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.
Neben etablierten Verfahren wie Western Blot, werden neue Verfahren benötigt wird, um schnell und leicht in experimentellen Modellen der Synucleopathien quantifizieren Krankheit assoziierten α-Synuclein (aS D). Eine transgene Mauslinie (M83) Überexpression des menschlichen A53T aS und spontan die Entwicklung einer dramatischen klinischen Phänotyp zwischen acht und 22 Monate alt sind, gekennzeichnet durch Symptome wie Gewichtsverlust, Erschöpfung, und schwere motorische Einschränkungen, wurde in dieser Studie verwendet. Für die molekulare Analyse von & alpha; S D (krankheitsassoziierten aS) in diesen Mäusen ein ELISA wurde entwickelt, um spezifisch zu quantifizieren aS D in kranken Mäusen. Analyse des Zentralnervensystems in diesem Mausmodell zeigte die Anwesenheit von & alpha; S D hauptsächlich in den kaudalen Hirnregionen und des Rückenmarks. Es gab keine Unterschiede in der & alpha; S D Verteilung zwischen verschiedenen experimentellen Bedingungen, die zu einer klinischen Erkrankung, dh in uninoculATED und normal alternden transgenen Mäusen und bei Mäusen mit Gehirnextrakten von kranken Mäusen eingeimpft. Der spezifische Nachweis von & alpha; S D Immunreaktivität unter Verwendung eines Antikörpers gegen Ser129 phosphoryliert aS durch ELISA im wesentlichen mit der durch Western-Blot und Immunhistochemie erhalten korreliert. Unerwarteterweise wurden ähnliche Ergebnisse mit mehreren anderen Antikörpern gegen den C-terminalen Teil des & alpha; S beobachtet. Die Ausbreitung von aS D, was auf die Beteiligung eines "Prion-ähnlichen" -Mechanismus kann somit leicht überprüft und in diesem Mausmodell unter Verwendung eines ELISA-Ansatz quantifiziert werden.
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.
Die Verwendung eines ELISA wurde gezeigt, spezifisch nachzuweisen aS D direkt von Maushirn-Homogenaten bei der Krankheit in den M83 transgenes Mausmodell. In der Tat, könnte dies ohne weiteres zu unterscheiden ELISA kranken Mäusen M83 M83 von gesunden Mäusen mit nur ganze Hirnhomogenaten in Hochsalzpuffer.
Die wichtigsten Schritte für eine erfolgreiche Ergebnisse mit diesen ELISA sind: richtig sezieren die verschiedenen Regionen der Maus-Gehirne von der Entwicklung der notwend…
The authors have nothing to disclose.
Die Autoren bedanken sich bei Damien Gaillard für Impfungen danken und Follow-up von Tierversuchen. Diese Arbeit wurde von ANSES (Französisch Agentur für Lebensmittel, Umwelt- und Arbeitsschutz) und durch einen Zuschuss von der Stiftung France Parkinson unterstützt.
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 |