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. 2024 Jan 17;29(2):453.
doi: 10.3390/molecules29020453.

Identification of GM1-Ganglioside Secondary Accumulation in Fibroblasts from Neuropathic Gaucher Patients and Effect of a Trivalent Trihydroxypiperidine Iminosugar Compound on Its Storage Reduction

Costanza Ceni  1   2 , Francesca Clemente  1 , Francesca Mangiavacchi  1 , Camilla Matassini  1 , Rodolfo Tonin  3 , Anna Caciotti  3 , Federica Feo  3 , Domenico Coviello  4 , Amelia Morrone  3   5 , Francesca Cardona  1 , Martino Calamai  2   6
Affiliations

Identification of GM1-Ganglioside Secondary Accumulation in Fibroblasts from Neuropathic Gaucher Patients and Effect of a Trivalent Trihydroxypiperidine Iminosugar Compound on Its Storage Reduction

Costanza Ceni et al. Molecules. .

Abstract

Gaucher disease (GD) is a rare genetic metabolic disorder characterized by a dysfunction of the lysosomal glycoside hydrolase glucocerebrosidase (GCase) due to mutations in the gene GBA1, leading to the cellular accumulation of glucosylceramide (GlcCer). While most of the current research focuses on the primary accumulated material, lesser attention has been paid to secondary storage materials and their reciprocal intertwining. By using a novel approach based on flow cytometry and fluorescent labelling, we monitored changes in storage materials directly in fibroblasts derived from GD patients carrying N370S/RecNcil and homozygous L444P or R131C mutations with respect to wild type. In L444P and R131C fibroblasts, we detected not only the primary accumulation of GlcCer accumulation but also a considerable secondary increase in GM1 storage, comparable with the one observed in infantile patients affected by GM1 gangliosidosis. In addition, the ability of a trivalent trihydroxypiperidine iminosugar compound (CV82), which previously showed good pharmacological chaperone activity on GCase enzyme, to reduce the levels of storage materials in L444P and R131C fibroblasts was tested. Interestingly, treatment with different concentrations of CV82 led to a significant reduction in GM1 accumulation only in L444P fibroblasts, without significantly affecting GlcCer levels. The compound CV82 was selective against the GCase enzyme with respect to the β-Galactosidase enzyme, which was responsible for the catabolism of GM1 ganglioside. The reduction in GM1-ganglioside level cannot be therefore ascribed to a direct action of CV82 on β-Galactosidase enzyme, suggesting that GM1 decrease is rather related to other unknown mechanisms that follow the direct action of CV82 on GCase. In conclusion, this work indicates that the tracking of secondary storages can represent a key step for a better understanding of the pathways involved in the severity of GD, also underlying the importance of developing drugs able to reduce both primary and secondary storage-material accumulations in GD.

Keywords: GM1; flow cytometry; glucocerebrosidase; glucosylceramide; lysosome; metabolic disorders; pharmacological chaperones.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Thawed primary cultures of fibroblasts isolated from control wild type (WT) and GD patients carrying N370S/RecNcil and L444P mutations were cultured, fixed, permeabilized, labelled with anti-glucosylceramide and secondary Alexa 568 (A) or CTXb-FITC (B), and imaged with confocal microscopy. A significant increase in anti-Glc and CTXb-FITC fluorescence intensities was observed in fibroblasts carrying the L444P mutation with respect to the control, highlighting a rise in cellular content of GlcCer and GM1. Scale bar 30 μm. A total of >20 cells were analysed for each condition. Error bar S.D. Student’s t-test: ** p ≤ 0.01 and *** p ≤ 0.001.
Figure 2
Figure 2
Flow-cytometry analysis of fibroblasts from WT and from GD patients carrying N370S and L444P mutations. Cells were fixed, permeabilized, and labelled with anti-GlcCer and secondary Alexa 647 antibodies (A) or CTXb-FITC (B). MFI/MFIWT mean values were obtained by dividing the MFI of a distribution by the mean MFI obtained from the controls. MFI/MFIWT mean values of anti-Glc (A) and CTXb-FITC (B) increased significantly in the fibroblasts carrying the L444P mutation with respect to the WT control and to fibroblasts carrying the N370S/RecNcil mutation, indicating a rise in content of GlcCer and GM1. A total of >4000 cells were analysed for each MFI dot. Student’s t-test: * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001. n ≥ 3 independent experiments.
Figure 3
Figure 3
(A,B) Primary cultures of fibroblasts isolated from patients carrying the L444P mutation were incubated for 4 days with three different concentrations of CV82 (10 µM, 1 µM, and 100 nM), fixed, permeabilized, labelled with anti-glucosylceramide and secondary Alexa 647 antibodies (A) or CTXb-FITC (B), and analysed with flow cytometry. Treatment with 10 µM and 100 nM of CV82 led to a significant reduction in cellular content of GM1 but not GlcCer. Error bar S.D. Student’s t-test: ** p ≤ 0.01, and *** p ≤ 0.001. A total of >4000 cells were analysed for each MFI dot. n ≥ 3 independent experiments.
Figure 4
Figure 4
(A) Activity of β-Gal in the presence of compound CV82 in human leukocyte homogenates derived from healthy donors. The corresponding calculated percentage of inhibition is indicated above bar. (B–D) Fibroblasts WT (B) and bearing p.Arg201His/p.Tyr83LeufsX8 (C) and p.Arg201His/p.Ile51Asn (D) mutations were incubated without or with different concentrations of compound CV82. The β-Gal activity was determined in lysates from the treated fibroblasts. Reported data are mean ± S.D. (n = 2).
Figure 5
Figure 5
Flow-cytometry analysis of primary cultures of fibroblasts isolated from WT and GD patients carrying the R131C mutation. Fibroblasts were fixed, permeabilized, labelled with anti-glucosylceramide and secondary Alexa 647 (A) or CTXb-FITC (B). A total of >4000 cells were analysed for each MFI dot. MFI/MFIWT mean values of anti-Glc (A) and CTXb-FITC (B) significantly increased in fibroblasts of patients carrying R131C mutation compared with WT, indicating an increase in GlcCer and GM1 storages. Treatment of primary cultures of fibroblasts isolated from patients for 4 days with different concentrations of CV82 led to a significant increase in cellular content of both GlcCer (C) and GM1 (D). Error bar S.D. Student’s t-test: * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001. (E) Relation between GlcCer and GM1 levels in WT and patients carrying N370S/RecNcil, for L444P and R131C mutations. A total of >4000 cells were analysed for each MFI dot. n ≥ 3 independent experiments.
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