Niemann-Pick type C2 (NPC2) disease is a fatal autosomal recessive neurovisceral degenerative disorder characterized by late endosomal-lysosomal sequestration of low-density lipoprotein derived cholesterol. The breach in intracellular cholesterol homeostasis is caused by deficiency of functional NPC2, a soluble sterol binding protein targeted to the lysosomes by binding the mannose-6-phosphate receptor. As currently there is no effective treatment for the disorder, we have investigated the efficacy of NPC2 replacement therapy in a murine gene-trap model of NPC2-disease generated on the 129P2/OlaHsd genetic background. NPC2 was purified from bovine milk and its functional competence assured in NPC2-deficient fibroblasts using the specific cholesterol fluorescent probe filipin. For evaluation of phenotype correction in vivo, three-week-old NPC2 −/− mice received two weekly intravenous injections of 5 mg/kg NPC2 until trial termination 66 days later. Whereas the saline treated NPC2 −/− mice exhibited massive visceral cholesterol storage as compared to their wild-type littermates, administration of NPC2 caused a marked reduction in cholesterol build up. The histological findings, indicating an amelioration of the disease pathology in liver, spleen, and lungs, corroborated the biochemical results. Little or no difference in the overall cholesterol levels was observed in the kidneys, blood, cerebral cortex and hippocampus when comparing NPC2 −/− and wild type mice. However, cerebellum cholesterol was increased about two fold in NPC2 −/− mice compared with wild-type littermates. Weight gain performance was slightly improved as a result of the NPC2 treatment but significant motor coordination deficits were still observed. Accordingly, ultrastructural cerebellar abnormalities were detected in both saline treated and NPC2 treated NPC2 −/− animals 87 days post partum. Our data indicate that protein replacement may be a beneficial therapeutic approach in the treatment of the visceral manifestations in NPC2 disease and further suggest that neurodegeneration is not secondary to visceral dysfunction.

The endocytic pathway is a complex network of highly dynamic organelles, which has been traditionally studied by quantitative fluorescence microscopy. The data generated by this method can be overwhelming and its analysis, even for the skilled microscopist, is tedious and error-prone. We developed SpatTrack, an open source, platform-independent program collecting a variety of methods for analysis of vesicle dynamics and distribution in living cells. SpatTrack performs Endocytosis of proteins from the cell surface and subsequent sorting of internalized cargo in endosomes is a complex and highly dynamic process. Traditionally, endocytosis has been studied by subcellular fractionation and by transmission electron microscopy after suitable labeling of endocytic ligands (1,2). In parallel, fluorescence microscopy has evolved as a major tool to study the endocytic pathway. This is a consequence of the high specificity and sensitivity of fluorescence and the relative ease of labeling endocytic cargo with small fluorescent probes.Moreover, only fluorescence studies of the endocytic pathway bear the potential of measuring biophysical properties of the endosome, like pH or concentration of ions like calcium and chloride (3,4). Ligands of endocytic receptors have several fates once arrived in the endosome; they can recycle to the cell surface from early sorting or recycling endosomes, like transferrin after releasing iron into the endosomal lumen (5). They can become targeted to the trans-Golgi network in a retrograde trafficking route, as furin or several plant toxins (5,6). Ligands can be also 1406 www.traffic.dk

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In the cell membrane complement receptor 3 (CR3) consists of one alpha chain (CD11b) and one beta chain (CD18). CR3 participates in many immunological processes, especially those involving cell migration, adhesion, and phagocytosis of complement-opsonized microbes. Recent findings of soluble CR3 in body fluids and in culture supernatant from experiments in vitro point to the involvement of ecto domain shedding as a part of the CR3 biology. To monitor such shedding on a quantitative basis, we have developed time-resolved immunofluorometric assays (TRIFMA) to detect soluble CD11b and CD18 in plasma or serum of either human or murine origin. Compared with most enzyme-linked immunosorbent assays methodologies, TRIFMA possesses prominent advantages, including better dynamic range and reproducibility. These assays may contribute to the understanding of the role of shedding of CR3 and other cell adhesion molecules in human disease and animal models involving inflammation.