Fig. 2

Lower Levels of Mature GCase Protein and Accumulation of Incompletely Glycosylated GCase in Brains from FTD-GRN Patients. a–c, Lysates of inferior frontal gyrus from FTD-GRN cases had lower levels of normal molecular-weight GCase protein than controls as detected by a rabbit polyclonal anti-GCase antibody (b, t test, p = 0.0059), and a mouse monoclonal anti-GCase antibody (c, t test, p = 0.0017). The polyclonal antibody also detected a lower molecular weight band that was present in all but one FTD-GRN case, but rarely in controls. d, To determine whether this lower molecular weight band could represent incompletely glycosylated GCase, lysates were treated with Endo H or PNGase F. The lower-molecular weight GCase band (red arrowhead) was approximately the same molecular weight as Endo H-treated GCase. Complete removal of glycosylation with PNGase F collapsed the multiple GCase bands into one band just over 50 kDa, indicating that the low-molecular weight form of GCase detected by the polyclonal antibody may consist of incompletely glycosylated GCase. The higher molecular weight band detected by the mouse monoclonal antibody was determined to be non-specific because it did not shift with PNGase F treatment (data not shown). e, Unlike normal molecular weight GCase, there were no group differences in deglycosylated (PNGase F-treated) GCase (t test, p = 0.9594), showing that while FTD-GRN brains contain less normal molecular weight, active GCase than controls, they have similar total levels of GCase protein. f, The low-molecular weight form of GCase accumulated in the sarkosyl-insoluble fraction of FTD-GRN brains, while normal molecular weight GCase was only detectable in the soluble fractions. Levels of sarkosyl-insoluble GCase did not statistically differ between controls and FTD-GRN patients (g, Mann-Whitney test, p = 0.1490), but the insoluble band was detectable in 6/7 FTD-GRN patients, but only reliably detectable in 1/5 controls. This stands in contrast to α-tubulin (f, h), which did not differ between groups. Levels of insoluble GCase were very high in some FTD-GRN patients, so data in g are shown in log scale. n = 5 controls, and 7 FTD-GRN. * = p < 0.05 and ** = p < 0.1 by Tukey’s post-hoc test. GCase = β-glucocerebrosidase, Endo = Endoglycosidase H, PNG = PNGase F