Sphingolipids in apoptosis, survival and regeneration in the nervous system

Biochimica et Biophysica Acta 1758 (2006) 1995 – 2015 www.elsevier.com/locate/bbamem

Review

Sphingolipids in apoptosis, survival and regeneration in the nervous system Elena I. Posse de Chaves ⁎ Centre for Alzheimer and Neurodegenerative Research, Signal Transduction Research Group and Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7 Received 14 June 2006; received in revised form 20 September 2006; accepted 21 September 2006 Available online 26 September 2006

Abstract Simple sphingolipids such as ceramide, sphingosine and sphingosine 1-phosphate are key regulators of diverse cellular functions. Their roles in the nervous system are supported by extensive evidence derived primarily from studies in cultured cells. More recently animal studies and studies with human samples have revealed the importance of ceramide and its metabolites in the development and progression of neurodegenerative disorders. The roles of sphingolipids in neurons and glial cells are complex, cell dependent, and many times contradictory. In this review I will summarize the effects elicited by ceramide and ceramide metabolites in cells of the nervous system, in particular those effects related to cell survival and death, emphasizing the molecular mechanisms involved. I also discuss recent evidence for the implication of sphingolipids in the development and progression of certain dementias. © 2006 Elsevier B.V. All rights reserved. Keywords: Sphingolipid; Ceramide; Apoptosis; Neuron; Sphingosine 1-phosphate; Alzheimer's disease

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ceramide metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . Origin of ceramide in neurons and glia. . . . . . . . . . . . . . . . . 3.1. De novo ceramide synthesis . . . . . . . . . . . . . . . . . . . 3.2. Sphingomyelin hydrolysis in neuronal cells . . . . . . . . . . . 3.2.1. Neutral sphingomyelinases . . . . . . . . . . . . . . . 3.2.2. Acid sphingomyelinases . . . . . . . . . . . . . . . . 3.3. Mixed mechanisms in ceramide generation . . . . . . . . . . . 3.4. Role of p75NTR in ceramide generation . . . . . . . . . . . . Bioactive sphingolipids: ceramide or its metabolites? . . . . . . . . . Ceramide and its metabolites in survival and apoptosis of neurons and 5.1. PC12 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Sympathetic neurons . . . . . . . . . . . . . . . . . . . . . . 5.3. DRG/sensory neurons . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: Aβ, amyloid β peptide; A-SMase, acid sphingomyelinase; BDNF, brain-derived neurotrophic factor; CNS, central nervous system; DAPK, death associated protein kinase; DIV, days in vitro; ER, endoplasmic reticulum; ERK, extracellular signal-regulated kinase; GlcCer, glucosylceramide; GSH, glutathione; GSK3, glycogen synthase kinase-3; JNK, c-Jun amino terminal kinase; MAPK, mitogen-activated protein kinase; NGF, nerve growth factor; NOE, N-oleoyl ethanolamine; N-SMase, neutral sphingomyelinase; PNS, peripheral nervous system; PI3K, phosphatidylinositide-3-kinase; PKC, protein kinase C; RA, retinoic acid; ROS, reactive oxygen species; SM, sphingomyelin; SMase, sphingomyelinase; SPh, sphingosine; SPhK, sphingosine kinase; S1P, sphingosine-1-phosphate; S1P1–5, sphingosine-1-P receptor 1–5; STP, serine palmitoyltransferase ⁎ 9-28 Medical Sciences Building, University of Alberta, Edmonton AB, T6G 2H7, Canada. Tel.: +1 780 492 5966; fax: +1 780 492 4325. E-mail address: [email protected]. 0005-2736/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamem.2006.09.018

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5.4. 5.5. 5.6. 5.7. 5.8. 5.9. 5.10.

