Phosphatidylcholine synthesis is elevated in neuronal models of Gaucher disease due to direct activation of CTP:phosphocholine cytidylyltransferase by glucosylceramide

Abstract

Glucosylceramide (GlcCer) accumulates in the inherited metabolic disorder, Gaucher disease, because of the defective activity of lysosomal glucocerebrosidase. We previously demonstrated that upon GlcCer accumulation, cultured hippocampal neurons exhibit modified growth patterns, altered endoplasmic reticulum density, and altered calcium release from intracellular stores. We here examined the relationship between GlcCer accumulation and phospholipid synthesis. After treatment of neurons with an active site-directed inhibitor of glucocerebrosidase, or in neurons obtained from a mouse model of Gaucher disease, [14C]methyl choline incorporation into [14C]phosphatidylcholine ([14C]PC) and [14C]sphingomyelin was elevated, as were [14C]CDP-choline levels, suggesting that CTP:phosphocholine cytidylyltransferase (CCT) is activated. Indeed, CCT activity was elevated in neurons that had accumulated GlcCer. GlcCer, but not galactosylceramide (GalCer), stimulated CCT activity in rat brain homogenates, and significantly higher levels of CCT were membrane associated in cortical homogenates from a mouse model of Gaucher disease compared with wild-type mice. Because CCT mRNA and protein levels were unaltered in either neurons or brain tissue that had accumulated GlcCer, it appeared likely that GlcCer activates CCT by a post-translational mechanism. This was verified by examination of the effect of GlcCer on CCT purified about 1200-fold from rat brain. GlcCer stimulated CCT activity, with stimulation observed at levels as low as 2.5 mol% and with maximal activation reached at 10 mol%. In contrast, GalCer had no effect. Together, these data demonstrate that GlcCer directly activates CCT, which results in elevated PC synthesis, which may account for some of the changes in growth rates observed upon neuronal GlcCer accumulation.

Glucosylceramide and Glucosylsphingosine Modulate Calcium Mobilization from Brain Microsomes via Different Mechanisms

“…5 However, lyso-GSLs accumulate at much higher levels in GSL storage diseases, such as Gaucher and Krabbe's disease, particularly in the brain, and the lyso-GSLs, rather than the GSLs, have been implicated in the mechanisms underlying disease pathology, especially neuropathology (34,64). Irrespective of the physiological relevance of lyso-GSLs in mobilizing Ca 2ϩ from microsomes, the specificity of GlcCer compared with both other GSLs and lyso-GSLs on Ca 2ϩ mobilization reported both in this study in microsomes and in our previous study in cultured neurons (22), and our recent study on activation of CTP:phosphocholine cytidylyltransferase by GlcCer (60), suggest that GlcCer is an important intracellular messenger that plays keys roles in both the regulation of phospholipid synthesis and in intracellular Ca 2ϩ homeostasis.…”

“…However, our understanding of the intracellular distribution of GSLs may need to be re-evaluated in light of recent findings demonstrating that GlcCer affects a number of activities associated with the ER and other intracellular organelles (59,60). Indeed, a recent study demonstrated that sphingolipid-specific glycosyltransferases are found in a mitochondrialassociated ER subcompartment of rat liver (61) which could not be ascribed to contaminating Golgi apparatus membranes.…”

Glucosylceramide transfer from lysosomes—the missing link in molecular pathology of glucosylceramidase deficiency: A hypothesis based on existing data

“…The neuronal degeneration has been shown to be associated with increased levels of GlcCer in the neuronal endoplasmic reticulum and with abnormally enhanced agonist-induced calcium release from GD type II brain microsomes, which served to make the neurons more sensitive to glutamate-induced neurotoxicity. Similarly, in the neuronal models of GD, it was shown that GlcCer directly activated phospholipid synthesis by activation of CTP:phosphocholine cytidylyltransferase (CCT), which was shifted from cytosol to the endoplasmic reticulum (Bodennec et al 2002;Korkotian et al 1999;Pelled et al 2005). All these phenomena could be explained by the transfer of the uncleaved lysosomal GlcCer into the neuronal endoplasmic reticulum (Bodennec et al 2002;Jmoudiak and Futerman 2005).…”

“…Similarly, in the neuronal models of GD, it was shown that GlcCer directly activated phospholipid synthesis by activation of CTP:phosphocholine cytidylyltransferase (CCT), which was shifted from cytosol to the endoplasmic reticulum (Bodennec et al 2002;Korkotian et al 1999;Pelled et al 2005). All these phenomena could be explained by the transfer of the uncleaved lysosomal GlcCer into the neuronal endoplasmic reticulum (Bodennec et al 2002;Jmoudiak and Futerman 2005). The role of GlcSph in neuronal degeneration is mentioned below.…”

Simultaneous quantification of lyso-neutral glycosphingolipids and neutral glycosphingolipids by N-acetylation with [3H]acetic anhydride

“…Individual embryos were genotyped (30). Cortices were removed from individual embryos on embryonic day 17 and lipids extracted as described below.…”