Detection of Disease-associated α-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated α-synuclein

Podsumowanie

An ELISA offering a novel quantitative approach is described. It specifically detects disease-associated α-synuclein (αS^D) 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.

Streszczenie

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated α-synuclein (αS^D) in experimental models of synucleopathies. A transgenic mouse line (M83) over-expressing the human A53T αS and spontaneously developing a dramatic clinical phenotype between eight and 22 months of age, characterized by symptoms including weight loss, prostration, and severe motor impairment, was used in this study. For molecular analyses of αS^D (disease-associated αS) in these mice, an ELISA was designed to specifically quantify αS^D in sick mice. Analysis of the central nervous system in this mouse model showed the presence of αS^D mainly in the caudal brain regions and the spinal cord. There were no differences in αS^D distribution between different experimental conditions leading to clinical disease, i.e., in uninoculated and normally aging transgenic mice and in mice inoculated with brain extracts from sick mice. The specific detection of αS^D immunoreactivity using an antibody against Ser129 phosphorylated αS by ELISA essentially correlated with that obtained by Western blot and immunohistochemistry. Unexpectedly, similar results were observed with several other antibodies against the C-terminal part of αS. The propagation of αS^D, suggesting the involvement of a “prion-like” mechanism, can thus be easily monitored and quantified in this mouse model using an ELISA approach.

Wprowadzenie

Most current methods for detecting disease-associated α-synuclein (αS^D) 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 αS^1,2 (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 patient^4,5. These studies reported the acceleration of a synucleinopathy by inoculating brain extracts containing αS^D 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 patients^7,8. 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 αS^D 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 αS^D (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 αS^D15. In this study, we report the detailed ELISA protocol used to quantitatively detect αS^D in sick mice (whether or not inoculated with αS^D from sick M83 mice) and more especially in the brain regions specifically targeted by the pathological process in this M83 transgenic mouse model⁴.

Protokół

All the procedures and protocols involving animals were in accordance with EC Directive 86/609/EEC and ratified by ComEth, the French national committee for consideration of ethics in animal experimentation (protocol 11-0043). The animals were housed and cared for in ANSES’s approved experimental facilities in Lyon (approval B 69387 0801).

1. Preparation of Mice

1. Euthanize mice by an intraperitoneal injection of lethal dose of sodium pentobarbital.
2. Retrieve the whole brain from the mouse skull and place it in a 35 mm plastic Petri dish on ice until extraction.
3. Extract the cervical spinal cord.
   NOTE: Extract αS either from one of the brain halves after sagittal sectioning or from dissected mouse brains, available after the experiments listed in Table 1.

Experiment	Mice Inoculum (brain equivalent)	Survival period (d.p.i.)	Median/maximal survival (days old)	αSd detection by ELISA /WB/IHC
1	Uninoculated mice	441 ± 166	419/736	8/8
2	Inoculated mice (0.2 mg)	150 ± 52	140/241	9/9

Table 1. List of experiments performed on M83 mice. Inoculations were performed at 6 weeks for experiment 2 in the striato-cortical area with 20 µl of a brain homogenate of a sick mouse (1% wt/vol in glucose 5%), after anesthesia of 6 weeks old homozygous M83 mice by 3% isoflurane inhalation. d.p.i.: days post inoculation.

2. αS Extraction from Brain Halves

1. Sagittally cut the brain to obtain two halves. Weigh each half in a ribolysis tube containing grinding balls.
2. Prepare High Salt (HS) buffer containing 50 mM Tris-HCl, pH 7.5, 750 mM NaCl, 5 mM EDTA, 1 mM DTT, 1% phosphatase and protease inhibitor cocktails. Add High Salt buffer to the brain halves to obtain 20% (weight/volume) homogenates.
3. Prepare samples from the brain halves using a mechanical homogenizer at 6.0 m/s for 23 sec twice. After the first 23 sec homogenization, place the tubes containing the homogenates on ice for 2 min before the second 23-sec cycle.
4. Centrifuge the samples at 1,000 x g for 5 min to eliminate unground brain fragments. Recover the supernatants, divide into 200 µl aliquots and keep them at -80 °C for subsequent ELISA analysis.