Hippocampal neurons . . . . . . . . . . . . . . . . . . . . . . . . Cerebellar neurons . . . . . . . . . . . . . . . . . . . . . . . . . . Cortical neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . Mesencephalic neurons . . . . . . . . . . . . . . . . . . . . . . . . Motoneurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neuroblastoma cells . . . . . . . . . . . . . . . . . . . . . . . . . Glial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.10.1. Oligodendrocytes . . . . . . . . . . . . . . . . . . . . . 5.10.2. Astrocytes . . . . . . . . . . . . . . . . . . . . . . . . 6. Ceramide and ceramide metabolites in neurodegenerative diseases . . . . . 6.1. Alzheimer’s disease . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.1. Modulation of APP cleavage and Aβ production by lipids . 6.1.2. Ceramide as a second-messenger in the cytotoxic effects of 6.2. HIV-associated dementia . . . . . . . . . . . . . . . . . . . . . . . 7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction In the past decades major findings have emphasized the significance of sphingolipids as bioactive molecules that control diverse cellular processes such as proliferation, differentiation, growth, senescence, migration and apoptosis. Several lines of evidence, derived mainly from studies using cultured cells, suggest that sphingolipids are also essential mediators of cell growth and stress response in the nervous system. Ceramide is at the centre of sphingolipid metabolism and has been recognized as a critical second messenger [1]. Ceramide accumulation is a common cellular response to various stimuli such as cytokines, ionizing radiation, heat shock, chemotherapeutic agents, exposure to receptor-specific ligands (TNFα, Fas ligand, 1,25-dihydroxyvitamin D3), and environmental factors such as stress, hypoxia/reperfusion, etc. This review focuses on the role of ceramide and its immediate metabolites in the regulation of neuronal functions, particularly survival, death and neurite extension. I also discuss the evidence directly implicating these sphingolipids in some neurodegenerative diseases and other disorders of the nervous system. A brief introduction of ceramide synthesis and metabolism is presented although a more detailed discussion is provided by other reviews in this issue. 2. Ceramide metabolism Ceramides are cellular precursors of more complex sphingolipids namely phosphosphingolipids, glucosphingolipids and galactosphingolipids. With exception of epidermis where ceramides form a barrier for water loss [3], most cells contain very low levels of ceramides under resting conditions. Our observations however indicate that ceramide mass in sympathetic neurons is exceptionally high [2], as it is in cerebellar granule cells (CGC) [4,5]. The implications of high levels of ceramide in neurons are unknown; but neuronal ceramide levels are still susceptible to augment. Numerous cellular stimuli induce transient, or sustained increase in ceramide with variable kinetics. Ceramide levels can reach up to 10 mol% of the total

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phospholipids [6] emphasizing the bioactive role of ceramide as a signaling molecule. Furthermore, since the formation of ceramide upon stimulation occurs in restricted cellular sites, the local concentration of ceramide could reach and exceed 25 mol % [7]. Ceramide is produced de novo, or by hydrolysis of sphingomyelin (SM) (Fig. 1). The pool of ceramide generated from the agonist-induced activation of sphingomyelinases (SMases) has long been involved in the signaling functions of ceramide. This pathway provides rapid increase of cellular ceramide levels in response to diverse stimuli. Comprehensive and extensive reviews of the different SMases and their role in cell signaling have been published [12,14–16]. The role of different SMases in the generation of ceramide in cells of neural origin is discussed below. On the other hand, ceramide generated from the de novo pathway has been recognized much later as a second messenger [8–11]. The discovery that many enzymes involved in the metabolism of sphingolipids are regulated in response to cellular stimuli has led to the concept of integrative signaling to explain the contribution of sphingolipid metabolic pathways in cell regulation [12]. Breakdown of ceramide occurs by the action of ceramidases (Fig. 1) [17,18]. Interestingly, some ceramidases can catalyze the reverse reaction and function as a ceramide synthase [19], therefore they can potentially increase ceramide concentration. Sphingosine (SPh) is converted to sphingosine 1-phosphate (S1P) by a family of sphingosine kinases (SPhKs), which are activated in response to stimulation by diverse agonists (see [20–22] for review). S1P is itself a very important bioactive lipid, implicated in several biological processes, often with effects opposed to those of ceramide. 3. Origin of ceramide in neurons and glia The involvement of the de novo or the SM breakdown pathway for ceramide generation in cells from the nervous system depends both on the stimulus and the cell type. The use of specific enzyme inhibitors (Fig. 1) has proven very helpful to discriminate between the two pathways of ceramide production.

