Characteristics of TBS-Extractable Hyperphosphorylated Tau Species: Aggregation Intermediates in rTg4510 Mouse Brain

Abstract

Conditional overexpression of four-repeat human tau containing the P301L missense mutation in the rTg4510 mouse model of tauopathy leads to progressive accumulation of neurofibrillary tangles and hyperphosphorylated, sarkosyl-insoluble tau species, which are biochemically comparable to abnormal tau characteristic of hereditary tauopathies termed FTDP-17. To fully understand the impact of tau species at different stages of self-assembly on neurodegeneration, we fractionated rTg4510 brain representing several stages of tauopathy to obtain TBS-extractable (S1), high salt/sarkosyl-extractable (S3), and sarkosyl-insoluble (P3) fractions. Under reducing condition, the S1 fraction was demonstrated by Western blotting to contain both 50–60 kDa normally-sized and 64 kDa tau. Both are thermo-stable, but the 64 kDa tau showed a higher degree of phosphorylation. Under non-reducing condition, nearly all TBS-extractable 64 kDa tau were detected as ~130 kDa species consistent with the size of dimer. Quantitative analysis showed ~80 times more 64 kDa tau in S1 than P3 fraction. Immunoelectron microscopy revealed tau-positive granules/short filaments in S1 fraction. These structures displayed MC1 immunoreactivities indicative of conformational/pathological change of tau. MC1 immunoreactivity was detected by dot blotting in samples from 2.5 month-old mice, whereas Ab39 immunoreactivity indicative of late stages of tau assembly was detected only in P3 fraction. Quantitative analysis also demonstrated a significant inverse correlation between brain weight and 64 kDa tau, but the level of TBS-extractable 64 kDa tau reflects neurodegeneration better than that of sarkosyl-insoluble 64 kDa tau. Together, the findings suggest that TBS-extractable 64 kDa tau production is a potential target for therapeutic intervention of tauopathies.

Mutant Tau knock-in mice display frontotemporal dementia relevant behaviour and histopathology

“…Abnormal elevations in tau phosphorylation at the PHF-1, CP-13 and AT-8 epitopes initially affected endogenous murine Tau, most prominently at the major 50 kDa species, but by 12 months of age progressed to encompass all remaining isoforms. Despite the elevation in tau phosphorylation there was no notable shift in electrophoretic migration nor the emergence of specific hyperphosphorylated species as reported in human AD cases (Flament and Delacourte, 1989) and in several FTD Tau mouse models (Berger et al, 2007;Sahara et al, 2013). The evidence present here suggests that elevations in tau phosphorylation may be sufficient to driving quantifiable behavioural phenotypes.…”

TOC1: A valuable tool in assessing disease progression in the rTg4510 mouse model of tauopathy

“…The cerebral cortex and hippocampus (ctx+hip) of the right hemisphere of each animal were quickly frozen on dry ice and stored at −80°C until use. Tissues were then homogenized in 10 volumes of Tris-buffered saline [TBS: 50 mM Tris/HCl (pH 7.4), 274 mM NaCl, 5 mM KCl, 1% protease inhibitor mixture (Sigma, St. Louis, MO), 1% phosphatase inhibitor cocktail I & II (Sigma), and 1 mM phenylmethylsulfonyl fluoride (PMSF)] (Berger et al, 2007; Sahara et al, 2012; Santacruz et al, 2005). The homogenates were centrifuged at 27,000 × g for 20 min at 4°C to obtain supernatant (S1) and pellet fractions.…”

Tau immunotherapy for Alzheimer's disease

“…The injected ALZ17 mice quickly developed solid filamentous tau pathology, and administration of immuno-depleted brain extracts from P301S mice or extracts from wild type mice did not induce tau pathology. Fractionation of the brain extracts in soluble (S1) and sarkosyl insoluble tau (P3) [19] and injection of these into ALZ17 mice demonstrated that the P3 fraction is most competent in inducing pathology. It contains most of the intracellular hyperphosphorylated filamentous tau.…”