3. αS Extraction from Dissected Brain Regions

1. Dissect a whole brain in a 35 mm plastic Petri dish on ice with a low power magnifier (8X magnification) using two forcipes whose ends are kept together except when dissecting the hippocampus. Do not exceed 10 min to preserve brain integrity. Place the brain right side up and retrieve the following brain regions in this order:
   1. Separate one of the two olfactory bulbs using forceps placed just behind the bulb. Detach it from the brain by a downwards movement. Repeat this operation for the second bulb.
   2. Gently wedge the forceps in between the two cortexes and move it forwards to facilitate dissociation of the two cortexes. Keeping the brain in place with one forceps, use another to separate the cortex from the hippocampus.
   3. Position the forceps 2 mm below the cortex. Maintain a gentle pressure on the forceps until the top of the hippocampus is visible. Peel off the first part of the cortex, and repeat with the second part. Use the forceps to separate the two cortexes starting at the hippocampus and moving towards the front of the brain.
   4. Position the open forceps around one of the hippocampi. Close the forceps at the bottom of the hippocampus then gently remove it, recovering as much as possible. Repeat the procedure for the second hippocampus.
   5. Position the open forceps below one of the striata and gently separate it from the brain. Use the forceps to remove any remaining cortex from the striatum. Repeat this procedure for the second striatum.
   6. Use the forceps to gently depress by 2 mm the contour of the cerebellum to facilitate separation of the cerebellum from the brain. Place the forceps just behind the cerebellum and remove it by moving the forceps forward.
   7. Use the wide part of the forceps to raise the mesencephalon in order to clearly see where it joins the brain stem. Make a vertical incision at the junction then remove the brain stem.
   8. Position the forceps behind the mesencephalon, which is composed of four rounded structures. Incise vertically until the mesencephalon has been completely separated from the remaining brain.
2. Prepare homogenates of variable % (weight/volume) in HS buffer, depending on the quantity of available tissues, i.e., 5% homogenate for a weight between 10 and 30 mg, 10% for a weight between 30 and 80 mg, and 20% for a weight above 80 mg.
   1. Add an adequate volume of HS buffer to the dissected brain regions to obtain the expected % of homogenate.
   2. Vortex and check that the tissues are fully immersed in the HS buffer.
   3. Homogenize samples prepared from the dissected brain regions or the cervical spinal cord with a tissue grinder composed of a borosilicate glass tube and two pestles, A and B.
   4. Pour each brain region to be crushed directly into the tube. Insert pestle A into the tube and retract it. Repeat this movement about ten times to dissociate the tissue. Then use pestle B to continue grinding the tissue with a further 20 movements. Transfer the homogenates into a 1.5 ml tube with a 1 ml transfer pipette.
3. Centrifuge the samples at 1,000 x g for 5 min at 4 °C to eliminate any unground brain fragments. Retrieve the supernatants, divide them up into 200 µl aliquots and keep them at -80 °C for subsequent ELISA analysis.

4. Detection of αS by ELISA

1. Dilute the coating antibodies to 0.01 ng/ml. Use either anti-αS rabbit polyclonal or monoclonal clone 42 antibody in 50 mM Na₂CO₃/NaHCO₃ buffer (pH 9.6).
2. Coat the 96-well microplates with 100 µl per well of this coating solution, and leave at 4 °C O/N. Use the anti-αS rabbit polyclonal antibody in the coating solution for ELISAs using detection antibodies syn514, clone 42, LB509, AS11, 4D6 or 8A5. Use the anti-αS monoclonal antibody clone 42 as a coating solution in combination with the anti-pSer129 αS detection antibody.
   NOTE: If necessary, the plates may be kept at 4 °C for one week before the ELISA is performed.
3. Use a plate washer to wash the plates five times with 300 µl of phosphate-buffered saline with 0.05% Tween 20 (PBST) per well. From this step onward, incubation is at RT.
4. Add 200 µl of T20 PBS blocking buffer per well. Shake for 1 hr at 150 rpm. Wash the plates five times with PBST.
5. Dilute the brain homogenates (dilution 1:100 of the 20% homogenates, 1:50 of the 10% homogenates and 1:25 of the 5% homogenates in PBST BSA 1%), and add 100 µl to each well. Then incubate for 2 hr while shaking at 150 rpm. Wash the plates five times with PBST.
6. Add the different αS detection antibodies in PBST with BSA 1% at the dilutions mentioned in the Materials List. Incubate for 1 hr at 150 rpm. Wash the plates five times with PBST.
7. Add either anti-mouse or anti-rabbit IgG HRP conjugates diluted 1:8,000 in PBST supplemented with BSA 1% for 1 hr while shaking at 150 rpm. Wash the plates five times with PBST.
8. Add 100 µl of 3,3’,5,5’-tetramethylbenzidine (TMB) solution to each well and incubate for 15 min in the dark while shaking at 150 rpm.
9. Stop the reaction by adding 100 µl of 1 N HCl per well then measure the absorbance at 450 nm with the microplate reader.
10. For data analysis, subtract the OD value obtained in a well with all the reagents except any mouse brain samples (blank well) from the OD values measured for each of the analyzed samples.

