인간 A53T를 과발현 형질 전환 마우스의 뇌에 강화 된 ELISA에 의한 질병 관련 α-synuclein의 검출은 α-synuclein의 돌연변이
Summary
An ELISA offering a novel quantitative approach is described. It specifically detects disease-associated α-synuclein (αSD) in a transgenic mouse model (M83) of synucleinopathy using several antibodies against either the Ser129 phosphorylated αS form or the C-terminal part of the protein.
Abstract
웨스턴 블롯 등의 설립 방법과 더불어, 새로운 방법을 쉽고 빠르게 synucleopathies의 실험 모델에서 질병 관련 α-synuclein의 (αS D)를 정량화하기 위해 필요하다. 과발현 인간 A53T의 αS를 자발적으로 체중 감소, 쇠약, 및 심각한 운동 장애 등의 증상을 특징으로 나이 여덟 22 개월 사이 극적인 임상 표현형을 개발하는 트랜스 제닉 마우스 선 (M83)는이 연구에 사용 하였다. 이 마우스 αS D (질병 관련 αS)의 분자 분석을 위해, ELISA 특별히 아픈 쥐에서 αS D를 정량화하기 위해 설계되었습니다. 이 마우스 모델에서 중추 신경계의 분석은 주로 꼬리 뇌 영역과 척수 αS D의 존재를 보여 주었다. 즉, 임상 질환으로 이어지는 다른 실험 조건 사이의 αS D 분포의 차이는 uninocul에 없었다ated 정상적으로 형질 전환 마우스를 노화와 병 마우스의 뇌 추출물 접종 생쥐에서. Ser129에 대한 항체를 사용하여 αS D 면역 반응의 구체적인 검출은 본질적으로 웨스턴 블롯 및 면역 조직 화학에 의해 얻어진 그 상관 관계를 ELISA로 αS를 인산화. 예기치 않게, 유사한 결과 αS의 C 말단 부위에 대해 여러 가지 다른 항체를 관찰 하였다. "프리온 같은"기구의 관련을 암시 αS D의 전파는, 쉽게 감시되고 따라서, ELISA 접근법을 사용하여이 마우스 모델에서 정량화 할 수있다.
Introduction
Most current methods for detecting disease-associated α-synuclein (αSD) in experimental models of Parkinson’s disease (PD), such as immunohistochemistry or Western blot, are time-consuming and not quantitative. This neurodegenerative disease is characterized by alpha-synuclein aggregation mainly in the form of inclusions containing an aggregated form of the normally soluble presynaptic protein αS1,2 (Lewy bodies and Lewy neurites). Normally only marginally phosphorylated, αS is hyperphosphorylated at its serine 129 residue in these inclusions3 and can be monitored by antibodies specifically directed against Ser129 phosphorylated αS, thus providing a reliable marker of the pathology.
Recent research suggests that a “prion-like” mechanism could be involved in the propagation of αS aggregation within the nervous system of an affected patient4,5. These studies reported the acceleration of a synucleinopathy by inoculating brain extracts containing αSD into a transgenic mouse model (M83) expressing an A53T mutated human αS protein associated with a severe motor impairment occurring as the mice age6. In the same manner, intra-cerebral inoculation of aggregated recombinant αS in the same M83 mouse model confirmed the acceleration of aggregation5. The induction of deposits of phosphorylated αS has also been reported after inoculation of C57Bl/6 wild-type mice with either fibrillar recombinant αS or brain extracts from human DLB patients7,8. Sacino et al.9 recently pointed out that after injection of fibrillar human αS, a widespread and progressive cerebral αS inclusion formation could be induced in M83 mice, but not in E46K transgenic mice or non-transgenic mice in which induced αS inclusions were transient, and mainly restricted to the site of injection. Recent studies on monkeys confirmed propagation of αS aggregates after inoculation of PD-derived extracts in species closer to humans10.
The link between αS alterations and Parkinson’s disease suggest that αSD is a potential biomarker for Parkinson’s disease11. A recent study showed the detection of oligomeric soluble aggregates of α-synuclein in human cerebro-spinal fluid (CSF) and plasma as a potential biomarker for Parkinson’s disease based on a conventional sandwich system ELISA using the same antibody to capture and detect αS12. Based on the same method, multimeric proteins were recognized in biological samples, including the brain, because there are multiple copies of epitopes present in the assembled forms13. Very recently, pathological αS in the CSF of patients with a proven Lewy body pathology was detected using both an ELISA kit with a highly specific antibody against αSD (5G4) and an immunoprecipitation assay14. These methods could differentiate patients with PD/DLB from other types of dementia.
