Ultrasensitive and selective detection of 3-repeat tau seeding activity in Pick disease brain and cerebrospinal fluid

Abstract

The diagnosis and treatment of diseases involving tau-based pathology such as Alzheimer disease and certain frontotemporal dementias is hampered by the inability to detect pathological forms of tau with sufficient sensitivity, specificity and practicality. In these neurodegenerative diseases, tau accumulates in self-seeding filaments. For example, Pick disease (PiD) is associated with frontotemporal degeneration and accumulation of 3-repeat (3R) tau isoforms in filaments constituting Pick bodies. Exploiting the self-seeding activity of tau deposits, and using a 3R tau fragment as a substrate, we have developed an assay (tau RT-QuIC) that can detect tau seeds in 2 µl aliquots of PiD brain dilutions down to 10-10. PiD seeding activities were 100-fold higher in frontal and temporal lobes compared to cerebellar cortex. Strikingly, this test was 10- to 10-fold less responsive when seeded with brain containing predominant 4-repeat (4R) tau aggregates from cases of corticobasal degeneration, argyrophilic grain disease, and progressive supranuclear palsy. Alzheimer disease brain, with 3R + 4R tau deposits, also gave much weaker responses than PiD brain. When applied to cerebrospinal fluid samples (5 µl), tau RT-QuIC analyses discriminated PiD from non-PiD cases. These findings demonstrate that abnormal tau aggregates can be detected with high sensitivity and disease-specificity in crude tissue and fluid samples. Accordingly, this tau RT-QuIC assay exemplifies a new approach to diagnosing tauopathies and monitoring therapeutic trials using aggregated tau itself as a biomarker.

Seeding selectivity and ultrasensitive detection of tau aggregate conformers of Alzheimer disease

“…K19CFh was prepared as described previously [37]. Another tau construct used in the study was designed to include the core part of the AD fibril [14], with a point mutation at residue 322 cysteine to serine called τ306 (residues 306–378 using the numbering for full-length human tau isoform htau40).…”

“…Both constructs were expressed in BL21(DE3) Escherichia coli following the protocol described in [37]. Briefly, expression was induced using the Overnight Express autoinduction method [42].…”

“…We recently developed an ultrasensitive cell-free assay for the tau aggregates of Pick disease [37]. PiD is characterized by the predominant accumulation of the human tau isoforms with three microtubule binding repeats (3R).…”

“…The assay is performed in multi-well plates, and the fibrillar products are detected using an amyloid fibril-sensitive fluorescent dye, thioflavin T (ThT) [37]. 3R tau RT-QuIC, which uses a 3R tau fragment (K19CFh) as the substrate, has strong selectivity for the 3R tau aggregates of PiD over the predominantly 4R or mixed 3R/4R tau aggregates of other diseases.…”

A Review of Biomarkers for Neurodegenerative Disease: Will They Swing Us Across the Valley?

“…Nascent biofluids methods currently impressing include high-sensitivity assays such as MagQu’s detection system utilizing magnetic nanoparticles [60, 61] and “seeding” and “quaking” assays that take advantage of the prion-like properties of pathological tau and alpha-synuclein [62–64]. Early reports of extremely high sensitivity and specificity have to be tempered against the reality that clinical diagnosis has so far been the only gold standard, reducing accuracies of 90% to less-stellar levels.…”