Inhibition of cysteine protease cathepsin L increases the level and activity of lysosomal glucocerebrosidase
Myung Jong Kim, … , Thomas Reinheckel, Dimitri Krainc
Myung Jong Kim, … , Thomas Reinheckel, Dimitri Krainc
Published February 8, 2024
Citation Information: JCI Insight. ;9(3):e169594. https://doi.org/10.1172/jci.insight.169594.
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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

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Figure 3

GCase protein stability and lysosomal proteolysis in cathepsin L–KO cells.

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GCase protein stability and lysosomal proteolysis in cathepsin L–KO cell...
(A) In vitro cleavage assay using recombinant cathepsin L and GCase. Recombinant GCase and cathepsin L were incubated as indicated. After 20 minutes of incubation at room temperature, protein samples were immunoblotted with antibodies against GCase and cathepsin L. (B) Time-course study of cathepsin L–mediated GCase cleavage in vitro. Recombinant GCase and cathepsin L incubated for indicated times. Protein samples were immunoblotted with antibodies against GCase and cathepsin L. (C) GCase stabilities in WT and cathepsin L–KO cells. WT and cathepsin L–KO cells were treated with 100 μM cycloheximide for indicated times. Cell lysates were immunoblotted with antibodies against GCase, cathepsin L, and tubulin. Band intensities were normalized with tubulin and compared with 0-hour samples. Data represent mean ± SEM. n = 3 independent experiments. Paired 2-tailed Student’s t test. *P < 0.05, **P < 0.01, compare with the same time point of WT cells. (D) Representative images of DQ-RED BSA proteolysis assay. WT and cathepsin L–KO cells were labeled with 10 µg/ mL DQ-RED BSA for 4 hours in OPTI-MEM. Graph shows as mean fluorescence intensity of cleaved DQ-RED BSA ± SEM. n = 50 microscopic fields from 4 dishes for WT and n = 53 microscopic fields from 4 dishes for cathepsin L–KO cells. Scale bar: 250 μm. Unpaired 2-tailed Student’s t test; ***P < 0.01.