The “prion-like” propagation of αS aggregation was further studied in transgenic mouse model M83 using an ELISA approach that was designed to specifically identify αSD15. In this study, we report the detailed ELISA protocol used to quantitatively detect αSD in sick mice (whether or not inoculated with αSD from sick M83 mice) and more especially in the brain regions specifically targeted by the pathological process in this M83 transgenic mouse model4.
Protocol
Representative Results
Discussion
ELISA의 사용은 구체적 M83 트랜스 제닉 마우스 모델에서 질병 중에 마우스 뇌 균질 물로부터 직접 αS D를 검출하는 것으로 입증되었다. 사실,이 ELISA 쉽게 높은 소금 버퍼 만 전체 뇌 균질를 사용하여 건강한 M83 마우스에서 아픈 M83 마우스를 구별 할 수있다.
이 ELISA를 사용하여 성공적인 결과를 위해 가장 중요한 단계는 다음과 같습니다 제대로 해부하는 동안 손상을 방…
Disclosures
The authors have nothing to disclose.
Acknowledgements
저자는 예방 접종에 대한 데미안 가일 라드 감사 및 후속 동물 실험을하고 싶습니다. 이 작품은 ANSES (식품, 환경 및 산업 보건 및 안전을위한 프랑스 기관)에 의해 재단 프랑스 파킨슨에서 교부금에 의해 지원되었다.
Materials
| LB509 | Abcam | ab27766 | Detection antibody 1/2000 | |
| AS11 | Produced at Anses | Detection antibody 1/1000 | ||
| 4D6 | Abcam | ab1903 | Detection antibody 1/2000 | |
| PSer129 | Abcam | ab59264 | Detection antibody 1/3000 | |
| PSer129 EP1536Y | Abcam | ab51253 | Detection antibody 1/1000 | |
| syn514 | Abcam | ab24717 | Detection antibody 1/500 | |
| clone 42 | BD Biosciences | 610787 | Coating and detection antibody (1/2000) | |
| 8A5 | Provided by Dr. Anderson | Detection antibody 1/2000 | ||
| polyclonal anti-αsyn antibody | Millipore | AB5038P | Coating antibody | |
| Anti-mouse IgG HRP conjugate | Southern Biotech | 1010-05 | ||
| Anti-rabbit IgG HRP conjugate | Southern Biotech | 4010-05 | ||
| Goat anti-mouse IgG HRP conjugate | Dianova | 115-035-164 | ||
| HS buffer | Tris-HCl 50 mM | Euromedex | 26-128-3094-B | Adjust at pH 7.5 and keep at 4°C |
| NaCl 750 mM | Euromedex | 1112-A | ||
| EDTA 5 mM | Euromedex | EU0007-B | ||
| DTT 1 mM | Sigma | 43815 | ||
| PBS | Na2HPO4 1 mM | Euromedex | 1309 | Adjust at pH 7.5 |
| KH2PO4 1,5 mM | Euromedex | 2018 | ||
| NaCl 137 mM | Euromedex | 1112-A | ||
| KCl 2,7 mM | Euromedex | P017 | ||
| Tween 20 | Euromedex | 2001-C | ||
| BSA | Sigma | A7906 | ||
| DTT 1 mM | Sigma | 43815 | stock solution 100 mM, toxic | |
| 1% phosphatase cocktail | Pierce | 78428 | ||
| 1% protease inhibitor cocktail | Roche | 04 693 132 001 | 50 X concentrated | |
| Microplate MaxiSorpTM | Thermo Scientific | 442404 | ||
| Tampon carbonate 50 mM pH 9.6 | Na2CO3, 10H2O | Sigma | 71360 | 2.86 g/L |
| NaHCO3 | Merk | 6329 | 3.36 g/L, pH9.6 | |
| Superblock T20 PBS blocking buffer | Pierce | E6423H | 10 X concentrated | |
| TMB | Sigma | T0440 | Used for ELISA | |
| TMB | Analytik Jena AG | 847-0104200302 | Used for epitope mapping | |
| HCl 1N | Chimie plus | 40030 | ||
| Ribolyser | Thermo | Fast prep FP120 | keep on ice at this step | |
| Grinding tubes | Biorad | 355-1197 | ||
| Plate washer | Tecan | Columbus Pro | ||
| Plate reader | Biorad | Model 680 | ||
| Low power magnifier | VWR | 630-1062 | X8 magnification | |
| Forceps Dumont#7 | WPI | 14097 | For dissection steps | |
| Transfer pipette 1ml Samso | Samso | 043231 | ||
| 1,5 ml tubes | Dutscher | 033290 |
References
- Goedert, M., Spillantini, M. G., Del Tredici, K., Braak, H. 100 years of Lewy pathology. Nat Rev Neurol. 9, 13-24 (2013).
