Chapelet, Guillaume

Derkinderen, Pascal

Letournel, Franck

Neunlist, Michel

Oullier, Thibauld

Prigent, Alice

English

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Optimizing filter trap assay for the detection of aggregated alpha-synuclein in brain samples.

Open Access-Zeitschriften an der WWU Münster (Westfälische Wilhelms-Universität)

Free Neuropathology 1:14 (2020) Thibauld Oullier et al. doi: https://doi.org/10.17879/freeneuropathology-2020-2749 page 1 of 4     Copyright: © 2020 The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, a link to the Creative Commons license is provided, and any changes are indicated. The Creative Commons Public Domain Dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.  Optimizing filter trap assay for the detection of aggregated alpha-synuclein in brain samples Thibauld Oullier 1*, Alice Prigent 1*, Guillaume Chapelet 1,2, Michel Neunlist 1, Franck Letournel 3, Pascal Derkinderen 1,4 1 Université de Nantes, Inserm, TENS, The Enteric Nervous System in Gut and Brain Diseases, IMAD, Nantes, France 2 CHU Nantes, Clinical Gerontology Department, Nantes, France 3 CHU Angers, Neurobiology and Neuropathology Laboratory, Angers, France 4 CHU Nantes, Department of Neurology, Nantes, France *These authors contributed equally to this work. Corresponding author: Pascal Derkinderen · Inserm U1235 Nantes · 1 rue Gaston Veil · 44035 Nantes · France · Tel: +33(0)240165202 · Fax: +33(0)240165203 pascal.derkinderen@chu-nantes.fr ; derkinderenp@yahoo.fr Additional resources and electronic supplementary material: supplementary material Submitted: 04 April 2020 · Accepted: 26 April 2020 · Copyedited by: Christian Thomas · Published: 28 April 2020 Keywords: Alpha-synuclein, Parkinson’s disease, Filter trap, Dot blot, Aggregates  Over the past two decades, the progress in mo-lecular genetics and neuropathology enabled a better understanding of the pathogenesis of neurodegenera-tive disorders. The observation that abnormal protein accumulation is characteristic of a particular disease has led to a neuropathological classification according to the composition of the abnormal protein aggregates [1]. Several methods have been developed for the detection of these insoluble protein aggregates including, for in-stance, dye binding assays and immunohistochemistry with proteinase K pretreatment [1]. Filter retardation assay is a simple and rapid method which detects pro-tein aggregates formed either in vivo or in vitro, includ-ing amyloid-beta/tau aggregates from Alzheimer’s dis-ease (AD) [2–4] and polyglutamine expansion from Hun-tington’s disease [5,6]. In this assay, ‘lysis buffer’-resistant protein aggregates, but not soluble monomeric species, are retained by a cellulose acetate membrane and subsequently detected by immunoblotting. As load-ing control, a ‘classical’ dot blot with either polyvinyli-dene fluoride (PVDF) or nitrocellulose membranes is run in parallel with the same lysates. In contrast to polyglutamine expansion, tau and beta-amyloid, there are only few studies on using filter retardation assays for the detection of alpha-synuclein aggregates, which are characteristic of Parkinson’s dis-ease (PD) and dementia with Lewy bodies (DLB) [7]. In addition, a full characterization of any filter-based assay for alpha-synuclein, including the effect of membrane composition, lysis procedure and antibodies has not yet been reported. We therefore set out the current study in which we characterize a vacuum-based 96-well format filter assay for the detection of aggregated alpha-synuclein in human brain specimens. Frozen brain sam-ples from neuropathologically confirmed cases of DLB, progressive supranuclear palsy (PSP) and AD were ob-Letter Free Neuropathology 1:14 (2020) Thibauld Oullier et al. doi: https://doi.org/10.17879/freeneuropathology-2020-2749 page 2 of 4    tained from the Neuropathology Department of Angers (Dr Franck Letournel). Brain samples were lysed with the “Precellys 24™” tissue homogenizer (Bertin technolo-gies, Cat# P000669-PR240-A, Montigny-le-Bretonneux, France) followed by a brief sonication (one minute, 5 seconds on, 5 seconds off, amplitude 70%; VCX130 ultra-sonic processor with a 6 mm probe, Sonics and Materi-als, Newton, CT, USA) either in (i) radioimmunoprecipita-tion assay (RIPA) buffer (Merck Millipore, Cat# 20-188, Molsheim, France) containing 2 mM orthovanadate (Sigma, Cat# S6505, Saint Quentin Fallavier, France), 1% (v/v) phosphatase inhibitor cocktail III (Sigma, Cat# P0044) and a protease inhibitors cocktail (Roche, Cat# 14424700, Meylan, France) at 4°C; (ii) ‘Sodium dodecyl sulfate (SDS) buffer’ containing 1% (w/v) SDS, 2 mM orthovanadate (Sigma, Cat# S6505) and 1% (v/v) phos-phatase inhibitor cocktail III (Sigma, Cat# P0044); (iii) ‘Urea buffer’ containing 7 M urea (Sigma, Cat# T7875), 2 M thiourea (Sigma, Cat# L3771), 4% (v/v) CHAPS (Sigma, Cat# C3023), 2 mM orthovanadate (Sigma, Cat# S6505), 1% (v/v) phosphatase inhibitor cocktail III (Sigma, Cat# P0044) and a protease inhibitors cocktail (Roche, Cat# 14424700). Lysates were centrifuged at 10,000 x g for 5 minutes; supernatants were recovered and protein con-centration determined using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific, Cat# 