amount of 46 phosphorylated MARCKS protein in a sample specimen taken from the temporal lobe of the subject is greater than the amount of 46 phosphorylated MARCKS protein in a sample specimen taken from the temporal lobe of a healthy human.
[12] The method according to [11], wherein the sample specimen is cerebrospinal fluid.
[13] The method according to [11] or [12], wherein the MARCKS protein phosphorylated at
[14] The method according to any one of [11] to [13], wherein the amount of 46-position phosphorylated MARCKS protein is a value obtained by normalizing a measured value of 46-position phosphorylated MARCKS protein with respect to the total amount of protein in a sample or the total amount of MARCKS protein in a sample.
[15] The method according to [11], wherein the 46-position phosphorylated MARCKS protein is measured by PET (positron emission tomography) using a PET tracer for imaging the 46-position phosphorylated MARCKS protein.
[16] The method according to [15], wherein the MARCKS protein phosphorylated at the 46-position imaging PET tracer is an antibody PET tracer which labels an antibody against the MARCKS protein phosphorylated at the 46-position with a positive electron nuclide.
The specification includes the disclosures of japanese patent application nos. 2018-043641 and 2018-138785, which are the basis of the priority of the present application.
Effects of the invention
According to the method of the present invention, a neurodegenerative disease selected from the group consisting of human AD (Alzheimer's disease), FTLD (frontotemporal lobar degeneration), ALS (amyotrophic lateral sclerosis), PD (Parkinson's disease) and DLB (Lewy body type dementia) can be detected with high sensitivity and high specificity.
Brief Description of Drawings
FIG. 1-1 is a graph showing the results of evaluation as a biomarker for the diagnosis of pSer46-MARCKS for cerebrospinal fluid (CSF). FIGS. 1-1A show values normalized to total protein for the amount of pSer46-MARCKS in each group of neurodegenerative diseases. FIGS. 1-1B show sensitivity and specificity.
FIG. 1-2 is a graph showing the results of evaluation as a biomarker for the diagnosis of pSer46-MARCKS for cerebrospinal fluid (CSF). FIGS. 1-2A show values normalized to total MARCKS protein for the amount of pSer46-MARCKS in each group of neurodegenerative diseases. FIGS. 1-2B show sensitivity and specificity.
FIG. 2-1 is a graph showing the results of evaluation as a biomarker for non-phosphorylated MARCKS diagnosis of cerebrospinal fluid (CSF). FIG. 2-1A shows values normalized to total protein for the amount of non-phosphorylated MARCKS in each of the groups of neurodegenerative diseases. FIG. 2-1B shows sensitivity and specificity.
FIG. 2-2 is a graph showing the results of evaluation as a biomarker for non-phosphorylated MARCKS diagnosis of cerebrospinal fluid (CSF). FIGS. 2-2A show values normalized to total MARCKS protein for the amount of non-phosphorylated MARCKS in each group of neurodegenerative diseases. FIGS. 2-2B show sensitivity and specificity.
FIG. 3-1 is a graph showing the amount of pSer46-MARCKS in AD, FTLD and ALS patients. FIG. 3-1A shows the amount normalized to total protein and FIG. 3-1B shows the value normalized to total MARCKS amount.
FIG. 3-2 is a graph showing the amount of non-phosphorylated MARCKS in AD, FTLD and ALS patients. FIGS. 3-2A show the normalized amount for total protein and FIGS. 3-2B show the normalized values for total MARCKS amount.
FIG. 4-1 is a graph showing the relationship between pSer46-MARCKS and non-phosphorylated MARCKS when normalized by total protein amount in AD, FTLD and ALS patients, expressed in terms of each disease group (A: control, B: AD, C: FTLD, D: ALS).
FIG. 4-2 is a view showing all the relationships A to D in FIG. 4-1.
FIGS. 4-3 are graphs showing the relationship of pSer46-MARCKS to non-phosphorylated MARCKS when normalized to the total MARCKS amount in AD, FTLD and ALS patients, expressed for each disease group (A: control, B: AD, C: FTLD, D: ALS).
FIG. 4-4 is a view showing all the relationships A to D in FIG. 4-3.
FIG. 5-1 is a graph showing the relationship of HMGB1 in cerebrospinal fluid (CSF) to pSer46-MARCKS (pSer46-MARCKS relative to total protein amount).
FIG. 5-2 is a graph showing the relationship of HMGB1 in cerebrospinal fluid (CSF) to pSer46-MARCKS (pSer46-MARCKS relative to the total MARCKS amount).
FIG. 6-1 is a diagram showing a pTDP-43 staining image of human FTLD.
FIG. 6-2 is a graph showing the results of immunohistochemical staining of human FTLD brain with anti-pSer 46-MARCKS antibody.
FIG. 6-3 is a graph showing the results of co-staining MAP2 and pSer46-MARCKS in human FTLD occipital lobe (occipital cortex).
Fig. 7 is a graph showing the loss of neurons and cavernous degeneration in frontal lobe (frontal cortex) and occipital lobe (occipital cortex) in human FTLD.
FIG. 8-1 is a graph showing the result of exploring the "upper normal limit of DO" which is optimal by numerical simulation.
FIG. 8-2 is a graph showing DO values in respective diseases.
FIG. 8-3 is a graph showing the number of cases judged to be abnormal in the control, AD, FTLD, and ALS groups, and the values of sensitivity and specificity when the subject score and DO range are optimized to the maximum.
FIG. 9 is a graph showing the correlation between DO in clinical symptoms and severity of disease.
Fig. 10A is a graph showing the results of a global phosphoproteome analysis of postmortem human AD and DLB brains, comparing the results of independent phosphorylation sites in AD and DLB.
FIG. 10B is a graph showing the results of a global phosphoproteome analysis of post-mortem human AD and DLB brains, a graph showing the changes in 4 different phosphorylation sites in MARCKS [ Ser27/Ser27, Ser46/Ser46, Ser145/Ser138 and Thr150/Thr143 (human/mouse) ] between human DLB patients and AD patients.
FIG. 11A shows immunohistochemistry of human DLB brain, showing simultaneous staining of pSer46-MARCKS with
Fig. 11B is a graph showing immunohistochemistry of human DLB brain. Staining of pSer129- α -Syn revealed multiple cytoplasmic inclusion bodies (Lewy bodies) in the same patient group.
FIG. 11C is a graph showing immunohistochemistry of human DLB brain, showing the results of simultaneous staining of pSer129- α -Syn with ubiquitin.
FIG. 11D is a graph showing immunohistochemistry of human DLB brain, showing the results of co-staining of pSer46-MARCKS with pSer129- α -Syn.
FIG. 11E shows immunohistochemistry of human DLB brain, and is a graph showing the results of Western blot analysis of non-DLB control patients and DLB patient-derived occipital lobes with antibodies against pSer46-MARCKS, total MARCKS and β -actin. The graph shows the quantification of pSer46-MARCKS from 5 patients and 5 non-neurological disease controls. The intensity of the bands was normalized to β -actin. Statistical analysis was performed using student's t-test. Denotes p < 0.05.
FIG. 12 is a graph showing chronological changes in pSer46-MARCKS in humans. alpha-Syn-BAC-Tg/GBA-heterozygous KO mice pSer46-MARCKS and pSer 129-alpha-Syn were stained simultaneously in human normal alpha-Syn-BAC-Tg/Glucosidase (GBA) -heterozygous-KO mice at 1, 6 and 24 months of age (3 male mice at each time point). Images were obtained using OlympusFV1200 IX83 confocal microscopy. All histograms represent mean and s.e.m. In each group, 3 mice were used, and signal intensity (average pixel intensity) was quantitatively analyzed in 10 visual fields (100 × 100 μm) selected at random from the brain region. Statistical analysis was performed using a binary ANOVA followed by student's t-test. Denotes p <0.05, denotes p < 0.01. Increased phosphorylation was detected first in the olfactory bulb and frontal cortex, followed by temporal and occipital cortex. The signal intensity of pSer46-MARCKS increased rapidly in the temporal and occipital regions, most notably in multiple brain regions.
FIG. 13A is a graph showing the results of co-staining of pSer46-MARCKS and pSer129- α -Syn in the olfactory bulb of human α -Syn-BAC-Tg/GBA-heterozygous KO mice (1 month of age).
FIG. 13B is a graph showing the results of co-staining of pSer46-MARCKS and pSer129- α -Syn in the olfactory bulb of human α -Syn-BAC-Tg/GBA-heterozygous KO mice (6 months of age).
FIG. 13C is a graph showing the results of co-staining of pSer46-MARCKS and pSer129- α -Syn in the olfactory bulb of human α -Syn-BAC-Tg/GBA-heterozygous KO mice (24 months of age).
FIG. 13D shows staining of ubiquitin in olfactory bulb of human α -Syn-BAC-Tg/GBA-heterozygous KO mice. Ubiquitin co-stained as a spotting structure or cytoplasmic aggregates in the cell subpopulation (yellow arrows), but spots or aggregates of pSer129- α -Syn positive/ubiquitin negative were also observed.
FIG. 14A shows immunohistochemistry of apical leaves of α -Syn-BAC-Tg/GBA-heterozygous KO mice. pSer46-MARCKS and pSer129- α -Syn were co-stained at 24 months of age at both the outer and inner pyramidal cell layers. pSer46-MARCKS was stained in both apical dendrites and cell bodies, but pSer129- α -Syn was stained in aggregates of cytoplasm.
FIG. 14B shows immunohistochemistry of apical leaves of α -Syn-BAC-Tg/GBA-hybrid KO mice, staining of mural cortical tissue derived from human α -Syn-BAC-Tg/GBA-hybrid KO mice. pSer129- α -Syn and pSer46-MARCKS or ubiquitin were co-stained at 1, 6 and 24 months of age. The same co-staining pattern as in olfactory bulb was confirmed in the top leaf.
