In vivo antisense oligonucleotide reduction of NPC1 expression as a novel mouse model for Niemann Pick type C- associated liver disease

Abstract

Niemann-Pick type C (NPC) is a fatal autosomal recessive lipidosis that is characterized by lysosomal storage of cholesterol and glycosphingolipids. Patients exhibit prolonged neonatal jaundice, hepatosplenomegaly, and progressive neurodegeneration that generally result in death by the teen years. Most clinical cases are caused by mutations in the NPC1 gene. Current mouse models of NPC are not well suited for studying the liver disease due to the rapidly progressing neurological disease. To facilitate study of NPC-associated liver dysfunction, we have developed a novel mouse model using antisense oligonucleotides to ablate NPC1 expression primarily in the liver. Here, we show that the NPC1 knockdown leads to a liver disease phenotype similar to that of patients with NPC and the NPC(nih) mouse model. Key features include hepatomegaly, lipid storage, elevated serum liver enzymes, and increased apoptosis.

Conclusion: This novel NPC1 antisense mouse model will allow delineation of the mechanism by which NPC1 dysfunction leads to liver cell death.

Fig. 1

Expression of NPC1, NPC1L1, and NPC2 in mismatched (MM) ASO–treated and NPC1 ASO–treated mice. (A) NPC1 western blot using liver homogenates from different time points and (B) multiple tissues at the 9 week time point. (C) NPC1L1 and NPC2 western blot with 9 week liver homogenate. Immunoblots of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and actin were used as internal controls.

Fig. 2

The effect of NPC1 knockdown on whole-body and liver weights. Absolute (A) body and (B) liver weights are shown of mice treated for 4, 6, and 9 weeks with mismatched (MM) ASOs and NPC1 ASOs. The weight of the liver (C) is also presented as a percentage of whole-body weight. Each bar represents the mean ± SD of nine to 13 animals in each treatment group. *P < 0.001, ł P < 0.05 when compared to mismatched ASO–treated mice as determined by Student t test.

Fig. 3

The effect of NPC1 knockdown on plasma liver enzyme levels. (A) Alanine aminotransferase (ALT) and (B) aspartate aminotransferase (AST) from mice treated with mismatched (MM) ASOs and NPC1 ASOs for 4, 6, and 9 weeks. Each bar represents the mean ± SD for 6 to 13 animals per treatment group. *P < 0.01, ł P < 0.05 when compared to mismatched ASO–treated mice as determined by Student t test.

Fig. 4

Hepatic lipid accumulation and fibrosis in mismatched (MM) ASO–treated and NPC1 ASO–treated mice. (A) Quantification of lipid laden cells where each bar represents the mean ± SD from three to five animals in each treatment group. *P < 0.001 when compared to mismatched ASO–treated as determined by Student t test. Hematoxylin-eosin–stained liver sections from mice treated for 9 weeks with mismatched (B) ASOs and (C) NPC1 ASOs. The arrowhead points to a cluster of lipid-laden cells; the arrow indicates a site of inflammation. Anti-Hepatocyte (D) and anti-vimentin (E) stained liver sections from mice treated for 9 weeks with NPC1 ASOs. The arrowhead (D) points to a hepatocyte positively stained with anti-hepatocyte and the black arrows in both (D) and (E) represent sites of lipid accumulation. Trichrome-staining of liver sections from mice treated for 9 weeks with (F) mismatched ASOs and (G) NPC1 ASOs reveals collagen deposition as indicated by blue staining. (H) Collagen is interspersed throughout clusters of lipid-laden cells. PV, portal vein; CV, central vein.

Fig. 5

NPC1 knockdown leads to increased apoptosis. Liver sections of mismatched (MM) ASO–treated and NPC1 ASO–treated mice were subjected to fluorometric TUNEL staining. (A) Quantification of apoptotic cells where each bar represents the mean ± SD from three to five animals in each treatment group. Liver sections from mice treated for 6 weeks with (B) mismatched ASOs and (C) NPC1 ASOs. *P < 0.0001, ł P < 0.01 when compared to mismatched ASO–treated mice as determined by Student t test.

Fig. 6

Cell proliferation is increased by NPC1 knockdown. (A) Quantification of proliferating cells where each bar represents the mean ± SD from three to five animals in each treatment group. Liver sections from mice treated for 9 weeks with (B) mismatched ASOs and (C) NPC1 ASOs. The arrowhead points to a stellate cell with anti-BrdU-HRP reaction product. Immunohistochemistry for α-SMA was performed on liver sections from mice treated for 9 weeks with (D) mismatched ASO and (E) NPC1 ASOs. The arrow points to an α-SMA–positive stellate cell. ł P < 0.01, * P < 0.00001 when compared to mismatched ASO–treated mice as determined by Student t test.