23227, Saint Herblain, France) along with the Varioskan™ multimode microplate reader (ThermoFisher Scientific, Cat# VL0L00D0); samples lysed in urea buffer were diluted 5 times for compatibility with the BCA assay (https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0068-Protein-assay-compatibility.pdf). Twenty µg of these supernatants were subjected to vacuum filtration through a 96-well dot blot apparatus (Bio-Rad, Cat# 1706545, Marnes-la-Coquette, France) with 0.45 µM pore size cellulose acetate (Sterlitech, Cat# CA0453001, Kent, USA), nitrocellulose (ThermoFisher Scientific, Cat# 77010) or PVDF membranes (Ther-moFisher Scientific, Cat# 88518). The resultant mem-branes were blocked for one hour at room temperature in Tris-buffered saline (TBS, Sigma, Cat# T5912) with 0.1% (v/v) Tween-20 (Sigma, Cat# P1379) and 5% (w/v) non-fat dry milk, then incubated overnight at 4°C with the primary antibodies listed in Table 1. Bound antibod-ies were detected with horseradish peroxidase-conjugated anti-rabbit (Life technologies Cat# 31460, diluted 1:5,000) or anti-mouse antibodies (Sigma, Cat# A9044, diluted 1:5,000) and visualized by enhanced chemiluminescent detection (SuperSignal™ West Pico PLUS Chemiluminescent Substrate, ThermoFisher Scien-tific, Cat# 34580). Oligomeric aggregates of alpha-synuclein, which were obtained by incubating monomer-ic recombinant alpha-synuclein (Sigma, Cat# 575001) in phosphate-buffered saline under agitation at a concen-tration of 10 mg/mL at 37°C for one week, served as a control [8]. When cellulose acetate membranes were used, re-combinant oligomeric alpha-synuclein and DLB lysates were barely or not detected by two of the total alpha-synuclein antibodies (Syn211 and Syn-1), regardless of the lysis procedure (Figure 1a). In contrast, MJFR1 anti-body efficiently detected both oligomeric alpha-synuclein and aggregated alpha-synuclein from RIPA-lysed DLB samples (Figure 1a). No specific signal was observed with this antibody when DLB samples were lysed with SDS or urea buffers (Figure 1a). Two of the phosphospecific antibodies (EP1536Y and to a lesser extent pSyn#64) detected aggregated alpha-synuclein in RIPA-DLB lysates. No signal was observed when PSP and AD lysates were filtered and analyzed with either MJFR1 or EP1536Y antibody. These findings show that sample lysis with RIPA sample lysis together with MJFR1 and/or EP1536Y immunoblotting is the most specific and repro-ducible approach. We therefore used these two antibod-ies and RIPA-lysed samples to perform additional dot blots with PVDF or nitrocellulose membranes, that could serve as a loading control. The results show that PVDF achieved better reproducibility and specificity for the detection of total and phosphorylated alpha-synuclein than nitrocellulose membranes (Figure 1b).   Table 1. Alpha-synuclein and phospho-alpha-synuclein antibodies used in the current study. Free Neuropathology 1:14 (2020) Thibauld Oullier et al. doi: https://doi.org/10.17879/freeneuropathology-2020-2749 page 3 of 4       Figure 1. Results from the filter trap and dot blot assays. (a) recombinant oligomeric alpha-synuclein (oligo) and samples from DLB, PSP and AD brains lysed in RIPA, SDS and Urea buffers were subjected to vacuum filtration through a 96-well dot blot apparatus with an acetate cellulose membrane; immunoblotting (IB) was then performed with 3 different antibodies to total alpha-synuclein (MJFR1, Syn-1 and Syn211) and 3 antibodies specific for the phosphorylated form of the protein (EP1536Y, pSyn#64 and Phospho S129). (b) recom-binant oligomeric alpha-synuclein (Oligo) and RIPA-lysed samples from DLB, PSP and AD brains were subjected to vacuum filtration through a 96-well dot blot apparatus with a PVDF or a nitrocellulose membrane ; immunoblotting (IB) was then performed with MJFR1 or EP1536Y antibodies. Results shown are representative of 3 to 5 independent experiments.  The results presented here, although preliminary, provide the basis for reproducible and specific detection of pathological alpha-synuclein in diseased brains using a filter trap assay. We show that the selection of primary antibody and lysis buffer has important effects on the performance of the assay. As an optimized protocol, we suggest that brain homogenates should be lysed in RIPA buffer and that immunoblotting should be performed with MJFR1 for the detection of total alpha-synuclein and EP1536Y for the detection of the phosphorylated form of the protein (a step by step standard operating procedure is provided as supplementary material). Ow-ing to the high-throughput capability of the filter trap assay, such an approach is not only potentially interest-ing for detecting aggregated alpha-synuclein in post mortem brain samples but also in the peripheral nervous system of living PD patients in order to develop original biomarkers of the disease [9]. 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Optimizing filter trap assay for the detection of aggregated alpha-synuclein in brain samples

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Optimizing filter trap assay for the detection of aggregated alpha-synuclein in brain samples

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