In vitro activity assay of GCase

Neurons were washed in PBS, harvested in 1% Triton lysis buffer, and activity was measured from whole-cell lysates as described previously (Mazzulli et al., 2011) using 4-methylumbelliferyl β-d-glucopyranoside (4MU-Gluc; Sigma-Aldrich). To determine whether 758 directly activates GCase in lysates, 758 was added at a final concentration of 10 μm into the activity assay buffer at the same time as the 4MU-Gluc substrate. Lysates [±1 μm conduritol-b-epoxide (CBE)] were incubated for 30 min, and the assay was stopped by addition of equi-volume 1 m glycine, pH 12.5. 4MU fluorescence was detected in white fluoro plates (Nunc, 136101) and fluorescence (ex = 355 nm, em = 460) was determined in a Molecular Devices i3 microplate reader. Relative fluorescent units (RFUs) from CBE-treated lysates were subtracted from non-CBE-treated lysates to obtain activity derived from GBA1, and RFUs were expressed as fold-change compared with DMSO control. The residual activity in CBE-treated samples was graphed as percentage of non-CBE-treated samples.