JP2018091844A - METHOD FOR DETECTING α-SYNUCLEIN - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting α-synuclein useful for differentiating and assisting the differentiation between α-synucleinopathy and other dementia, and a method for assisting and assisting the diagnosis of α-synucleinopathy. To provide. SOLUTION: (1) A step of mixing a biological sample and a normal α-synuclein in a buffer to obtain a reaction mixture; (2) Incubating the reaction mixture to obtain an abnormal α-synuclein in the biological sample. A method for detecting abnormal α-synuclein in a biological sample, which comprises a step of forming an agglomerate of α-synuclein seeded with the above; and (3) a step of detecting an agglomerate of the formed α-synuclein. [Selection diagram] None

Description

  The present invention relates to a method for detecting α-synuclein, particularly to a method for detecting aggregates of fibrotic α-synuclein, and a method for assisting in the diagnosis of α-synucleinopathies such as Lewy body disease.

  Accumulation of large amounts of misfolded proteins in the brain is a critical feature of most neurodegenerative diseases. Lewy body diseases (LBD) such as dementia with Lewy bodies (DLB) and Parkinson's disease (PD) are mainly composed of aggregated α-synuclein (αSyn) and are Lewy bodies which are cytoplasmic fibrous inclusions. It is characterized by the presence of (LB). Although the pathogenic mechanism has not been fully elucidated, LBD is thought to be caused by accumulation of LB in neurons, ie aggregated αSyn.

  Most of the α-Syn aggregates in LB are phosphorylated by serine 129 (Ser129), whereas αSyn in normal brain is hardly phosphorylated. Phosphorylation at Ser129 is a pathological feature of LBD and may be crucial for LB formation and development of LBD. In the report, polymerization of recombinant αSyn (r-αSyn) was promoted by phosphorylation of Ser129 (Fujiwara et al., 2002 (Non-patent Document 1)). Overexpression of wild-type (WT) r-αSyn induced significant accumulation of LB-like inclusion bodies in cultured cells, but not with non-phosphorylated mutants in which Ser129 was replaced with alanine (S129A). (Smith et al., 2005 (non-patent document 2)). Ser129 phosphorylation inhibits r-αSyn fibrosis (Paleologou et al., 2008 (Non-Patent Document 3)), and mutation of S129A increases inclusion body formation in the Drosophila model (Chen and Feany, 2005 (Non-patent Document) 4)) was proved by other groups. Studies with Drosophila have also shown that the number of inclusion bodies is inversely correlated with toxicity to dopaminergic neurons, indicating that inclusion bodies prevent neurotoxicity (Chen and Feany). 2005 (Non-Patent Document 4)). However, studies in rat models using recombinant adeno-associated virus (rAAV) to overexpress WT αSyn and mutant αSyn have shown that non-phosphorylated αSyn is more severe for dopaminergic neurons than phosphorylated. (Azeredo da Silveira et al., 2009 (Non-Patent Document 5); Gorbatyuk et al., 2008 (Non-Patent Document 6)), and there is no significant difference in neurotoxicity between the two types ( McFarland et al., 2009 (Non-Patent Document 7)). Therefore, the importance of Ser129 phosphorylation of αSyn in the development of LBD remains largely unknown.

  Postmortem studies by Braak and co-workers have shown that LB lesions first occur in the medulla and olfactory bulb, then extend to the midbrain and limbic regions, and then spread to the neocortical regions in PD ( Braak et al., 2003 (Non-Patent Document 8)). Furthermore, the pathological stage seems to be closely linked to lesion progression and clinical symptoms (Braak et al., 2005). The spread of LB in the brain suggests that pathological αSyn retains the ability to propagate like a prion. Transplantation studies have provided evidence that LB is present in normal neurons of the fetal mesencephalon transplanted into the striatum of PD patients, which led to healthy healthy transplanted lesions from the host brain. It is shown that it can propagate to the brain (Kordower et al., 2008 (Non-Patent Document 10); Li et al., 2008 (Non-Patent Document 11)). In addition, WT (Luk et al., 2012b (Non-patent Document 12)) and A53T Tg mice (Luk et al.) Of lesion brain homogenates derived from A53T mutant transgenic (Tg) mice of human αSyn showing abnormal movements. , 2012b (Non-patent document 12); Mougenot et al., 2012 (Non-patent document 13); Watts et al., 2013 (Non-patent document 14)), and inoculation with endogenous αSyn-derived LB-like aggregates and cells. Diffusion occurred, reducing the survival time exhibited by A53T Tg mice (Luk et al., 2012b). WT mice inoculated with brain extracts from DLB patients (Masuda-Suzukike et al., 2013) and A53T Tg mice inoculated with brain homogenates from multiple system atrophy (MSA) patients (Watts et al., 2013 (Non-Patent Document 14)), a similar prion-like phenomenon was induced. Furthermore, by introducing artificially formed fibrotic r-αSyn, primary neurons (Volpicelli-Dalley et al., 2011 (Non-patent Document 16)) and WT mice (Luk et al., 2012a (Non-patent Document 17) )), Cell-to-cell transmission of the lesion αSyn was induced and dopamine neuron loss resulting in movement disorders developed. These reports suggested that αSyn behaves like a prion.

  We have previously named “Real-time quaking-induced conversion (RT-QUIIC)”, an in vitro amplification technique for prion detection in tissues and body fluids. Developed. In this assay, the abnormal prion protein as a seed and the recombinant prion protein as a substrate are mixed, and then a continuous reaction by stirring is performed to induce amyloid formation, which is considered to be a mechanism of prion propagation. It became possible to observe the autocatalytic reaction over time (Atarashi et al., 2011 (Non-patent Document 18)).

  Recently, a method using an RT-QUIIC assay to detect the prion-like structural transformation of r-αSyn has been reported (Fairfoul, G. et al., 2016 (Non-patent Document 19)). Currently, at least 48 hours are required, and more rapid detection is required.

Fujiwara, H .; Hasegawa, M .; Dohmae, N .; , Kawashima, A .; Masliah, E .; Goldberg, M .; S. , Shen, J .; Takao, K .; , And Iwatsubo, T .; (2002). alpha-Synnuclein is phosphorylated in syndrome pathions. Nature cell biology 4, 160-164. Smith, W.M. W. Margollis, R .; L. Li, X .; , Troncoso, J .; C. Lee, M .; K. Dawson, V .; L. Dawson, T .; M.M. , Iwatsubo, T .; and Ross, C.I. A. (2005). Alpha-synuclein phosphorescence enhancement eosinophilic cytoplasmic inclusion formation in SH-SY5Y cells. The Journal of neuroscience: the official journal of the Society for Neuroscience 25, 5544-5552. The Journal of neuroscience: the official journal of the Society for Neuroscience 25, 5544-5552. Paleologou, K .; E. Schmid, A .; W. Rospigliosi, C.I. C. Kim, H .; Y. , Lamberto, GR. Fredenburg, R .; A. Lanbury, P .; T.A. , Jr. , Fernandez, C.I. O. Elliezer, D .; , Zwecketter, M .; , Et al. (2008). Phosphorylation at Ser-129 but not the phophomics S129E / D inhibits the fibration of alpha-synclein. The Journal of biologic chemistry 283, 16895-16905. Chen, L .; , And Feany, M .; B. (2005). Alpha-synuclein phosphorylation control neurotoxicity and inclusion formation in a Drosophila model of Parkinson disease. Nature neuroscience 8, 657-663. Azeredo da Silveira, S.A. Schneider, B .; L. Cifentes-Diaz, C.I. Sage, D .; Abbas-Terki, T .; , Iwatsubo, T .; Unser, M .; , And Aebischer, P.A. (2009). Phosphorylation dos not prompt, the prevent, the formation of alpha-synucleic toxins in a rat model of Parkinson's disease. Human molecular genetics 18, 872-887. Gorbatyuuk. O. S. Li, S .; Sullivan, L .; F. Chen, W .; , Kondrikova, G .; Manfredsson, F .; P. Mandel, R .; J. et al. , And Muzyczka, N .; (2008). The phosphorylation state of Ser-129 in human alpha-synclein determines neurodegeneration in a rat model of Parkinson disease. Proceedings of the National Academy of Sciences of the United States of America 105, 763-768. McFarland, N.M. R. , Fan, Z. Xu, K .; , Schwartzschild, M .; A. , Feany, M .; B. Hyman, B .; T.A. , And McLean, P.M. J. et al. (2009). Alpha-synclein S129 phosphoration mutants do not alter tactical proximity in a rat model of Parkinson disease. Journal of neuropathology and experimental neurology 68, 515-524. Braak, H .; Del Tredici, K .; Rub, U .; , De Vos, R .; A. Jansen Steur, E .; N. , And Braak, E .; (2003). Staging of brain pathology related to sporadic Parkinson's disease. Neurobiology of aging 24, 197-211. Braak, H .; Rub, U .; Jansen Steur, E .; N. Del Tredici, K .; , And de Vos, R .; A. (2005). Cognitive status correlates with neuropathological stage in Parkinson disease. Neurology 64, 1401-1410. Kordower, J .; H. , Chu, Y .; , Hauser, R .; A. , Freeman, T .; B. , And Onowow, C.I. W. (2008). Lewy body-like pathology in long-term embryonic transients in Parkinson's disease. Nature medicine 14, 504-506. Li, J. et al. Y. Englund, E .; Holton, J .; L. , Soulet, D .; Hagel, P .; Lees, A .; J. et al. Lashley, T .; , Quinn, N .; P. Rehncrona, S .; , Bjorkland, A .; , Et al. (2008). Lewy bodies in grafted neurons in subjects with Parkinson's disease suggest host to-graft disease propagation. Nature medicine 14, 501-503. Luk, K .; C. Kehm, V .; M.M. Zhang, B .; O'Brien, P .; , Trojanowski, J. et al. Q. , And Lee, V .; M.M. (2012b). Intracerebral inoculation of pathological alpha-synchronin initiates a rapidly progressive neurodegeneratively in the world. Mougenot, A.M. L. , Nicot, S .; , Bencsik, A .; , Morignat, E .; Verchere, J .; Lakhdar, L .; , Legastelois, S .; , And Baron, T .; (2012). Prion-like acceleratio of a synthinopathy in a transgenic mouse model. Neurobiology of aging 33, 2225-2228. Watts, J .; C. Giles, K .; Oehler, A .; Middleton, L., et al. Dexter, D .; T.A. Gentleman, S .; M.M. , DeArmond, S.M. J. et al. , And Prusiner, S .; B. (2013). Transmission of multiple system atropri- tions to transgenic rice. Proceedings of the National Academy of Sciences of the United States of America 110, 19555-19560. Masuda-Suzuki, M .; , Nonaka, T .; Hosokawa, M .; , Oikawa, T .; , Arai, T .; , Akiyama, H .; Mann, D .; M.M. , And Hasegawa, M .; (2013). Prion-like spreading of pathological alpha-synuclein in brain. Brain: a journal of neurology 136, 1128-1138. Volpicelli-Dalley, L.M. A. Luk, K .; C. Patel, T .; P. , Tanik, S .; A. Riddle, D .; M.M. , Steiber, A .; , Meaney, D .; F. , Trojanowski, J. et al. Q. and Lee, V .; M.M. (2011). Exogenous alpha-synclein fibrils induce Lewy body pathology leading to synthetic dysfunction and neuron death. Neuron 72, 57-71. Luk, K .; C. Kehm, V .; Carroll, J .; Zhang, B .; O'Brien, P .; , Trojanowski, J. et al. Q. , And Lee, V .; M.M. (2012a). Pathological alpha-synuclein transmission initiations Parkinson-like neurodegeneration in nontransgenic mice, Science (New York, NY) 338, 949-953. Atarashi, R.A. Satoh, K .; Sano, K .; Fuse, T .; Yamaguchi, N .; Ishibashi, D .; , Matsubara, T .; Nakagaki, T .; Yamanaka, H .; Shirabe, S .; , Et. al. (2011). Ultrasensitive human prion detection in cerebrospinal fluid by real-time queuing-induced conversion. Nature medicine 17, 175-178. Fairfoul, G.M. et al. , (2016). Alpha-synclein RT-QuIC in the CSF of patients with alpha-syncleinopathies. Anals of Clinical and Translational Neurology doi: 10.1002 / acn3.338.

  In the case of Alzheimer's disease, amyloid may be detected by examination of cerebrospinal fluid or positron emission tomography (PET) even before the onset of symptoms. In Alzheimer's disease, amyloid aggregation occurs early, so the usefulness of PET examination is known. On the other hand, α-synucleinopathies such as dementia with Lewy bodies are currently being developed to differentiate from other diseases based on symptoms, but it is not possible to differentiate quickly before the onset of symptoms. Have difficulty. The present invention enables early and rapid diagnosis of α-synucleinopathies such as Lewy body disease, α-synuclein from a subject, particularly polymerized or fibrotic abnormal aggregation It is an object of the present invention to provide a method for detecting α-synuclein.

