Abstract 417: Elevation Of Serum Hdl And Apoe Level In A Novel Vps35l Deficient Mouse Model

Abstract

Aim: Defect of the endosomal protein sorting factor VPS35L causes novel congenital anomalies syndrome, in which cholesterol dysmetabolism is one of the symptoms. To reveal the biochemical mechanisms, the a model mouse was developed and characterized. Methods: Loss-of-function mutation, N995del, VPS35L knock-in (KI) mice were generated. Serum lipoprotein profiles were assessed by LipoSEARCH™ (Immuno-Biological Laboratories, Japan). For biochemical assays, 70 micro-L of serum was sub-fractionated with Superose 6 column in PBS and total and free cholesterol detected using LabAssay™cholesterol (Fujifilm Wako Chemicals, Japan) and Pureauto™S F-CHO-N (SEKISUI MEDICAL, Japan) assay kits. Negative images of fractioned lipoproteins were observed by TEM (JEM-1400Plus, JEOL, Japan). ApoA-I and apoE in the fractions were determined by use of a mouse apoA1 ELISA PRO kit and a mouse apoE ELISA PRO kit (Mabtech AB, Sweden). Mouse liver total RNA was isolated by ISOGEN (Nippon Gene, Japan) and first-strand cDNA generated using the Invitrogen™ SuperScript™IV First-Strand Synthesis system with random hexamers. For quantitative real-time PCR, the StepOnePlus™ real-time PCR system (Thermo Fisher) was used, and the PCR end product of each well confirmed by the melt curve analysis. Results: Lipoprotein analysis indicated the KI mice showed 1.9-fold higher total cholesterol than WT mice. LDL-C and HDL-C were increased 2.9- and 1.9-fold compared to WT mice, respectively. ApoA-I levels in the large HDL and HDL fractions were all significantly increased 1.5-, and 1.6-fold, whereas apoE levels were elevated 25-, and 8.4-fold, respectively. Conclusions: Ablation of VPS35L in MEF(3T3) cells caused loss of endocytosis of the LDL receptor and LRP1, and perhaps even SR-BI, which may lead to increased serum LDL and HDL levels in the loss-of-function KI model mice. Surprisingly. a high level of apoE was detected in KI mice, especially in the HDL fraction. In this case, apoE catabolism became abnormal due to the lack of LRP1 at the cell surface. Abnormal metabolism of apoE may affect the central nervous system or neuron generation. Further examination of the phenotype of this this model mouse line are warranted.