Altered dynamics of a lipid raft associated protein in a kidney model of Fabry disease

Abstract

Accumulation of globotriaosylceramide (Gb3) and other neutral glycosphingolipids with galactosyl residues is the hallmark of Fabry disease, a lysosomal storage disorder caused by deficiency of the enzyme alpha-galactosidase A (α-gal A). These lipids are incorporated into the plasma membrane and intracellular membranes, with a preference for lipid rafts. Disruption of raft mediated cell processes is implicated in the pathogenesis of several human diseases, but little is known about the effects of the accumulation of glycosphingolipids on raft dynamics in the context of Fabry disease. Using siRNA technology, we have generated a polarized renal epithelial cell model of Fabry disease in Madin-Darby canine kidney cells. These cells present increased levels of Gb3 and enlarged lysosomes, and progressively accumulate zebra bodies. The polarized delivery of both raft-associated and raft-independent proteins was unaffected by α-gal A knockdown, suggesting that accumulation of Gb3 does not disrupt biosynthetic trafficking pathways. To assess the effect of α-gal A silencing on lipid raft dynamics, we employed number and brightness (N&B) analysis to measure the oligomeric status and mobility of the model glycosylphosphatidylinositol (GPI)-anchored protein GFP-GPI. We observed a significant increase in the oligomeric size of antibody-induced clusters of GFP-GPI at the plasma membrane of α-gal A silenced cells compared with control cells. Our results suggest that the interaction of GFP-GPI with lipid rafts may be altered in the presence of accumulated Gb3. The implications of our results with respect to the pathogenesis of Fabry disease are discussed.

Ion channels and pain in Fabry disease

“…21 This is particularly interesting to understand the underlying disease mechanism and provide a cellular and molecular platform for developing novel treatment strategies. 22 In vitro cell models (Table 1), initially, used cells obtained from FD patients especially of target organs in FD: endothelial cells [23][24][25][26][27]29 kidney cells [48][49][50][52][53][54] to determine glycosphingolipids accumulation and a-Gal A activity, and obtain lines for further research. Fibroblast 32,34 and bone marrow cells 30,31 were subjected to different techniques to incorporate an a-Gal A for enzyme expression, as well as for the analysis of different mutations present in FD patients.…”

How membrane dysfunction influences neuronal survival pathways in sphingolipid storage disorders

“…Such an example is found in a mouse model of Krabbe's disease, the twitcher mouse, in which toxic accumulation of galactosylsphingosine (psychosine) disrupts lipid rafts, resulting in the inhibition of survival signals such as PKC, Akt, and ERK; defects in axonal transport and synaptic maintenance; and neuronal degeneration through a dying‐back mechanism (White et al, ; Castelvetri et al, ; Cantuti Castelvetri et al, ; Hawkins‐Salsbury et al, ; Teixeira et al, ; Cantuti‐Castelvetri et al, ). Glucosylceramide accumulation in Gaucher's disease (Hattersley et al, ), sulfatide accumulation in metachromatic leukodystrophy (Moyano et al, ), globotriaosylceramide accumulation in Fabry's disease (Labilloy et al, ), and cholesterol accumulation in Niemann‐Pick type C (Vainio et al, ) also preferentially take place in lipid rafts and cause altered lipid raft dynamics. Therefore, it is conceivable that they involve neuronal vulnerability through a similar mechanism that includes membrane disruption as a root cause for downregulation of survival signals leading to neurodegeneration.…”

Detectors for Super-Resolution & Single-Molecule Fluorescence Microscopies

“…N&B analysis was performed on wildtype and alpha-galactosidase deficient cells, which act as a model for Fabry's disease. Antibodyinduced clustering of a model lipid raft protein was increased in the mutant compared to control cells [110]. These results suggested that accumulated Gb3 may alter lipid raft protein interactions in membranes of alpha-galactosidase deficient cells.…”

“…Hybrid detectors have been employed to measure the formation of lipid rafts in a cell model of Fabry's disease [110]. In Fabry's disease, lysosomal function is disrupted due to reduced activity of a specific enzyme (alpha-galactosidase A) that leads to accumulation of neutral glycosphingolipids such as globotriaosylceramide (Gb3).…”