Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

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

The accumulation of intracellular tau amyloids defines tauopathies such as Alzheimer's disease. In early disease stages, pathology is generally localized to discrete regions of the brain, but with disease progression, pathology invariably spreads along distinct neural networks^1-5. Accumulating evidence suggests transcellular propagation of toxic protein aggregates underlies this pathology (reviewed in ^6-10). In this model, proteopathic seeds (e.g., tau) are released from donor cells and enter neighboring cells, transforming native tau protein into the misfolded form via templated conformational change^11-15. The assay descr....

Protocol

NOTE: This protocol emphasizes the use of FRET flow cytometry for detecting seeding activity from mouse biological samples. It is also compatible with recombinant fibrils and human biological samples. Mouse euthanasia and brain harvesting was performed in accordance with IACUC-approved procedures.

1. Brain Extraction

1. Following deep anesthetization with isoflurane (2%), perfuse a mouse with ice-cold PBS containing 0.03% heparin, and extract the brain following the details described in Gage et al.¹⁸
2. Place extracted tissue in a cryo-vial, and snap freeze by placing in liquid nitrogen. Alternatively, freeze tissue on dry ic....

Results

FRET flow cytometry enables sensitive, quantitative, and rapid detection of seeding activity from recombinant or biological samples. Assay setup is facile: monoclonal-derived stable cell lines expressing tau-RD-CFP/YFP are transduced with seed material, incubated for 24-48 hr, and subjected to flow cytometry analysis (Figure 1A). In the absence of seeds, biosensor cells maintain tau in a soluble, monomeric form (Figure 1B). In the presence of seeds, however, biosensor cells convert tau i.......

Discussion

The FRET flow cytometry system described here is a powerful tool for quickly and quantitatively assessing tau seeding activity. It requires only moderate cell culture experience and a working knowledge of FRET and flow cytometry. Other seeding assays, such as Thioflavin T - which exhibits enhanced fluorescence when bound to beta sheet structure - are laborious and require a pure, recombinant protein substrate. Additionally, in vitro seeding assays for tau are only semi-quantitative and generally insensitive to s.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 μl pipet tips	Rainin	GPS-L10
200 μl pipet tips	Rainin	GPS-250
1 ml pipet tips	Rainin	GPS-1000
200 μl 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