The ceramide synthase 2 catalytic lag1p motif and homeodomain and their different influence on brain function and metabolism

Abstract

Among the different lipid classes, sphingolipids are known as essential components of every cell’s membrane formation. The backbone of each sphingolipid is ceramide, which is catalyzed by enzymes called ceramide synthases (CerS). In mammals are six isoforms of CerS found, which show tissue- and substrate-specificity. The ER-membrane bound enzymes produce ceramides from the substrates sphinganine, a long chain base, and fatty acid acyl-CoAs of specific chain lengths. Research of the last decades unraveled important functions of these enzymes and their products for general organism homeostasis and in developmental processes. The homeostasis between long chain C16 and very long chain C24 ceramides and sphingolipids has become the focus of recent research, as an imbalance between these mentioned species has extensive consequences for cellular function and metabolism. For example, catalytic inactivity of CerS2, the most abundant and ubiquitously expressed CerS in mammals, leads to loss of C24 ceramide but compensatory increase of C16 ceramide, thereby promoting pro-apoptotic and pro-inflammatory processes. This results in a vast amount of phenotypes in CerS2 loss of function mice showing developmental delay, early lethality, hepatocellular carcinoma, myelin sheath defects and much more.<br />In this study, the exact reasons for early lethality and developmental delay should be figured out of the abundance of severe phenotypes by use of tissue-specific expression of catalytic inactive CerS2 protein in mice. Because hepatocyte-specific expression resulted in assumed liver tumor formation but not in expected developmental delay and early lethality, the focus was set on the central nervous system. CerS2 is mainly expressed by the myelinating oligodendrocytes of the brain, but tremendous effects could be also observed on other glial cell types and on neurons: Conditioned media experiments demonstrated influence of secreted or lack of unknown factors on astrocytic function and, by sandwich-culture approaches, a so far unknown lipid accumulating phenotype of primary neurons could be identified upon CerS2 catalytic inactivity.<br />Next to the catalytic domain, most CerS enzymes harbor a homeodomain with ambiguous function. Previous studies in <em>Drosophila melanogaster</em> could demonstrate DNA-binding and gene expression regulating functions by the homeodomain of its CerS, called Schlank. Due to six homologous CerS in mammals, in contrast to single one in Drosophila, any influences of a mutated CerS homeodomain on organism homeostasis were not or only minor expected. To investigate in this, a new transgenic mouse line was generated in context of this thesis, ubiquitously expressing a point mutation within the nuclear localization signal 2 motif of CerS2 homeodomain. The characterization of this new mouse line revealed an unexpected aging phenotype and also possible evidence for gene expression regulating functions of CerS2 in mammals: Differentially expressed lipid metabolism genes and increased susceptibility to lipid accumulation in hepatocytes was found, in contrast to catalytic inactive CerS2 mice.