FIG. 15 is a graph showing the results of Western blot analysis of olfactory bulbs of α -Syn-BAC-Tg/GBA-hybrid KO mice at various time points. The left panel (column 3) shows the results of western blots of all cortices (3 males at each time point) using antibodies against pSer46-MARCKS, pSer129- α -Syn and ubiquitin. The right panel shows the results of quantitative analysis of 3 independent blots of pSer46-MARCKS, pSer129- α -Syn and ubiquitin. The intensity of the bands was normalized to β -actin. Statistical analysis was performed using a binary ANOVA followed by student's t-test; testing by student t; p <0.05, p < 0.01.
FIG. 16 is a graph showing the interaction of MARKCS with alpha-syn. FIG. 16A shows the results of anti-pSer 46-MARCKS or anti-pSer 129- α -Syn antibody immunoprecipitates from brain samples from α -Syn-BAC-Tg/GBA-hybrid KO mice blotted with anti-pSer 129- α -Syn antibody or anti-pSer 46-MARCKS antibody at 1, 6 and 24 months, and FIG. 16B shows the results of examining the same co-precipitates from the occipital cortex of human DLB patients.
FIG. 17A is a graph showing Erk1/2 activation in cortical neurons from mouse PD/DLB models and human DLB patients. Identification was performed by co-staining of antibodies against the Erk1/2 active type (pThr202/Tyr204-Erk1 and pThr185/Tyr187-Erk2) and antibodies against pSer46-MARCKS against occipital cortex 914 tissue from 1, 6 and 24 month old α -Syn-BAC-Tg/GBA-hybrid KO (Tg) or non-transgenic sibling control (non-Tg) mice. The right panel shows the quantitative analysis of the pErk1/2 signal intensity in 3 mice (average of 10 fields for each mouse). Statistical analysis was performed using a binary ANOVA followed by student's t-test. P <0.05, p < 0.01.
FIG. 17B is a graph showing Erk1/2 activation in cortical neurons in mouse PD/DLB models and human DLB patients, showing the results of concurrent staining of pSer46-MARCKS with MAP2 or GFAP in mouse cortex.
FIG. 17C shows Erk1/2 activation in cortical neurons in mouse PD/DLB models and human DLB patients, and is a graph showing the same simultaneous staining of pErk1/2 and pSer46-MARCKS in human occipital leaves from DLB patients and non-DLB control patients (non-DLB patients).
FIG. 17D shows activation of Erk1/2 in cortical neurons in mouse PD/DLB models and human DLB patients, is a graph showing the results of Western blot analysis of
FIG. 17E shows Erk1/2 activation in cortical neurons in mouse PD/DLB models and human DLB patients, and is a graph showing the results of the same Western blot analysis of human DLB patient-derived occipital lobes.
FIG. 18 is a graph showing pSer46-MARCKS in 1-month-old human α -Syn-BAC-Tg/GBA-hybrid KO mice. pSer46-MARCKS and pSer129- α -Syn were co-stained in 1 month old human normal α -Syn-BAC-Tg/glucosidase-heterozygous KO mice (3 in each group). The signal intensity is significantly higher in the yellow region.
FIG. 19 is a graph showing pSer46-MARCKS in 6-month-old human α -Syn-BAC-Tg/GBA-hybrid KO mice. pSer46-MARCKS and pSer129- α -Syn were co-stained in 1 month old human normal α -Syn-BAC-Tg/glucosidase-heterozygous KO mice (3 in each group). The signal intensity is significantly higher in the yellow region.
FIG. 20 is a graph showing pSer46-MARCKS in 24-month-old human α -Syn-BAC-Tg/GBA-hybrid KO mice. pSer46-MARCKS and pSer129- α -Syn were co-stained in 1 month old human normal α -Syn-BAC-Tg/glucosidase-heterozygous KO mice (3 in each group). The signal intensity is significantly higher in the yellow region.
FIG. 21 is a graph showing a comparison of the protein levels of pSer46-MARCKS between Peripheral Blood Cells (PBC) and whole cerebral cortex (brain) in alpha-Syn-BAC-Tg/GBA-hybrid KO (Tg) mice or non-transgenic sibling control (non-Tg) mice at 6 months of age.
Modes for carrying out the invention
The present invention will be described in detail below.
The invention is a method for detecting degenerative diseases of the nervous system. In the present invention, the detection of a neurodegenerative disease refers to the judgment that a subject is suffering from a neurodegenerative disease or the judgment that a subject is at risk of developing a neurodegenerative disease.
In addition, the invention also includes a method for obtaining auxiliary data for detecting a degenerative disease of the nervous system. In the present invention, the degenerative diseases of the nervous system include: alzheimer's Disease (AD), Frontotemporal lobar degeneration (FTLD), Amyotrophic Lateral Sclerosis (ALS), Parkinson's Disease (PD), and Lewy body Dementia (DLB).
"Alzheimer disease" refers to a degenerative disease of the nervous system also called dementia of the Alzheimer type, and includes "familial Alzheimer disease" and "hereditary Alzheimer disease" caused by gene mutation, and "sporadic Alzheimer disease" caused by environmental factors such as lifestyle habits and stress.
"frontotemporal lobar degeneration disease," also known as FTLD, refers to a non-Alzheimer's disease type of neurodegenerative disease that presents atrophy in the frontal/temporal lobes at an early stage and progresses to global atrophy of the brain at an advanced stage. That is, frontotemporal lobar degeneration is classified into 3 diseases including frontotemporal dementia (FTD), progressive aphasia (PNFA) and Semantic Dementia (SD) in clinical features, and also 4 diseases classified into FTLD-Tau, FTLD-TDP, FTLD-UPS and FTLD-FUS pathologically, depending on the kind of proteins accumulated as abnormal proteins in cells.
Furthermore, FTLD-Tau is classified into 3R Tau type, 4R Tau type and 3/4R Tau type according to the number of repeats of the microtubule binding region in Tau protein mainly accumulated in cells. Furthermore, the 3R Tau type includes FTLD (Pick disease) accompanied by Pick cell, FTLD (FTLD-17) accompanied by MAPT (microtubule-associated protein Tau) gene mutation, and the like; the 4R Tau type includes cerebral cortical basal nucleus degeneration, progressive supranuclear palsy, multi-system Tau proteinopathy accompanied by dementia, silver-loving granule dementia (silver-loving granule disease), FTLD (FTLD-17) accompanied by MAPT gene mutation, etc.; 3/4R Tau includes neurofibrillary change dementia, FTLD (FTLD-17) accompanied by MAPT gene mutation, and the like. On the other hand, the FTLD group with tau negative and ubiquitin positive inclusion bodies is called FTLD-U, and includes FTLD-TDP, FTLD-UPS and FTLD-FUS as described above.
FTLD-TDP refers to a disease in which TDP-43 is positive in FTLD-U, and the disease includes FTLD with PGRN (progranulin gene) mutation, FTLD with sporadic FTLD-TDP/FTLD-U, TARDBP (TDP-43 gene) mutation, FTLD with VCP (containing valosin protein gene) mutation, FTLD linked with chromosome 9, and the like. Furthermore, FTLD-FUS refers to a disease in which TDP-43 is negative and FUS (fused in sarcoma fusion gene) is positive in FTLD-U, and the disease includes neurocytocytic intermediate filament inclusion body disease, atypical FTLD-U, basophilic inclusion body disease, FTLD accompanied with FUS mutation, and the like.
In addition, "TLD-UPS" is a kind of FTLD-U in which TDP-43 is negative, and this disease includes FTLD accompanied by CHMP2B (charged multivesicular protein 2B gene) mutation, and the like.
Amyotrophic lateral sclerosis is a type of motor neuron disease, a neurodegenerative disease characterized by severe muscle atrophy and muscle strength decline. About 20 genes have been reported as causative genes of amyotrophic lateral sclerosis, and abnormal accumulation of TDP-43 causes the disease.
"Parkinson's disease" refers to a progressive degenerative disease that is mainly caused by degeneration of nigral dopamine nerve cells. It is observed that the loss of pigment from the substantia nigra and the locus coeruleus of the midbrain leads to the loss of nerve cells such as substantia nigra, locus coeruleus, dorsal nucleus of vagus nerve, hypothalamus, sympathetic ganglion, etc., which results in the deficiency of dopamine, a conductive substance. Characteristic inclusion bodies such as Lewy bodies were observed in the residual nerve cells or a part of the projections thereof.
"dementia of the lewy body type" refers to degenerative dementia in which lewy bodies appear in the whole cerebral cortex and which shows hallucinogenic symptoms and parkinsonism in addition to progressive cognitive dysfunction.
Protein aggregation is a recognized feature of degenerative disorders of the nervous system including Alzheimer's Disease (AD), parkinson's disease, dementia of the lewy body type, frontotemporal lobar degeneration (FTLD), Huntington's Disease (HD), spinocerebellar disorder (SCA), and Amyotrophic Lateral Sclerosis (ALS). Generally, disease-associated proteins are thought to have misfolded structures that then transition to structures that are prone to aggregation, including β -sheets.
However, little is known about the details of the temporal and/or random variation of these structures, leading to controversy regarding the identity of the actual toxic substance and whether the aggregated or soluble protein is toxic.
Clinical trials of AD treatment have had a significant impact on these issues. Passive immunization with anti-a β antibodies was successful in reducing the presence of extracellular a β plaques in the brain.
However, in these tests, a difference between improvement of a β -PET (positron emission tomography) and improvement of clinical symptoms was reported.
Thus, the elucidation of early-stage disease states is an urgent problem to be solved for elucidation of causes of AD and development of therapeutics, and is basically the same in other nervous system degenerative diseases including PD/DLB.