5. Epitope Mapping

1. Perform epitope mapping according to the method described by Osman¹⁶. Briefly, spot peptides of the human α-synuclein sequence containing 12 amino acids on nitrocellulose with 10 overlapping amino acids.
2. Block with 50 mM Tris/150 mM NaCl buffer pH 10 containing 0.05% Tween 20 and 5% milk powder. Incubate the antibody in blocking solution at a concentration of 2 µg antibody per ml at 2-10 °C O/N.
3. Wash the membrane three times using 50 mM Tris/150 mM NaCl buffer pH 10 containing 0.05% Tween 20. Incubate with the goat anti-mouse IgG HRP conjugate. Wash the membrane another five times using the same buffer then stain using a Western blot TMB staining kit.

6. Statistical Analysis

1. Use the R software and nlme package to perform mixed-effects regressions to model OD. For each comparison, perform a mixed effect regression model. Use a fixed effect to distinguish symptomatic from asymptomatic groups.
2. Use a random effect to reflect the variability of repetitions for a given mouse. Check homoscedasticity by examining the residuals and if needed, use the variance functions to model the variance structure of within-group errors in keeping with Pinheiro and Bates¹⁷. Set 0.05 as the significance threshold of P.

Wyniki

In this study, the ELISAs used specifically identified disease-associated αS (αS^D) in brain homogenates prepared in a High Salt buffer from sick M83 mice. Using an antibody specifically recognizing pSer129 αS (p = 0.0074), the ELISA readily distinguishes old, sick mice (> 8 months old) from young (2-5 months old), healthy M83 mice (Figure 1). Several other antibodies showed similarly high signals (> 0.6 OD) only in brain homogenates from sick mice. This is the case for 4...

Dyskusje

The use of an ELISA was demonstrated to specifically detect αS^D directly from mouse brain homogenates during the disease in the M83 transgenic mouse model. Indeed, this ELISA could readily distinguish sick M83 mice from healthy M83 mice using only whole brain homogenates in High Salt buffer.

The most critical steps for successful results using this ELISA are: correctly dissecting the different regions of the mouse brains by developing the necessary manual dexterity to prevent d...

Materiały

Name	Company	Catalog Number	Comments
LB509	Abcam	ab27766	Detection antibody 1/2,000
AS11	Produced at Anses		Detection antibody 1/1,000
4D6	Abcam	ab1903	Detection antibody 1/2,000
PSer129	Abcam	ab59264	Detection antibody 1/3,000
PSer129 EP1536Y	Abcam	ab51253	Detection antibody 1/1,000
syn514	Abcam	ab24717	Detection antibody 1/500
clone 42	BD Biosciences	610787	Coating and detection antibody (1/2,000)
8A5	Provided by Dr. Anderson		Detection antibody 1/2,000
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			Adjust at pH 7.5 and keep at 4 °C
Tris-HCl 50 mM	Euromedex	26-128-3094-B
NaCl 750 mM	Euromedex	1112-A
EDTA 5 mM	Euromedex	EU0007-B
DTT 1 mM	Sigma	43815
PBS			Adjust at pH 7.5
Na2HPO4 1 mM	Euromedex	1309
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	50x 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, pH 9.6
Superblock T20 PBS blocking buffer	Pierce	E6423H	10x concentrated
TMB	Sigma	T0440	Used for ELISA
TMB	Analytik Jena AG	847-0104200302	Used for epitope mapping
HCl 1 N	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	8X magnification
Forceps Dumont#7	WPI	14097	For dissection steps
Transfer pipette 1ml Samso	Samso	043231
1.5 ml tubes	Dutscher	033290