Gaucher Disease Psychosine Mis-Traffics Endocytosed Cargo to a Non-Functional, Pre-Lysosomal Compartment.

Gaucher’s disease is a rare metabolic disorder resulting from a deficiency in glucocerebrosidase activity. It is characterized by the formation of large multinucleate macrophages which accumulate enzyme substrate - principally glucosylsphingosine (psychosine) and glucosylceramide - as well as unrelated products such as cholesterol, lipofuscin, glycopeptides and oligosaccharides. The mechanism of this storage remains obscure. Psychosines have been shown to inhibit cytokinesis (Kanazawa et al., 2000) in cells expressing a G-protein linked receptor: GPR65 (Im et al., 2001). Signalling via GPR65 was shown to affect phosphorylation and calcium release as well as result in the formation of intracellular actin clots. Could such psychosine-triggered signalling account for the secondary accumulation of lipids that are not substrates for glucocerebrosidase in Gaucher disease? We investigated the effects of psychosines and other lysosphingolipids on human macrophages and monocyte-related cell lines expressing GPR65. Trafficking was evaluated by pulse-chase experiments using fluorescent lipids and markers of pH. Changes induced by lysosphingolipids were visualized by electron microscopy and compared with Gaucher cells from affected spleen. Confocal fluorescence and electron microscopy were used to identify sub-cellular compartments. GPR65 expression in Gaucher spleen and other cells was confirmed by RT- PCR.

At sub-pathophysiological concentrations (1-5μM), psychosines but not ceramides disrupt endocytosis: mis-trafficked membrane and ingested materials accumulate in a large vesicular compartment. This multivesicular body bears most lysosomal markers including LAMP1 and LAMP2 but has a neutral pH and does not contain acid phosphatase or Cathepsin D -consistent instead with late endosomes. The ultrastructure of the psychosine-treated monocytic cells closely resembles naturally occurring splenic Gaucher cells and both are also of course multinucleate. This suggests that psychosines dysregulate intracellular trafficking in Gaucher disease. Recycling is perturbed and membrane lipids and other cargo accumulate in a pre-lysosomal compartment where, with glucosylceramide, they may be preferentially concentrated. Since related lysolipid metabolites are present in several other sphingolipidoses, we propose that a common trafficking mechanism accounts for the cellular pathology seen in this class of diseases.

In addition, an active role for psychosines in pathogenesis may also begin to suggest mechanisms to explain why Gaucher patients carry a high relative risk for developing myeloma and other B-cell dyscrasias (Rosenbloom et al., 2005). The majority of these malignancies are associated with mutations at position 14q32. Receptor GPR65 is also located at that position and its regulation may predispose to mutation and eventual transformation.