Comprehensive phosphoprotein analysis of brain samples from mouse AD models and human AD patients showed that changes in phosphorylation of several proteins had begun before extracellular A β plaques could be detected histologically (Tagawa et al (2015) Hum MolGenet 24: 540-558).
MARCKS phosphorylation at Ser46 is induced by a TLR 4-mediated damage-associated molecular pattern (DAMP), in particular by HMGB1 (Fujita et al, (2016) Sci Rep 6: 31895).
This time, MARCKS phosphorylation at Ser46 was detected in BAC-Tg mice overexpressing human normal alpha-synuclein (alpha-Syn) in the Glucosidase (GBA) -hybrid Knockout (KO) background (human alpha-Syn-BAC-T cells) as observed in human DLB patients. The histological features and chronological order of MARCKS phosphorylation on serine 46(pSer46-MARCKS) were the same as in AD and PD/DLB. Interestingly, this marker of nervous system degeneration was positive prior to the formation of histologically detectable alpha-Syn aggregates.
These results clearly show the relationship between neuronal degeneration and protein aggregation, indicating that the onset of neural mutations precedes the formation of protein aggregates.
pSer46-MARCKS can be used as a biomarker capable of detecting molecular pathology in PD/DLB in the very early (pre-aggregation/preclinical) stage.
1. Detection of neurodegenerative diseases based on the amount of MARCKS phosphorylated on serine at position 46(pSer46-MARCKS)
In the present invention, the presence or absence of AD, FTLD or ALS in a subject is detected using MARCKS phosphorylation in a human biological sample as an index. "detection" may also be referred to as "determination" or "evaluation".
MARCKS refers to myristoylated alanine-rich protein kinase C substrate, and includes: human-derived proteins identified by RefSeq ID NP-002347 (NP-002347.5). Furthermore, typical examples of human sources of MARCKS-encoding nucleic acids include: nucleic acid comprising the coding region (CDS) described in RefSeqID, NM-002356 (NM-002356.6).
Examples of the biological sample used as the sample include body fluid, tissue, cells, and the like (for example, cerebrospinal fluid, cranial nerve tissue (particularly, neurological biopsy tissue), blood, plasma, lymph, urine, and saliva) collected from the body of a subject, and among them, cerebrospinal fluid is preferable.
In the present invention, phosphorylation of serine (Ser) at
The method for detecting phosphorylated MARCKS is not limited, and any known method may be used. Examples thereof include: mass spectrometry, and immunological assay.
Mass spectrometry can be performed using a mass spectrometer. The mass spectrometer includes a sample introduction unit, an ionization chamber, an analysis unit, a detection unit, a recording unit, and the like. As the ionization method, a matrix-assisted laser desorption (MALDI) method, an electrospray ionization (ESI) method, or the like may be used. The analysis unit may use a dual focus mass spectrometer, a Quadrupole Mass Spectrometer (QMS), a time-of-flight mass spectrometer (TOF), a fourier transform mass spectrometer (FT), an ion cyclotron mass spectrometer (ICR), or the like. A tandem mass spectrometer (MS/MS) combining 2 mass spectrometers together can also be used for precision analysis. The mass spectrometer may be used alone, or may be connected to a separation instrument such as a liquid chromatography instrument or a measurement instrument, and may perform analysis by liquid chromatography mass spectrometry (LC/MS ) combined with high-speed liquid chromatography.
The immunological assay may be carried out by an immunological assay using an anti-pSer 46-MARCKS antibody which recognizes serine at position 46Acid phosphorylated MARCKS (pSer 46-MARCKS). Examples of immunological measurement methods include: solid phase Immunoassay (RIA, EIA, FIA, CLIA, etc.), dot blot method, latex aggregation method (LA: LatexAgglutination-Turbidimetric Immunoassay, latex agglutination-Turbidimetric Immunoassay), immunochromatography, and the like. The antibody may be immobilized on a substrate for use. Among them, from the viewpoint of quantitativeness, an Enzyme-Linked ImmunoSorbent Assay (ELISA) method, which is one of EIA (Enzyme Immunoassay) methods, is preferable. In the ELISA method, a sample is added to a well of a microtiter plate immobilized with an antibody to perform an antigen-antibody reaction, an enzyme-labeled antibody is further added to perform an antigen-antibody reaction, and after washing, the reaction with an enzyme substrate and color development are performed to measure absorbance, thereby detecting a marker protein or a partial peptide in the sample, and the concentration of the protein or the partial peptide in the sample can be calculated from the measured value. Alternatively, fluorescence may be measured after antigen-antibody reaction using a fluorescent-labeled antibody. The antigen-antibody reaction may be carried out at 4 to 45 ℃, more preferably 20 to 40 ℃, and still more preferably 25 to 38 ℃, and the reaction time may be about 10 minutes to 18 hours, more preferably 10 minutes to 1 hour, and still more preferably 30 minutes to 1 hour. The antibody used in the immunological method may be a monoclonal antibody, a polyclonal antibody, or Fab, F (ab') of a monoclonal antibody2And the like bind to the active fragment.
Furthermore, MARCKS phosphorylated at serine 46(pSer46-MARCKS) was also detected by PET (positron emission tomography). In the case of detection by PET, the subject may be administered a tracer for imaging pSer46-MARCKS and pSer46-MARCKS in the subject may be detected by PET (positron emission tomography). As tracers for imaging pSer46-MARCKS, mention may be made, for example, of: antibody PET tracer labeled with a positive electron nuclide for antibodies directed against phosphorylated MARCKS protein at
In the present invention, a sample collected from a healthy person can be simultaneously measured as a negative control. Here, "healthy" refers to a state in which the subject does not suffer from a neurodegenerative disease such as Alzheimer Disease (AD), frontotemporal lobar degeneration (FTLD), Amyotrophic Lateral Sclerosis (ALS), PD (parkinson's disease), and DLB (dementia with lewy bodies). In this case, when the subject suffers from or is at risk of developing a neurodegenerative disease such as Alzheimer's Disease (AD), frontotemporal lobar degeneration (FTLD), Amyotrophic Lateral Sclerosis (ALS), or Parkinson's Disease (PD), and lewy body Dementia (DLB), the phosphorylation degree of serine at
In this case, pSer46-MARCKS can be quantified by the above-described method, and the resulting quantified values can be normalized and judged. Normalization can be performed, for example, by dividing the pSer46-MARCKS concentration by the total protein concentration in the sample. In this case, for example, the amount of pSer46-MARCKS can be expressed in terms of concentration (ppm) relative to the total protein amount. In addition, pSer46-MARCKS concentration can also be quantified, divided by the total MARCKS in the sample (total amount of phosphorylated and non-phosphorylated MARCKS). In this case, the amount of pSer46-MARCKS can be expressed in% relative to the total MARCKS.
For example, when the value of pSer46-MARCKS in a subject sample is 1.3 times or more, preferably 1.5 times or more, more preferably 2.0 times or more, and particularly preferably 3.0 times or more the amount in a healthy human sample, it is possible to determine that a neurological disorder such as Alzheimer's Disease (AD), frontotemporal lobar degeneration (FTLD), Amyotrophic Lateral Sclerosis (ALS), Parkinson's Disease (PD), and lewy body Dementia (DLB) is detected in the subject.
In addition, the amount of pSer46-MARCKS in a sample of a healthy person was measured in advance, and a cut-off value (threshold) was determined based on the measured value of the amount of pSer 46-MARCKS. When the amount of pSer46-MARCKS in the sample collected from the subject is determined to be greater than the amount of pSer46-MARCKS in the sample collected from a healthy person on the basis of the critical value, it can be determined that a neurological disorder such as Alzheimer's Disease (AD), frontotemporal lobar degeneration (FTLD), Amyotrophic Lateral Sclerosis (ALS), Parkinson's Disease (PD), and Lewy body Dementia (DLB) is detected in the subject.
The cut-off value is set to, for example, the average +2SD of healthy human control groups, and when the cut-off value is larger than the average +2SD, it can be judged that a neurodegenerative disease such as Alzheimer Disease (AD), frontotemporal lobar degeneration (FTLD), Amyotrophic Lateral Sclerosis (ALS), Parkinson Disease (PD), and lewy body Dementia (DLB) is detected in the subject.
2. Detection of neurodegenerative diseases based on DO (Distance from origin) values
In the present invention, neurodegenerative diseases such as Alzheimer's Disease (AD), frontotemporal lobar degeneration (FTLD), and Amyotrophic Lateral Sclerosis (ALS) are detected using DO (distance from origin) value, which is a new parameter obtained by integrating pSer46-MARCKS with non-phosphorylated MARCKS, as an index. DO is a parameter integrating the amounts of pSer46-MARCKS and non-phosphorylated MARCKS, both of which are used as indicators. DO is a value reflecting the distance of the coordinate of the plotted point from the origin when the amount of pSer46-MARCKS and the amount of non-phosphorylated MARCKS protein are plotted on (x, y) coordinates, and DO is the square root of the value of the sum of the power of 2 of the amount of pSer46-MARCKS and the power of 2 of the amount of non-phosphorylated MARCKS, which is represented by the following formula.
[ formula 1]
In the formula, "pSer 46-MARCKS" represents the amount of phosphorylated MARCKS protein at
The DO value enables detection of a neurodegenerative disease such as Alzheimer Disease (AD), frontotemporal lobar degeneration (FTLD), or Amyotrophic Lateral Sclerosis (ALS) with higher sensitivity and high specificity than when pSer46-MARCKS alone is used as a marker. DO values by performing numerical simulations as shown in FIG. 8-1, the sensitivity and specificity can be determined as good upper normal values (cut-off values). As shown in fig. 8-1, other parameters such as sensitivity of 3 diseases (maximum 1.0 in each disease, and therefore maximum 3.0 in 3 diseases) of Alzheimer's Disease (AD), frontotemporal lobar degeneration (FTLD), and Amyotrophic Lateral Sclerosis (ALS) and a total of specificity common to 3 diseases (maximum 1.0 as a representative of 3 diseases), that is, an "objective score" may be used in numerical simulation, and a DO range that yields the highest objective score may be selected. For example, when values normalized by the total protein concentration in a sample are used as the amounts of pSer46-MARCKS and non-phosphorylated MARCKS, the DO value has a cut-off value of 50 to 55(ppm), a sensitivity of 0.8 to 1.0, and a specificity of 0.8 or more, preferably 0.8.
