Følsomme Påvisning af Proteopathic Seeding Aktivitet med FRET flowcytometri
Summary
Cell-to-cell transfer of protein aggregates, or proteopathic seeds, may underlie the progression of pathology in neurodegenerative diseases. Here, a novel FRET flow cytometry assay is described that enables specific and sensitive detection of seeding activity from recombinant or biological samples.
Abstract
Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer’s disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples.
By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as α-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.
Introduction
Ophobningen af intracellulære tau amyloider definerer tauopatier såsom Alzheimers sygdom. I tidlige stadier sygdom, patologi generelt lokaliseret til diskrete områder af hjernen, men med sygdomsprogression, patologi altid spredes langs forskellige neurale netværk 1-5. Akkumulere tyder transcellulær formering af giftige proteinaggregater ligger til grund for denne patologi (revideret i 6-10). I denne model er proteopathic frø (f.eks tau) frigives fra donorceller og indtast naboceller, omdanne native tau-protein i misfoldet form via template konformationsændring 11-15. Den her beskrevne assay blev udviklet til at påvise en sådan følsomt seeding. Den er kompatibel med rekombinant protein og biologiske prøver og muliggør kvantificering af små niveauer af proteopathic seeding aktivitet 16.
HEK 293T-celler, der stabilt udtrykker tau repeat domain (RD) indeholdende sygdomsassocieret P301S mutation fusioneret til enten CFP eller YFP (i det følgende benævnt tau-RD-CFP / YFP celler) fungere som en fast biosensor podningstidspunktet aktivitet. I mangel af proteopathic frø, cellerne fastholde tau som en opløselig monomer, og har ingen mærkbar baggrund FRET. Spontan optagelse eller liposommedieret transduktion af tau frø i celler resulterer imidlertid i RD-CFP og RD-YFP aggregering, som frembringer et FRET signal, der er målt inden for de enkelte celler via flowcytometri.
Talrige elementer i dette assay blev fremstillet til at øge følsomheden og reducere variabilitet. Et monoklonalt cellelinje med en 1: 1 RD-CFP / YFP udtryk forholdet blev valgt, da den giver optimal signal: støj. For at øge følsomheden, er phospholipider anvendes til at indføre frøene direkte ind i celler (selvom at studere biologiske mekanismer af optagelse, kan dette punkt udelades). Endelig flowcytometri skærme FRET på populationen og en enkelt celle niveau, i modsætning til andre proteinaggregering assays. Det endelige resultat foranstaltning, integreret FRET densitet, er meget kvantitativ og redegørelser for både antallet af celler med aggregering og i hvilken grad aggregering er opstået inden for hver celle. Alle disse optimerede parametre forbedre følsomheden og sikre reproducerbarhed.
Dette system blev for nylig ansat i en omfattende undersøgelse i transgene P301S tauopati mus 17, der evaluerede den tidsmæssige debut og progression af tau såning aktivitet i forhold til andre almindeligt anvendte tau patologiske markører (f.eks MC1, AT8, PG5 og ThioflavinS). Seeding er langt den tidligste og mest robuste markering af tau patologi vurderes, forud histologisk påvisning af mindst 6 uger. Såning aktivitet vises ved 1,5 måneder og stiger gradvist med alderen, hvilket tyder på en kausal rolle proteopathic frø i starten og / eller progression af neurodegeneration 16.
