İnsan A53T aşırı eksprese eden transjenik farelerin beyninde Geliştirilmiş ELISA ile Hastalık ile ilişkili α-sinüklein tespiti α-sinüklein Mutasyona uğramış
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
Western blot gibi bilinen metotlara ek olarak, yeni yöntemler hızlı ve kolay bir sinükleopatiler deneysel modellerinde hastalıkla ilişkili α-sinüklein (aS D) ölçmek için ihtiyaç vardır. Aşırı ifade eden insan A53T aS ve kendiliğinden kilo kaybı, bitkinlik, ve ağır motor bozukluğu dahil olmak üzere semptomlarla karakterize Sekiz ve 22 ay arasında bir dramatik klinik fenotip üreten bir transjenik fare soyu (M83), bu çalışmada kullanılmıştır. Bu farelerde aS D (hastalıkla ilişkili aS) moleküler analiz için, bir ELISA, özellikle hasta farelerde aS D ölçmek için tasarlanmıştır. Bu fare modeli, merkezi sinir sistemi analizi esas kaudal beyin bölgelerinde ve omurilikteki aS D varlığını göstermiştir. Yani klinik hastalığa yol açan farklı deneysel koşullar arasında aS D dağılımında farklılıklar uninocul de vardıated ve normal transgenik fareler yaşlanma ve hastalık farelerden alınan beyin özleri ile aşılanmış farelerde. Ser129 karşı bir antikor kullanılarak aS D imünoreaktivite spesifik tespiti esas olarak Western Blot ve immünohistokimya ile elde edilen korelasyon ELISA ile aS fosforile. Beklenmedik bir şekilde, benzer bir sonuç aS C-terminal kısmına karşı birkaç diğer antikorlar ile gözlendi. Bir "prion gibi" mekanizmasının rolünü düşündürmüştür aS D yayılması, böylece kolayca izlenebilir ve bir ELISA yaklaşımı kullanarak bu fare modelinde belirlenebilir.
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
bir ELISA kullanımı özellikle M83 transjenik fare modelinde hastalıktan sırasında fare beyni homojenatlarında şirketinden aS D tespit etmek için ortaya konmuştur. Nitekim, bu ELISA kolayca Yüksek Tuz tamponunda sadece tüm beyin homojenatları kullanarak sağlıklı M83 farelerden elde edilen hasta M83 fareler ayırt olabilir.
Bu ELISA kullanılarak başarılı sonuçlar için en kritik adımlar şunlardır: doğru diseksiyon sırasında zarar görmesini önlemek için …
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Yazarlar aşılar için Damien Gaillard teşekkür ve takibinde hayvan deneyleri istiyoruz. Bu eser Anses (Gıda, Çevre ve İş Sağlığı ve Güvenliği Fransız Ajansı) tarafından ve Vakıf Fransa Parkinson bir hibe ile desteklenmiştir.
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 |
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