In addition, the amount of pSer46-MARCKS and the amount of non-phosphorylated MARCKS in a sample of a healthy person may be measured in advance, a DO value may be calculated for the healthy person, and a cut-off value (threshold) may be determined for the DO value based on the calculated value. When the DO value is greater than the threshold value based on the threshold value, it can be determined that the subject has or is at risk of developing a neurodegenerative disease, such as Alzheimer's Disease (AD), frontotemporal lobar degeneration (FTLD), or Amyotrophic Lateral Sclerosis (ALS).
Moreover, it can be judged that in Alzheimer's Disease (AD) and frontotemporal lobar degeneration (FTLD), the severity of the disease is associated with the DO value, and the higher the DO value, the higher the severity of the disease.
In PD (Parkinson's disease) and DLB (Lewy body type dementia), pSer46-MARCKS levels in brain tissue were also elevated as well as Alzheimer's Disease (AD) and frontotemporal lobar degeneration (FTLD). Thus, in PD (Parkinson's disease) and DLB (dementia with Lewy bodies), it is also possible to detect nervous system degeneration by elevated levels of pSer46-MARCKS in the sample, and to reflect severity.
3. Treatment of degenerative diseases of the nervous system
When a neurodegenerative disease is detected by the method of the present invention, Alzheimer's Disease (AD), frontotemporal lobar degeneration (FTLD), Amyotrophic Lateral Sclerosis (ALS), Parkinson's Disease (PD), or dementia of the lewy body type (DLB) may be treated or symptom thereof may be improved by the following method.
Alzheimer's Disease (AD)
Administering drugs that promote information transfer between neurons, such as donepezil, rivastigmine, galantamine, and amantadine; treatment with an antibody drug such as Bapineuzumab (Bapineuzumab) or perizumab (Solanezumab) that can remove amyloid β that causes alzheimer's disease from the brain.
Frontotemporal lobar degeneration disease (FTLD)
Administration of a selective serotonin reuptake inhibitor and a therapy targeting a gene such as Tau gene, TDP-43 gene, and granule protein precursor gene as a pathogenic gene.
Amyotrophic Lateral Sclerosis (ALS)
Administering riluzole, edaravone, methylcobalamin, retigabine, BIIB067, MN-166, etc.; therapy using genes such as SOD1 gene, TDP-43 gene, FUS gene, and C9orf72 gene, which are reported as pathogenic genes, as targets, and therapy using regenerative therapy in which human pluripotent stem cells are used to regenerate nerve cells.
Parkinson's Disease (PD)
There are pharmacotherapy, surgical therapy, pharmacologic therapy, as pharmacotherapy oral L-dopa and dopamine agonists, as surgical therapy for deep brain stimulation therapy (subthalamic nucleus stimulation, globus pallidus stimulation, thalamus stimulation) and localized destruction (thalamus destruction, globus pallidus destruction) or deep brain stimulation therapy (DBS).
Dementia with lewy body type (DLB)
Donepezil and the like are administered.
Examples
The present invention is specifically illustrated by the following examples, but the present invention is not limited to these examples.
EXAMPLE 1 Studies on Alzheimer's Disease (AD), frontotemporal lobar degeneration (FTLD) and Amyotrophic Lateral Sclerosis (ALS)
Method of producing a composite material
Human patient
Cerebrospinal fluid from 8 AD patients, 7 FTLD patients and 10 ALS patients was used in the celebrity house university and northeast university by neuroimaging diagnostics including clinical symptoms, electrophysiology examination, MRI, SPECT and PET.
Control subjects were 6 men (mean ages 72.6 years, 55-83 years) and 4 women (mean ages 75.5 years, 69-79 years). The simple mental state examination (MMSE) score is 25 or more (average 28.3, 26-30) at the time point of collecting cerebrospinal fluid (CSF). AD patients were male 2 (54 and 80 years) and female 6 (64.5, 57-75 years on average). The MMSE score of these patients did not exceed 25 (mean 17.1, 4-25), and the mean sum of FAB scores ranged from 9.3 and 6-13. FTLD patients are male 5,
Preparation of samples
Samples were prepared from cerebrospinal fluid (CSF) for proteomic analysis. That is, 50. mu.l of human patient-derived cerebrospinal fluid was added to 5. mu.l of a buffer containing 100mM Tris-HCl (pH7.5), 2% SDS and 1mM DTT, and incubated at 100 ℃ for 15 minutes.
The sample solution was centrifuged at 16,000g for 10 minutes at 4 ℃ and the supernatant was filtered using a 0.22 μm PVDF filter (Millipore). Aliquots (55. mu.L) were added to 25. mu.L of 1M triethylammonium bicarbonate (TEAB) (pH8.5), 0.75. mu.L of 10% SDS, and 7.5. mu.L of 50mM tricarboxyethylphosphine and incubated for 1 hour at 60 ℃. The cysteine residues were blocked with 10mM methyl methylthiosulphonate at 25 ℃ for 10 minutes. Next, the sample was incubated with 24mM CaCl21.5. mu.g of trypsin (10: 1 ═ protein: enzyme, w/w) in (E) was digested at 37 ℃ for 24 hours, desalted by C18 spin columns (MonoSpin C18, GL Sciences Inc.), dried and dissolved in 0.1% formic acidIn 35. mu.l.
Mass Spectrometry of SWATH-cerebrospinal fluid
A30. mu.l aliquot was analyzed with an Eksigent NanoLC-Ultra 1D Plus system (Sciex Inc.) in a C18 column (0.1 mm. times.100 mm, KYA technologies corporation) containing solution A (0.1% formic acid) at a flow rate of 300 nl/min in a gradient of 2% to 41% solution A and B (99.9% acetonitrile and 0.1% formic acid) and supplied to the Triple TOF 5600 system (Sciex) at an ion spray voltage of 2.3 kV. Setting IDA (information-dependent acquisition) with 2-5 times of electrification as 400-1000 m/z, wherein the MS/MS scanning range of generated ions is 100-1600 Da, and the accumulation time of a mass spectrum library is 100 MS. SWATH (registered trademark) (sequential window acquisition of all the theoretical mass spectra, collected in window order) was performed through 24 consecutive windows of 25Da from 400Da to 1000 Da. The SWATH acquisition of MS/MS spectral data was performed at 100 MS/window using analysttf1.6 software (Sciex Inc.) and a MS/MS mass spectral library was prepared using Protein Pilot software (version 4.5). MS/MS spectral data peptide data were correlated with LC retention time using Peakview software (version 1.2.0.3, sciexeinc.). The MS/MS product ions from the same peptide were added and used as the amount of peptide.
Quality data analysis
MS/MS spectra raw data were processed using the SWATH 1.0 application of the peavview1.2 software, at 5 minutes of the extracted ion chromatography (XIC) extraction window and 0.01Da width of XIC, to extract well-characterized species spectra data. XIC is shown as a plot of LC retention time and relative ionic strength over a small range of m/z. The fragment ions XICs are added to obtain a peptide peak region, and the areas of the peptides of each protein are added to obtain a protein area. Mass spectral data for peptides were normalized by the total amount of all proteins detected or the total amount of peptides associated with MARCKS proteins for each individual subject (these are referred to as "total protein" or "total MARCKS", respectively). For calculation of sensitivity and specificity, abnormal phosphorylation was judged by the mean amount of control group ± 2 SD.
Immunohistochemical method
For immunohistochemistry, paraffin-embedded human brain sections were dewaxed and rehydrated, microwaved 5 times in 10mM citrate buffer (pH6.0, 100 ℃) followed by 5
Nissel staining
Paraffin-embedded sections of human brain were dewaxed and rehydrated. After washing with distilled water, the slices were immersed in a tar violet (Cresyl violet) solution (300 mL of 0.1% tar violet (Cresyl violet) was added to distilled water along with 5 drops of 10% acetic acid solution at 37 ℃ followed by filtration before use) for 15 minutes. The sections were then dehydrated with ethanol and washed with xylene and covered with a coverslip. Images were obtained by light microscopy (Olympus, BX 53).
Numerical simulation
As parameters for maximization in numerical simulation of the upper normal limit, sensitivity values from 3 diseases (maximum 1.0 in each disease, maximum 3.0 in 3 diseases) and the total specificity of 3 diseases (specificity common to 3 diseases (maximum 1.0 as a representative of 3 diseases) were used. In addition, a raw numerical simulator using the R programming language (version 3.4.0, R statistical calculations, https:// www.R-project. org /) was developed to optimize the upper normal limit for maximizing the parameters. The DO value is entered into the program with the main class for that class. In the initial trial run, the program was run to determine the range of intrinsic analysis, and the upper limit of the simulated DO at a sensitivity score of 0 for 3 diseases was determined to be 1,000. Within this range, the numerical simulation is repeated to find the best DO that maximizes the objective score.
Statistical analysis
The biological distance between the disease group and the control group was tested by Wilcoxon rank sum test. To evaluate the correlation between pSer46 and the non-phosphorylated peptide in each subject, Pearson correlation coefficients were calculated. Using Pearson correlation coefficients, the correlation of HMGB1 concentration in each subject to the pSer46-MARCKS signal was also calculated.
