Glycosphingolipids and mitochondria: Role in apoptosis and disease

Glycosphingolipids (GSLs) comprise a class of lipids with important structural and signaling functions. Synthesized from ceramide in the Golgi, they are subsequently distributed to different compartments, most predominantly in the plasma membrane where they integrate signaling platforms. A recently characterized trafficking of ganglioside GD3 (GD3), a GSLs with two sialic-acid residues, to mitochondria has revealed a novel function of this lipid as a death effector. In addition to the interaction of GD3 with mitochondria recruiting these organelles to apoptotic pathways, GD3 disables survival paths dependent on NF-B, thus favoring the balance towards cell death. The present review gathers the evidence documenting this emerging function of GSLs in cell death and their involvement in pathological states. Published in 2004..     

The role of mitochondria in apoptosis

Apoptosis (programmed cell death) is a cellular self-destruction mechanism that is essential for a variety of biological events, such as developmental sculpturing, tissue homeostasis, and the removal of unwanted cells. Mitochondria play a crucial role in regulating cell death. Ca2+ has long been recognized as a participant in apoptotic pathways. Mitochondria are known to modulate and synchronize Ca2+ signaling. Massive accumulation of Ca2+ in the mitochondria leads to apoptosis. The Ca2+ dynamics of ER and mitochondria appear to be modulated by the Bcl-2 family proteins, key factors involved in apoptosis. The number and morphology of mitochondria are precisely controlled through mitochondrial fusion and fission process by numerous mitochondria-shaping proteins. Mitochondrial fission accompanies apoptotic cell death and appears to be important for progression of the apoptotic pathway. Here, we highlight and discuss the role of mitochondrial calcium handling and mitochondrial fusion and fission machinery in apoptosis.     

Key role of mitochondria in cerulenin-mediated apoptosis

Cerulenin, a fungal metabolite, is known to be a specific inhibitor of fatty acid synthase. Here we report that cerulenin is an effective inducer of apoptosis in different wild-type p53 and mutant p53 tumor cell lines, whereas normal human keratinocytes and fibroblasts are resistant to the apoptotic effect. To get more insight into the mechanisms of how cerulenin induces apoptosis we investigated several signal transduction molecules, including p53, p73, p21/WAF1, Bax, cytochrome c, and caspases 3 and 9. Our data strongly indicate that mitochondria play a key role in the cerulenin-mediated pathway. Bax overexpression correlated with the extent of apoptosis and appears to be regulated in a p53-independent manner. The significance of the mitochondrial pathway for the cerulenin-mediated apoptosis was confirmed by the rapid mitochondrial release of cytochrome c both in wild-type p53 and mutant cell lines. Interestingly, the rapid release of cytochrome c was not accompanied by a breakdown of the mitochondrial potential. Instead, the complete disruption of the mitochondrial function coincided with the appearance of a p18 kDa cleavage product of Bax.     

Functional characterization of mitochondria in neutrophils: a role restricted to apoptosis

Mitochondria are known to combine life-supporting functions with participation in apoptosis by controlling caspase activity. Here, we report that in human blood neutrophils the mitochondria are different, because they preserve mainly death-mediating abilities. Neutrophil mitochondria hardly participate in ATP synthesis, and have a very low activity of the tested marker enzymes. The presence of mitochondria in neutrophils was confirmed by quantification of mitochondrial DNA copy number, by detection of mitochondrial porin, and by JC-1 measurement of Deltapsi(m). During neutrophilic differentiation, HL-60 cells demonstrated a profound cytochrome c depletion and mitochondrial shape change reminiscent of neutrophils. However, blood neutrophils containing extremely low amounts of cytochrome c displayed strong caspase-9 activation during apoptosis, which was also observed in apoptotic neutrophil-derived cytoplasts lacking any detectable cytochrome c. We suggest that other proapoptotic factors such as Smac/DIABLO and HtrA2/Omi, which are massively released from the mitochondria, have an important role in neutrophil apoptosis.     

Bcl-2-family proteins and the role of mitochondria in apoptosis

Mitochondria are central to many forms of cell death, usually via the release of pro-apoptotic proteins from the mitochondrial intermembrane space. Some intermembrane space proteins, including cytochrome c, Smac/DIABLO, and Omi/Htra2, can induce or enhance caspase activation, whereas others, such as AIF and endonuclease G, might act in a caspase-independent manner. Intermembrane space protein release is often regulated by Bcl-2-family proteins. Recent evidence suggests that pro-apoptotic members of this family, by themselves, can permeabilize the outer mitochondrial membrane without otherwise damaging mitochondria. Mitochondria can contribute to cell death in other ways. For example, they can respond to calcium release from the endoplasmic reticulum by undergoing the mitochondrial permeability transition, which in turn causes outer membrane rupture and the release of intermembrane space proteins. Bcl-2-family proteins can influence the levels of releasable Ca(2+) in the endoplasmic reticulum, and thus determine whether the released Ca(2+) is sufficient to overload mitochondria and induce cell death.     