  In order to solve the above-mentioned problems, the present inventors have conducted intensive studies and investigated whether prion-like structural transformation of r-αSyn is induced by brain tissue derived from a DLB patient using an RT-QUIIC assay. Researched. Furthermore, the present inventors verified whether Ser129 phosphorylation of the substrate αSyn is involved in the formation of aggregates. Then, the biological sample of the subject and r-αSyn are mixed in a solution under specific conditions, and the fibrogenic substrate r-αSyn is detected to diagnose α-synucleinopathies such as Lewy body disease Found useful. αSyn fibril formation was specifically detected only when a biological sample of a subject suffering from Lewy body disease was used, and it was found that the fiber was useful for differentiation. Thus, the present invention was completed.

That is, the present invention is as follows.
[1] (1) A step of mixing a biological sample and normal α-synuclein in a buffer solution to obtain a reaction mixture;
(2) Incubating the reaction mixture to form an α-synuclein aggregate using abnormal α-synuclein in a biological sample as a seed; and (3) detecting the formed α-synuclein aggregate. Process,
A method for detecting abnormal α-synuclein in a biological sample, comprising:
[2] (1) A step of mixing a biological sample and normal α-synuclein in a buffer solution to obtain a reaction mixture;
(2) Incubating the reaction mixture to form an α-synuclein aggregate using abnormal α-synuclein in a biological sample as a seed; and (3) detecting the formed α-synuclein aggregate. Process,
A method for assisting diagnosis of α-synucleinopathy, comprising:
[3] The method according to [1] or [2], wherein the normal α-synuclein is recombinant α-synuclein or purified α-synuclein.
[4] The method according to any one of [1] to [3], wherein the biological sample is a brain tissue sample.
[5] The method according to any one of [1] to [3], wherein the biological sample is blood, plasma, serum, leukocyte, or cerebrospinal fluid sample.
[6] The method according to any one of [1] to [5], wherein the concentration of normal α-synuclein in the reaction mixture is 120 μg / mL to 150 μg / mL.
[7] The method according to any one of [1] to [6], wherein the buffer solution is a buffer solution having a pH of 7.2 to 7.8.
[8] The method according to any one of [1] to [7], wherein the buffer is a HEPES buffer.
[9] The method according to any one of [1] to [8], wherein in the step (2), the reaction mixture is shaken.
[10] The method according to [9], wherein the formation of α-synuclein aggregates is carried out in a cycle of a shaking period and a shaking period.
[11] The method according to [10], wherein the cycle is 3 minutes in total.
[12] The method according to any one of [1] to [11], wherein an α-synuclein aggregate is detected using an antibody.
[13] The method according to [12], wherein the antibody is an anti-α-synuclein polyclonal antibody, an anti-α-synuclein monoclonal antibody, or an anti-Ser129 phosphorylated α-synuclein monoclonal antibody.
[14] The method according to any one of [1] to [13], wherein the step (3) comprises quantifying the formed α-synuclein aggregate.
[15] The method according to any one of [1] to [14], wherein the step (3) includes Western blotting.
[16] The method according to any one of [1] to [15], wherein the step (3) includes fluorescence spectroscopy.
[17] The method according to [16], wherein the step of detecting an α-synuclein aggregate comprises the use of thioflavin T (ThT).
[18] The method of [16] or [17], wherein the detection is performed every 10 minutes.
[19] The method according to any one of [2] to [18], which is a method for assisting in differentiation between α-synucleinopathy and other diseases associated with cognitive impairment.
[20] The method according to any one of [2] to [19], wherein the α-synucleinopathy is Lewy body disease or multiple system atrophy.
[21] The method according to [20], wherein the Lewy body disease is Lewy body dementia, Parkinson's disease, or diffuse neocortical Lewy body dementia.

  According to the present invention, abnormal α-synuclein can be detected with high sensitivity and speed from a biological sample of a subject. According to the present invention, it is possible to detect abnormal α-synuclein from a patient before the onset of symptoms, and thus it is possible to diagnose whether or not a subject suffers from α-synucleinopathy earlier. Become. In general, dementia has a different treatment policy depending on its type, but by using the present invention to differentiate α-synucleinopathy at an early stage, it becomes possible to receive more appropriate treatment from an early stage.

FIG. 1 is a diagram showing purification and structural analysis of recombinant α-synuclein. (A, B) Purified His-tagged human recombinant αSyn (His-r-αSyn), His-r-αSyn (His-r-αSyn + DAPase) after DAPase treatment, and His-r-αSyn and DAPase were removed. The later product (r-αSyn) was verified by Western blot analysis with (A) CBB stained SDS-PAGE and (B) anti-α-Syn polyclonal antibody D119 (upper panel) and anti-His antibody (lower panel). (A) CBB-stained SDS-PAGE analysis shows that the size of His-r-αSyn after DAPase treatment is shifted from 18 kDa to 17 kDa, and after removal of His-r-αSyn and DAPase, a single protein The most abundant band was observed. (B) The same molecular size shift of His-r-αSyn was observed by immunoblotting using anti-α-Syn polyclonal antibody D119. It was confirmed that purified r-αSyn was not detected by immunoblotting using an anti-His tag antibody. The molecular weight marker is shown in kilodaltons (kDa) on the left side of each panel. (C) Circular dichroism (CD) spectrum of r-αSyn. The CD spectrum showed the minimum mean residue ellipticity at 119 nm characteristic of denatured proteins. (D) Fourier transform infrared spectroscopy (FTIR) spectrum of r-αSyn. The FTIR spectrum showed a prominent band at 1650 cm ^{−1 that} was attributed to the modified structure. FIG. 2 is a diagram of characterization of human brain-derived α-synuclein by histochemical analysis and Western blot analysis. (A) Hematoxylin and eosin staining and phosphorylated αSyn in the substantia nigra and frontal cortex from 2 DN-DLB patients (cases # 1 and # 2), Li-DLB patients, and non-DLB cases (breast cancer patients) Immunohistochemical staining with antibodies. Arrows and arrowheads indicate LB and pSer129-αSyn, respectively. Scale bar is 50 μm. (BD) DN-DLB (cases # 1 and # 2), Li-DLB, schizophrenia, Alzheimer's disease (AD) (cases # 1 and # 2), arcuate Creutzfeldt-Jakob disease (sCJD) Brain homogenate (BH) samples from patients with type 1 (sCJD MM1) (cases # 1 and # 2), sCJD2 type (sCJD MM2), and Gerstman-Streisler-Scheinker syndrome (GSS), (B) anti-α Immunoblotting with Syn polyclonal antibody D119, (C) anti-pSer129-αSyn monoclonal antibody, and (D) anti-Ser87 phosphorylated α-Syn polyclonal antibody. In the immunoblot analysis for pSer129-αSyn presented in (C), samples were detected with a 30 second short exposure (upper panel) and a 2 minute long exposure (lower panel). The molecular weight marker is shown in kilodaltons (kDa) on the left side of each panel. The arrow indicates the top of the concentrated gel. FIG. 3 is a diagram showing fibril formation of r-αSyn in RT-QUIIC reaction. (A) Human r-αSyn was diluted with the indicated dilutions of DN-DLB (cases # 1 and # 2), Li-DLB, schizophrenia (Sc), AD (cases # 1 and # 2), sCJD MM1 (cases) RT-QUIIC was performed with # 1 and # 2), sCJD MM2, and BSS of GSS patients or without seed (No-seeded). The curve represents the average ThT fluorescence kinetics of all 6 replicate wells. See also FIG. (B) The values of maximum fluorescence intensity and lag phase obtained in individual samples after 96 hours reaction are plotted in the upper and lower graphs, respectively. The lag phase was defined as the time required for the fluorescence intensity to reach> 120 arbitrary units. Horizontal bars indicate mean ± standard deviation. Data for maximum fluorescence intensity was analyzed by one-way analysis of variance followed by Tukey-Kramer test. Data analysis for the lag phase was performed by log rank test and Tukey-Kramer test. ^** , P <0.01 (compare with no seed); ^* , P <0.05 (compare with no seed). (C) The sample was verified by TEM. Bar is 100 nm. (D) The sample was subjected to FTIR analysis. Samples of DN-DLB # 1, DN-DLB # 2, Sc, and AD # 2 of (C) and (D) were seeded with 5 × 10 ⁻⁶ diluted BH. FIG. 4 shows Western blot analysis and RT-QUIIC analysis of four additional DN-DLB brains. (A) BH samples (cases # 3, # 4, # 5 and # 6) from DN-DLB patients were treated with anti-α-Syn polyclonal antibody D119 (left panel) and anti-pSer129-αSyn monoclonal antibody (right panel). Immunoblot. The molecular weight marker is shown in kilodaltons (kDa) on the left side of each panel. The arrow indicates the top of the concentrated gel. (B) RT-QUIIC was performed using human r-αSyn with the indicated dilutions of DN-DLB-derived BH. The curve represents the average ThT fluorescence kinetics of all 6 replicate wells. See also FIG. (C) The values of maximum fluorescence intensity and lag phase obtained in individual samples after 96 hours reaction are plotted in the upper and lower graphs, respectively. The lag phase was defined as the time required for the fluorescence intensity to reach> 120 arbitrary units. Horizontal bars indicate mean ± standard deviation. Data for maximum fluorescence intensity was analyzed by one-way analysis of variance followed by Tukey-Kramer test. Data analysis for the lag phase was performed by log rank test and Tukey-Kramer test. ^** , P <0.01 (compare with no seed); ^* , P <0.05 (compare with no seed). FIG. 5 shows the seeding activity of r-αSyn insoluble aggregates induced by incubation. WT or S129A r-αSyn (14 μg) in the presence of 200 μM ATP in 140 U casein kinase 2 (CK2) or 35 μl reaction buffer (20 mM Tris-HCl, pH 7.5, 50 mM KCl and 10 mM MgCl ₂ ) (+) Or in the absence (-) and incubated at 37 ° C. (A) After incubation at 0 (left panel), 72 (middle panel) or 264 hours (right panel), samples were immunoblotted with anti-α-Syn polyclonal antibody D119 and anti-pSer129-αSyn monoclonal antibody. The molecular weight marker is shown in kilodaltons (kDa) on the left side of each panel. The arrow indicates the top of the concentrated gel. (B) WT r-αSyn mixed with ^{CK2 in the} absence (WT ^CK2 ) or presence (WT ^{CK2 + ATP} ) and S129r-αSyn mixed with CK2 and ATP after incubation for 0, 72, 168 and 264 hours The ThT fluorescence level of (S129A ^{CK2 + ATP} ) was measured. Data are expressed as mean ± standard deviation (n = 4). (C) WT or S129A r-αSyn incubated with CK2 and ATP for 0 hour (WT-0h and S129A-0h) or 264 hours (WT-264h and S129A-264h) were subjected to FTIR analysis. (D) The sample was verified by TEM. Bar is 200 nm. (E) The seeding activity of the samples of WT-0h, WT-264h, S129A-0h, and S129A-264h was evaluated by dilution at 2 × 10 ⁻² and 2 × 10 ⁻⁴ by RT-QUIC. The curve represents the average ThT fluorescence kinetics of 3 or 4 replicate wells. FIG. 6 is a diagram showing the seeding activity of r-αSyn oligomers produced by RT-QUIIC. (A) WT or S129A r-αSyn without seed, by RT-QUIIC in the presence of 125 U CK2 alone (WT ^CK2 and S129A ^CK2 ) or both 125 U CK2 and 200 μM ATP (WT ^{CK2 + ATP} and S129A ^{CK2 + ATP} ) (12.5 μg) of fibril formation was induced. The curve represents the average ThT fluorescence kinetics of triplicate wells. (B) RT-QUIIC samples (WT ^CK2 , WT ^{CK2 + ATP} , S129A ^CK2 , and S129A ^{CK2 + ATP} ) were immunoblotted with anti-α-Syn polyclonal antibody D119 and anti-pSer129-αSyn monoclonal antibody. The molecular weight marker is shown in kilodaltons (kDa) on the left side of each panel. The arrow indicates the top of the concentrated gel. (C) RT-QUIIC samples (WT ^CK2 , WT ^{CK2 + ATP} , S129A ^CK2 , and S129A ^{CK2 + ATP} ) were subjected to FTIR analysis. (D) The sample was verified by TEM. Bar is 50 nm. (E) The seeding activity of RT-QUIIC samples (WT ^CK2 , WT ^{CK2 + ATP} , S129A ^CK2 , and S129A ^{CK2 + ATP} ) was evaluated by a dilution of 2 × 10 ⁻⁴ to 2 × 10 ⁻⁸ by subsequent RT-QUIC tests. did. The curve represents the average ThT fluorescence kinetics of replicate wells (n = 3-6). (F) Maximum fluorescence intensity values and lag phase values obtained in individual samples after 72 hours of reaction were plotted in the right and left graphs, respectively. The lag phase was defined as the time required for the fluorescence intensity to reach> 120 arbitrary units. Horizontal bars indicate mean ± standard deviation. Data for maximum fluorescence intensity was analyzed by one-way analysis of variance followed by Tukey-Kramer test. Data analysis for the lag phase was performed by log rank test and Tukey-Kramer test. ^**, (compared to 2 × ^{10 -4} dilution WT- ^mock) P <0.01; *, (compared to 2 × ^{10 -4} dilution WT- mock) P <0.05;##, P < 0.01 (compared to the same dilution of WT ^{CK2 + ATP} ). FIG. 7 is a diagram showing estimation of Ser129 phosphorylated α-synuclein concentration in DLB brain by immunoblotting after Phos-tag SDS-PAGE. (A) In order to generate Ser129 phosphorylated r-αSyn (pSer129-r-αSyn) as a reference for dot blot analysis, r-αSyn was phosphorylated by incubation in the presence of casein kinase 2 (CK2) and ATP. Oxidized. The measurement of pSer129-r-αSyn concentration was performed by Zn ²⁺ -Phos-tag SDS-PAGE followed by Western blotting. Immunoblotting using anti-αSyn antibody D119 reveals that the sample (1 μg of total r-αSyn) is divided into three different states (a, b, c) in a manner dependent on the amount of CK2. (Left panel). It should be noted that the anti-pSer129 monoclonal antibody specifically recognizes types (a) and (b), whereby protein (b) is mono (Ser129) -phosphorylated r-αSyn, Protein (a) is shown to be diphosphorylated at Ser129 and another amino acid (right panel). The pSer129-r-αSyn concentration in the sample treated with 500 U of CK2 was calculated as 460 ng / μg of the total r-αSyn from the ratio of the bands (a) and (b) (left panel). Although phos-tag SDS-PAGE does not depend on the molecular weight, a molecular weight marker was used as a criterion for protein migration. The molecular weight marker is shown in kilodaltons (kDa) on the left side of each panel. (B) Predetermined amounts of r-αSyn and pSer129-r-αSyn were used as criteria for quantitative analysis of the amount of αSyn (upper left panel) and pSer129-r-αSyn (lower left panel) in BH, respectively. . Symbols for the reference r-αSyn are as follows (upper left panel): a1, 0 ng; a2, 125 ng; a3, 250 ng; a4, 500 ng; a5, 1000 ng; a6, 2000 ng. Symbols for BH samples are as follows (upper left panel): b1, DN-DLB case 1 (0.2% wt / vol); b2, DN-DLB case 2 (0.2% wt / vol) B3, Li-DLB (2% wt / vol); b4, schizophrenia (2% wt / vol); b5, CJD1 case 1 (2% wt / vol); b6, CJD1 case 2 (5% wt / vol); c5, CJD type 2 (2% wt / vol); c6, GSS (2% wt / vol); d5, AD case 1 (2% wt / vol); d6, AD case 2 (2% wt / vol). The symbols for the reference pSer129-r-αSyn are as follows (lower left panel): a1, 0 ng; a2, 50 ng; a3, 100 ng; a4, 200 ng; a5, 400 ng. Symbols for BH samples are as follows (lower left panel): b1-3, DN-DLB case 1 (0.1% wt / vol); c1-3, DN-DLB case 2 (0.2 D 1-3, Li-DLB (20% wt / vol); b4, schizophrenia (20% wt / vol); b5, CJD1 case 1 (20% wt / vol); b6 CJD type 1 case 2 (20% wt / vol); c5, CJD type 2 (20% wt / vol); c6, GSS (20% wt / vol); d5, AD case 1 (20% wt / vol) D6, AD case 2 (20% wt / vol). A 20 μl sample was applied to a nitrocellulose membrane (GE Healthcare Life Sciences). Linear regression between the dot intensity and a reference, in the upper right diagram for ^{r-αSyn (r 2 = 0.9018} ), and shown in the right figure for ^{pSer129-r-αSyn (r 2} = 0.9781). In BH derived from 2 cases of DN-DLB and 1 case of Li-DLB, αSyn accumulation was detected by anti-αSyn antibody D119, but nothing was detected in non-DLB cases (upper panel). Immunoreactivity with antibodies against pSer129-αSyn was observed only in 2 cases of DN-DLB and 1 case of Li-DLB (lower panel). The levels of pSer129-αSyn in DN-DLB cases # 1 and # 2 are 13.5 ± 0.4 mg / g brain and 3.7 ± 0.2 mg / g brain, respectively, and the level of Li-DLB is 0. Estimated to be 06 ± 0.02 mg / g brain. The ratio of pSer129-αSyn to total αSyn was as follows: DN-DLB case 1 (57.9 ± 1.5%), DN-DLB case 2 (22.6 ± 1.2%), And Li-DLB (6.1 ± 1.7%). FIG. 8 is a diagram showing an RT-QUIIC reaction in a reaction buffer containing a recombinant human prion protein or a reaction buffer containing no substrate protein. (A) Reaction buffer with 5 × 10 ⁻⁶ dilution of DN-DLB case # 1 in reaction buffer containing recombinant human prion protein (rPrP), or without seed, or without substrate protein RT-QUIIC was performed in solution using 5 × 10 ⁻⁶ diluted DN-DLB case # 1. The final concentration of reaction buffer components was 50 mM HEPES (pH 7.5) and 10 μM Thioflavin T (ThT). The concentration of rPrP was 150 μg / ml. The curve represents the average ThT fluorescence kinetics of all 6 replicate wells. (B) Maximum fluorescence intensity values and lag phase values obtained in individual samples after 96 hours of reaction are plotted in the left and right graphs, respectively. The lag phase was defined as the time required to reach> 120 arbitrary units of fluorescence intensity. Horizontal bars indicate mean ± standard deviation. There was no significant difference in maximum fluorescence intensity or lag phase in rPrP fibril formation between with and without seeding. In reactions containing no substrate, ThT fluorescence did not increase. FIG. 9 shows the dynamics of r-αSyn fibril formation by seeds derived from DN-DLB (cases # 1 and # 2), Li-DLB, schizophrenia, AD, CJD, and GSS. All reactions were performed in 6 replicates. The curve represents the ThT fluorescence kinetics from individual reactions seeded with the same BH. FIG. 10 is a diagram showing the dynamics of r-αSyn fibril formation by seeding with BH derived from DN-DLB (cases # 3, # 4, # 5 and # 6). All reactions were performed in 6 replicates. The curve represents the ThT fluorescence kinetics from individual reactions seeded with the same BH. FIG. 11 is a diagram schematically showing fibril formation of α-synuclein. FIG. 12 is a diagram showing a comparison of experimental methods in cerebrospinal fluid (CSF) derived from a DLB patient. FIG. 13 is a diagram showing a differential diagnosis auxiliary test between prion disease and DLB.