Results
pSer46-MARCKS in cerebrospinal fluid (CSF) of neurodegenerative diseases
Using mass spectrometry, pSer46-MARCKS of cerebrospinal fluid (CSF) from human patients of 3 neurodegenerative diseases of Alzheimer's Disease (AD), frontotemporal lobar degeneration (FTLD) and Amyotrophic Lateral Sclerosis (ALS) from human patients was quantified, as well as from controls. To compare the relative amounts of phosphorylated peptides in the disease groups or in each subject, the amount of total protein or total MARCKS protein detected by mass spectrometry was normalized for each subject. Peptides were identified with a confidence of 95% or more.
To evaluate the biomarker as a diagnosis of pSer46-MARCKS for cerebrospinal fluid (CSF), the sensitivity and specificity of pSer46-MARCKS normalized to total protein (FIGS. 1-1A and B) or total MARCKS protein (FIGS. 1-2A and B) in the 3 disease groups was calculated. FIGS. 1-1A are graphs plotting the values of pSer46-MARCKS, normalized by total protein amount per subject, for each disease group. FIGS. 1-2A are graphs plotting the value of pSer46-MARCKS, normalized by the total MARCKS protein amount per subject, for each disease group. The rectangular box sections below fig. 1-1A and fig. 1-2A represent the mean of the control group ± 2SD, and the lines in the box represent the mean of the healthy controls. FIGS. 1-1B and 1-2B show sensitivity and specificity. An abnormality is determined when the patient value is greater than the average +2SD of the control group. In this state, it was confirmed that the specificity of pSer46-MARCKS was very high in all 3 groups of neurodegenerative diseases (FIGS. 1-1B and 1-2B). This indicates that the subject was indeed abnormal when pSer46-MARCKS was higher than the mean +2SD of the control.
On the other hand, the sensitivity of pSer46-MARCKS for diagnosis was relatively low in all disease groups. When pSer46-MARCKS was normalized to the protein population, the sensitivity was 0.50 in AD, 0.57 in FTLD and 0.40 in ALS (FIGS. 1-1B). pSer46-MARCKS normalized by total MARCKS, the values were smaller (FIGS. 1-2B). Thus, it is considered appropriate that the use of pSer46-MARCKS is restricted during this analysis stage, and that pSer46-MARCKS is used just as a progression biomarker, rather than as a disease-specific biomarker or screening biomarker.
In the same manner, non-phosphorylated MARCKS was quantified in cerebrospinal fluid (CSF) from human patients of 3 neurodegenerative diseases (FIGS. 2-1 and 2-2). FIG. 2-1A is a graph plotting the amount of non-phosphorylated MARCKS for each disease group, values normalized by total protein. Fig. 2-2A is a graph plotting the amount of non-phosphorylated MARCKS for each disease group, values normalized by total MARCKS protein amount. The rectangular box portion below fig. 2-1A and 2-2A represents the mean of the control group ± 2SD, and the line in the box represents the mean of the healthy controls. FIGS. 2-1B and 2-2B show sensitivity and specificity. In this case, the specificity in AD and ALS was not 100%, and the sensitivity was 0. Thus, non-phosphorylated MARCKS alone is clearly not suitable for use in diagnostic biomarkers.
Elevation of pSer46-MARCKS of cerebrospinal fluid (CSF) in AD, FTLD and ALS
Next, the values of pSer46-MARCKS were compared between the disease groups and the controls. The amount of pSer46-MARCKS in each subject is shown in FIG. 3-1, and the amount of non-phosphorylated MARCKS in each subject is shown in FIG. 3-2. The amounts of FIGS. 3-1A and 3-2A were normalized by total protein amount, and the amounts of FIGS. 3-1B and 3-2B were normalized by total MARCKS amount. The lines in the graph represent the median values for each group. A nonparametric Wilcoxon rank-sum test was performed for comparing the values of the groups, considering values higher than or equal to those determined in the disease group, and statistical differences were found between AD patients and controls (p-0.02941), FTLD and controls (p-0.00358) and ALS and controls (p-0.0007) (fig. 3-1A). Results varied by the method of normalization of pSer46-MARCKS, but differences between FTLD patients and controls (p 0.0128) remained if pSer46-MARCKS was normalized to total MARCKS (fig. 3-1B). Furthermore, the values of non-phosphorylated MARCKS were also statistically different when values were normalized by total protein between AD patients and controls (p ═ 0.0455) and ALS patients and controls (p ═ 0.0212) (fig. 3-2A). Furthermore, it was shown that there was a statistical difference between FTLD patients and controls (p ═ 0.0217) when values were normalized by total MARCS (fig. 3-2B).
Relationship between pSer46-MARCKS and non-phosphorylated MARCKS
Next, pSer46-MARCKS was analyzed for the relationship (ratio) to non-phosphorylated MARCKS in each group (FIGS. 4-1 to 4-3). FIG. 4-1 is a graph plotting normalized values by total protein amount for each AD, FTLD and ALS disease group, and FIG. 4-2 is a graph combining the plots in FIG. 4-1. Fig. 4-3 is a graph plotting normalized values for total MARCKS amount for each AD, FTLD, and ALS disease group, and fig. 4-4 is a graph in which the plots in fig. 4-3 are merged. The control group showed a clear positive correlation between pSer46-MARCKS and non-phosphorylated MARCKS (FIGS. 4-1 and 4-3), the AD group showed a weak negative correlation, and neither the FTLD nor ALS groups showed any correlation. This result suggests that MARCKS phosphorylation in Ser46 is pathologically affected in AD and FTLD.
Another point that should be noted is that the disease groups are plotted essentially all above the regression line determined for the control group of the graphs (x, y — unphosphorylated MARCKS, pSer46-MARCKS) (fig. 4-2 and 4-4). This indicates that there was a trend of increasing the ratio of pSer46-MARCKS to non-phosphorylated MARCKS in AD, FTLD and ALS patients, regardless of whether pSer46-MARCKS was normalized by total protein or total MARCKS (FIGS. 4-1-4). Based on these analyses, it was tested whether the ratio of pSer46-MARCKS to non-phosphorylated MARCKS could be a biomarker for pathological changes. However, the differences between groups were not statistically significant (FIGS. 4-1 to 4-4). Since the scores were very different (FIGS. 4-1-4), this could be due to heterogeneity in the patient groups that mixed the early and late disease stages.
Relationship of HMGB1 in cerebrospinal fluid (CSF) to pSer46-MARCKS
The inventors previously established that HMGB1, a representative molecule to which the damage-associated molecular pattern (DAMP) belongs, induces MARCKS phosphorylation at Ser46 downstream of the signaling pathway of TLR4, and antibodies against HMGB1 may potentially delay the onset of AD 13. Based on a series of assumptions, the relationship of HMGB1 in cerebrospinal fluid (CSF) of AD patients to pSer46-MARCKS was tested using previous data (FIGS. 5-1 and 5-2). FIG. 5-1 is pSer46-MARCKS relative to total protein amount, and FIG. 5-2 is pSer46-MARCKS relative to total MARCKS amount. In order to know whether the HMGB1 and pSer46-MARCKS related hypotheses do apply in vivo, retrospective analysis of previously published data (by other examiners) must be non-bias tests.
There are technical limits to the HMGB1-ELISA sensitivity threshold, with HMGB1 values below detection levels in most patients, but a significantly higher than expected positive correlation was found between cerebrospinal fluid (CSF) and PSER46-MARCKS in AD patients (fig. 5-1 and 5-2).
Elevation of pSer46-MARCKS in dystrophic neurites of human FTLD
Next, in the AD mouse model and AD human patients, whether pSer46-MARCKS increased in neurites in human FTLD brain like dystrophic neurites was tested (Fujita, k.et al, sci. rep.6,31895 (2016)). Pathologically diagnosed brains of human FTLD (fig. 6-1) were used for immunohistochemistry using anti-pSer 46-MARCKS antibody (fig. 6-2A and B). FIG. 6-1 shows a pTDP-43 stained image of human FTLD, and it was found that TDP43 stained the abnormal distribution of TDP43 and the cytoplasmic encapsulation of TDP 43. In addition, FIGS. 6-2A and B show immunohistochemistry using anti-pSer 46-MARCKS antibody. As shown in fig. 6-2A, neural degeneration (layer IV) was seen in the FTLD occipital lobe (occipital cortex) and frontal lobe (frontal cortex). In addition, both the dendrites and cell bodies at the tip of the occipital lobe were well stained. However, as shown in fig. 6-2B, little staining was seen in the most severely affected frontal lobe. After single staining with anti-pSer 46-MARCKS antibody and secondary Cy 3-labeled anti-rabbit IgG antibody, it was visible under excitation light at 552nm and fluorescence at 570nm, but not under excitation light at 498nm and fluorescence at 520nm (fig. 6-2B), or excitation light at 552nm and fluorescence at 520nm (data not shown), indicating site specificity of staining without autofluorescence. The staining pattern was very similar to that of human AD in the previous report (Fujita, k.et al., sci.rep.6,31895 (2016)). pSer46-MARCKS positive neurites were frequently observed in occipital leaves where FTLD was relatively unaffected, but were rarely observed in frontal leaves, which were most severely affected. The findings leading to this debate showed pSer46-MARCKS to be an early stage specific marker, with the progression of disease phase abrogated. FIGS. 6-3A show the results of co-staining MAP2 and pSer46-MARCKS in human FTLD occipital leaves, with staining of neurons with anti-pSer 46-MARCKS antibody. In the brain of human FTLD, the protrusions and cell bodies stained by pSer46-MARCKS were also positive for MAP2, indicating that they are neurons and neurites (FIGS. 6-3A). Next, it was confirmed that pSer46-MARCKS positive cells represent abnormal cytoplasmic distribution of neurons with aggregates or TDP 43. That is, FIG. 6-3B shows that neurons with cytoplasmic phosphorylated TDP43 co-stained with pSer 46-MARCK. Interestingly, pSer46-MARCKS and phospho TDP43 co-stained in cytoplasmic aggregates (FIGS. 6-3C).
