Action myoclonus-renal failure syndrome: diagnostic applications of activity-based probes and lipid analysis.

Lysosomal integral membrane protein-2 (LIMP2) mediates trafficking of glucocerebrosidase (GBA) to lysosomes. Deficiency of LIMP2 causes action myoclonus-renal failure syndrome (AMRF). LIMP2-deficient fibroblasts virtually lack GBA like the cells of patients with Gaucher disease (GD), a lysosomal storage disorder caused by mutations in the GBA gene. While GD is characterized by the presence of glucosylceramide-laden macrophages, AMRF patients do not show these. We studied the fate of GBA in relation to LIMP2 deficiency by employing recently designed activity-based probes labeling active GBA molecules. We demonstrate that GBA is almost absent in lysosomes of AMRF fibroblasts. However, white blood cells contain considerable amounts of residual enzyme. Consequently, AMRF patients do not acquire lipid-laden macrophages and do not show increased plasma levels of macrophage markers, such as chitotriosidase, in contrast to GD patients. We next investigated the consequences of LIMP2 deficiency with respect to plasma glycosphingolipid levels. Plasma glucosylceramide concentration was normal in the AMRF patients investigated as well as in LIMP2-deficient mice. However, a marked increase in the sphingoid base, glucosylsphingosine, was observed in AMRF patients and LIMP2-deficient mice. Our results suggest that combined measurements of chitotriosidase and glucosylsphingosine can be used for convenient differential laboratory diagnosis of GD and AMRF.

A shortcut to the lysosome: the mannose-6-phosphate-independent pathway.

Lysosomal hydrolases have long been known to be responsible for the degradation of different substrates in the cell. These acid hydrolases are synthesized in the rough endoplasmic reticulum and transported through the Golgi apparatus to the trans-Golgi network (TGN). From there, they are delivered to endosomal/lysosomal compartments, where they finally become active due to the acidic pH characteristic of the lysosomal compartment. The majority of the enzymes leave the TGN after modification with mannose-6-phosphate (M6P) residues, which are specifically recognized by M6P receptors (MPRs), ensuring their transport to the endosomal/lysosomal system. Although M6P receptors play a major role in the intracellular transport of newly synthesized lysosomal enzymes in mammalian cells, several lines of evidence suggest the existence of alternative processes of lysosomal targeting. Among them, the two that are mediated by the M6P alternative receptors, lysosomal integral membrane protein (LIMP-2) and sortilin, have gained unequivocal support. LIMP-2 was shown to be implicated in the delivery of beta-glucocerebrosidase (GCase) to the lysosomes, whereas sortilin has been suggested to be a multifunctional receptor capable of binding several different ligands, including neurotensin and receptor-associated protein (RAP), and of targeting several proteins to the lysosome, including sphingolipid activator proteins (prosaposin and GM2 activator protein), acid sphingomyelinase and cathepsins D and H. Here, we review the current knowledge on these two proteins: their discovery, study, structural features and cellular function, with special attention to their role as alternative receptors to lysosomal trafficking. Recent studies associating both LIMP2 and sortilin to disease are also extensively reviewed.

Progressive myoclonus epilepsy with nephropathy C1q due to SCARB2/LIMP-2 deficiency: clinical report of two siblings.

Action myoclonus-renal failure syndrome (AMRF) is considered a rare form of progressive myoclonus epilepsy (PME) associated with renal failure. A mutation on the gene encoding the lysosomal integral membrane protein type 2-LIMP-2 (SCARB2), the receptor responsible for targeting glucocerebrosidase to the lysosomes, was recently described, allowing a better understanding of its etiopathogenesis. We describe clinically two sisters with AMRF that resulted from a mutation in the SCARB2 gene. The renal involvement was due to nephropathy C1q. When substrate-reduction therapy, to correct the possible glucocerebroside storage in the cells with glucocerebrosidase deficiency, was administered to one of the siblings, a significant improvement was observed. This report points out a rational for a therapeutical approach to this new lysossomopathy.

Deletion of the murine scavenger receptor CD68.