Hereinafter, the present invention will be described in detail.
In the present invention, α-synucleinopathy refers to an eosinophilic cell inclusion body in which α-synuclein, which is a kind of synaptic protein, is aggregated in specific cells in brain tissue. It refers to a disease of the brain characterized by being. It is known that α-synuclein having a normal function is involved in the function of the Golgi apparatus and the function of vesicle transport in neurons, and is localized at the axon end in the brain and easily solubilized by proteolytic enzymes. It has been.
Examples of α-synucleinopathies in the present invention include Lewy body disease and multiple system atrophy. Lewy body diseases include Parkinson's disease and Lewy body dementia. In Lewy body disease, this cell inclusion body is called Lewy Body. Lewy bodies are found in neurons in the substantia nigra and the locus coeruleus, which are part of the brain stem in Parkinson's disease, but are also found in neurons in the limbic system and cortex in Lewy bodies . In multiple system atrophy, the inclusion bodies are called glial intracellular inclusions (Gial Cytoplasmic Inclusion), and in addition to the striatum, oligodendrocytes such as the substantia nigra, cerebellar cortex, pontine nucleus, olive nucleus, cerebral cortical motor area, etc. It is found in glial cells and in neuronal cytoplasm.

In the present invention, Parkinson's disease is a progressive degenerative disease of the extrapyramidal system that presents four symptoms: resting tremor, muscle rigidity, ataxia, and posture reflex disorder. Degeneration of dopaminergic neurons that project from the midbrain substantia nigra to the striatum results in malfunction of the extrapyramidal system.
The number of Japanese patients is about 50 to 100 per 100,000 (approximately 200 per 65 years for a population of 65 and over).

  In the present invention, Lewy body dementia mainly occurs in early age or old age, and in addition to progressive cognitive dysfunction, Parkinsonism (generally two or more of the above four symptoms of Parkinson's disease). It refers to a degenerative disease that shows a specific psychiatric condition (called “parkinsonism”). It typically develops when over 60 years old, and the number of patients in Japan is considered to be about 500,000. It is known to be the second most common dementia after Alzheimer's disease. In Lewy body dementia, neurotransmitter concentrations and neuronal pathways from the striatum to the neocortex become abnormal. The essential symptoms of Lewy body dementia include cognitive dysfunction that impairs social and daily life functions. Examples of cognitive dysfunction include memory impairment, attention disorder, visuospatial disorder, and executive dysfunction. Core symptoms of Lewy body dementia include perturbed cognitive function, hallucinations, and Parkinsonism. There are pathological subtypes in dementia with Lewy bodies, which are classified into diffuse cortical DLB (DN-DLB), limbic DLB (Li-DLB), etc., depending on the distribution of Lewy bodies.

  Moreover, compared with Alzheimer's disease, Lewy body dementia has characteristics such as psychiatric symptoms such as hallucinations, delusions, depression, and motor symptoms such as Parkinson's disease from an early stage. In addition, it is known that donepezil, which is a therapeutic agent for Alzheimer's disease, may be effective in a small amount for Lewy body dementia.

  In the case of Alzheimer's disease, amyloid may be detected by examination of cerebrospinal fluid or positron emission tomography (PET) even before the onset of symptoms. On the other hand, in Lewy body dementia, it was difficult to quickly differentiate before the onset of symptoms.

The present invention relates to a method for rapidly detecting abnormal α-synuclein in a biological sample effective for diagnosis of α-synucleinopathies. Specifically, (1) a biological sample and normal α-synuclein in a buffer solution. And obtaining a reaction mixture;
(2) a step of incubating the reaction mixture to form an α-synuclein aggregate using the polymerized and fibrotic α-synuclein as a seed in a biological sample; and (3) the formed α-synuclein aggregate. Detecting step,
A method for detecting polymerized and fibrotic α-synuclein in a biological sample is provided.

  In the present invention, α-synuclein is classified into normal α-synuclein and abnormal α-synuclein in relation to α-synucleinopathy. Normal α-synuclein becomes abnormal α-synuclein in the presence of a polymerization nucleus (also referred to as a seed in the present specification), and can form an aggregate. Normal α-synuclein itself refers to a monomer of α-synuclein that does not have seeding activity in aggregate formation (FIG. 11).

  Abnormal α-synuclein is an aggregated α-synuclein detected in Lewy bodies and has a seeding activity in the formation of α-synuclein aggregates. It takes a β-sheet-like three-dimensional structure different from normal α-synuclein and aggregates to form a stable oligomer (polymerization) -like granular form, and in one embodiment, it becomes a higher molecular weight fibrosis (FIG. 11). In the present specification, polymerized and fibrotic α-synuclein may be referred to as “abnormal α-synuclein”.

  In the method of the present invention, α-synuclein aggregates are formed by abnormal α-synuclein present in a biological sample as a seed, normal α-synuclein serving as a substrate undergoes structural conversion and polymerization. In one embodiment, the aggregates form a polymeric fibrous structure. In one embodiment, the aggregate is insoluble. In the present specification, an aggregate of α-synuclein is sometimes simply referred to as “aggregate”.

  The normal α-synuclein used in the present invention includes proteins annotated as α-synuclein in various protein databases. Specifically, a protein containing a human α-synuclein amino acid sequence (Genbank Accession No. EAX06035.1) represented by SEQ ID NO: 2 and a splicing variant thereof (for example, SEQ ID NO: 3 (Genbank Accession No. AAA98493. 1) and 4 (Genbank Accession No. AAA98487.1)), these constituent amino acid residues may be mutated as long as they can be aggregated and structurally transformed in the presence of a seed, In the amino acid sequence represented by SEQ ID NO: 2, it may be a protein comprising an amino acid sequence in which amino acids are deleted, substituted or added. The amino acid sequence of α-synuclein in the present invention is preferably the amino acid sequence represented by SEQ ID NO: 2.

  The normal α-synuclein in the present invention may be one in which constituent amino acids have been modified as long as they aggregate and undergo structural conversion in the presence of seeds. The modification may be naturally occurring or artificial. Examples of modifications include glycosylation, ubiquitination, tyrosine nitration, biotinylation and the like.

