Propagation of prions causing synucleinopathies in cultured cells

Abstract

Increasingly, evidence argues that many neurodegenerative diseases, including progressive supranuclear palsy (PSP), are caused by prions, which are alternatively folded proteins undergoing selfpropagation. In earlier studies, PSP prions were detected by infecting human embryonic kidney (HEK) cells expressing a tau fragment [TauRD(LM)] fused to yellow fluorescent protein (YFP). Here, we report on an improved bioassay using selective precipitation of tau prions from human PSP brain homogenates before infection of the HEK cells. Tau prions were measured by counting the number of cells with TauRD(LM)-YFP aggregates using confocal fluorescence microscopy. In parallel studies, we fused α-synuclein to YFP to bioassay α-synuclein prions in the brains of patients who died of multiple system atrophy (MSA). Previously, MSA prion detection required ∼120 d for transmission into transgenic mice, whereas our cultured cell assay needed only 4 d. Variation in MSA prion levels in four different brain regions from three patients provided evidence for three different MSA prion strains. Attempts to demonstrate α-synuclein prions in brain homogenates from Parkinson's disease patients were unsuccessful, identifying an important biological difference between the two synucleinopathies. Partial purification of tau and α-synuclein prions facilitated measuring the levels of these protein pathogens in human brains. Our studies should facilitate investigations of the pathogenesis of both tau and α-synuclein prion disorders as well as help decipher the basic biology of those prions that attack the CNS.

Tau prions from Alzheimer’s disease and chronic traumatic encephalopathy patients propagate in cultured cells

“…Previously, we described an enhanced cellular assay to quantify tau prions in PSP patient samples (32) (32). To improve the robustness of the assay and increase cell infection, we precipitated tau prions with sodium phosphotungstate (PTA), which selectively isolates aggregated proteins, including PrP Sc , tau, and α-synuclein, from soluble proteins (32,33).…”

“…However, although intracerebral injection of tau fibrils into Tg mice expressing tau transgenes resulted in tau neuropathology, such experiments were inconclusive with respect to assessing the kinetics and accumulation of tau prions (40)(41)(42)(43)(44). Instead, measurements made from cells expressing the tau-YFP fusion proteins (31,32,45) have been more informative.…”

“…SDS/PAGE studies demonstrated tau isoform specificity in detergent-extracted samples obtained from AD, PiD, AGD, CBD, and PSP patient samples more than a decade ago (46), but the more recent development of a rapid tau prion bioassay using Tau(4RD*LM)-YFP(1) cells (31), followed by modifications to the bioassay conditions (32), has facilitated the rapid detection of tau prions. Using this powerful approach, we posited that homotypic interactions in tau prion replication were just as critical as those demonstrated in PrP Tg mice described above (52).…”

“…These findings demonstrate that tau prions feature in the pathogenesis of both AD and CTE. Importantly, the rapid and highly reproducible fusion protein bioassays described here have allowed us, and others, to begin unraveling the molecular pathogenesis of several neurodegenerative diseases (31,32). Moreover, the insights gained from these assays make it possible to focus drug discovery efforts on the mechanism responsible for the propagation of these diseases, increasing the likelihood of developing effective therapeutics.…”

“…Previously, we and others used HEK293 cells expressing the RD of 4R tau, fused to yellow fluorescent protein (YFP), with the two familial mutations P301L and V337M [originally named TauRD(LM)-YFP cells but denoted here as Tau(4RD*LM)-YFP(1) cells] (Table S1) (31) to quantify tau prions isolated from PSP patient samples (32). To investigate the role of the RD in tau prion strains, we developed a panel of HEK293T cell lines expressing several variations of tau isoform fusion proteins: the RD of 3R tau, the RD of 4R tau, and the combination of both.…”