Figure 4

PTI-00703 cat’s claw also inhibited tau protein tangles and filament formation, and reduced preformed tau fibrils. (a) Tau protein isoforms and a purified tau 4-repeat domain. Diagram of six tau isoforms aligned with a “new” tau 4-repeat domain that was generated (top of image). (b) SDS-PAGE and silver staining showed purified tau 4-repeat domain protein at ~15 kDa (250 ng of protein loaded). (c,d) Electron microscopy showed paired helical filaments (i.e. tangles) generated from in vitro aggregated tau 4-repeat domain (10 µM) in the presence of heparin. Scale bars = 50 nm. (e) Inhibition of tau protein fibril formation by PTI-00703 cat’s claw using a Thioflavin S binding assay. n = 4. (f) PTI-00703 cat’s claw also disrupted/disaggregated pre-formed tau protein filaments and fibrils using a Thioflavin S binding assay. n = 4 (g) PTI-00703 cat’s claw inhibited tau protein fibril formation in a dose-dependent manner as determined by CD spectroscopy. Tau protein alone (black bar) showed a characteristic beta-sheet CD spectra with a minima at a wavelength of 218 nm. Increasing concentrations of PTI-00703 cat’s claw demonstrated a dose-dependent reduction of the tau protein beta-sheet secondary folding as shown by a dose-dependent flattening of the 218 nm minima. n = 3. (h) PTI-00703 cat’s claw inhibited tau protein fibril formation in a dose-dependent manner as shown by EM. Increasing concentrations of PTI-00703 cat’s claw (i.e. 10 µg/ml, 50 µg/ml and 200 µg/ml) demonstrated reduction and disaggregation of tau protein filaments and fibrils into primarily amorphous non-fibrillar material. Scale bars = 200 nm.