  Normal α-synuclein may be isolated and purified from bacterial tissue or mammalian tissue, or may be chemically synthesized. Normal α-synuclein is produced by genetic engineering according to a method known per se, by culturing a transformant containing a nucleic acid encoding the same and isolating and purifying α-synuclein from the resulting culture. Alternative α-synuclein may be used. Recombinant α-synuclein is preferably used because it can be stably supplied.

  The normal α-synuclein used in the present invention is preferably isolated or purified. “Isolated or purified” means that an operation for removing components other than the target component from a natural state has been performed. The purity of the isolated or purified α-synuclein (the ratio of the weight of the antibody that specifically recognizes α-synuclein to the total protein weight) is usually 50% or more, preferably 70% or more, more preferably 90%. Above, most preferably 95% or more (for example, substantially 100%).

  When recombinant α-synuclein is used as normal α-synuclein, recombinant α-synuclein can be prepared, for example, by the following method. a polynucleotide encoding an α-synuclein amino acid sequence (for example, in the case of human α-synuclein, a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 (Genbank Accession No. EAX06035.1 CDS)) It is incorporated into an expression vector, inserted into an appropriate host, and transformed, and the desired recombinant α-synuclein can be obtained from the disrupted product of the transformed cell. The host cell is not particularly limited, and various host cells conventionally used in genetic engineering techniques such as Escherichia coli, Bacillus subtilis, yeast, plant or animal cells can be used.

  When produced by genetic engineering, a tag sequence may be added to the α-synuclein polypeptide in order to facilitate purification after synthesis. As tags, Flag tag, histidine tag, c-Myc tag, HA tag, AU1 tag, GST tag, MBP tag, fluorescent protein tag (for example, GFP, YFP, RFP, CFP, BFP, etc.), immunoglobulin Fc tag, etc. It can be illustrated. The position where the tag sequence is added is preferably the N-terminus or C-terminus of the α-synuclein polypeptide. Moreover, the α-synuclein polypeptide having these tag sequences may be used as it is in the method of the present invention, or the tag may be removed before that.

  Examples of biological samples that can be used in the method of the present invention include blood, brain tissue, cerebrospinal fluid, and the like. Seeding activity for α-synuclein fibril formation associated with α-synucleinopathy As long as it can be detected, there is no particular limitation. Brain tissue (for example, brain tissue in the frontal lobe region, substantia nigra) is preferably used in the method of the present invention in the form of a homogenate by crushing in an appropriate buffer solution. As blood, blood derived from any tissue can be assumed, but peripheral blood is usually used because of easy collection. As a blood collection method, a method known per se can be applied. The collected blood may be used in this step as it is, but it may be used as a liquid component (plasma) separated from cell components (erythrocytes, leukocytes, platelets, etc.) using methods known per se, such as centrifugation, filtration, etc. It is preferable to use in the process. It is also preferable to use in this step as a liquid component (serum) obtained by coagulating blood to separate platelets and coagulation factors. It is also preferable to use leukocytes as a biological sample. Cerebrospinal fluid fills and circulates brain tissue and is thought to reflect the state of the brain. Cerebrospinal fluid is preferably collected by a method such as lumbar puncture method, suboccipital puncture method, ventricular puncture method, and the like, and is preferably used in the method of the present invention.

  The biological sample is taken from the subject. A “subject” is a “subject suspected of suffering from α-synucleinopathy”. In the present specification, the “subject suspected of suffering from α-synucleinopathy” refers to a subject exhibiting symptoms suspected of suffering from α-synucleinopathy. Other diseases with cognitive impairment (infectious spongiform encephalopathy (TSE or prion disease) (Kreuzfeld-Jakob disease (CJD) in humans and its variants (spontaneous Creutzfeldt-Jakob disease (sCJD)) Etc.), Gerstmann-Streisler-Scheinker syndrome (GSS)), schizophrenia and Alzheimer's disease, etc .; may be referred to as “dementia other than α-synucleinopathy” in this specification) It refers to mammals, preferably humans, for which discrimination is sought.

  In a further aspect, the “subject” can be a “subject suffering from α-synucleinopathy”. Specifically, it is a subject that has recognized clinical findings defining that it is α-synucleinopathy, and a biological sample from the subject can be used as a positive control in the present invention.

  In another aspect, a “subject” can be a “subject not afflicted with α-synucleinopathy”. Specifically, a subject who does not recognize clinical findings that define α-synucleinopathy, and a biological sample from the subject can be used as a negative control in the present invention. Examples of the subject include healthy subjects and subjects suffering from dementia other than α-synucleinopathy.

  In the present invention, the “mammal” includes laboratory animals such as rodents and rabbits such as mice, rats, hamsters, and guinea pigs, domestic animals such as pigs, cows, goats, horses, sheep, pets such as dogs and cats, Primates such as humans, monkeys, cynomolgus monkeys, rhesus monkeys, marmosets, orangutans and chimpanzees are preferred, and humans are particularly preferred.

  As a container used for mixing a biological sample and normal α-synuclein, for example, flask, dish, petri dish, multi-dish, microplate, microwell plate, multiplate, multiwell plate (6, 12, 24, 48 or 96-well multi-well plate), petri dishes, tubes, and trays.

  Normal α-synuclein to be mixed with a biological sample is used in an excessive amount with respect to abnormal α-synuclein, which is a seed that would normally be present in the biological sample, and the concentration is not limited, but for example, 1 μg / ml to 10 mg / ml, preferably 10 μg / ml to 1 mg / ml, more preferably 10 μg / ml to 500 μg / ml, even more preferably 80 μg / ml to 250 μg / ml, even more preferably 100 μg / ml to 200 μg / ml, most Preferably, it is 120 μg / ml to 150 μg / ml.

  As for the concentration of the biological sample, for example, a dilution series is prepared by diluting the biological sample with an appropriate buffer, and the method of the present invention is performed on each of them to appropriately detect or quantify abnormal α-synuclein. .

  When cerebrospinal fluid is used as the biological sample, the concentration of cerebrospinal fluid in the reaction mixture is, for example, 1% (v / v) to 30% in the total reaction mixture (converted to the stock solution of cerebrospinal fluid). (V / v)%, 5% (v / v) to 20 (v / v)%, 7 (v / v)% to 25 (v / v)%, 10 (v / v)% to 15 (v / V)%.

  The buffer solution used for mixing the biological sample and normal α-synuclein is not particularly limited as long as it can maintain a certain range of pH, and various buffers (eg, phosphate buffer solution, phosphate buffered saline). , Tris-HCl buffer, borate buffer, citrate buffer, acetate buffer, etc.). Buffers are HEPPSO (N- (2-hydroxyethyl) piperazine-N ′-(2-hydroxypropanesulfonic acid)), POPSO (piperazine-1,4-bis- (2-hydroxy-propane-sulfonic acid) anhydrous Product), HEPES (4- (2-hydroxyethyl) piperazine-1-ethanesulfonic acid), HEPPS (EPPS) (4- (2-hydroxyethyl) piperazine-1-propanesulfonic acid), TES (N- [Tris (Hydroxymethyl) methyl] -2-aminoethanesulfonic acid) and combinations thereof. Preferably, HEPES is used.

  The pH in the above buffer is usually 6.5 to 8.5, preferably 7.0 to 8.0, more preferably 7.2 to 7.8, and even more preferably 7.3 to 7. 7, more preferably 7.4 to 7.6, most preferably 7.5. If the pH is less than 6.5 or more than 8.5, there is a possibility of deviating from the pH of the environment around α-synuclein present in the living body.

  In the method of the present invention, mixing of the biological sample and normal α-synuclein may be performed with or without adding beads such as zirconium / silica beads to the reaction mixture. Preferably, mixing is performed without adding beads.

  In the method of the present invention, the reaction mixture may contain a salt. Examples of such salts include sodium chloride, magnesium chloride, calcium chloride, potassium chloride, aluminum chloride, lithium chloride, strontium chloride, cobalt chloride, zinc chloride, iron chloride, copper chloride, magnesium sulfate, sodium sulfate, magnesium sulfate, and ammonium sulfate. , Magnesium nitrate, sodium nitrate, magnesium nitrate, ammonium nitrate, sodium carbonate, magnesium carbonate, sodium hydrogen carbonate, sodium monohydrogen phosphate, calcium lactate and the like.

  As a density | concentration in the liquid mixture of the said salt, 1 micromol-1M, 100 micromol-800 mM, 1 mM-600 mM, 10 mM-500 mM, 100 mM-500 mM, 200 mM-400 mM, 250 mM-350 mM are mentioned, for example.

  The step (2) of forming an aggregate is performed by incubating the reaction mixture. The incubation temperature is, for example, 32 ° C to 50 ° C, preferably 35 ° C to 45 ° C, more preferably 38 ° C to 42 ° C, and most preferably 40 ° C. If the temperature is lower than 32 ° C., there is a possibility that the change in the three-dimensional structure of the normal α-synuclein molecule is suppressed in the formation of the aggregate, and if it exceeds 50 ° C., the change in the three-dimensional structure of the normal α-synuclein molecule independent of the formation of the aggregate. May be triggered.

  The incubation time is not particularly limited as long as an aggregate is formed to the extent that it can be detected, but for example, 5 hours or more, 10 hours or more, 12 hours or more, 14 hours or more, 16 hours or more, 20 hours or more, 24 hours or more. 48 hours or more. In addition, the amount of aggregate formed even after a long incubation is constant, and in view of the purpose of rapid measurement, the incubation is usually performed for 96 hours or less, 72 hours or less, or 48 hours or less.

  In the step (2) for promoting the formation of aggregates, the reaction mixture may be shaken. The shaking is performed using, for example, a commercially available shaking incubator, for example, 200 rpm to 1,000 rpm, 300 rpm to 800 rpm, 400 rpm to 500 rpm, 500 rpm to 800 rpm, 800 rpm to 1,200 rpm, preferably 420 rpm to 480 rpm, most preferably 432 rpm. To do.

  Step (2) may include a cycle in which a constant shaking period and a constant shaking period are repeated. For example, step (2) includes a 60 second shaking period and a 60 second no shaking period cycle. In another example, step (2) includes a 30 second shaking period and a 30 second no shaking period cycle. The period may be varied, such as a 45 second shaking period and a 45 second no shaking period or a 70 second shaking period and a 70 second shaking period. In other embodiments, the cycle of the shaking period and no shaking period is, for example, 1 minute, 2 minutes, preferably 3 minutes in length throughout the cycle. In step (2), the shaking period and the non-shaking period may be changed depending on the length of the entire cycle.

  The time for repeating the cycle of the shaking period / no shaking period is not particularly limited as long as aggregates are formed to the extent that they are detected, but the total is, for example, 10 hours or more, 12 hours or more, 14 hours or more, 16 hours. These are 20 hours or more, 24 hours or more, 48 hours or more. In addition, the amount of aggregates formed is constant even when carried out for a long time, and in view of the purpose of rapid measurement, the cycle is usually repeated for 96 hours or less, 72 hours or less, or 48 hours or less.

  The aggregate formed in the method of the present invention is a polymer, and the reaction mixture containing the aggregate may be used as it is for detection, or the polymer fraction may be partially purified and used. . The molecular weight of the aggregate to be detected is, for example, 200 kDa or more, preferably 250 kDa or more, more preferably 280 kDa or more. Partial purification can be performed, for example, by various types of chromatography or ultrafiltration.

  Examples of the detection method in the present invention include enzyme immunoassay (EIA method), immunochromatography, latex agglutination, radioimmunoassay (RIA), fluorescence immunoassay (FIA), luminescence immunoassay, surface A plasmon resonance measurement method (SPR method), Western blotting, electron microscopy, fluorescence microscopy, fluorescence spectroscopy, and the like can be used. Among these, Western blotting and fluorescence spectroscopy are preferable from the viewpoint of ease of operation and rapidity.

  When the detection is performed using an antibody, the antibody may be any antibody that is capable of specifically binding to an α-synuclein aggregate, and may be a polyclonal antibody or a monoclonal antibody. Such antibodies also include binding fragments such as chimeric antibodies, single chain antibodies, or F (ab ') 2, Fab', or Fab fractions of antibody molecules. As these antibodies, antibodies prepared by a method known per se using α-synuclein as an immunogen can be used, and commercially available antibodies can also be used.

  Examples of commercially available anti-α-synuclein antibodies include anti-α-synuclein (α-Syn) polyclonal antibody D119 (Bioworld Technology, Inc.), anti-pSer129-αSyn monoclonal antibody against α-synuclein phosphorylated at the 129th serine, And anti-Ser87 phosphorylated α-Syn polyclonal antibody and the like, and anti-α-Syn polyclonal antibody D119 and anti-pSer129-αSyn (anti-Ser129 phosphorylated α-synuclein) monoclonal antibody are preferable.