A metabolic role for mitochondria in palmitate-induced cardiac myocyte apoptosis

After cardiac ischemia, long-chain fatty acids, such as palmitate, increase in plasma and heart. Palmitate has previously been shown to cause apoptosis in cardiac myocytes. Cultured neonatal rat cardiac myocytes were studied to assess mitochondrial alterations during apoptosis. Phosphatidylserine translocation and caspase 3-like activity confirmed the apoptotic action of palmitate. Cytosolic cytochrome c was detected at 8 h and plateaued at 12 h. The mitochondrial membrane potential (DeltaPsi) in tetramethylrhodamine ethyl ester-loaded cardiac myocytes decreased significantly in individual mitochondria by 8 h. This loss was heterogeneous, with a few energized mitochondria per myocyte remaining at 24 h. Total ATP levels remained high at 16 h. The DeltaPsi loss was delayed by cyclosporin A, a mitochondrial permeability transition inhibitor. Mitochondrial swelling accompanied changes in DeltaPsi. Carnitine palmitoyltransferase I activity fell at 16 h; this decline was accompanied by ceramide increases that paralleled decreased complex III activity. We conclude that carnitine palmitoyltransferase I inhibition, ceramide accumulation, and complex III inhibition are downstream events in cardiac apoptosis mediated by palmitate and occur independent of events leading to caspase 3-like activation.     

Zinc induced apoptosis in HEP-2 cancer cells: the role of oxidative stress and mitochondria

Induction of apoptosis by zinc sulfate was investigated during 96 h exposure on the cancer Hep-2 cell line. During 48 h of exposure, zinc translocated into mitochondria and stimulated production of reactive oxygen species (ROS), affected cellular GSH management and induced moderate activation of p53 and dissipation of mitochondrial membrane potential. In Zn-exposed cells, mitochondria released cytochrome c and AIF, whose translocation to the cytoplasm or the nucleus coincided with the activation of apoptosis. The use of various pharmacological inhibitors inhibiting particular apoptotic targets (antioxidants such as N-acetyl-cysteine and coenzyme Q, the caspase inhibitors z-DEVD-fmk and z-VAD-fmk, cyclosporin A and bonkgrekic acid) proved that Zn acts both directly and indirectly on mitochondria and observed apoptosis is executed by caspase-dependent and caspase-independent pathways.     

Metabolic reprogramming and the role of mitochondria in polycystic kidney disease

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a slowly progressive disease characterized by the relentless growth of renal cysts throughout the life of affected individuals. Early evidence suggested that the epithelia lining the cysts share neoplastic features, leading to the definition of PKD as a “neoplasm in disguise”. Recent work from our and other laboratories has identified a profound metabolic reprogramming in PKD, similar to the one reported in cancer and consistent with the reported increased proliferation. Multiple lines of evidence suggest that aerobic glycolysis (a Warburg-like effect) is present in the disease, along with other metabolic dysfunctions such as an increase in the pentose phosphate pathway, in glutamine anaplerosis and fatty acid biosynthesis, while fatty acid oxidation and oxidative phosphorylation (OXPHOS) are decreased. In addition to glutamine, other amino acid-related pathways appear altered, including asparagine and arginine. The precise origin of the metabolic alterations is not entirely clear, but two hypotheses can be formulated, not mutually exclusive. First, the polycystins have been recently shown to regulate directly mitochondrial function and structure either by regulating Ca²⁺ uptake in mitochondria at the Mitochondria Associated Membranes (MAMs) of the Endoplasmic Reticulum, or by a direct translocation of a small fragment of the protein into the matrix of mitochondria. One alternative possibility is that metabolic and mitochondrial dysfunctions in ADPKD are secondary to the de-regulation of proliferation, driven by the multiple signaling pathways identified in the disease, which include mTORC1 and AMPK among the most relevant. While the precise mechanisms underlying these novel alterations identified in ADPKD will need further investigation, it is evident that they offer a great opportunity for novel interventions in the disease.     

PKB and the mitochondria: AKTing on apoptosis

Cellular homeostasis depends upon the strict regulation of responses to external stimuli, such as signalling cascades triggered by nutrients and growth factors, and upon cellular metabolism. One of the major molecules coordinating complex signalling pathways is protein kinase B (PKB), a serine/threonine kinase also known as Akt. The number of substrates known to be phosphorylated by PKB and its interacting partners, as well as our broad understanding of how PKB is implicated in responses to growth factors, metabolic pathways, proliferation, and cell death via apoptosis is constantly increasing. Activated by the insulin/growth factor-phosphatidylinositol 3-kinase (PI3K) cascade, PKB triggers events that promote cell survival and prevent apoptosis. It is also now widely accepted that mitochondria are not just suppliers of ATP, but that they participate in regulatory and signalling events, responding to multiple physiological inputs and genetic stresses, and regulate both cell proliferation and death. Thus, mitochondria are recognized as important players in apoptotic events and it is logical to predict some form of interplay with PKB. In this review, we will summarize mechanisms by which PKB mediates its anti-apoptotic activities in cells and survey recent developments in understanding mitochondrial dynamics and their role during apoptosis.     

