Niemann-Pick C2 protein regulates sterol transport between plasma membrane and late endosomes in human fibroblasts.

Niemann-Pick disease type C2 is a lipid storage disorder in which mutations in the NPC2 protein cause accumulation of lipoprotein-derived cholesterol in late endosomes and lysosomes (LE/LYSs). Whether cholesterol delivered by other means to NPC2 deficient cells also accumulates in LE/LYSs is currently unknown. We show that the close cholesterol analog dehydroergosterol (DHE), when delivered to the plasma membrane (PM) accumulates in LE/LYSs of human fibroblasts lacking functional NPC2. We measured two different time scales of sterol diffusion; while DHE rich LE/LYSs moved by slow anomalous diffusion in disease cells (D ∼ 4.6∙10-4 µm2/sec; α∼0.76), a small pool of sterol could exchange rapidly with D ∼ 3 µm2/s between LE/LYSs, as shown by fluorescence recovery after photobleaching (FRAP). By quantitative lipid mass spectrometry we found that esterification of 13C-labeled cholesterol but not of DHE is reduced 10-fold in disease fibroblasts compared to control cells. Internalized NPC2 rescued the sterol storage phenotype and strongly expanded the dynamic sterol pool seen in FRAP experiments. Together, our study shows that cholesterol esterification and trafficking of sterols between the PM and LE/LYSs depends on a functional NPC2 protein. NPC2 likely acts inside LE/LYSs from where it increases non-vesicular sterol exchange with other organelles.

Ficolin B in Diabetic Kidney Disease in a Mouse Model of Type 1 Diabetes.

BACKGROUND: The innate immune system may have adverse effects in diabetes and cardiovascular disease. The complement system seems to play a key role through erroneous complement activation via hyperglycaemia-induced neoepitopes. Recently mannan-binding lectin (MBL) was shown to worsen diabetic kidney changes. We hypothesize that mouse ficolin B exerts detrimental effects in the diabetic kidney as seen for MBL. METHODS: We induced diabetes with streptozotocin in female wild-type mice and ficolin B knockout mice and included two similar nondiabetic groups. Renal hypertrophy and excretion of urinary albumin and creatinine were quantified to assess diabetic kidney damage. RESULTS: In the wild-type groups, the kidney weighed 24% more in the diabetic mice compared to the controls. The diabetes-induced increase in kidney weight was 29% in the ficolin B knockout mice, that is, equal to wild-type animals (two-way ANOVA, P = 0.60). In the wild-type mice the albumin-to-creatinine ratio (ACR) was 32.5 mg/g higher in the diabetic mice compared to the controls. The difference was 62.5 mg/g in the ficolin B knockout mice, but this was not significantly different from the wild-type animals (two-way ANOVA, P = 0.21). CONCLUSIONS: In conclusion, the diabetes-induced effects on kidney weight and ACR were not modified by the presence or absence of ficolin B.

Detection of soluble CR3 (CD11b/CD18) by time-resolved immunofluorometry.

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.

Protein replacement therapy partially corrects the cholesterol-storage phenotype in a mouse model of Niemann-Pick type C2 disease.

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.