The antibody may be directly or indirectly labeled with a labeling substance. Labeling substances include fluorescent substances (eg, FITC, rhodamine), radioactive substances (eg, ¹⁴ C, ³ H, ¹²⁵ I), enzymes (eg, alkaline phosphatase, peroxidase), colored particles (eg, metal colloid particles, colored) Latex), biotin and the like.

  If it is such an antibody, only 1 type of antibody may be used in this process, and 2 or more types may be used.

  The “antibody specifically recognizing α-synuclein” can be used in the form of an aqueous solution. In this case, α-synuclein aggregates may be bound to a solid phase. Such “solid phases” include plates (eg, microwell plates), tubes, beads (eg, plastic beads, magnetic beads), chromatographic carriers (eg, Sepharose ™), membranes (eg, nitrocellulose membrane) , PVDF film), gel (eg, polyacrylamide gel), metal film (eg, gold film) and the like. Of these, plates, beads, membranes and metal membranes are preferably used, and membranes are most preferably used because of easy handling. Furthermore, in order to suppress non-specific adsorption and non-specific reaction, it is general to bring a buffer solution into contact with the solid phase and block the solid phase surface portion not coated with the antibody with the BSA or bovine milk protein. Done.

  The contact between “an antibody specifically recognizing α-synuclein” and “α-synuclein aggregate” in the detection specifically recognizes α-synuclein aggregate and α-synuclein in the reaction vessel. There are no particular limitations on the embodiment, the order, the specific method, and the like as long as they can interact with each other by mixing the antibodies. When the antibody is used in the form of an aqueous solution, the contact is performed, for example, by adding the antibody to a reaction solution in which a membrane on which an aggregate is immobilized is immersed.

  The time for maintaining such contact is not particularly limited as long as the antibody specifically recognizes α-synuclein and an aggregate of α-synuclein are combined to form a complex. In general, it is several seconds to several tens of hours, and from the viewpoint of promptly determining whether or not it is α-synucleinopathy, it is preferably 1 minute to 2 hours, and most preferably 2 minutes to 30 minutes. . Moreover, as temperature conditions which perform a contact, it is 4 to 50 degreeC normally, 4 to 37 degreeC is preferable and the room temperature of about 15 to 30 degreeC is the most preferable. Furthermore, the pH condition for carrying out the reaction is preferably 5.0 to 9.0, particularly preferably the neutral range of 6.0 to 8.0.

  When Western blotting is selected as the detection method, for example, the formed α-synuclein aggregate is mixed with an SDS loading buffer, boiled, denatured, and a sample solution is prepared. The sample solution is separated by SDS polyacrylamide electrophoresis, transferred to a membrane such as a nitrocellulose membrane or a polyvinylidene difluoride (PVDF) membrane, and solidified. The membrane is immersed in an appropriate reaction solution (eg, TBST containing 5% skim milk), blocked, and then reacted with a primary antibody (anti-α-synuclein antibody) in an appropriate reaction solution (eg, TBST). . The time required for the reaction is preferably 1 minute to 2 hours, more preferably 2 minutes to 30 minutes, from the viewpoint that rapid measurement is required. Next, the membrane is washed and reacted with a secondary antibody (eg, horseradish peroxidase-conjugated secondary antibody) in an appropriate reaction solution (eg, TBST). The time required for the reaction is preferably 1 minute to 2 hours, more preferably 2 minutes to 30 minutes, from the viewpoint that rapid measurement is required. The membrane is washed again, and the membrane is contained together with a detection buffer in an appropriate container such as a hybrid bag, and detected by an appropriate detection system.

  When a spectrophotometer is selected as the detection method, a fluorescent dye that binds to amyloid fibrils and emits fluorescence is added to a reaction mixture obtained by mixing a biological sample and normal α-synuclein to form an aggregate. Perform the above reaction. Since the fluorescent dye binds to the formed fibrous aggregate and emits fluorescence, the reaction mixture is irradiated with excitation light having an appropriate wavelength, and the fluorescence is detected with a spectrophotometer or the like.

  Examples of fluorescent dyes that emit fluorescence by binding to amyloid fibrils include Thioflavin T (ThT), 1-Bromo-2,5-bis (3-carboxy-4-hydroxystyryl) benzene, 1-Fluoro-2,5 -Bis (3-carbyl-4-hydroxystyryl) benzene, ProteoStat (registered trademark) amyloid detection reagent, and the like.

  In one embodiment, the step (3) includes quantifying α-synuclein aggregates formed using abnormal α-synuclein as a seed. For example, when Western blotting is selected as the detection method, the amount of aggregate can be quantified by measuring the intensity of the detected (smear) band with an image analyzer or the like. When a spectrophotometer is selected as the detection method, the amount of aggregate can be quantified by measuring the fluorescence intensity of the fluorescent dye bound to the aggregate.

  In one embodiment, the detection in the step (3) is performed while the formation of the aggregate is in progress, and then the step of further forming the aggregate continues. In one embodiment, the detection in step (3) is intermittently performed a plurality of times while the formation of aggregates proceeds. For example, the detection in step (3) is performed immediately after one cycle of the shaking period / no shaking period in step (2). However, the detection need not be performed every cycle, for example, once every 16 minutes, preferably once every 10 minutes, for example, every 5 cycles, preferably every 3 cycles.

The method of the present invention may include the following steps after (2).
(3) A step of mixing a part of the formed α-synuclein aggregate and normal α-synuclein in a buffer solution to obtain a further reaction mixture;
(4) Incubating a further reaction mixture to form an α-synuclein aggregate using abnormal α-synuclein as a seed; and (5) Repeating steps (3) to (4) as required.

  The aggregate formed by the steps (1) to (2) may be further formed by mixing a part thereof with normal α-synuclein to obtain a further mixed solution and incubation. it can. Even if the amount of abnormal α-synuclein in the biological sample is small and the amount of α-synuclein aggregate formed directly from the biological sample by steps (1) to (2) is small, steps (3) to (4) Alternatively, by including the steps (3) to (5), the amount of α-synuclein aggregate formed can be amplified. As a result, abnormal α-synuclein detection sensitivity in a biological sample can be increased.

  The present invention also provides a method for assisting diagnosis of α-synucleinopathy using the method for detecting abnormal α-synuclein of the present invention.

  According to the diagnosis assisting method of the present invention, the determination is performed as follows, for example. A biological sample (eg, cerebrospinal fluid) is collected from a healthy subject or a patient with dementia other than α-synucleinopathy (negative control) and a patient with α-synucleinopathy (positive control), and the subject. The presence or absence of α-synuclein aggregates formed by the biological sample collected from the positive control and negative control is confirmed.

  And from the confirmation result, when an aggregate is detected when a biological sample derived from the subject is used, the subject is relatively likely to have α-synucleinopathy. Can be determined. Conversely, if aggregates are not detectable, it can be determined that the subject is relatively unlikely to suffer from α-synucleinopathy. Further, when using the method of the present invention to differentiate between α-synucleinopathy and dementia other than α-synucleinopathy, when an aggregate is detected, the subject is α -It can be determined that the possibility of suffering from synucleinopathy is relatively high. Conversely, if no aggregate is detected, it can be determined that the subject is likely not α-synucleinopathic.

  In another embodiment, for example, a biological sample (eg, cerebrospinal cord) from a healthy person or a patient with dementia other than α-synucleinopathy (negative control) and a patient with α-synucleinopathy (positive control). The amount of α-synuclein aggregates formed by the biological sample collected from the subject is compared with that of the positive control and the negative control.

  If the amount of aggregates formed from the biological sample derived from the subject is relatively large based on the comparison result of the amount of α-synuclein aggregates, the subject suffers from α-synucleinopathy. It is possible to determine that there is a relatively high possibility. Conversely, when the amount of aggregate is relatively small, it can be determined that the subject is relatively unlikely to suffer from α-synucleinopathy. In addition, when using the method of the present invention to differentiate between α-synucleinopathy and dementia other than α-synucleinopathy, when the amount of aggregate is relatively large, the subject Can be determined to be relatively likely to have α-synucleinopathies. On the contrary, when the amount of aggregate is relatively small, it can be determined that the subject is not likely to be α-synucleinopathy.

  Alternatively, a cut-off value of the amount of α-synuclein aggregates in the reaction mixture may be set in advance, and the measured amount of α-synuclein aggregates may be compared with this cut-off value. it can. For example, when the amount of aggregate is not less than the cut-off value, it can be determined that the subject is highly likely to suffer from α-synucleinopathy. Conversely, if the amount of aggregate is below the cut-off value, it can be determined that the subject is unlikely to suffer from α-synucleinopathy. In addition, when using the method of the present invention to differentiate between α-synucleinopathy and dementia other than α-synucleinopathy, when the amount of aggregate is not less than the cutoff value, It can be determined that the subject is relatively likely to have α-synucleinopathy. Conversely, if the amount of aggregate is below the cut-off value, it can be determined that the subject is likely not α-synucleinopathic.

  In another embodiment, for example, a biological sample (eg, brain) from a healthy subject or a patient with dementia other than α-synucleinopathy (negative control) and a patient with α-synucleinopathy (positive control). Spinal fluid) is collected, and the amount of α-synuclein aggregate formed using a biological sample collected from the subject is measured continuously (eg, in real time), and α-synuclein aggregate is measured from the start of the reaction. Is compared to that of the positive and negative controls.

  From the comparison result, when the time until the aggregate is formed is relatively short, the subject can determine that the amount of abnormal α-synuclein is relatively large. On the other hand, when the time until an aggregate is formed is relatively long, the subject can determine that the amount of abnormal α-synuclein is relatively small. In addition, when differentiating α-synucleinopathy and dementia other than α-synucleinopathy using the method of the present invention, when the negative control is negative, an aggregate is formed. Regardless of whether the time is relatively long or short, it can be determined that the subject is relatively likely to have α-synucleinopathies.

  Alternatively, in yet another aspect, a cutoff value is set in advance for the time until the α-synuclein aggregate is formed in the reaction mixture, and the measured α-synuclein aggregate is formed. This time can also be compared with this cut-off value. For example, when the time until an aggregate is formed is shorter than the cut-off value, it can be determined that the subject is highly likely to suffer from α-synucleinopathy. Conversely, if the time until the formation of the aggregate is the same as or longer than the cutoff value, it is determined that the subject is unlikely to have α-synucleinopathy. Can do. When using the method of the present invention to differentiate between α-synucleinopathy and dementia other than α-synucleinopathy, the time until an aggregate is formed is less than the cutoff value. If short, it can be determined that the subject is relatively likely to have α-synucleinopathy. On the contrary, when the time until an aggregate is formed is equal to or longer than the cutoff value, it can be determined that the subject is not likely to be α-synucleinopathy.

  The “cut-off value” is a value that can satisfy both high diagnostic sensitivity (prevalence of prevalence of disease) and high specificity of diagnosis (prevalence of nondiagnosticity) when determining the disease or condition based on that value. is there. For example, the amount of α-synuclein aggregates showing a high positive rate in patients with α-synucleinopathy and a high negative rate in patients with dementia other than healthy subjects or α-synucleinopathies Alternatively, the time until formation of α-synuclein aggregates can be set as a cutoff value. The cut-off value can be obtained by creating a receiver operating characteristic curve (ROC curve) that represents the relationship between the sensitivity and specificity of diagnosis based on the detection and quantification results in individual subjects.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited at all by these Examples.

Materials and Methods Patients DLB brain tissue was obtained at necropsy from 7 patients whose clinical diagnosis was confirmed histopathologically. According to the Bragg staging, 6 of these subjects were classified as having diffuse cortical DLB (DN-DLB) and the remaining 1 case was limbic DLB (Li-DNB) there were. Prion disease brain tissue was obtained from autopsy from 3 sporadic CJD (sCJD) patients and 1 GSS patient associated with a Pro to Leu mutation at codon 102 of PRNP. The sCJD subtype was diagnosed according to the genotype at codon 129 of the PRNP gene and the physiological properties of the aberrant prion protein (PrP ^Sc ). They included two cases of type 1, codon 129MM (MM1), type 2, one case of codon 129MM (MM2). AD brain tissue was obtained at autopsy from two patients who had undergone neuropathological diagnosis of neurofibrillary tangles and the presence of senile plaques. The brain specimens were pure AD with little or no LBD coexistence. Brain tissue from patients with schizophrenia and breast cancer without histopathological changes in the brain was used as a non-degenerative case. Written informed consent was received from the patient's family about joining the study. The study protocol was approved by the Nagasaki University Hospital Ethics Committee (ID: 10049823) and registered with the University Hospital Medical Information Network (ID: UMIN000010001).