In Nissl staining of human FTLD, few frontal neurons were detected and only spongiform degeneration was seen in the cell mass (fig. 7A). On the other hand, neurons with abnormal characteristics such as chromatin lysis or no pigment were observed in the occipital lobe (fig. 7B). In the brain not subjected to the control of the neurodegenerative disease, such abnormal changes were not seen with respect to the neuron density, the neural network morphology, and the like (fig. 7C).
Integration of pSer46-MARCKS as biomarker with non-phosphorylated MARCKS
If the plots are viewed (FIGS. 4-2 and 4-4), the (x, y) coordinate position of the control subjects is known to be relatively close to the origin (0, 0). Thus, the degree of change in integrated pSer46-MARCKS and non-phosphorylated MARCKS is expected to distinguish pathological changes from normal controls, and the square root of the
In order to effectively distinguish pathological DO values from normal DO values, an "upper normal limit of DO" is required that can simultaneously produce both maximum sensitivity and specificity. The optimal "upper normal limit for DO" was explored by numerical simulation (FIG. 8-1). Here, an attempt was made to find a DO range that yields the highest objective score using other parameters such as the sensitivity of 3 diseases (maximum of 1.0 among the diseases, and therefore maximum of 3.0 among the 3 diseases) and the sum of the specificities common to the 3 diseases (maximum of 1.0 as a representative of the 3 diseases), that is, "objective score" (fig. 8-1). FIG. 8-1 is a graph showing objective DO scores of 0-1000. As a result, it was found that the highest objective score of 3.532 was obtained in DO having an upper limit of 50.26 to 54.24 (FIGS. 8-1 and 8-2). FIG. 8-2 shows the DO values in each disease. In the figure, the DO value is represented by a block diagram. The upper normal limit is determined by numerical simulation. The boxes of each group represent values from lower to upper. The median value is also shown in the box. FIG. 8-3 shows the number of cases judged abnormal in the control, AD, FTLD, and ALS groups, and the values of sensitivity and specificity when the subject scores and DO range were optimized to the maximum. The threshold of fig. 8-1 is temporarily represented as a DO of 50.262, but in the DO range of 50.262 to 54.239, sensitivity and specificity did not change. As shown in fig. 8-3, by setting the upper limit of the normal value to 50.26, it is possible to determine abnormal values with sensitivities of 0.875, 0.857, and 1.00 in AD, FTLD, and ALS (fig. 8-3, fig. 9). Specificity was 0.800 in all diseases (FIGS. 8-3, FIG. 9). The specificity was increased to 0.889 (in parentheses in FIGS. 8-3) by judging abnormal cases from the control group and excluding cases accompanied by acute cognitive decline.
Correlation of DO with severity of disease
Finally, the correlation of DO in clinical symptoms with severity of disease was examined (fig. 9). In AD (fig. 9A and B) and FTLD (fig. 9C and D), it can be seen that MMSE and DO (fig. 9A and C) or FAB (Frontal Assessment battle scale) are negatively correlated with DO (fig. 9B and D), indicating that DO reflects the severity of clinical symptoms in these dementias. However, no association of ALS was detected between the ALS function score scale (ALSFRS-R) and DO (fig. 9E). The cause is not clear, but may be associated with the rapid progression of ALS more rapidly than AD and FTLD. For example, the actual rate of progression of the pathology sometimes does not reflect the severity of dyskinesia at a particular time point in ALS.
From the results of the present embodiment, the following is made clear.
MARCKS phosphorylation at Ser46 is reported to occur in neurites at very early stages of AD via the HMGB1-TLR4 signaling pathway induced by the injury-warning molecule HMGB1 released by neurons that accumulate in cells with nearby intracellular Α β (Fujita, k.et al, sci.rep.6,31895 (2016)). In this example, the evaluation of MARCKS phosphorylated at Ser46 was carried out, and based on the findings so far, MARCKS phosphorylated at Ser46 were expected to be biomarkers as indicators of degeneration of neurites available at the early pathological stage in 3 kinds of cerebrospinal fluid (CSF) including AD, FTLD, and ALS.
pSer46-MARCKS of cerebrospinal fluid (CSF) is highly specific and relatively insensitive in all neurodegenerative diseases (FIGS. 1-1 and 1-2). To overcome the difficulty of pSer46-MARCKS as a biomarker, the values of non-phosphorylated MARCKS were integrated with pSer46-MARCKS, creating a new parameter like DO (FIG. 8-1). It was confirmed that DO is a good parameter with both high sensitivity and high specificity, and that DO reflects disease severity well in AD and FTLD. Using numerical simulations, to optimize sensitivity and specificity in 3 neurodegenerative diseases, the optimal values of the upper normal limit for DO were further retrieved. As a result, the sensitivity of AD, FTLD and ALS was 0.875, 0.857 and 1.00 using the "upper limit of normal DO" of 50.26 to 54.24, respectively, and the specificity of 0.800 was obtained in all diseases (FIGS. 8-1 to 8-3 and 9).
Surprisingly, DO also undergoes common changes in multiple nervous system degenerative diseases. In addition, DO and pSer46-MARCKS were themselves higher in FTLD than AD. This result indicates that DO and pSer46-MARCKS are not disease-specific markers, and they are indicators of pathological processes common in 3 neurodegenerative diseases. Previous studies have shown that pSer46-MARCKS has been abnormally increased even before the formation of histological Α β aggregates (Tagawa, k.et al, hum.mol.genet.24,540-58 (2015); Fujita, k.et al, sci.rep.6,31895(2016)), and thus DO and pSer46-MARCKS may reflect neural mutability during the early stages of AD and FTLD and ALS.
The biological implications that the value of non-phosphorylated MARCKS should be integrated in order to generate better biomarkers are not clear. However, both pSer46-MARCKS and non-phosphorylated MARCKS were released from the decomposed neurites, active phosphorylation of MARCKS did not occur in terminal neurons, and thus DO values obtained by integration with non-phosphorylated MARCKS were better markers than pSer-MARCKS alone.
EXAMPLE 2 Studies in Parkinson's Disease (PD) and dementia with Lewy bodies (DLB)
Method of producing a composite material
Mouse PD/DLB model
Normal human α -Syn-BAC-Tg mice were prepared according to the method described in Yamakado et al (2012) Neurosci Res 73: 173-177.
Specifically, the BAC-Tg construct (containing 28kb of 5 '-flanking sequence and 50kb of 3' -flanking sequence in addition to PAC AF163864 and BAC AC097478, all human genes) was microinjected into the C57BL6/J ovum, resulting in homozygous α -Syn-Tg mice. GBA-heterozygous KO mice were purchased from The Jackson Laboratory (B6.129S6-Gbatm1Nsb/J, stock number 003321) and mated with human α -Syn-BAC-Tg mice. The resulting normal human α -Syn-BAC-Tg/GBA-heterozygous-KO (homozygous/heterozygous) mice were maintained as strains.
α -Syn-BAC-Tg/GBA-heterozygous-KO mice were mated for more than 10 generations and used for immunohistochemistry and biological analysis at 1, 6 and 24 months of age (N ═ 3).
Immunohistochemistry
For immunohistochemistry, mouse or human brains were fixed with 4% paraformaldehyde and embedded in paraffin.
Paraffin-embedded brain sections were dewaxed and rehydrated, antigen activated (microwaved at 100 ℃ for 5 minutes in 10mM citrate buffer, pH 6.0. the process was repeated 3 times), and cooled to Room Temperature (RT). To stain for phospho- α -Syn, sections were further activated with 98% formic acid (WAKO, 066-00461) for 5 minutes at room temperature.
Sections were washed 2 times in PBST (PBS containing 0.1% Tween-20) for 5 minutes, treated with PBS containing 0.5% Triton X-100 for 20 minutes, and then washed 3 times in PBST for 5 minutes. After blocking (incubation with 10% FBS for 30 min at 37 ℃), incubations were carried out successively as follows: sections were incubated with 2% FBS and 0.1% Triton X-100, primary antibody (mouse anti-phosphorylated-. alpha. -Syn (Ser129) (1:2000, WAKO, 015-reservoir 25191)) for 60 min at 37 ℃; incubation with mouse anti-alpha-Syn (1:1000, Abcam, ab27766) for 12 hours at 4 ℃; incubation with either rabbit anti-phospho-MARCKS (Ser46) (1:1000, glbiochem (shanghai) Ltd.) or mouse anti-ubiquitin antibody (1:1000, Cell Signaling Technology, 3936S) for 120 min at 37 ℃; the quality was evaluated in accordance with the method described in Thr203/Tyr205-ERK1/Thr183/Tyr185-ERK2 for detection of rabbit anti-phosphorylation-
For double labeling of pSer129- α -Syn and ubiquitin, anti-pSer 129- α -Syn antibodies were labeled with the Zenon AlexaFluor 488 mouse IgG1 labeling kit (Z-25002, Invitrogen). Nuclei were stained with DAPI (syngeneic institute, D523). The image, Olympus FV1200 IX83(Olympus), was obtained by confocal microscopy.
