Inhibition of cysteine protease cathepsin L increases the level and activity of lysosomal glucocerebrosidase
Myung Jong Kim, Soojin Kim, Thomas Reinheckel, Dimitri Krainc
Myung Jong Kim, Soojin Kim, Thomas Reinheckel, Dimitri Krainc
View: Text | PDF
Research Article Cell biology Neuroscience

Inhibition of cysteine protease cathepsin L increases the level and activity of lysosomal glucocerebrosidase

Abstract

The glucocerebrosidase (GCase) encoded by the GBA1 gene hydrolyzes glucosylceramide (GluCer) to ceramide and glucose in lysosomes. Homozygous or compound heterozygous GBA1 mutations cause the lysosomal storage disease Gaucher disease (GD) due to severe loss of GCase activity. Loss-of-function variants in the GBA1 gene are also the most common genetic risk factor for Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). Restoring lysosomal GCase activity represents an important therapeutic approach for GBA1-associated diseases. We hypothesized that increasing the stability of lysosomal GCase protein could correct deficient GCase activity in these conditions. However, it remains unknown how GCase stability is regulated in the lysosome. We found that cathepsin L, a lysosomal cysteine protease, cleaves GCase and regulates its stability. In support of these data, GCase protein was elevated in the brain of cathepsin L–KO mice. Chemical inhibition of cathepsin L increased both GCase levels and activity in fibroblasts from patients with GD. Importantly, inhibition of cathepsin L in dopaminergic neurons from a patient GBA1-PD led to increased GCase levels and activity as well as reduced phosphorylated α-synuclein. These results suggest that targeting cathepsin L–mediated GCase degradation represents a potential therapeutic strategy for GCase deficiency in PD and related disorders that exhibit decreased GCase activity.

Authors

Myung Jong Kim, Soojin Kim, Thomas Reinheckel, Dimitri Krainc

×

Figure 2

Lysosomal GCase activity is increased and GluCer levels are reduced in cathepsin L–KO cells.

Options: View larger image (or click on image) Download as PowerPoint
Lysosomal GCase activity is increased and GluCer levels are reduced in c...
(A) In vitro GCase activity in WT HEK293-FT and cathepsin L–KO cell lysates. Lysates from WT or cathepsin L–KO cells were assayed for in vitro GCase assay using 4-MUG substrates. Data are mean ± SEM. One-way ANOVA followed by Dunnett’s test; ***P < 0.001. n = 6. (B) PFB-FDGlu GCase activity of WT HEK293-FT and cathepsin L–KO cells. Live WT HEK293-FT and cathepsin L–KO cells were labeled with 100 μM PFB-Glu for 2 hours in OPTI-MEM media. After washing with PBS twice, cells were lysed with RIPA buffer and fluorescence intensities were measured. Data normalized with protein concentration of cell lysates and compared with the WT. n = 4 for WT, n = 4 for KO cells. **P < 0.01; 2-tailed unpaired t test. (C) LysoLive-GCase activity of WT HEK293-FT and cathepsin L–KO cells. Representative fluorescence images from LysoLive-GCase assay are shown. Graph shows mean fluorescence intensity from the LysoLive-GCase assay. Data were compared with the WT cells. n = 27 microscopic fields from 3 independent dishes for WT, 30 microscopic fields from 3 independent dishes for cathepsin L–KO cells. Two-tailed unpaired t test, ***P < 0.001. Scale bar: 250 μm. (D) GluCer staining of WT HEK293-FT and cathepsin L–KO cells. Cells were stained with anti-GluCer antibody. Graph shows mean fluorescence intensity of GluCer staining. n = 15 microscopic fields from 2 coverslips for WT, n = 15 microscopic fields from 2 coverslips for cathepsin L–KO cells. Two-tailed unpaired t test; **P < 0.01. (E) Lipidomic analysis of GluCer species in WT HEK293-FT cells and cathepsin L–KO#1 cells. GluCer were quantified and expressed as p mol/n mole inorganic phosphate (Pi). Two-tailed unpaired t test; *P < 0.05, **P < 0.01, ***P < 0.001. n = 5 for WT and n = 4 for KO#1.