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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 5, 2002 as doi:10.1096/fj.02-0149fje. |
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Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel
2Correspondence: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel. E-mail: tony.futerman{at}weizmann.ac.il
SPECIFIC AIMS
This study was designed to determine whether there is a correlation between the rate of phospholipid synthesis and intracellular accumulation of glucosylceramide (GlcCer), the glycosphingolipid that accumulates in the inherited metabolic disorder Gaucher disease due to defective activity of the lysosomal enzyme glucocerebrosidase. We have already shown that on stimulation of its synthesis or on inhibition of its degradation, GlcCer stimulates neuronal growth and development. Since phospholipids are the major structural components of neuronal membranes, we examined whether GlcCer directly influences the rate of phospholipid synthesis.
PRINCIPAL FINDINGS
1. GlcCer accumulation stimulates axonal growth and phosphatidylcholine synthesis
Hippocampal neurons cultured from embryonic rats were incubated for > 4 days with conduritol-B-epoxide (CBE), an inhibitor of lysosomal glucocerebrosidase, or were cultured from a mouse model of Gaucher disease (the Gba-/- mouse). In both cases, an 
four- to eightfold increase in intracellular GlcCer levels was detected, and axons were significantly longer after 3 days in culture. A significant increase in [14C]methyl choline incorporation into [14C]sphingomyelin and [14C]phosphatidylcholine (PC) was observed after 2 days of CBE treatment and remained elevated for at least 8 days. A similar increase was observed in Gba-/- neurons (Table 1
). The increase in levels of the water-soluble metabolite [14C]CDP-choline (Table 1)
, the decrease in [14C]phosphorylcholine levels (Table 1)
, and the lack of a difference in the rate of [14C]PC degradation suggested that CTP:phosphocholine cytidylyltransferase (CCT), the rate-limiting enzyme in PC synthesis, is activated in neurons upon GlcCer accumulation.
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2. CCT activity is up-regulated upon GlcCer accumulation
A two- to threefold increase in CCT activity was detected in rat hippocampal neurons that had been incubated with CBE for 4 days and a decrease was detected in neurons treated with D-threo-1-phenyl-2-decanoyl-amino-3-morpholino-1-propanol, an inhibitor of GlcCer synthase. No elevation in mRNA levels of either the 
or ß1/2 CCT isoforms could be detected by RT-PCR after 8 days of CBE treatment or in Gba-/- (or Gba+/-) neurons. Together with the lack of change in CCT protein levels, determined by Western blotting using anti-CCT and anti-CCTß1/2 specific antibodies in Gba-/- brain homogenates compared to Gba+/+ brain, this demonstrates that CCT is not transcriptionally up-regulated on GlcCer accumulation but is probably up-regulated by a post-transcriptional mechanism.
No comprehensive study comparing the effect of different glycosphingolipids, particularly glucose-based glycosphingolipids such as GlcCer, on CCT activity has been performed before. When rat brain cortical homogenates were incubated with dioleylphosphatidylcholine (DOPC) liposomes containing increasing amounts of GlcCer, a dose-dependent increase in CDP-choline synthesis was observed, with a > twofold increase using 200 µM GlcCer. No other glycosphingolipids or lyso-glycosphingolipids, including galactosylceramide, caused an increase in CCT activity, although some inhibited CCT, as previously reported. Significantly more CCT activity was membrane associated in Gba-/- brains than in Gba+/+ brains. Together this suggests that PC synthesis is up-regulated in neurons that accumulate GlcCer by a post-translational mechanism in which GlcCer stimulates the translocation or binding of CCT to membranes.
3. GlcCer directly activates purified brain CCT
We next purified CCT from rat brain according to procedures developed for rat liver. This involves passing an n-octyl-ß-D-glucopyranoside extract through DEAE-Sepharose CL-6B and hydroxyapatite columns. A 1200-fold purification was obtained.
When purified rat brain CCT was incubated with DOPC liposomes containing increasing amounts of GlcCer, a significant activation of CCT was observed even at the lowest GlcCer concentration (2.5 mol % of GlcCer), with a maximal three- to fourfold activation at 10 mol % of GlcCer (Fig. 1
A). In contrast and similar to that observed in rat brain homogenates, galactosylceramide had no effect on purified CCT (Fig. 1A
). Likewise, a small but significant increase in CCT activity was obtained by including GlcCer in a liposome mixture of DOPC and oleic acid. However, CCT activity was 10-fold higher in DOPC/oleic acid liposomes than in DOPC liposomes alone; the level of CCT activation obtained by inclusion of GlcCer in DOPC/oleic acid liposomes was lower than in liposomes containing DOPC alone (Fig. 1B
). Again, no significant effect of galactosylceramide on CCT activity was observed when it was included in DOPC/oleic acid liposome mixtures.
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CONCLUSIONS AND SIGNIFICANCE
The major findings of this study are that 1) PC synthesis is increased in neurons that accumulate GlcCer, due to activation of CCT, which is correlated with increased axonal growth rates, and 2) purified rat brain CCT is directly activated by GlcCer. Activation of CCT is highly specific for GlcCer, since galactosylceramide failed to stimulate CCT. We are now attempting to define the mechanism of CCT activation by GlcCer not only at the biochemical level using purified CCT, but also in intact cells, in which CCT becomes associated with the endoplasmic reticulum (ER) upon activation of its activity (see Fig. 2
). However, GlcCer is believed to accumulate in lysosomes in Gaucher disease, implying that GlcCer exits lysosomes to interact with CCT. GlcCer normally is not associated with the ER, as it is made distal to the ER (in the Golgi apparatus). However, our view of the intracellular distribution of glycosphingolipids is under challenge by a number of unexpected observations, such as its occurrence in cytoplasmic inclusions in multidrug-resistant cells. The specificity of GlcCer activation of CCT might even suggest that it is a normal physiological regulator of this key enzyme in phospholipid synthesis.
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Our results may help delineate the biochemical basis of neuronal dysfunction in neuronopathic forms of Gaucher disease. There is no evidence that neuronal growth and/or development is altered in neuronopathic forms of Gaucher disease, but this may be due to the lack of systematic studies on postmortem brain tissue and to the lack of a viable mouse model. However, changes in neuronal morphology have been observed, including dilated and distended smooth and rough ER in brains from neuronopathic forms of Gaucher disease, in the brains of mice fed CBE and in cultured neurons treated with CBE, consistent with the changes observed in PC synthesis that occur in the ER. Several studies have shown that GlcCer accumulation in non-neuronal tissues is accompanied by cell growth and/or proliferation. Thus, GlcCer accumulates in some tumor cells such as multidrug-resistant cells and, in tumorigenesis can be associated with activation of the CDP-choline pathway of PC biosynthesis. In Gaucher disease, macrophages become enlarged, as do the spleen and liver. The observation that GlcCer directly stimulates CCT activity and, as a result, PC synthesis might account at least partially for increased rates of cell growth in various cell types similar to that observed in Gba mouse axons.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0149fje; to cite this article, use FASEB J. (September 5, 2002) 10.1096/fj.02-0149fje 
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