Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

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.

Tau reduction prevents neuronal loss and reverses pathological tau deposition and seeding in mice with tauopathy

“…One in vitro measure of the ability of pathological tau aggregates to induce misfolding of naïve monomeric tau, i.e. tau “seeding” activity, is a cell-based assay that relies on flow cytometry detection of Forster resonance energy transfer (FRET) created by intracellular aggregation of endogenously expressed repeat domain of tau (Tau RD ) fused with cyan fluorescent protein (Tau RD -CFP) and yellow fluorescent protein (Tau RD -YFP) in stably expressing human embryonic kidney (HEK-293) cells (30, 31) (Fig. 4A).…”

Widespread tau seeding activity at early Braak stages

“…The FRET flow cytometry assay we have previously described sensitively and specifically quantifies tau seeding activity in biological samples [17, 21]. We have never observed non-specific seeding of tau by heterologous fibrillar species, suggesting that it is both sensitive and specific [21, 31].…”

“…Tau seeding activity was quantified as detailed previously [17, 21]. HEK 293T cells stably expressing the aggregation-prone repeat domain (RD) of tau containing the disease-associated P301S mutation tagged to either CFP or YFP were transduced with human brain homogenates.…”

“…FRET flow cytometry was performed as described previously [17, 21]. Following 24 hr treatment, cells were trypsinized, centrifuged, and fixed in 2% paraformaldehyde (Electron Microscopy Sciences).…”

“…Gating strategy was similar to that described previously [17, 21]. CFP and YFP spillover into the FRET channel were excluded using compensation and the “False FRET” gate.…”