e_content "> Præcis kvantificering af små niveauer af frø materiale fra biologiske prøver kan fremme undersøgelser, der overvåger tidligt sygdomsprogression. Ved at forkorte retssagen varighed og muliggør brug af yngre dyr, kan det øge effektiviteten og nøjagtigheden af prækliniske dyreforsøg. For eksempel i den P301S musen tidligere beskrevet, kunne blyforbindelser leveres så tidligt som 4-6 uger (umiddelbart før eller i starten af såning aktivitet), og overvåges for effektivitet 2-4 uger senere. Analysen bør nøjagtigt kvantificere eventuelle nedsættelser i såning FRET flowcytometri har kan testes hurtigt for deres evne til at blokere såning induktion direkte i kultur, enten ved hjælp af rekombinant tau in vitro screening ansøgninger. For eksempel anti-tau reagenser (f.eks, antistoffer, små molekyler, osv) aktivitet. aggregater eller hjerne-afledt lysater som frøkilde (figur 5). Med denne opsætning, når podemateriale fremstilles en exeksperimentere tager kun tre dage til at fuldføre, herunder dataanalyse. Den hurtige kvantificering af proteopathic såning aktivitet kan således fremme mange undersøgelser af neurodegeneration.Protocol
Representative Results
Discussion
FRET flowcytometri her beskrevne system er et kraftfuldt værktøj til hurtigt og kvantitativt at vurdere tau seeding. Det kræver kun moderat cellekultur erfaring og et praktisk kendskab til FRET og flowcytometri. Andre seeding assays, såsom thioflavin T– som udviser forbedret fluorescens, når de er bundet til beta-sheet struktur – er besværlig og kræver en ren, rekombinant protein substrat. Derudover in vitro såning analyser for tau er kun semi-kvantitativ og generelt ufølsom for subnanomolære niveaue…
Declarações
The authors have nothing to disclose.
Acknowledgements
This work was supported by the Tau Consortium (M.I.D); National Institutes of Health Grant 1R01NS071835 (M.I.D.), a Department of Defense Grant PT110816 (to M.I.D.), 1F32NS087805 (to J.L.F.), and 1F31NS079039 (to B.B.H.).
Materials
| TBS | Sigma | T5912 |
| cOmplete Protease Inhibitors (EDTA-free) | Roche | 4693159001 |
| Cryo-vials | Sarstedt | 72.694.006 |
| Analytical Balance | Mettler Toledo | XSE 105DU |
| Weighing Boats | Fisher Scientific | 13-735-743 |
| 15 mL conical tube | USA Scientific | 1475-0501 |
| Omni Sonic Ruptor Ultrasonic Homogenizer | Omni International | 18-000-115 |
| Micro-Tip for Ultrasonic Homogenizer | Omni International | OR-T-156 |
| 2-Propanol | Fisher Scientific | A451 |
| Noise Cancelling Ear Muffs | Fisher Scientific | 19-145-412 |
| Kimwipes | Fisher Scientific | S47299 |
| 1.5 mL tubes | USA Scientific | 1615-5510 |
| Microcentrifuge | Eppendorf | 5424 000.215 |
| DPBS | Life Technologies | 14190-136 |
| DMEM | Life Technologies | 11965-084 |
| Fetal Bovine Serum | HyClone | SH30071.03 |
| Penicillin-Streptomycin | Life Technologies | 15140-122 |
| GlutaMax | Life Technologies | 35050-061 |
| Trypsin-EDTA | Life Technologies | 25300-054 |
| 50 mL Conical Tubes | Phenix Research | SS-PH15 |
| 25 mL reagent resevoirs | VWR | 41428-954 |
| Multi channel pipet | Fisher Scientific | TI13-690-049 |
| 96 well flat bottom plates | Corning | 3603 |
| Opti-MEM | Life Technologies | 31985-070 |
| Lipofectamine 2000 | Invitrogen | 11668019 |
| 96 well round bottom plates | Corning | 3788 |
| 16% Paraformaldehyde | Electron Microscopy Sciences | RT 15710 |
| PBS | Sigma-Aldrich | P5493 |
| EDTA | Sigma-Aldrich | ED2SS |
| HBSS | Life Technologies | 14185-052 |
| Sorvall ST 40 Centrifuge | Thermo Scientific | 75004509 |
| BIOLiner Swinging Bucket Rotor | Thermo Scientific | 75003796 |
| Hemacytometer | VWR | 15170-172 |
| MACSQuant VYB Flow Cytomter | Miltenyi Biotec | 130-096-116 |
| Chill 96 Rack | Miltenyi Biotec | 130-094-459 |
| Flow Jo analysis software | Flow Jo | |
| 20 uL pipet tips | Rainin | GPS-L10 |
| 200 uL pipet tips | Rainin | GPS-250 |
| 1 mL pipet tips | Rainin | GPS-1000 |
| 200 uL pipet tips | USA Scientific | 1111-1800 |
| 5 mL serological pipett | Phenix Research | SPG-606180 |
| 10 mL serological pipett | Phenix Research | SPG-607180 |
| 25 mL Serological pipett | Phenix Research | SPG-760180 |
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