Implication of mitochondria in apoptosis

The induction phase of programmed cell death (PCD) or apoptosis is characterized by an extreme heterogeneity of potential PCD-triggering signal transduction pathways. During the subsequent effector phase, the numerous PCD-inducing stimuli converge into a few stereotypical pathways and cells pass a 'point of no return', thus becoming irreversibly committed to death. Evidence is accumulating that cytoplasmic structures, including mitochondria, participate in the critical effector stage and that alterations usually considered to define apoptosis, as nuclear chromatolysis and cytolysis, have to be ascribed to the late degradation phase. We and others have recently shown that nuclear features of apoptosis are preceded by alterations in mitochondrial function and structure. The importance of these alterations for the apoptotic process and also the possible link between, these observations, the permeability transition pore and the programmed cell death, are dicussed. (Mol Cell Biochem 174: 185–188, 1997)     

A potential role for apoptosis in neurodegeneration and Alzheimer's disease

Previous studies have shown that β-amyloid (Aβ) peptides are neurotoxic. Recent data suggest that neurons undergoing Aβ-induced cell death exhibit characteristics that correspond to the classical features of apoptosis, suggesting that these cells may initiate a program of cell death. This chapter explores the criteria and precautions that must be applied to evaluate mechanisms of cell death in vitro and in vivo, discusses the evidence supporting an apoptotic mechanism of cell death in response to Aβ in cultured neurons, and describes potential correlations for these findings in the Alzheimer's disease brain. In addition, cellular signaling pathways that may be associated with apoptosis in response to Aβ are examined, and support for apoptosis as a mechanism of cell death for other neurodegeneration-inducing stimuli (e.g., oxidative injury) is described. The connection of multiple stimuli that induce neuronal cell death to an apoptotic mechanism suggests that apoptosis could play a central role in neurodegeneration in the brain.     

Shaping the role of mitochondria in the pathogenesis of Huntington's disease

Intense research on the pathogenesis of Huntington's disease (HD), a genetic neurodegenerative disease caused by a polyglutamine expansion in the Huntingtin (Htt) protein, revealed multiple potential mechanisms, among which mitochondrial alterations had emerged as key determinants of the natural history of the disease. Pharmacological and genetic animal models of mitochondrial dysfunction in the striatum, which is mostly affected in HD corroborated a key role for these organelles in the pathogenesis of the disease. Here, we will give an account of the recent evidence indicating that the mitochondria-shaping machinery is altered in HD models and patients. Since its correction can counteract HD mitochondrial dysfunction and cellular damage, drugs impacting on mitochondrial shape are emerging as a new possibility of treatment for this devastating condition.     

Glycosphingolipids and lysosomal storage disorders as illustrated by gaucher disease

Glycosphingolipids are important building blocks of the outer leaflet of the cell membrane. They are continuously recycled, involving fragmentation inside lysosomes by glycosidases. Inherited defects in degradation cause lysosomal glycosphingolipid storage disorders. The relatively common glycosphingolipidosis Gaucher disease is highlighted here to discuss new insights in the molecular basis and pathophysiology of glycosphingolipidoses reached by fundamental research increasingly using chemical biology tools. We discuss improvements in the detection of glycosphingolipid metabolites by mass spectrometry and review new developments in laboratory diagnosis and disease monitoring as well as therapeutic interventions.     

Morphology of mitochondria during apoptosis: worms-to-beetles in worms

Although mitochondria are crucial for most pathways of mammalian cell apoptosis, evidence for their role in classic invertebrate models of programmed cell death has been frustratingly scant. New work showing that inhibition of mitochondrial fragmentation during C. elegans development inhibits programmed cell death bridges this gap and should advance a more detailed understanding of the role of mitochondria in caspase activation.     

Glycosphingolipids in membrane architecture

As part of a program to investigate the behavior and interactions of glycolipids in biological membranes we have synthesized spin-labeled derivatives of 2 families of carbohydrate-bearing ceramides (glycosphingolipids): simple neutral glycolipids and gangliosides. Galactosyl ceramide has been synthesized with the spin label at 3 different positions on the fatty acid chain. It has been studied in bilayers of various different lipids and lipid mixtures and compared to the corresponding phospholipid spin labels. Considerable similarity has been found between the behavior of galactosyl ceramide and phosphatidylcholine. These similarities include a negligible flip-flop rate, a flexibility gradient in the acyl chains, and exclusion from phosphatidylserine domains in the face of a Ca²⁺-induced lateral phase separation. Evidence for dramatic clustering of simple neutral glycolipids has not been found. Glycosphingolipids do seem to have the capacity to increase rigidity in fluid lipid bilayers. A general procedure has been developed for covalent attachment of a nitroxide spin label to the headgroup region of complex glycolipids such as gangliosides. Studies of beef brain gangliosides labeled in this manner and incorporated into bilayers of phosphatidylcholine indicate that the headgroup oligosaccharides are in rapid, random motion as opposed to being in any way immobilized. This headgroup mobility depends very little on the fluidity or rigidity of the bilayer. However, headgroup mobility decreases, perhaps as a result of cooperative headgroup interactions, with increasing bilayer concentration of unlabeled ganglioside.