Protein Replacement Therapy Partially Corrects the Cholesterol-Storage Phenotype in a Mouse Model of Niemann-Pict 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

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

Histochemical analysis of NPC2 replacement therapy in murine spleen sections.

<p>Hematoxylin<i>-</i>eosin (H&E) staining of spleen from saline treated wild type mice (<i>left panel</i>), saline treated <i>NPC2^−/−</i> mice (<i>middle panel</i>), and NPC2 treated <i>NPC2^−/−</i> mice (<i>right panel</i>). Massive accumulation of lipid droplets was most prominent observed in the spleens of saline treated NPC2<i>^−/−</i> mice. Data are representative of three separate experiments. <i>n</i> = 3 animals in each experimental group. Scale bares represent 100 µm.</p

Immune response to NPC2 in 129P2 wild type- and <i>NPC2^−/−</i> mice.

<p>NPC2 coated microtiter wells were incubated with serial dilutions of immunized serum as indicated. Bound antibodies were detected by TRIFMA as described in Materials and Methods. An arbitrary concentration of anti-NPC2 antibodies was set to 1000 mU/ml in positive control serum prepared by subcutaneously injections of NPC2 with Freund's complete adjuvant as immune potentiator. Sera from saline treated healthy mice were similarly tested and served as negative controls. (<b>A</b>) Anti-NPC2 antibody concentration in intraperitoneal injected wild type mice, (<b>B</b>) Anti-NPC2 antibody concentration in intravenous injected wild type mice, and (<b>C</b>) Anti-NPC2 antibody concentration in intravenous NPC2 treated <i>NPC2^−/−</i> mice. Data represents mean values of triplicate wells.</p

Effect of NPC2 treatment on animal body weight.

<p>(<b>A</b>) Males (n = 4) and (<b>B</b>) females (n = 6), respectively, of saline-treated wild type mice (•), saline-treated <i>NPC2^−/−</i> mice (○), and NPC2 treated <i>NPC2^−/−</i> mice (▾). The mice were weighed weekly from P21 to P87. Each animal was injected twice weekly with saline or NPC2 (5 mg/kg). Values are means ± SEM.</p

Effect of purified bovine NPC2 on cholesterol accumulation in wild type and <i>NPC2^−/−</i> fibroblasts.

<p>(<b>A</b>) SDS-PAGE of 5 µg purified NPC2 resolved in a 10–20% gradient gel under nonreducing conditions and stained with Coomassie Brilliant Blue (<i>lane 1</i>). Molecular mass markers are shown on the left (<i>lane M</i>). (<b>B</b>) Upper row human- (Hu) and lower row murine (Mu) fibroblasts cultivated for 48 hours in complete medium (DMEM + 10% FBS). Wild type fibroblasts (<i>left panels</i>), <i>NPC2^-/-</i> fibroblasts (<i>middle panels</i>), <i>NPC2^-/-</i> fibroblasts supplemented with 600 nM NPC2 (<i>right panels</i>). Cells were fixed with 10% phosphate buffered formalin, pH 7.4 and stained with Filipin III and visualized using fluorescence microscope.</p

Effect of NPC2 replacement therapy on murine brain cholesterol storage.

<p>Total cholesterol levels in cerebellum, cortex, and hippocampus was measured postmortem in saline treated wild type mice (<i>black bars</i>), saline treated <i>NPC2^−/−</i> mice (<i>light gray bars</i>), and NPC2 treated <i>NPC2^−/−</i> mice (<i>dark gray</i>). Each bar represents the mean ± SEM for 6 animals in each of the three groups. Bars not sharing a letter within a given panel are significantly different (<i>P</i><0.05).</p