Inactivation of Ceramide Synthase 6 in Mice Results in an Altered Sphingolipid Metabolism and Behavioral Abnormalities

Philipp Ebel,Katharina Vom Dorp,Elisabeth Petrasch-Parwez,Armin Zlomuzica,Kiyoka Kinugawa,Jean Mariani,David Minich,Christina Ginkel,Jochen Welcker,Joachim Degen,Matthias Eckhardt,Ekrem Dere,Peter Dörmann,Klaus Willecke

doi:10.1074/jbc.m113.479907

Abstract

The N-acyl chain length of ceramides is determined by the specificity of different ceramide synthases (CerS). The CerS family in mammals consists of six members with different substrate specificities and expression patterns. We have generated and characterized a mouse line harboring an enzymatically inactive ceramide synthase 6 (CerS6KO) gene and lacz reporter cDNA coding for β-galactosidase directed by the CerS6 promoter. These mice display a decrease in C16:0 containing sphingolipids. Relative to wild type tissues the amount of C16:0 containing sphingomyelin in kidney is ∼35%, whereas we find a reduction of C16:0 ceramide content in the small intestine to about 25%. The CerS6KO mice show behavioral abnormalities including a clasping abnormality of their hind limbs and a habituation deficit. LacZ reporter expression in the brain reveals CerS6 expression in hippocampus, cortex, and the Purkinje cell layer of the cerebellum. Using newly developed antibodies that specifically recognize the CerS6 protein we show that the endogenous CerS6 protein is N-glycosylated and expressed in several tissues of mice, mainly kidney, small and large intestine, and brain.

Highlights

Generation of Ceramide Synthase 6-deficient Mice—The catalytically active domain of CerS6 is encoded by DNA sequences starting in exon 6 and ending in exon 8
We decided to minimize the impact on the genomic locus, since deletion of the whole coding region including the introns might lead to unintended loss of possible regulatory sequences
We used the non-conditional vector shown in Fig. 1A, which consists of the splice acceptor site of intron 6, the first 9 base pairs of the coding region of exon 7 and the integrated stop codon, followed by an internal ribosomal entry site, the lacz reporter cDNA, the last 9 base pairs of exon 7, and the splice donor site of intron 7 resulting in a truncated enzymatically inactive CerS6 protein

Summary

Background

Ceramide synthases N-acylate (dihydro-)sphingosine to (dihydro-)ceramide in mammals. Results: Enzymatically inactive ceramide synthase 6 in mice (CerS6KO) results in an altered sphingolipid metabolism and behavioral abnormalities. The N-acyl chain length of ceramides is determined by the specificity of different ceramide synthases (CerS). We have generated and characterized a mouse line harboring an enzymatically inactive ceramide synthase 6 (CerS6KO) gene and lacz reporter cDNA coding for ␤-galactosidase directed by the CerS6 promoter. These mice display a decrease in C16:0 containing sphingolipids. All CerS harbor at least five transmembrane domains and are suggested to be located in the endoplasmic reticulum [4, 5] They differ regarding their tissue distribution as well as their acyl chain length specificity [6, 7]. CerS3, which is mostly expressed in testis and skin, produces ceramides with mainly ultra-long chain fatty

The abbreviations used are

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION