Assessment of glucocerebrosidase activity

For each of the N = 7 mice per genotype from the Group 1 (biochemistry) aged experimental cohorts described previously, half of the tissue samples (one hemisphere of brain and one lobe of liver) were shipped to Amicus Therapeutics and the other brain hemisphere and liver lobe were shipped to Pfizer, Inc. for independent, contemporaneous analyses. Both brain and liver were divided into 8 samples and frozen at Charles River Labs (formerly WIL Research) prior to analyses. Brain and liver samples of each mouse were prepared in duplicate and assigned a unique sample number such that the 7 mice were treated as 14 blinded samples. Brain hemispheres were thoroughly minced and mixed before sampling. In duplicate, samples were analyzed for GCase activity determination by the conduritol-B epoxide (CBE)/4-Methylumbelliferyl β-D-glucopyranoside (4-MUG) method at Amicus and the MDW941 activity probe method at Pfizer. Moreover, two samples were used for independent lipid extractions for glucosylceramide (GlcCer) analysis, and one sample was used for lipid extraction for GlcSph analysis.

These methods apply specifically to the sample preparation and analyses at Amicus for assessment of brain and liver tissue for GCase activity by the CBE/4-MU method. Tissue samples were homogenized in GCase enzyme buffer (McIlvaine citrate/phosphate pH 5.2 containing 0.25% Na-taurocholate and 0.1% TX-100). GCase activity was determined in triplicate from two independent samples of each homogenate, and protein was determined with the BCA assay in singlet from each independent sample of homogenate using BSA as a standard. GCase activity was measured in a 30 min reaction at 37°C in GCase enzyme buffer supplemented to 300 μM N-(n-Butyl) deoxygalactonojirimycin and with or without the covalent GCase inhibitor CBE using 4-MUG as the substrate. CBE-inhibitable GCase activity was converted to nM 4-MU released by comparison with a 4-MU standard curve run with each assay. These levels were normalized to protein weight and hour. Two-Way ANOVA tests were performed for statistical analysis to understand the effect of genotype and age with Bonferroni post hoc tests. Individual data points with mean and standard error of the mean are represented on each graph.

These methods apply specifically to sample preparation at Pfizer for assessment of brain and liver tissue for GCase activity by the MDW941 probe method. Lysates were prepared by excision of cortex from the frozen brain hemisphere and from tissue obtained from the frozen liver lobe. Tissues were homogenized in 10 volumes of GCase lysis buffer containing 0.25% Triton X-100 using Qiagen Tissuelyser at 25 Hz for 4 min x 2. Samples were then sonicated with a Branson Ultrasonics 450 Digital Sonifier (Branson) for 10 seconds at 80 watts at 20 kHz and diluted to 2 mg/mL with lysis buffer. The GCase activity probe MDW941 was added to the samples to a final concentration of 25 nM probe, 5mM citric acid. Lysate and MDW941 probe were incubated at 37°C for 2 hours. Samples were centrifuged at 21,000 x g for 2 minutes to remove acid precipitates, the supernatant was retained. NuPAGE LDS (4X) sample buffer (ThermoFisher) containing NuPAGE Sample Reducing Agent (dithiothreitol) was added and the mixtures were incubated at 70°C for 10 min. Protein concentration was determined by BCA (Pierce). 30 μg of protein lysate samples were loaded onto a 4–12% gel NuPAGE (ThermoFisher) with TAMRA labelled fluorescent ladder and probe labelled 5 ng recombinant GCase and run at 150 V in MES buffer until the dye front reached the bottom of the gel. A 20% methanol solution was used to wash away unbound probe.

Gels were visualized using a GE Typhoon at 532 nm excitation and 575 nM Long Pass Filter emission, PMT 1000. Bands were quantified for relative fluorescence units by ImageQuant software (GE). Two-Way ANOVA tests were performed for statistical analysis to understand the effect of genotype and age. When significance was identified by the Two-Way ANOVA test, Bonferroni post hoc tests were used to understand the significance of the individual comparisons. Individual data points with mean and standard error of the mean are represented on each graph.