Expression and Purification of Recombinant Human α-Synuclein A DNA sequence encoding N-terminal residues 1 to 140 of His-tagged wild type human αSyn was obtained from a human cDNA (Toyobo) by using a forward primer (5′-ggaattccatgacatata SEQ ID NO: 5)) and reverse primer (5′-ctagcttagctagtaggtctcaggttcgttagcttt-3 ′ (SEQ ID NO: 6)). The S129A mutant is composed of a forward primer (5′-ggaattccatgacatacatcatcatcatcaccataggagtgtattcatgaagagg-3 ′ (SEQ ID NO: 7)) and a reverse primer (5′-ctagctaggctcgtcgtcgtcgtcgtcgtcgtcgtcgtcgtcgtcgtcgtcgtcgtcgtcgtcgtcgtgtgt A termination point for dipeptidyl peptidase I that digests the N-terminal His tag was introduced into the protein sequence by inserting a glutamine codon into the expression construct. The amplified PCR fragment was inserted into the NdeI and NheI sites of the pET11a vector (Novagen) and confirmed by sequencing analysis. The plasmid was transformed into B21 DE3 E. coli. After transformation into E. coli competent cells (BioDynamics Laboratory), MagicMedia E. coli. The recombinant protein was expressed using E. coli Expression Medium (Invitrogen). The cell pellet was suspended in CelLytic B (Sigma-Aldrich) in the presence of lysozyme (Wako) and benzonase nuclease (Novagen). The lysate was centrifuged at 3000 rpm for 15 minutes and the supernatant was incubated with Ni-NTA Superflow resin (Qiagen) for 30 minutes at room temperature. The protein was eluted with a buffer containing 300 mM NaCl, 50 mM Tris-HCl (pH 8.0), 250 mM imidazole and dialyzed against 10 mM phosphate buffer (pH 7.0). As shown in FIGS. 1A and 1B, tag removal from N-terminal His-tagged human α-synuclein was performed using TAGZyme system (Qiagen). Purified His-r-Syn contains a glutamine termination point for the N-terminal exopeptidase DAPase (Qiagen) between the His tag sequence and the first amino acid of αSyn. The tag was cleaved with DAPase in the presence of the glutamine cyclotransferase Qcyclase (Qiagen), and the product was applied to Ni-NTA resin (Qiagen) to remove the uncleaved His-tagged protein. In the presence of Qcyclase, glutamine residues are converted to pyroglutamic acid, which acts as a termination point for DAPase degradation. Human αSyn was obtained by removing pyroglutamic acid by the action of the pyroglutamyl aminopeptidase pGAPase (Qiagen). Since DAPase, Qcyclase, and pGAPase have a His tag at their C-terminal, they were removed using Ni-NTA resin. The final protein was dialyzed against 10 mM sodium phosphate buffer (pH 7.0) and filtered through a 2.0 μm syringe filter. The purity of the protein sample was estimated to be 98% > by SDS-PAGE and immunoblotting. Analysis by circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) showed the modified structure of r-αSyn (FIGS. 1C and 1D). After purification, equal aliquots of protein were stored at −80 ° C. until use.

RT-QUIIC Experiment We prepared a final total volume of 100 μl of reaction mixture in black 96 well fluorescent plates (Nunc). To prevent contamination, we prepared all materials inside the biological safety cabinet and used aerosol resistant chips. The final concentration of reaction buffer components was 50 mM HEPES (pH 7.5) and 10 μM Thioflavin T (ThT). The concentration of r-αSyn was 100 μg / ml to 150 μg / ml, and only freshly thawed r-αSyn was used. We observed slight variations in the optimal r-αSyn concentration (100 μg / ml to 150 μg / ml) among r-αSyn lots, but the final sensitivity was generally the same. Using a multi-bead shocker, brain tissue (frontal lobe region) was homogenized with 10% in ice-cold PBS supplemented with a protease inhibitor mixture (Roche). After centrifugation at 2000 × g for 2 minutes, the supernatant was collected and frozen at −80 ° C. until use. Brain homogenate was diluted with PBS prior to reaction. Cover 96-well plate with sealing tape (Nunc) and shake intermittently in plate reader (Infinite M200 fluorescence plate reader; TECAN) (circular shaking at maximum speed for 40 seconds, no shaking for 20 seconds, then fluorescence measurement For 2 minutes) and incubated at 40 ° C. Using a monochromator with excitation light and fluorescence wavelengths of 440 nm and 485 nm, respectively, the fluorescence intensity at the bottom of the plate was read every 10 minutes to monitor the kinetics of amyloid formation. Each replicate brain homogenate sample was measured in 6 replicates. Each diluted sample of insoluble aggregates and r-αSyn oligomer was subjected to 3-4 replicate and 3-6 replicate assays, respectively. We determined that the reaction with a fluorescence intensity of more than 120 arbitrary units was a positive reaction, the seed dose (SD ₅₀ ) that showed a positive reaction in 50% of the replicate reactions, the Spearman-Kelver method (Wilham et al., 2010) ) Calculated using as described.

Western blotting Brain tissue (frontal lobe region) was washed with Triton-deoxycholic acid (DOC) lysis buffer (50 mM Tris-HCl, pH 7.5 (150 mM NaCl, 0.5% Triton X-100, 0.5% deoxychol). Sodium sulfate, 2 mM EDTA, and a protease inhibitor) were dissolved at 4 ° C. for 30 minutes. After centrifugation at 2000 × g for 2 minutes, the supernatant was collected and the total protein concentration was measured using a bicinchoninic acid (BCA) protein assay kit (Pierce). Samples were loaded with sodium dodecyl sulfate (SDS) loading buffer (62.5 mM Tris-HCl, PH 6.8 (containing 5% 2-mercaptoethanol, 2% SDS, 5% sucrose, and 0.005% bromophenol blue). And boiled at 95 ° C. for 5 minutes, and subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE). Protein was transferred to Immobilon-P membrane (Millipore) at 300 mA for 2 hours in transcription buffer containing 15% methanol; membranes were TBST (10 mM Tris-HCl, pH 7.8, 100 mM NaCl, 0.1% Blocked with 5% nonfat dry milk in Tween 20) at 4 ° C. for 2 hours and reacted with diluted primary antibody. The immunoreacted band was visualized with HRP-conjugated secondary antibody using an enhanced chemiluminescence system (GE healthcare Life Sciences).

Transmission Electron Microscopy Negative staining was performed on a carbon support membrane grid (glow discharge before staining). A 5 μl aliquot of the sample was adsorbed onto the grid, and the remaining liquid was absorbed with filter paper. The grid was stained with 5 μl of freshly filtered staining solution (2% uranium acetate). When dried, the sample was observed with a transmission electron microscope (JEM-1400PLUS; JEOL).

CD
Circular dichroism (CD) spectra were measured with a JASCO J-820 spectropolarimeter (JASCO) using a quartz cell with a path length of 1 mm. CD spectra were obtained by averaging four scans in the wavelength range of 195 nm to 250 nm. r-αSyn was dissolved in a buffer of 20 mM sodium phosphate (pH 6.5) and 150 mM NaCl. The concentration of r-αSyn was 300 μg / ml.

FTIR
Fourier Transform Infrared Spectroscopy (FTIR) spectra were measured with a Bruker Sensor 27 FTIR instrument (Bruker Optics) equipped with a mercury cadmium tellurium (MCT) detector cooled with liquid nitrogen. A 20 μl aliquot of the sample was loaded into a BioATRcell II attenuated total reflection type reflection unit. All 128 scans at 4 cm ⁻¹ resolution were collected for each sample under a constant nitrogen purge, corrected for water vapor, and the buffer background spectrum subtracted.

Histopathology and immunohistochemical staining Brain tissue was fixed in 20% neutral buffered formalin and 8 μm paraffin sections were prepared on a slide glass with a microtome. After deparaffinization and rehydration, tissue sections were subjected to staining with hematoxylin and eosin and immunohistochemical staining using anti-Ser129 phosphorylated α-Syn antibody (1: 3000 dilution; Wako). To enhance immunogenicity, sections were prepared by heating at 98 ° C. for 40 minutes prior to incubation with the primary antibody. The binding of the primary antibody was detected by a labeled streptavidin-biotin method (DAKO). Peroxidase-conjugated streptavidin was visualized using 3'3-diaminobenzidine (Wako) as a chromogen. Immunostained sections were lightly counterstained with Mayer's hematoxylin.

Statistical analysis Data for maximum fluorescence intensity were analyzed by one-way analysis of variance followed by Tukey-Kramer test. Data analysis for the lag phase was subjected to log rank test and Tukey-Kramer test. P <0.05 or P <0.01 was used to show statistical significance.

Supplemental Experimental Procedure In Vitro Phosphorylation of Recombinant α-Synuclein r-αSyn (3 μg) was added to 20 μl of reaction buffer (20 mM Tris-HCl, pH 7.5, 50 mM KCl, and 10 mM MgCl ₂ ). Incubation with kinase 2 (New England Biolabs) and 200 μM ATP (Sigma) at 37 ° C. for 5 hours. The reaction was stopped by boiling at 95 ° C. for 10 minutes.

Phos-tag SDS-PAGE and Western blotting Phos-tag SDS-PAGE was performed based on the tricine-SDS-PAGE method. The polyacrylamide separation gel contained 1 M Tris-HCl (pH 8.45), 0.1% SDS, 13.3% glycerol, 100 μM Phos-tag, and 400 μM ZnCl ₂ . The concentration of the anode buffer component was 200 mM Tris-HCl (pH 8.9). The concentration of the cathode buffer component was 100 mM Tris, 100 mM Tricine, and 0.1% SDS. With SDS loading buffer (62.5 mM Tris-HCl, pH 6.8 containing 5% 2-mercaptoethanol, 2% SDS, 5% sucrose, and 0.005% bromophenol blue) at 95 ° C. It was boiled for 5 minutes and subjected to Phos-tag SDS-polyacrylamide electrophoresis. After electrophoresis, the gel was washed with a transfer buffer containing 10 mM EDTA to chelate Zn ²⁺ ions. The protein was transferred onto Immobilon-P membrane (Millipore) at 300 mA for 2 hours in transcription buffer containing 15% methanol. The membrane was blocked with 5% non-fat dry milk in TBST (10 mM Tris-HCl, pH 7.8, 100 mM NaCl, 0.1% Tween 20) for 2 hours at 4 ° C. and reacted with diluted primary antibody. Immunoreactive bands were visualized with HRP-conjugated secondary antibody using an enhanced chemiluminescence system (Amersham). The intensity of the band was measured using ImageJ 1.41.

Dot blot BH and r-αSyn were boiled at 95 ° C. for 10 minutes with SDS loading buffer (62.5 mM Tris-HCl, pH 6.8 containing 5% 2-mercaptoethanol and 2% SDS). Samples were blotted onto nitrocellulose membranes under mild vacuum-enhanced conditions using bio-blots (Bio-Rad, Hercules, CA, USA). After washing with TBST (10 mM Tris-HCl, pH 7.8, 100 mM NaCl, 0.1% Tween 20) and blocking with 5% nonfat dry milk in TBST for 2 hours, the membrane was probed with diluted primary antibody. Immunoreactive bands were visualized with HRP-conjugated secondary antibody using an enhanced chemiluminescence system (Amersham). The intensity of the dots was measured using ImageJ 1.41.

Results α-synuclein phosphorylated at Ser129 is abundant in the brain derived from dementia with Lewy bodies.
First, we confirmed the presence of αSyn (pSer129-αSyn) phosphorylated by LB and Ser129 in the brains of DLB patients by hematoxylin and eosin staining and immunohistochemical staining, respectively (FIG. 2A). In sections of the substantia nigra and frontal cortex obtained at autopsy from two diffuse neocortical DLB (DN-DLB) patients and one limbic DLB (Li-DLB) patient, LB and pSer129- Both αSyn were observed. pSer129-αSyn appeared to be present in LB. In the cortex, LB containing pSer129-αSyn in DN-DLB was clearly larger than that in Li-LDB. In contrast, histochemical analysis revealed no pathological abnormalities in the brains of non-DLB cases. Tannickal et al. Consistent with previous reports by (2007), cells containing melanin granules in the substantia nigra were usually lost due to neurodegeneration in DN-DLB and Li-DLB patients, but in non-DLB It wasn't.