Western blotting method
Mouse cerebral cortex and human temporal lobe tissue were dissolved in extraction buffer containing 2% SDS, 1mM DTT and 10mM Tris-HCl (pH7.5) and homogenized on ice using a 20-stroke Dounce glass homogenizer. The crude extract was centrifuged at 16,000g for 10 minutes at 4 ℃. The cells were separated by equal volumes of loading buffer (0.1M Tris-HCl pH7.5, 4% SDS, 20% glycerol, 12% beta-mercaptoethanol and 1% SDS-PAGE, transferred onto polyvinylidene fluoride membranes (Immobilon-P, Merck Millipore) by semidry method, blocked with 5% milk or 2% broth (broth). BSA dissolved in TBST (10mM Tris/HCl pH8.0,150mM NaCl and 0.05% Tween-20) was reacted with the following primary and secondary antibodies diluted with Can Get Signal solution (Toyobo.) and diluted with mouse anti-phosphorylation-alpha-Syn (Ser129) (1:5000, WAKO, 015-91), mouse anti-phosphorylation-alpha-Syn (1:5000, Abcam, 27766) for 12 hours at 4 ℃ and rabbit anti-phosphorylation-Ser (355000, Abcam, 27766) for 12 hours (1:5000, Abcam, 2725: Ser 1: 120: Hash) (GL 1: Lchemi; Ser 25: 25100; Ser 1: 5000; 29: 25: 25100: 25: GL; Biochem; 2515.), cell Signaling Technology, 3936S) at 4 ℃ for 12 hours; rabbit anti-phosphorylation-ERK 1/2(Thr203/Tyr205 (mouse) -ERK1/Thr183/Tyr185 (mouse) -ERK2, Thr202/Tyr204 (human) -ERK1/Thr185/Tyr187 (human) -ERK2) (1:10,000, Cell Signaling technology, # 4370); or rabbit anti-ERK 1/2(1:5,000, Cell Signaling Technology, # 4695S); anti-mouse IgG HRP conjugates (1:3000, GE Healthcare, NA931VA) and anti-rabbit IgG (1:3000, GE Healthcare, NA934 VS). For detecting the bands using LAS4000(GE Healthcare), ECL Prime (GE Healthcare, RPN2232) or SCL Select (GE Healthcare, RPN2235) was used.
Immunoprecipitation
Mouse and human brain samples were dissolved in TNE buffer (10mM Tris-HCl (pH7.5), 10mM NaCl, 1mM EDTA, 1% NP-40, 0.5% protease inhibitor cocktail, 0.5% phosphatase inhibitor cocktail). Aliquots were incubated with 50% slurry of Protein G agarose beads (Protein G Sepharose beads) (GE Healthcare) followed by centrifugation (2000 Xg) for 3 minutes. The supernatant was incubated with 1. mu.g of rabbit anti-pSer 46-MARCKS or rabbit anti-pSer 129-tau-synuclein antibody overnight at 4 ℃. Incubated with protein G agarose beads (GE Healthcare) for 4 hours, washed with TNE buffer, and eluted with loading buffer.
Peripheral blood cell collection from AD model mice
Peripheral blood cells (polymorphonuclear leukocytes (PMNs) and Monocytes (MCs)) were collected using polymorphrep solution (ale Technologies AS) according to the manufacturer's instructions. Briefly, 1mL of venous blood containing EDTA (final concentration 2.0mM) was carefully overlaid on 1mL of Polymorphprep in a 15mL tube. Centrifugation at 500 Xg for 30 min at room temperature followed by removal of plasma yielded a PMN and MC containing layer. The collected aliquots were diluted with 0.45% NaCl and centrifuged at 400 Xg for 10 minutes, thereby obtaining cell pellets. The pellet was dissolved in a dissolution buffer (10mM Tris-HCl (pH7.5), 0.2% SDS, 0.5% protease inhibitor mixture and 0.5% phosphatase inhibitor mixture), and added to a mixture of 62.5mM Tris-HCl, pH6.8, 2SDS, 2.5% (v/v) 2-mercaptoethanol, 5% (v/v) glycerol and 0.0025% (w/v) bromophenol blue to carry out SDS-PAGE.
Mass spectrometry
For phosphoprotein analysis, phosphorylated peptides were prepared according to the method described in Fujita et al, (2016) Sci Rep 6:31895 and Tagawa et al, (2015) Hum Mol Genet 24: 540-.
Specifically, brain extracts of mouse and human cerebral cortex sources are denatured by surfactant and heat treatment, followed by reduction of cysteine residues, blocking by alkylation. The protein sample was digested with trypsin.
Labeling was performed using iTRAQ reagent multiplexing kit (SCIEX Ins), separation was performed using strong cation exchange chromatography, and phosphopeptides were enriched using titanphere Phos-TiO kit (GL Sciences Inc). Each fraction was analyzed using an LC-MS/MS system (Triple TOF 5600 system, Eksigent LC system, SCIEX Ins.) and a C18 column (0.1X 100 mm; KYATECOLOGIEs Corporation). The ion spray voltage was 2.3kV, and IDA (information-dependent acquisition) was set to 400-1250 m/z.
Data analysis
Mass spectral data of the peptides were acquired and analyzed by analysis TF (version 1.6) (AB SCIEXA). The result corresponds to a protein retrieved from UniProtKB/Swiss-Prot (downloaded from http:// www.uniprot.org on
The allowable deviation for peptide retrieval by ProteinPilot was set at 0.05Da for MS and 0.10Da for MS/MS analysis. Proteins identified as redundant were removed using the Pro Group algorithm (AB SCIEX).
Confidence scores for protein or peptide identification were calculated by ProteinPilot and used as confidence thresholds. The threshold for detection was set at 95% confidence and peptides with greater than 95% confidence were accepted as identified peptides.
The quantification of proteins was performed by analysis of iTRAQ reporter groups in MS/MS spectral data generated upon cleavage in a mass spectrometer. For quantification of peptides and proteins, bias corrections were used between different iTRAQ reports to normalize the signals, assuming that the total amount of signal from each iTRAQ had to be equal. For quantification of peptides, bias correction options were used to normalize the different iTRAQ signals.
Peptide ratios were calculated as the ratio of the reported signal in the disease model after bias correction relative to the control model. Details of this formulation are set forth in the handbook of AB SCIEX.
Summary results of peptides from ProteinPilot were exported as Excel files for further data analysis. The amount of peptide fragments was calculated as the geometric mean of the signal intensity of multiple MS/MS fragments containing phosphorylation sites.
All disease groups were evaluated for biological differences from the control group by the Welch test. To correct for multiple tests, the p-value was adjusted using the Benjamini-Hochberg program.
Phosphopeptides that changed in multiple disease groups were identified and selected for further analysis.
Human brain samples of temporal and occipital lobe for inter-human brain proteome analysis were dissected from 5 human AD, 5 human DLB and 5 human age-matched normal control patients and cryogenically frozen (-80 ℃) within 1 hour after death.
Results
Elevation of pSer46-MARCKS levels in the phosphorylated proteome of human AD and DLB
Following previous analysis of brain samples from AD mouse models and human AD patients (Tagawa et al, (2015) HumMol Genet 24: 540-. The front ends of the occipital and temporal lobes of pathologically simple AD (5 men) and DLB (5 women) brains were used.
In this case, postmortem brains with simple pathology were selected, but intracellular α -Syn aggregates were localized within neurons, without extracellular Α β aggregates or cytoplasmic Tau/TDP43 lesions. From this comparison, it was found that the phosphorylation sites were shared between the AD sample and the DLB sample.
pSer46-MARCKS, one of such modifications, was elevated in a mouse model of AD and in post-mortem human AD brain (Fujita et al, (2016) Sci Rep 6:31895) (FIG. 10A).
Interestingly, pSer46-MARCKS levels were not elevated in the occipital and temporal lobes of DLB, but were elevated in the opposite pattern (i.e., in the occipital and not in the temporal lobes) in AD (fig. 10B). In 5 × FAD mice, the elevation of pSer46-MARCKS levels at the early stage of the disease (preclinical/pre-aggregation stage) normalized at the later stage (Fujita et al, (2016) SciRep 6: 31895).
Thus, the difference in pSer46-MARCKS levels between 2 lobes likely reflects the preference of the neurologically degenerated lobe for each disease. Mass spectrometry results suggest that a common change occurs in the AD and PD/DLB human brains, indicating that it is necessary to investigate whether pSer46-MARCKS reflects the preclinical/pre-aggregation phase in PD/DLB.
Increase of pSer46-MARCKS in immunohistochemistry of human DLB
Next, it was examined whether pSer46-MARCKS was elevated in human post-mortem DLB brains. For this purpose, the inventors found that occipital leaf samples from DLB patients were stained and apical dendrites labeled with MAP2 were co-stained by pSer46-MARCKS (fig. 11A).
On the other hand, it was confirmed that most of the brain region contained ubiquitinated pSer129- α -Syn cytoplasmic inclusion bodies (FIG. 11B) (FIG. 11C). Since cytoplasmic staining of pSer46-MARCKS was considered to be aggregates (FIG. 11A), examining whether they were alpha-Syn aggregates, it was found that half of the cytoplasmic pSer46-MARCKS positive structures of neurons were stained by pSer 129-alpha-Syn (yellow arrows). However, the other half of them was not strongly stained by pSer129- α -Syn (FIG. 11D, red arrow).
It was suggested that pSer46-MARCKS positive/pSer 129- α -Syn negative cells could correspond to indirectly affected peripheral neurons via HMGB1 released by injured neurons accumulating pSer129- α -Syn, and that pathology continued in the human brain even at the end of DLB.
To confirm the above results of immunohistochemistry, the present inventors performed western blotting of pSer46-MARCKS and total MARCKS of occipital lobe with human post-mortem DLB patients and non-DLB control patients (fig. 11E). Levels of pSer46-298 MARCKS were increased in DLB patients.
Increase of pSer46-MARCKS in humanized α -Syn-Tg mice
To determine the initial time point of increase of pSer46-MARCKS in the brain, human α -Syn-BAC-Tg/GBA-heterozygous-KO mice of 1, 6 and 24 months of age, showing no motor dysfunction or behavioural abnormality in the animals, were analyzed.
Mice slightly elevated the signal of pSer46-MARCKS in the olfactory bulb, frontal cortex and parietal cortex at 1 month (fig. 12, fig. 18). Then, the area of positive staining of pSer46-MARCKS expanded to the parietal and occipital cortex at 6 months (fig. 12, fig. 19), but did not increase in the hippocampus by 24 months (fig. 12, fig. 20).
