Objective: The aim of this study was to investigate if GCase deficiency in macrophage model of GD lead to lysosphingolipids accumulation.

Background: Mutations in the GBA gene lead to a deficiency of glucocerebrosidase (GCase) enzymatic activity and to the development of Gaucher disease (GD) which belongs to lysosomal storage diseases. At the same time GBA mutations increase the risk of Parkinson’s disease (PD) in 7-8 times. Developing of the appropriate cellular models of GD are crucial for the new therapeutic strategies for GD and PD treatment.

Methods: Lysosphingolipid (hexosylsphingosine HexSph (glucosylsphingosine GlcSph+ galactosylsphingosine GalSph)) levels and GCase enzymatic activity were measured by liquid chromatography tandem-mass spectrometry (LC-MS/MS) in dry blood spots (DBS) and dry macrophage cell spots with the concentration of 2×10^6 cells/ml in GD patients (n=4) and healthy controls (n=10)The effect of dose-dependent inhibition of GCase enzymatic activity with conduritol-B-epoxide (CBE) (150 and 450 ng/ml) on HexSph level was estimated in macrophages of 9 healthy individuals. Mononuclear fraction was isolated using the Ficoll gradient centrifugation. The cells were differentiated into macrophages using RPMI supplemented with 10% bovine serum, 1% streptomycin-penicillin and 10 ng/ml M-CSF for 4 days, with daily media changes. GCase enzymatic activity was inhibited with CBE (0, 150 and 450 ng/ml) for 4 days.

Results: We showed a significant GCase enzymatic activity decrease in patients with GD compared to control group both in DBS and in cultivated macrophages (p<0.001 and p<0.001, respectively) as well as increase in HexSph levels (p<0.001 and p<0.001, respectively ). A dose-dependent increase in macrophage HexSph level was shown: CBE concentrations: 150 and 450 ng/ml compared to cells without CBE (p<0.001 and p<0.001, respectively), and in cell with 450 ng/ml CBE concentration compared to 150 ng/ml CBE (p=0.036) [figure1].

Conclusions: We showed that GCase enzymatic activity and HexSph levels in cultured macrophage estimated by LC-MC/MC from GD patents reflect the same parameters in blood. A dose-dependent GCase activity inhibition with CBE in macrophages of healthy individuals leads to dose dependent HexSph accumulation. We showed that macrophages, showing GCase dysfunction, accumulate lysospingolipids in vitro that supports the possibility of using macrophage model of GD in exploring new therapies for GD and related disorders.

« Back to 2018 International Congress

MDS Abstracts - https://www.mdsabstracts.org/abstract/lysosphingolipids-accumulation-in-macrophage-model-of-gaucher-disease/