In this study, we showed that CERT, a protein that transports ceramide from the ER to the Golgi, is essential for survival in mice. Mice lacking CERT die at E11.5 because of cardiac abnormalities manifesting as a result of underlying structural and functional defects in ER and mitochondria. The in vivo significance of ceramide transfer from the ER to the Golgi was studied recently using a Drosophila
model (Rao et al., 2007
). Flies lacking a functional CERT protein are viable and fertile but die prematurely as a result of accelerated aging. The different outcomes in these organisms stem not only from the differences in their developmental programs but also from differences in the underlying biochemical changes accompanying the CERT deficiency.
CERT mutants in both organisms have decreased sphingomyelin levels (CPE in Drosophila
). Although there is a fourfold decrease in the Drosophila
adult, there is slightly more than a twofold decrease in the mutant mouse embryos. This is probably because the mouse embryos, unlike Drosophila
, developed within the uterus of a heterozygous mother, where sphingomyelin, a component of plasma lipoprotein fraction, can be delivered to the developing embryos through the amniotic fluid (Liu et al., 1992
; Subbaiah and Liu, 1993
). In an analogous situation, CERT-deficient CHO cells (Ly-A), when grown in the presence of serum, do not show changes in steady-state sphingomyelin levels because they can incorporate the metabolites from the medium (Fukasawa et al., 1999
; Hanada et al., 2003
). However, experimental evidence for this explanation of the differences in sphingomyelin levels is lacking. In any case, the effects of the decrease differ in mice and Drosophila
: the very significant decrease in the CPE levels of Drosophila
CERT mutants affects plasma membrane fluidity and initiates oxidative stress; the smaller decrease in sphingomyelin in the CERT-deficient mouse embryo did not alter plasma membrane fluidity. In Drosophila
, total ceramide levels decrease by 80%, whereas they remain unchanged in mice. However, in the mouse embryo, a 200% increase in ceramide levels in the ER instigates a series of pathophysiological changes and ultimately causes early embryonic lethality.
The most important finding of this study is the impact of deficient ceramide transport on mitochondrial structure and function in mice. Mass spectrometric data indicated increased levels of ceramide in the mitochondria and ER, and electron micrographic pictures showed progressive degeneration of the mitochondria. The changes in ceramide levels and in the structure and function of mitochondria that accompany the CERT deficiency in mice support the idea that ceramide is normally directly transferred from the ER to the mitochondria. Studies have demonstrated a close functional link between the ER and mitochondrial physiology (Copeland and Dalton, 1959
; Ruby et al., 1969
; Morre et al., 1971
; Meier et al., 1978
; Eggens et al., 1979
; Vance, 1991
; Rizzuto et al., 2004
; Franzini-Armstrong, 2007
). Several of these studies have even demonstrated a physical link between the ER and mitochondria. ER membranes called mitochondria-associated membranes are physically and functionally allied with mitochondria and have been implicated in the integration of several aspects of ER and mitochondrial function. Mitochondria-associated membranes are also involved in multiple mechanisms of the cooperative synthesis by the two organelles of the mitochondrial cytochrome c
oxidase and phospho- and glycerosphingolipids (Shore and Tata, 1977
; Vance, 1991
; van Meer and Lisman, 2002
). A recent electron tomography study demonstrated the existence of 10-nm and 25-nm tethers connecting smooth and rough ER, respectively, to mitochondria (Csordas et al., 2006
). Furthermore, although sphingolipid levels are low in mitochondria, some enzymes involved in the de novo steps of the sphingolipid biosynthetic pathway, including ceramide synthase and ceramidase, have been reported to localize to mitochondria (Bionda et al., 2004
). Our data support the idea that some mechanism exists for the transfer of ceramide from the ER to the mitochondria, either by physical continuity between the organelles or by a transfer protein. Although delivering sphingolipid metabolites to the mitochondria may serve a useful function in healthy cells, it might also serve to mitigate the effect of the sphingolipids accumulating in the ER during stressful or pathological conditions.
In a recent study probing the mechanism of drug resistance in tumor cells, it was shown that down-regulation of CERT sensitized these cells to the cytotoxic effects of paclitaxel and potentiated paclitaxel-induced ER stress (Swanton et al., 2007
). However, in this study, we demonstrate that under physiological conditions, in vivo loss of CERT induces a state of chronic stress in the ER and, most importantly, compromises mitochondrial structure and function. The state of chronic stress in CERT mutant embryos is comparable with that of cells that are exposed to chronic ER stress but that have adapted to the stress. In a study of different states of ER stress, cells exposed to low concentrations of tunicamycin showed UPR activation and ER perturbation, but the cells still proliferated, although at a slower rate than untreated cells, and did not show any signs of apoptosis (Rutkowski et al., 2006
). The chronically stressed cells achieved confluence and could be passaged for as long as their untreated counterparts (and they eventually achieved a near-normal growth rate) despite the continued presence of tunicamycin in the culture. Thus, the decreased proliferation of the tunicamycin-treated cells was caused by a growth delay, and chronic ER stress by exogenous pharmacological manipulation does not necessarily result in cell death, although death could be caused by a secondary mitigating event. Similarly, physiological activation of the UPR is observed in B lymphocytes as a result of increased demands of protein secretion (Wu and Kaufman, 2006
The lack of CERT function in mice did not result in cell death. The biochemical data suggest that the cells mounted an adaptation response to the ER and mitochondrial stress in the mutant embryos. Because CERT performs a fundamental function in sphingolipid metabolism, it is present in all cells and tissues. Perhaps surprisingly, although none of the cells in the Certgt/gt mice could transport ceramide, they could still divide at a compromised rate, and they progressed through a nearly normal developmental program until about E10–11.5.
This stage is a remarkable period in mouse development. Beginning around E8 and lasting until around E11.5, there is a sudden increase in the growth and complexity of the embryo (Viragh and Challice, 1973
). The sudden shift in the pace of division and differentiation imposed on the CERT mutant cells at these stages apparently outstrips the ability of these compromised cells to respond to developmental signals. This notion is partly supported by our EM data that showed at E9.5 the beginnings of changes to the mitochondria and ER that were much more obvious at E10.5. That the animals died of cardiovascular defects is very reasonable in this context. During the 24-h window between E8.0 and E9.0, the linear dimensions of the heart increase four- to fivefold, which apparently pushed the mutant cells beyond their capacity to adapt. Because a functional circulatory system is an early requirement for survival and growth, its developmental failure resulted in early death attributable to the lack of CERT function. However, the underlying pathology existed in all of the embryonic cells, as indicated by the similar mitochondrial and ER changes observed in the cells of the developing eye.
Our study clearly demonstrates that manipulation of sphingolipid flux in vivo and the consequent accumulation of ceramide within cellular compartments do not necessarily end in apoptotic cell death. In the present study, it leads to defects in cell cycle and differentiation caused by the stress imposed on the ER and mitochondria. In sum, our combined approach has identified the essential role of CERT in mammalian embryogenesis and provided molecular insight into the role of the de novo sphingolipid biosynthetic pathway in influencing the ER-dependent structure/function of mitochondria.