In vitro tau aggregation

All proteins in assay were dialyzed overnight at 4 °C into assay buffer (Dulbecco’s PBS pH7.4, 2 mM MgCl₂, 1 mM DTT). Tau (10 μM) was pre-incubated in the presence or absence of chaperones (2.5 μM-20 μM) for 30 min at 37 °C. Thioflavin T (Sigma) at a final concentration of 10 μM was added and aggregation was induced by the addition of a freshly prepared heparin sodium salt solution (Santa Cruz) at a final concentration of 44 ug/ml. For non-induced controls, assay buffer was added in place of heparin solution. In assays using arachadonic acid as an aggregation inducer, heparin was replaced with arachadonic acid (150 μM) in 5% ethanol and non-induced controls contained 5% ethanol. Aggregation reactions were carried out at 37°C with continuous shaking and monitored via Thioflavin T fluorescence (excitation=444 nm, emission=485 nm, cutoff=480 nm) in a Spectramax M5 microplate reader (Molecular Devices). Readings were taken every 5 min for a minimum of 24 h. Note that for experiments with Hsp72_SBD, assays were performed at 30°C, well below its observed protein melting point (42°C). Chaperones with poor solubility in standard screen conditions as indicated in the text were assayed in reactions with the following modifications: the tau concentration was reduced to 7.5 uM and the aggregation reaction was carried out in 96 well plates with total volumes of 200 ul per well.

For data processing, baseline curves of non-induced controls (3 replicates) were subtracted from induced samples (3 replicates). Baseline subtracted aggregation curves were analyzed in Grace plotting program (http://plasma-gate.weizmann.ac.il/Grace/) and fit to the Goempertz function ^62,63:

where y= the fluorescence at time t, A is the amplitude of curve, t_i is the inflection point, t_i-b = lag time, and b = 1/k_app where k_app is the apparent elongation rate constant. Amplitude values are represented relative to 0N4R tau^WT controls for each experiment to adjust for day to day fluctuations in absolute ThT signal intensity. Curves with final amplitude values <15% of the internal tau alone controls were classified as non-aggregating. For the primary aggregation screen, each experiment contained multiple reference controls including Hsc70 NBD and Hsc70 (at each concentrated tested) and 0N4R tau^WT tau (10 μM). The effect of each chaperone on aggregation kinetics is normalized to the values for the internal Hsc70_NBD and Hsc70 controls. Heat maps for chaperone effects on aggregation kinetics were generated in R ⁶⁴. For experiments involving chaperone addition after lag phase period, the aggregation curve for 4 h immediately following addition was fit with linear regression to calculate the slope.