  Next, we verified the biochemical properties of αSyn in brain homogenates (BH) from DLB patients by Western blotting using anti-αSyn antibody D119 and compared them with those from non-DLB cases (FIG. 2B). Sample immunoblot analysis from all patients showed a natural αSyn band at 18 kDa, a 36 kDa dimer form, or both. Two cases of DN-DLB and one case of Li-DLB contained significantly bulky multimeric αSyn with a molecular weight range of> 250 kDa. Insoluble multimers were more abundant in DN-DLB than Li-DLB, indicating that αSyn multimer formation is crucial for disease progression. This band was not observed in non-DLB cases, but a relatively small amount of polymer was observed in GSS. In all cases, bands of various sizes were shown in the molecular weight range of about 50 kDa to 250 kDa. These bands were not significantly different between cases, presumably due to oligomeric αSyn and / or ubiquitinated αSyn. The antibody against pSer129-αSyn detected an apparent band of high molecular weight (> 250 kDa) in two cases of DN-DLB (FIG. 2C). A similar sized band was observed in one case of Li-DLB, but the intensity of immunoreactivity was much weaker than that of DN-DLB. In contrast, non-DLB cases did not show immunoreactivity to antibodies against pSer129-αSyn. These observations indicated the presence of insoluble multimers of αSyn (mostly phosphorylated with Ser129) in DLB brain,> 250 kDa. Furthermore, we estimated the level of pSer129-αSyn in the brains of DLB cases by quantitative dot blot immunoassay following Phos-tag SDS-PAGE (FIG. 7). The levels of pSer129-αSyn in DN-DLB cases # 1 and # 2 are 13.5 ± 0.4 mg / g brain and 3.7 ± 0.2 mg / g brain, respectively, and the level in Li-DLB is 0. 06 ± 0.02 mg / g brain. The ratio of pSer129-αSyn to total αSyn in DN-DLB was higher than that in Li-DLB: DN-DLB case # 1 (57.9% ± 1.5%), DN-DLB # 2 (22. 6% ± 1.2%), and Li-DLB (6.1% ± 1.7%). These results suggest that pSer129-αSyn is associated with disease progression. Previous studies have shown that the level of αSyn phosphorylated at Ser87 is also increased in the LBD brain (Paleologou et al., 2010). However, the antibody against Ser87 phosphorylated αSyn detected a 36 kDa dimer band, and there was no significant difference between cases in the level of Ser87 phosphorylated form (FIG. 2D).

Conversion of soluble recombinant human α-synuclein to amyloid fibrils by RT-QUIIC We next formed r-αSyn fibril formation in RT-QUIIC when BH from DLB patients was added to the reaction. Was able to be induced by monitoring the ThT fluorescence level (FIGS. 3A and 3B). When BH derived from 2 cases of DN-DLB diluted at 5 × 10 ⁻⁵ and 5 × 10 ⁻⁶ was added, ThT fluorescence was positive within 24 hours at the earliest and within 96 hours in all the reaction solutions. At 5 × 10 ⁻⁷ dilution, the assay was positive in 4 out of 6 replicate wells in case # 1 and 3 out of 6 wells in case # 2. At 5 × 10 ⁻⁸ dilution, 2 out of 6 responses for case # 1 were positive, but no increase in fluorescence was observed in case # 2. The reaction at 5 × 10 ⁻⁹ dilution was all negative in case # 1. For Li-DLB cases, 2 out of 6 reactions at 5 × 10 ⁻⁵ dilution showed a positive reaction, but no reaction was observed at thinner dilutions. We also verified the effect of 5 × 10 ⁻⁴ dilution on Li-DLB, but no increase in fluorescence was observed (data not shown). The negative response was probably due to high levels of various components in BH that inhibit r-αSyn fibril formation in the reaction. In contrast, reactions with unseeded controls and 5 × 10 ⁻⁵ and 5 × 10 ⁻⁶ dilutions of schizophrenic BH did not produce a response over 96 hours. Maximum fluorescence intensity, as compared to the control without seed, DN-DLB patients # 5 × ^{10 -5} and 5 × ^{10 -6} dilution of 1, DN-DLB patients # 2 of 5 × ¹⁰ -5 ~5 × ^{10 -} Significantly stronger with reactions in the range of ⁷ and with 5 × 10 ⁻⁵ dilution of Li-DLB (FIG. 3B). The lag phase was significantly shorter in the response at 5 × 10 ⁻⁵ dilution from 2 cases of DN-DLB compared to the seedless control (FIG. 3B). The values of SD ₅₀ / g brain for DN-DLB cases # 1 and # 2 were 10 ^7.8 and 10 ^7.3 , respectively. We were unable to accurately calculate the seeding dose of Li-DLB, but the value was 5.1 (log SD ⁵⁰ / g based on the assumption that 5 × 10 ⁻⁴ dilution showed 100% positive. Estimated to be less than (brain). In the reaction using the recombinant human prion protein as a substrate or in the reaction without the protein, specific detection of DLB was not observed (FIG. 8), but this led to seeding of r-Syn in BH derived from DLB cases. But the ability to seed other proteins has not been proven. To further evaluate the impact of other protein misfolding and degenerative diseases on RT-QUIIC, we have Alzheimer's disease (AD), sporadic Creutzfeldt-Jakob disease (sCJD) types 1 and 2, and BH from patients with GSS was applied to the assay. In AD, sCJD (types 1 and 2) and GSS, all 5 × 10 ⁻⁵ and 5 × 10 ⁻⁶ dilution reactions resulted in a negative response within 96 hours (FIGS. 3A and 3B). . These observations indicate that RT-QUIIC induces the formation of r-αSyn fibrils only in the presence of BH from DLB cases and that the seeding activity of DN-DLB is higher than that of Li-DLB. Indicated. These findings suggest that r-αSyn can be converted to amyloid fibrils by a prion-like mechanism.

To characterize the fiber structure of r-αSyn, samples were verified by negative staining transmission electron microscopy (TEM). Electron micrographs of r-αSyn fibers seeded in DN-DLB case # 1 revealed long, thin and branched fiber bundles, but fibers were observed in the negative control reaction without seeding. There was no (Figure 3C). Fourier Transform Infrared Spectroscopy (FTIR) showed that there was almost no difference in the most prominent band at 1650 cm ⁻¹ attributed to the denatured structure between DLB and non-DLB cases (FIG. 3D). The results suggest that only a small amount of r-αSyn fibers were produced in the reaction seeded with DLB BH. To further confirm the authenticity of RT-QUIIC, we further analyzed BH samples from 4 DN-DLB patients (FIGS. 4B and 4C). Similar to DN-DLB cases # 1 and # 2, immunoblot analysis of all patient-derived samples showed> 250 kDa insoluble αSyn multimers. pSer129-αSyn was detected only in multimeric size (FIG. 4A). When 5 × 10 ⁻⁶ and 5 × 10 ⁻⁷ dilutions of these cases were added, the RT-QUIIC reaction all showed positive. Cases # 5 and # 6 were all positive at 5 × 10 ⁻⁸ dilution. Cases # 3 and # 4 showed a positive response in 5 out of 6 reactions at 5 × 10 ⁻⁸ dilution. At 5 × 10 ⁻⁹ dilution, the assay was positive in 4 out of 6 wells for case # 4, all wells for case # 5, and 4 out of 6 wells for case # 6, and case # 3 All of the wells were negative. At 5 × 10 ⁻¹⁰ dilution, all reactions in cases # 4, # 5 and # 6 were negative. Maximum fluorescence intensity ranges from 5 × 10 ^{−5 to} 5 × 10 ⁻⁸ for case # 3, # 4 and # 6 and 5 × 10 ⁻⁶ to case # 5 compared to the control without seeding. It was significantly stronger in reactions with dilutions in the range of 5 × 10 ⁻⁹ (FIG. 4C). Lag phase, as compared to the control without seeding, case # 3 and # 4 of 5 × ¹⁰ -6 ~5 × ^{10 -8} and case # 5 and ^{# 6 5 × 10 -6 ~5 ×} 10 the ^{- It} was significantly shorter in reactions with a range of ⁹ dilutions (Figure 4C). The SD ₅₀ / g brain values were as follows: 10 ^8.6 (case # 3) 10 ^9.3 (case # 4) 10 ^9.8 (case # 5) and 10 ^9.5 (case) # 6). Thus, we were able to detect brain seeding activity from other DN-DLB patients.

Insoluble aggregates of α-synuclein have little or no seeding activity. Next, we used Ser129 phosphorylated r-αSyn (pSer129-r-αSyn), and Ser129 phosphorylation was a prion. It was examined whether it is extremely important for αSyn fibril formation by a similar mechanism. WT r-αSyn was phosphorylated at Ser129 only by incubation in the presence of both casein kinase 2 (CK2) and ATP, whereas the S129A mutant was not phosphorylated under the same conditions (FIG. 5A). As with DLB BH, insoluble pSer129-r-αSyn was only observed in the molecular weight range of> 250 kDa after 72 and 264 hours of incubation. Significantly increased ThT fluorescence levels between WT r-αSyn and CK2 incubated with CK2 and S129A r-αSyn (S129A ^{CK2 + ATP} ) incubated with ^{CK2 in the} absence (WT ^CK2 ) or presence (WT ^{CK2 + ATP} ) in the presence or absence of ^ATP Although there was no difference (FIG. 5B), after 72 hours incubation, aggregate formation of pSer129-r-αSyn was induced more efficiently than that of unphosphorylated r-αSyn (FIG. 5A). These results suggested that Ser129 phosphorylation promotes the polymerization of r-Syn. Consistent with previous reports (Vlad et al., 2011), a 13 kDa band of lower molecular weight than full-length r-αSyn was observed in all samples after 72 hours incubation, which resulted in r- It has been shown that αSyn aggregate formation is mediated by cleavage and / or degradation of r-αSyn. FTIR spectra of WT (WT-264h) and mutant r-αSyn (S129A-264h) incubated with CK2 and ATP for 264 hours show slightly lower wavenumbers compared to before incubation (WT-0h and S129A-0h) This showed a modest increase in β-sheet content (1630-1610 cm ⁻¹ ) (FIG. 5C). There was little difference in the infrared spectrum between WT and mutant r-αSyn before and after incubation (FIG. 5C). TEM analysis showed that WT-264h and S129A-264h consist exclusively of amorphous aggregates (FIG. 5D). We next examined in RT-QUIIC whether r-αSyn could be newly converted to amyloid fibrils in the presence of amorphous r-αSyn aggregates. Surprisingly, all reactions with WT-264h or S129A-264h produced negative results in the RT-QUIIC assay at 2 × 10 ⁻² and 2 × 10 ⁻⁴ dilutions (FIG. 5E). Therefore, the insoluble aggregates of r-αSyn did not have prion-like seeding activity, regardless of whether they were phosphorylated with Ser129.

oligomer-like form of α- synuclein by WT r-αSyn RT-QUIC with eliciting a prion-like propagation, in the reaction with CK2 in the presence of ^{ATP (WT CK2 +} ATP), in its absence ^{(WT CK2)} Resulted in a rapid increase in fluorescence intensity and a higher level of fluorescence intensity than in FIG. 6, but there was no difference in the ThT binding kinetics of S129A r-αSyn between the two conditions (S129A ^CK2 and S129A ^{CK2 + ATP} ) (FIG. 6A). ). The antibody against pSer129-αSyn detected a dominant band at 16 kDa and a thin band in the molecular weight range> 250 kDa only in WT ^{CK2 + ATP} (FIG. 6B). These results suggest that Ser129 phosphorylation promotes r-αSyn fibril formation in RT-QUICK. Unlike the insoluble aggregates produced without shaking, as shown in FIG. 5A, all of the reactions showed a dominant band of monomeric αSyn at 16 kDa. Furthermore, polymers over 250 kDa were barely detected in WT ^{CK2 + ATP} and WT ^CK2 . The difference in r-αSyn aggregate size from FIG. 5A is probably due to agitation that can cause fiber fragmentation. FTIR analysis showed that there was almost no difference in the dominant band at 1650 cm ⁻¹ attributable to the denatured structure between all reactions (FIG. 6C). TEM analysis of WT ^{CK2 + ATP} and S129A ^{CK2 + ATP} revealed an oligomeric granular form of r-αSyn (FIG. 6D). To verify whether these oligomeric species showed seeding activity, we performed a second passage of RT-QUIIC samples (FIGS. 6E and 6F). WT ^{CK2 + ATP} , WT ^CK2 and S129A ^{CK2 + ATP} diluted 2 × 10 ⁻⁴ and 2 × 10 ⁻⁵ showed 100% positive in all reactions. The 2 × ^{10 -6 dilution,} in 5 out of 6 wells for ^{WT CK2 + ATP,} with 2 out of 6 wells for ^{WT CK2,} for ^{S129a CK2 + ATP} in 5 out of 6 wells, assay were positive. At the 2 × 10 ⁻⁷ dilution, all reactions with WT ^{CK2 + ATP} and WT ^CK2 were negative, but at this concentration, 3 out of 6 reactions with S129A ^{CK2 + ATP} were positive. The 5 × 10 ⁻⁸ dilution showed a negative result in all wells with these three samples. The phosphorylated oligomer-like molecular species resulted in an SD ₅₀ value of 10 ^4.9 / μg r-αSyn (WT ^{CK2 + ATP} ), and the non-phosphorylated oligomer-like molecular species yielded 10 ^4.4 / μg r-αSyn (WT ^CK2). ) And 10 ^5.4 / μg r-αSyn (S129A ^{CK2 + ATP} ) showed SD ₅₀ values. In contrast, we contained mock samples (WT-mock) containing the same components as WT ^{CK2 + ATP} and prepared without shaking just prior to the assay at dilutions ranging from 2 × 10 ⁻⁴ to 2 × 10 ⁻⁸ . No increase in fluorescence was observed in any reaction. Maximum fluorescence intensity is compared to WT-mock for reactions with WT ^{CK2 + ATP} and WT ^CK2 at 2 × 10 ⁻⁵ dilution and with S129A ^{CK2 + ATP} for dilutions ranging from 2 × 10 ⁻⁴ to 2 × 10 ⁻⁶ It was significantly stronger (FIG. 6F). Lag phase, reaction with ^{WT CK2} dilution range of reactions and 2 × ^{10 -4} and 2 × ^{10 -5} and 2 × ¹⁰ -4 to 2 × dilution range of ^{10 -6 WT CK2 + ATP} and ^{S129a CK2 + ATP} Therefore, it was significantly shorter than WT-mock (FIG. 6F). Our results showed that r-αSyn oligomeric species show seeding activity with or without phosphorylation at Ser129.