No significant cytoplasmic alpha-Syn aggregates stained with anti-phosphorylated alpha-Syn (pSer 129-alpha-Syn) antibody were detected in neurons from the inner embryonic leaf layer and the mitral cell layer of the olfactory bulb until 24 months of age (FIGS. 13A-C). FIGS. 13A-C show high-magnification images of olfactory bulbs of human α -Syn-BAC-Tg/GBA-hybrid KO mice co-stained with antibodies against pSer46-MARCKS and pSer129- α -Syn. Spot-stained cytoplasmic staining of pSer46-MARCKS (white arrow) and pSer129- α -Syn (Star stamp) was detected from 1 month of age, but cytoplasmic aggregates of pSer129- α -Syn were detected only at 24 months of age. alpha-Syn-skein-like or Lewy neurite-like structures are present in these layers from 1 month of age, increasing substantially in the course of aging. These structures were not stained simultaneously by anti-ubiquitin antibodies at 1 month, but were clearly co-stained after 6 months (fig. 13D).
Interestingly, pSer46-MARCKS positive neurites were more than p- α -Syn-co-stained neurites.
Taken together, these results indicate that neurite changes detected by pSer46-MARCKS preceded the formation of α -Syn ubiquitin-positive aggregates, at least in this PD/DLB mouse model (table 1).
Cytoplasmic α -Syn aggregates were also detected in the mural cortex at 24 months of age (fig. 14A). The relationship of pMARCKS to p- α -Syn and p- α -Syn to ubiquitin in aging cortical neurons was the same as that of olfactory neurons (FIG. 14B).
To confirm these results, the present inventors performed Western blot analysis of pSer46-MARCKS, pSer129- α -Syn and ubiquitin with cerebral cortical tissues aged 1, 6 and 24 months (FIG. 15). The level of phosphorylated MARCKS was elevated at 1, 6 and 24 months of age, but phosphorylated a-Syn and ubiquitin were produced and increased substantially after 6 months (fig. 15).
The present inventors also tested the level of pSer46-MARCKS in peripheral blood cells, found to be much lower than in the brain (FIG. 21).
Interaction of MARKCS with alpha-Syn in DLB brain
Considering that half of the cytoplasmic aggregates were double positive for pSer46-MARCKS and pSer129- α -Syn (FIG. 11D), the inventors examined the biochemical interactions of 2 phosphoproteins. Immunoprecipitation of simultaneous pellet of anti-pSer 46-MARCKS and pSer129-a-Syn from 24-month-old α -Syn-BAC-Tg/GBA-heterozygous KO mice (FIG. 16A) (whole cerebral cortex) (FIG. 16A) and human DLB patients (FIG. 16B) was performed. Reverse precipitation of anti-pSer 129-a-Syn antibody also co-precipitated pSer46-MARCKS in both mouse models and human patients (FIGS. 16A and B). The results indicate biochemical interactions of 2 phosphoproteins.
Activation of upflow kinase in DLB brain
Finally, an upstream kinase that phosphorylates MARCKS at Ser46 was examined. MARCKS is a representative substrate for PKC known as a substrate for myristoylated alanine-rich protein kinase C. PKA α has been reported to phosphorylate Ser159, Ser163 and Ser 170. On the other hand, Erk1/Erk2(═ MAPK3/MAPK1) is known to phosphorylate MARCKS on Ser46 instead of PKC. Consistently, other groups also showed that MAPK phosphorylates MARCKS in hippocampal neurons, but the exact phosphorylation site of MAPK was determined in these studies.
Thus, activation of Erk1/2 by immunohistochemistry and western blotting was examined using rabbit monoclonal antibodies for detection of Erk1 phosphorylation in Thr202/Tyr204 and Erk2 phosphorylation in Thr185/Tyr187 (fig. 17A).
As expected, the immunostaining of pErk1/2 was increased in the cerebral cortex of α -Syn-BAC-Tg/GBA-heterozygous KO mice (FIG. 17A). Cells were simultaneously stained with MAP2, but were not co-stained at GFAP, indicating neurons (fig. 17B). Staining with pErk1/2 was consistent with their enzyme-substrate relationship with pSer46-MARCKS co-localization in the same neuron (FIG. 8A). In the occipital cortex of DLB patients, the same co-staining was demonstrated with increased pSer46-MARCKS and pErk1/2 (FIG. 17C). Western blot analysis also confirmed an increase in pErk1/2 in the cerebral cortex of α -Syn-BAC-Tg/GBA-heterozygous KO mice (FIG. 17D) and DLB patients (FIG. 17E). Together, these analyses demonstrated an abnormal increase in Erk1/2 phosphorylation and an age-dependent increase in cortical neurons under PD/DLB pathology (fig. 17A-E).
From the results of the above examples, the following is clear.
It was shown that the level of MARCKS phosphorylated at pSer46, a feature of neural mutation in the pre-aggregate stage of AD pathology, was also elevated in PD/DLB pathology in both mouse models and human patients.
Immunohistochemical staining patterns of pSer46-MARCKS were also similar in AD and PD/DLB lesions. Neural mutations identified by pSer46-MARCKS in the mouse model were initially detected in the olfactory bulb and then became apparent in the occipital and temporal cortex (table 1). Table 1 shows a summary of pSer46-MARCKS, pSer 129-alpha-Syn and ubiquitin staining patterns in human alpha-Syn-BAC-Tg/GBA-hybrid KO mice. "+/-" indicates that in background mice, positive staining was less than 10% of the cells and not observed, "+" indicates that positive staining was observed in 10% to 50% of the cells of Tg mice, and "+ +" indicates that positive staining was observed in more than 50% of the cells of Tg mice.
This pattern is consistent with the proposed progression of nervous system degeneration in human PD/DLB pathology, with the earliest lesions appearing in the olfactory bulb, with occipital lobes eventually predominating.
[ Table 1]
Summary of pathological changes in alpha-Syn-BAC-Tg/GBA-hybrid KO mice
The second important conclusion of this study is that neural mutations precede disease-associated protein aggregation at the histological level. In the mouse model, pSer46-MARCKS increased prior to formation of ubiquitinated α -Syn aggregates (Table 1).
The time-series of the PD/DLB mouse model of pSer46-MARCKS formed prior to the histological aggregates confirmed the staining detected by pSer46-MARCKS in the cytoplasm and neurites of neurons (similar to the previously observed extracellular A β aggregates in AD model mice).
Using this model, the inventors confirmed the importance of pSer46-MARCKS at the biochemical level. These results strongly suggest that intracellular misfolded α -Syn, not in aggregate but in monomeric or oligomeric form, plays a more important role in the onset of neural degeneration.
Interestingly, in the human postmortem brain, pSer46-MARCKS was not elevated in the severely affected areas (occipital lobe of DLB, temporal lobe of AD) but in areas of the brain relatively unaffected by disease (temporal lobe of DLB, occipital lobe of AD).
The reason for this is not yet established. One possibility is that the neurites in the severe brain regions "disappear" metabolically and fail to maintain a high level of pSer46-MARCKS ".
Based on these observations, it is worthwhile to develop pSer46-MARCKS as a biomarker capable of detecting molecular pathology in PD/DLB at the very early (pre-aggregation/preclinical) stage.
Furthermore, it is useful to develop mass spectrometry-based detection and ELISA-based detection and/or PET. If such a highly sensitive detection system could be utilized, it is considered to be directly linked to the detection of an ultra-early stage pathology in a population at risk of a neurodegenerative disease.
Preliminarily, the present inventors investigated whether or not Peripheral Blood Cells (PBC) such as erythrocytes, granulocytes or lymphocytes express pSer46-MARCKS by Western blotting. If so, it is difficult to distinguish between PBC-derived pSer46-MARCKS and brain-derived pSer46-MARCKS, and more skill is required when using pSer46-MARCKS as a biomarker for extracerebral diagnosis. Fortunately, PBC did not express detectable levels of pSer46-MARCKS comparable to brain tissue of 5xFAD mice (fig. 21). Thereby supporting the ability to develop pSer46-MARCKS as a blood biomarker.
The inventors found co-localization in cytoplasmic aggregates (FIG. 13, FIG. 14A, FIG. 14B) and biochemical interactions (FIG. 16) of pSer46-MARCKS and pSer129- α -Syn. Interestingly, both α -Syn and MARCKS are considered proteins (IDPs) that are disordered in nature. In previous studies, it was shown that α -Syn has naturally denatured properties.
On the other hand, previous reports predicting MARCKS to be IDP analyzed by bioinformatics of available algorithms (RONN v3.2, https:// www.strubi.ox.ac.uk/RONN and IUPred, http:// iuupred. enzim. hu) supported this idea by structural biology experiments. The same tendency of denaturation for α -Syn and MARCKS is likely to underlie their biochemical interactions. Furthermore, both α -Syn and MARCKS are restricted to dystrophic neurites and are important in axonal growth and actin network regulation of such axonal end functions.
Since the pathological cycle from neural degeneration to cell death repeatedly occurs in the brain with neurodegenerative disease before all neurons to the brain die, the elevation of pSer46-MARCKS and DO can be used as a quantitative biomarker for the in vivo activity of nervous system degeneration, although not disease-specific, as well as GOT and GPT, which are general quantitative indicators of hepatocellular disorders in multiple liver disease, in symptomatic patients and presumably non-symptomatic subjects. This example provides novel biomarkers that reflect neural mutational activity of degenerative diseases of the nervous system.
Industrial applicability
The method of the invention can be used for the detection of a neurodegenerative disease selected from the group consisting of human AD (alzheimer's disease), FTLD (frontotemporal lobar degeneration), ALS (amyotrophic lateral sclerosis), PD (parkinson's disease) and DLB (dementia of the lewy body type).
All publications, patents and patent applications cited in this specification are herein incorporated in their entirety by reference.
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