Scavenger receptors (ScRs) are a structurally unrelated family of receptors with the ability to bind modified low density lipoprotein (LDL) as well as a broad range of polyanionic ligands. CD68, whose expression is restricted to mononuclear phagocytes, is a unique ScR family member, owing to its lysosome associated membrane protein (LAMP)-like domain and predominant endosomal distribution. Knockout (ko) mice were generated to directly evaluate the role murine CD68 may play in oxidized LDL (Ox-LDL) uptake. However, CD68⁻/⁻ macrophages took up Ox-LDL robustly. Likewise, no defects were observed in the ability of CD68⁻/⁻ mononuclear phagocytes to take up or mount an effective innate response against a number of microbes. Curiously, CD68⁻/⁻ mononuclear phagocytes exhibited a trend toward enhanced antigen presentation to CD4⁺ T-cells, raising the possibility that CD68 may function either to negatively regulate antigen uptake, loading, or major histocompatibility complex class II (MHC-II) trafficking.

Phagocytosis of fungal agents and yeast via macrophage cell surface scavenger receptors.

BACKGROUND: Macrophages mediate phagocytosis via cell-surface pattern-recognition-receptors (PRRs) known to recognize certain fixed patterns on pathogens. Of these PRRs, scavenger receptors class A I and II (SR-A I and II) are known to mediate the binding and internalization of a large variety of Gram +ve and Gram -ve bacteria. Their role in phagocytic clearance of fungal agents has not been described. METHODS: Fluorescence microscopy and phagocytosis assays were used on murine macrophage cell lines RAW264.7. Chinese hamster ovarian cell lines (CHO) transfected with SR-A-I or SRA-II and known ligands that block SRA-uptake were used to test the ability of these cells to bind fungal agents. Macrophages from mice genetically deficient in SRA (MSR-knockouts) were used to establish whether absence of these receptors affects fungal uptake. RESULTS: We show for the first time that the SR-A I and II on macrophages are involved in both the binding and phagocytosis of S. cerevisiae and Candida albicans. SRA-mediated binding and internalization of these pathogens is specifically inhibited by known ligands of SRA (Fucoidan and Poly G) in a dose-titratable manner. Further, CHO cells transfected with either SR-A-I or SRA-II show an increased ability to bind and internalize S. cerevisiae compared with the non-transfected parental cells. In contrast, the macrophages that are deficient in the scavenger receptor (obtained from MSR-/- mice) do not show a decreased ability to phagocytose fungal agents. CONCLUSIONS: Scavenger receptors mediate phagocytosis of fungal agents, representing perhaps an alternative, fall back mechanism.

Uncoupling scavenger receptor A-mediated phagocytosis of bacteria from endotoxic shock resistance.

Unresolved infection by gram-negative bacteria can result in the potentially lethal condition known as endotoxic shock, whereby uncontrolled inflammation can lead to multiple organ failure and death of the infected host. Previous results have demonstrated that animals deficient in class A scavenger receptor (SRA), a trafficking receptor for bacteria and bacterium-derived molecules, are more susceptible to endotoxic shock. This has been proposed to be a result of impaired SRA-dependent phagocytic clearance of bacteria resulting in stronger proinflammatory stimuli. In this report, we test the hypothesis that there is an obligate reciprocal relationship between SRA-mediated phagocytosis of bacteria and susceptibility to endotoxic shock. Here, we demonstrate that both SRA-dependent and -independent gram-negative bacterial strains elicit SRA-dependent increased cytokine production in vitro and in vivo and increased susceptibility to endotoxic shock in SRA-deficient mice. This is the first evidence showing that SRA-mediated clearance of LPS is functionally distinct from the role of SRA in bacterial phagocytosis and is a formal demonstration that the SRA-dependent cytokine responses and the resultant endotoxic shock are not coupled to SRA-mediated clearance of bacteria.

Class A scavenger receptors regulate tolerance against apoptotic cells, and autoantibodies against these receptors are predictive of systemic lupus.

Apoptotic cells are considered to be a major source for autoantigens in autoimmune diseases such as systemic lupus erythematosus (SLE). In agreement with this, defective clearance of apoptotic cells has been shown to increase disease susceptibility. Still, little is known about how apoptotic cell-derived self-antigens activate autoreactive B cells and where this takes place. In this study, we find that apoptotic cells are taken up by specific scavenger receptors expressed on macrophages in the splenic marginal zone and that mice deficient in these receptors have a lower threshold for autoantibody responses. Furthermore, antibodies against scavenger receptors are found before the onset of clinical symptoms in SLE-prone mice, and they are also found in diagnosed SLE patients. Our findings describe a novel mechanism where autoantibodies toward scavenger receptors can alter the response to apoptotic cells, affect tolerance, and thus promote disease progression. Because the autoantibodies can be detected before onset of disease in mice, they could have predictive value as early indicators of SLE.