Furthermore, the maximum fluorescence intensity of the reaction with S129A ^{CK2 + ATP} at 2 × 10 ⁻⁴ and 2 × 10 ⁻⁵ dilutions was significantly stronger than that of WT ^{CK2 + ATP} (FIG. 6F). Moreover, the lag phase of the reaction with 2 × 10 ⁻⁵ dilution of S129A ^{CK2 + ATP} was significantly shorter than that of WT ^{CK2 + ATP} (FIG. 6F). Therefore, it was shown that the RT-QUIIC reaction liquid containing S129A ^{CK2 + ATP} has higher seeding ability than those containing WT ^{CK2 + ATP} or WT ^CK2 . The exact reason for these differences is not yet known, but it is likely that subtle structural differences between WT r-αSyn and S129A r-αSyn are related.

Discussion The results of this study demonstrated for the first time that the formation of r-αSyn fibrils is induced by RT-QUIIC with soluble r-αSyn only in the presence of BH from DLB patients (FIGS. 3A and 3). 3B). Sodium dodecyl sulfate (SDS) insoluble aggregates larger than 250 kDa, detected only in DLB cases, are specifically phosphorylated at Ser129 (FIG. 2C), so we have insoluble aggregates of pSer129-αSyn. Was assumed to give prion-like seeding activity. However, surprisingly, insoluble aggregates of r-αSyn accompanied by an increase in β-sheet structure had little seeding activity in both phosphorylated and non-phosphorylated states (FIG. 5E). Instead, r-αSyn profibril oligomers exhibited seeding activity, both with and without phosphorylation (FIGS. 6E and 6F). Our findings suggest that αSyn, which is a soluble oligomer but not completely fibrillar, is an in vitro seeding species. Previous studies have shown that oligomeric molecular species of r-αSyn are internalized by primary neurons or neuronal cell lines and induce aggregation of endogenous αSyn (Danzer et al., 2007; Danzer et al. , 2009). Furthermore, there is considerable evidence to suggest that the oligomeric form of αSyn is responsible for neuronal cell death and neurodegeneration (Brown, 2010; Vekrellis., 2011). In postmortem studies, soluble αSyn oligomer levels in the brains of DLB patients were significantly higher than AD patients and controls, while there was no significant difference in total αSyn levels between the three groups (Paleogouou et al.,). 2009). These reports support the suggestion that profibrillar αSyn oligomers rather than mature fibrils or amorphous aggregates represent pathogenic molecular species with prion behavior in LBD. A similar approach using monomeric amyloid β (Aβ) allows the protein misfolding circular amplification (PMCA) assay to detect seeding activity associated with Aβ oligomers present in CSF from AD patients. (Salvadores at al., 2014). DN-DLB from brain tissue, in cases # 1 and # 2, ^{respectively,} have a ^{10 3.4} / [mu] g total aSyn and ^{10 3.1} / ^μg _{SD 50} values of all aSyn, in Li-LDB is 10 ^2.1 / μg total αSyn (FIGS. 3A and 3B). Meanwhile, the value of the WT r-aSyn oligomers generated by RT-QUIC was ^{10 4.4 μg / r-αSyn~10 4.9} μg / r-αSyn ( FIGS. 6E and 6F). In the RT-QUIIC reaction, it is not clear whether all r-αSyn is present in oligomeric form, but if so, in the brains of DN-DLB cases # 1 and # 2, 3.2% of total αSyn, respectively. It is estimated that 10% and 1.6% to 5.0% are oligomers and 0.2% to 0.5% are oligomers in the Li-LDB brain. Although the exact role of insoluble LB aggregates remains unclear, cytoprotective and neuroprotective roles have been studied using cell lines (Tanaka et al., 2004) and Drosophila (Chen and Feany, 2005). It is reported in. The seeding activity of the S129A oligomer was higher than that of the WT oligomer regardless of the phosphorylation state (FIG. 6F), and a small amount of insoluble aggregates was detected in the reaction solution containing the WT oligomer in Western blot analysis. However, this was not the case in the reaction solution containing the S129A oligomer (FIG. 6B). These results suggest that insoluble aggregates provide protection from prion-like propagation of αSyn. Although there was no significant difference in the seeding activity of the r-αSyn oligomer between the Ser129 phosphorylated and non-phosphorylated forms, we found that phosphorylation of r-αSyn promoted self-aggregation. (FIGS. 5A and 6A). This is consistent with previous reports (Fujiwara et al., 2002). According to previous studies, elevated pSer129-αSyn levels precede the appearance of LB in the brains of LBD patients (Lue et al., 2012), and oxidative stress, mitochondrial dysfunction (Perfeitoto) observed in the pathology of LBD. at al., 2014) and proteasome inhibition (Chau et al., 2009). Furthermore, pSer129-αSyn has been reported to have a protective effect against neuronal dysfunction (Gorbatyuk et al., 2008; Kuwahara et al., 2012). These results suggest that pSer129-αSyn is due to a defense mechanism against neural dysfunction. pSer129-αSyn appears to facilitate the initiation of self-aggregation into pre-fiber oligomers and mature fibers thereafter.

RT-QUIIC made it possible to differentiate DLB from other degenerative diseases such as AD and prion diseases and non-degenerative cases, which allowed r-αSyn to be differentiated from other misfolded proteins: It is suggested that amyloid β, tau and PrP ^Sc are almost unaffected by the ability to induce heterologous cross seeding. There are reports of cross-seeding interactions between αSyn and other misfolded proteins in vitro and in vivo (Morales et al., 2013), but seems to have little effect on RT-QUIIC responses. In the differentiation of DLB from other degenerative diseases, the possibility of applying specific detection using RT-QUIIC to differential diagnosis arises. The brain from DN-DLB patients was estimated to have an SD ₅₀ value of 10 ^{7 to 10} / g brain, and the brain from Li-DLB patients was estimated to have an SD ₅₀ value of approximately 10 ^5.1 / g brain. . Thus, RT-QUIIC is more sensitive to detection of DLB compared to tests for pSer129-αSyn using Western blot or ELISA, resulting in more accurate brain biopsy or autopsy diagnosis Was suggested. Several studies using ELISA or bead-based flow cytometry assays investigated CSF and blood total αSyn levels in patients with DLB and other synucleinopathies, but the results are inconclusive And contradictory (Kasuga et al., 2012). On the other hand, in the CSF and blood of DLB and PD, soluble αSyn oligomer levels were shown to be increased compared to those of AD and control (Hansson et al., 2014; Tokuda et al.,). 2010). These reports support the suggestion that oligomeric αSyn is an important and promising target for LBD diagnosis, and the diagnostic potential of RT-QUIIC by seeding CSF and blood-derived oligomers in LBD patients Is suggested. Therefore, using RT-QUIIC together with an existing ELISA specific for αSyn oligomers is considered to be particularly advantageous for differential diagnosis of LBD. Further studies are needed to determine whether RT-QUIIC can be used to differentiate between different types of LBD and whether this assay can be used with body fluids or other tissues.

  In this study, we have demonstrated the feasibility of using RT-QUIIC to detect a candidate αSyn seeding species in the laboratory. Our data provide further support for the suggestion that the oligomeric form of αSyn is a pathogenic molecular species that triggers prion-like transmission and plays an important role in the development of LBD. We believe this new method will be a solid tool for clinical diagnosis, drug candidate screening, and our understanding of the role of αSyn as a prion-like protein in LBD.

Differentiation between α-synucleinopathy and other dementias using additional experimental cerebrospinal fluid samples. Autopsy confirms dementia with Lewy bodies (DLB) and arcuate Creutzfeldt-Jakob disease (CJD). Cerebrospinal fluid samples of patients who had been treated were collected, and RT-QUIIC for α-synucleinopathy was performed on each (FIG. 12). The reaction mixture contained 300 mM NaCl. FIG. 12 shows the results of 5 cases (cases # 1 to 5) for DLB and 1 case (case CJD # 1) for CJD. Signals were detected specifically only in samples of DLB patients in autopsy cases (certain cases). No signal was detected in samples of CJD patients and unseeded controls (PBS).

Differential diagnosis support test (prion disease vs DLB)
In cases where it was difficult to diagnose prion disease (CJD) and DLB, RT-QUIC test was performed on the collected cerebrospinal fluid, and it was positive by the synuclein / QUIC method (FIG. 13; control without seed (PBS)) No signal was detected, and the RT-QUIIC test for CJD (Non-patent Document 18) was negative). In this experiment, NaCl was not included in the reaction mixture. In subsequent examinations, DLB was diagnosed from the results of MIBG myocardial scintigraphy and cerebral blood flow scintigraphy, which was consistent with the results of RT-QUIIC method.

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  According to the present invention, it is possible to assist in the diagnosis of whether or not a subject is suffering from α-synucleinopathy. This method can be a powerful method that can assist in the clear differentiation between α-synucleinopathies and other dementias. By this method, it is possible to select an appropriate treatment method for suppressing the progression of α-synucleinopathy by early differentiation.

Claims (21)

(1) A step of mixing a biological sample and normal α-synuclein in a buffer solution to obtain a reaction mixture;
(2) Incubating the reaction mixture to form an α-synuclein aggregate using abnormal α-synuclein in a biological sample as a seed; and (3) detecting the formed α-synuclein aggregate. Process,
A method for detecting abnormal α-synuclein in a biological sample, comprising: (1) A step of mixing a biological sample and normal α-synuclein in a buffer solution to obtain a reaction mixture;
(2) Incubating the reaction mixture to form an α-synuclein aggregate using abnormal α-synuclein in a biological sample as a seed; and (3) detecting the formed α-synuclein aggregate. Process,
A method for assisting diagnosis of α-synucleinopathy, comprising:   The method according to claim 1 or 2, wherein the normal α-synuclein is recombinant α-synuclein or purified α-synuclein.   The method according to claim 1, wherein the biological sample is a brain tissue sample.   The method according to any one of claims 1 to 3, wherein the biological sample is a blood, plasma, serum, leukocyte or cerebrospinal fluid sample.   The method according to claim 1, wherein the concentration of normal α-synuclein in the reaction mixture is 120 μg / mL to 150 μg / mL.   The method according to any one of claims 1 to 6, wherein the buffer solution is a buffer solution having a pH of 7.2 to 7.8.   The method according to claim 1, wherein the buffer solution is a HEPES buffer solution.   The method according to claim 1, wherein the reaction mixture is shaken in the step (2).   10. The method of claim 9, wherein the formation of α-synuclein aggregates is performed in a cycle of shaking and no shaking periods.   The method of claim 10, wherein the cycle is a total of 3 minutes.   The method according to any one of claims 1 to 11, wherein α-synuclein aggregates are detected using an antibody.   The method according to claim 12, wherein the antibody is an anti-α-synuclein polyclonal antibody, an anti-α-synuclein monoclonal antibody, or an anti-Ser129 phosphorylated α-synuclein monoclonal antibody.   The method according to claim 1, wherein the step (3) comprises quantifying the formed α-synuclein aggregate.   The method according to any one of claims 1 to 14, wherein the step (3) comprises a Western blot method.   The method according to claim 1, wherein the step (3) includes fluorescence spectroscopy.   17. The method of claim 16, wherein detecting α-synuclein aggregates comprises the use of thioflavin T (ThT).   The method according to claim 16 or 17, wherein the detection is performed every 10 minutes.   The method according to any one of claims 2 to 18, which is a method for assisting in the differentiation of α-synucleinopathy from other diseases associated with cognitive impairment.   The method according to any one of claims 2 to 19, wherein the α-synucleinopathy is Lewy body disease or multiple system atrophy.   21. The method of claim 20, wherein the Lewy body disease is Lewy body dementia, Parkinson's disease, or diffuse neocortical Lewy body dementia.