Assay for GCase Enzymatic Activity

The assay for GCase enzymatic activity was performed as described[11 (link)]. Medaka brains were homogenized in 40 μl sample buffer (10 mM Tris-HCl, 150 mM NaCl, 1% (v/v) Triton X-100, pH 7.4), sonicated, and centrifuged at 10,000 ×g at 4°C for 5 min. Aliquots containing 50 μg protein were incubated in assay buffer (5 mM 4-Methylumbelliferyl β-d-glucopyranoside (Wako, #324–37441), 1% (w/v) sodium taurocholate (Wako, #197–10033), 50 mM sodium citrate, 50 mM sodium phosphate, pH 5.0) in the presence or absence of 2 mM Conduritol B epoxide (Toronto Research Chemicals, #C666000) in a total volume of 100 μl at 37°C for 4 hr. The reaction was stopped by adding 100 μl of 0.4 M glycine, pH 10.8, and the fluorescence at 460 nm (emission 355 nm) was measured with Fluoroskan Ascent FL (Thermo Fisher).