- Waxman, E. A., Giasson, B. I. Specificity and regulation of casein kinase-mediated phosphorylation of alpha-synuclein. J Neuropathol Exp Neurol. 67, 402-416 (2008).
- Anderson, J. P., et al. Phosphorylation of Ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic Lewy body disease. J Biol Chem. 281, 29739-29752 (2006).
- Mougenot, A. L., et al. Prion-like acceleration of a synucleinopathy in a transgenic mouse model. Neurobiol Aging. 33, 2225-2228 (2012).
- Luk, K. C., et al. Intracerebral inoculation of pathological alpha-synuclein initiates a rapidly progressive neurodegenerative alpha-synucleinopathy in mice. J Exp Med. 209, 975-986 (2012).
- Giasson, B. I., et al. Neuronal alpha-synucleinopathy with severe movement disorder in mice expressing A53T human alpha-synuclein. Neuron. 34, 521-533 (2002).
- Luk, K. C., et al. Pathological alpha-synuclein transmission initiates Parkinson-like neurodegeneration in nontransgenic mice. Science. 338, 949-953 (2012).
- Masuda-Suzukake, M., et al. Prion-like spreading of pathological alpha-synuclein in brain. Brain : a journal of neurology. 136, 1128-1138 (2013).
- Sacino, A. N., et al. Amyloidogenic alpha-synuclein seeds do not invariably induce rapid, widespread pathology in mice. Acta neuropathologica. 127, 645-665 (2014).
- Recasens, A., et al. Lewy body extracts from parkinson’s disease brains trigger alpha-synuclein pathology and neurodegeneration in mice and monkeys. Ann Neurol. , (2013).
- Foulds, P. G., et al. Phosphorylated alpha-synuclein can be detected in blood plasma and is potentially a useful biomarker for Parkinson’s disease. FASEB J. 25, 4127-4137 (2011).
- El-Agnaf, O. M., et al. Detection of oligomeric forms of alpha-synuclein protein in human plasma as a potential biomarker for Parkinson’s disease. Faseb J. 20, 419-425 (2006).
- Lee, H. J., et al. Enzyme-linked immunosorbent assays for alpha-synuclein with species and multimeric state specificities. J Neruosci Meth. 199, 249-257 (2011).
- Unterberger, U., et al. Detection of disease-associated alpha-synuclein in the cerebrospinal fluid: a feasibility study. Clin Neuropathol. 33, 329-334 (2014).
- Betemps, D., et al. Alpha-synuclein spreading in M83 mice brain revealed by detection of pathological alpha-synuclein by enhanced ELISA. Acta Neuropathol. (Berl). 2, 29 (2014).
- Osman, A. A., et al. A monoclonal antibody that recognizes a potential coeliac-toxic repetitive pentapeptide epitope in gliadins). Eur J Gastroenterol Hepatol. 13, 1189-1193 (2001).
- Pinheiro, J. C., Bates, D. M., Chambers, J. Ch. 5. Mixed-Effects Models in S and S-PLUS. 5, 206-225 (2000).
- Mougenot, A. L., et al. Production of a monoclonal antibody, against human alpha-synuclein, in a subpopulation of C57BL/6J mice, presenting a deletion of the alpha-synuclein locus. J Neruosci Meth. 192, 268-276 (2010).
- Specht, C. G., Schoepfer, R. Deletion of the alpha-synuclein locus in a subpopulation of C57BL/6J inbred mice. BMC Neurosci. 2, 11 (2001).
- Lee, B. R., Matsuo, Y., Cashikar, A. G., Kamitani, T. Role of Ser129 phosphorylation of alpha-synuclein in melanoma cells. J Cell Sci. 126, 696-704 (2013).
- Perrin, R. J., et al. Epitope mapping and specificity of the anti-alpha-synuclein monoclonal antibody Syn-1 in mouse brain and cultured cell lines. Neurosci Lett. 349, 133-135 (2003).
- Emmanouilidou, E., et al. Assessment of alpha-synuclein secretion in mouse and human brain parenchyma. PLoS One. 6, e22225 (2011).
- Mougenot, A. L., et al. Transmission of prion strains in a transgenic mouse models overexpressing human A53T mutated alpha-synuclein. J Neuropathol Exp Neurol. 70, 377-385 (2011).
- Foulds, P. G., et al. A longitudinal study on alpha-synuclein in blood plasma as a biomarker for Parkinson’s disease. Sci Rep. 3, 2540 (2013).
