Detecção de doença associada α-sinucleína por ELISA reforçada no cérebro de ratinhos transgénicos que sobre-expressam A53T Humano Mutante α-sinucleína
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
Além dos métodos estabelecidos como Western blot, são necessários novos métodos para quantificar rapidamente e facilmente associada a doença α-sinucleína (D aS) em modelos experimentais de synucleopathies. Uma linha de ratinhos transgénicos (M83) que sobre-expressam os aS A53T humanos e o desenvolvimento de um fenótipo clínico espontaneamente dramática entre oito e 22 meses de idade, caracterizada por sintomas que incluem a perda de peso, prostração, e deficiência motora grave, foi utilizada neste estudo. Para análises moleculares de aS D (aS associada a doença) nestes ratos, um ensaio ELISA foi desenvolvido para quantificar especificamente aS D em ratinhos doentes. Análise do sistema nervoso central neste modelo de ratinho revelou a presença de D aS principalmente nas regiões do cérebro caudal e da medula espinhal. Não houve diferenças na distribuição aS D entre diferentes condições experimentais que conduzem à doença clínica, isto é, em uninoculATED e normalmente envelhecimento ratinhos transgénicos e em ratinhos inoculados com extractos de cérebro de ratinhos doentes. A detecção específica de aS D imunorreactividade usando um anticorpo contra a Ser129 fosforilada aS por ELISA essencialmente correlacionados com os valores obtidos por transferência de Western e imuno-histoquímica. Inesperadamente, resultados semelhantes foram observados com vários outros anticorpos contra a parte C-terminal de aS. A propagação de aS D, sugerindo o envolvimento de um mecanismo de "prião-like", pode, assim, ser facilmente controlado e quantificado neste modelo de ratinho utilizando uma abordagem de ELISA.
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
Foi demonstrada a utilização de um ELISA para detectar especificamente aS D directamente a partir de homogenatos do cérebro do rato no decorrer da doença no modelo de ratinho transgénico para M83. Na verdade, este ELISA poderia facilmente distinguir os ratos doentes M83 M83 de camundongos saudáveis utilizando homogeneizados única inteiras cerebrais em tampão de alta sal.
Os passos mais críticos para os resultados bem sucedidos utilizando este ELISA são: dissecting …
Declarações
The authors have nothing to disclose.
Acknowledgements
Os autores gostariam de agradecer Damien Gaillard para inoculações e acompanhamento de experiências com animais. Este trabalho foi apoiado por ANSES (Agência Francesa de Alimentos, Saúde Ambiental e Ocupacional e Segurança) e por uma bolsa da Fundação France Parkinson.
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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