Opsporing van de ziekte-geassocieerde α-synucleïne door Enhanced ELISA in de hersenen van transgene muizen door overexpressie van Human A53T Gemuteerde α-synucleïne
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
Naast gevestigde werkwijzen zoals Western blot, zijn nieuwe methoden nodig om snel en gemakkelijk te kwantificeren ziektegeassocieerde α-synucleïne (aS D) in experimentele modellen van synucleopathies. Een transgene muis lijn (M83) overexpressie menselijke A53T aS spontaan ontwikkelen van een dramatische klinische fenotype acht tot 22 maanden oud, gekenmerkt door symptomen zoals gewichtsverlies, uitputting en ernstige motorische beschadiging, werd gebruikt in deze studie. Voor moleculaire analyses van aS D (ziekte-geassocieerde aS) in deze muizen, werd een ELISA ontworpen om specifiek te kwantificeren aS D in zieke muizen. Analyse van het centrale zenuwstelsel in dit muismodel toonde de aanwezigheid van aS D voornamelijk in de caudale hersenen en het ruggenmerg. Er waren geen verschillen in aS D verdeling over de verschillende experimentele omstandigheden die leiden tot klinische ziekte, dat wil zeggen in uninoculated en normaal veroudering transgene muizen en in muizen geënt met hersenen extracten van zieke muizen. De specifieke detectie van aS D immunoreactiviteit met behulp van een antilichaam tegen gefosforyleerd Ser129 aS door ELISA hoofdzaak gecorreleerd met die welke wordt verkregen door middel van Western blot en immunohistochemie. Onverwacht werden vergelijkbare resultaten waargenomen met diverse andere antilichamen tegen het C-terminale deel van aS. De voortplanting van aS D, wat een rol van een "prion-achtige" -mechanisme, kunnen dus gemakkelijk worden gecontroleerd en gekwantificeerd in dit muizenmodel met gebruik van een ELISA benadering.
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
Het gebruik van een ELISA werd aangetoond om specifiek te detecteren aS D rechtstreeks van muizenhersenen homogenaat gedurende de ziekte bij de M83 transgeen muismodel. Inderdaad, kan dit ELISA gemakkelijk onderscheiden ziek M83 muizen van gezonde M83 muizen met alleen hele hersenhomogenaten in High Salt buffer.
De meest kritische stappen voor succesvolle resultaten met deze ELISA zijn: kunnen ontleden de verschillende gebieden van de muizenhersenen door de nodige handvaardigheid …
Disclosures
The authors have nothing to disclose.
Acknowledgements
De auteurs willen graag Damien Gaillard bedanken voor inentingen en de follow-up van dierproeven. Dit werk werd ondersteund door ANSES (Franse agentschap voor Voedsel, Milieu en Occupational Health & Safety) en door een subsidie van de Stichting 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 |
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).