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Fig. 1. Ceramide synthesis and metabolism. The scheme represents the reactions that take place in ceramide synthesis and ceramide conversion into main metabolites. The enzymes involved and some of the identified inhibitors are also depicted.

The origin of ceramide determines the subcellular location of ceramide accumulation, hence it influences the direct targets acting downstream of ceramide. 3.1. De novo ceramide synthesis If de novo synthesis provides the bioactive pool of ceramide, inhibition of this pathway should block ceramide actions. The enzymes most targeted for inhibition are SPT and ceramide synthase (Fig. 1). Several inhibitors of SPT are available namely myriocin [23], cycloserine [24], β-chloroalanine [25], and others [24,26,27]. In addition, the inhibitor of ceramide synthesis fumonisin B1 (FB1) [13,28] has been extensively used. Treatment with FB1 inhibits the generation of ceramide and apoptosis induced by suramine in sensory neurons [29]. Likewise ceramide elevation and apoptosis induced by retinoic acid (RA) in PCC7-Mzl cells are blocked by treatment with FB1 but not by inhibition of SMase activity, suggesting that the pool of ceramide originates from the de novo synthesis [30]. 3.2. Sphingomyelin hydrolysis in neuronal cells Apoptotic and other stress stimuli usually activate the Mg2+dependent neutral SMase (N-SMase) and the lysosomal acidic SMase (A-SMase). The importance of this mechanism for ceramide generation in the nervous system was underscored by the discovery of the activation of SMase upon ligand binding to the neurotrophin receptor p75 (p75NTR) (see below).

3.2.1. Neutral sphingomyelinases Mammalian Mg2+ -dependent N-SMases are membrane proteins [16] especially enriched in neural tissues [31], and in cells of neuronal origin [32,33]. During the first 2 weeks of the neonatal period in the ontogenesis of rat brain the specific activity of N-SMase rapidly increases in parallel with neuronal maturation [34], suggesting that ceramide generated from SM by N-SMase may contribute to normal neuronal survival, growth and maturation of the CNS. Although the physiological role of ceramide in brain development remains undefined, mounting evidence from studies in neuronal and glial cells indicate that ceramide generated by N-SMase acts as second messenger in numerous cellular processes. Activation of N-SMase is responsible for generation of a pool of ceramide that mediates diverse neuronal responses ranging from neurite outgrowth and apoptosis in hippocampal neurons [35,36] to death elicited by the Human Immunodeficiency Virus 1 (HIV) glycoprotein gp120 [37]. In addition, studies in human neurons [38], oligodendrocytes [39] and brain sections [40] provided direct demonstration that the amyloid peptide (Aβ) that accumulates in Alzheimer's disease (AD) causes N-SMase activation and ceramide accumulation. A drop in the levels of the major antioxidant glutathione (GSH) causes activation of at least one type of Mg2+-dependent N-SMase, and leads to rapid increase in cellular ceramide levels [41]. The critical role of GSH depletion and N-SMase activation in cell death has been originally demonstrated by Hannun's group in human leukemia cells [42] and was later extended to cells of the nervous system or neuronal models. Cytokine

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stimulation of astrocytes or oligodendrocytes induces shortterm production of oxidants, which transiently deplete GSH leading to ceramide accumulation and oligodendrocyte apoptosis [43]. Likewise, a truncated version of Aβ (Aβ25–35) induces cell death by causing GSH depletion and activating the N-SMase pathway in oligodendrocytes [44] and in C6 glioma cells [45]. In addition, in naïve and differentiated PC12 cells hypoxia induces N-SMase activation, which is inhibited by GSH [46]. Supporting the role of GSH in the regulation of ceramide generation and apoptosis in the nervous system, brain from patients with multiple sclerosis and X-adrenoleukodystrophy showed DNA fragmentation and contained lower levels of GSH and higher levels of ceramide compared to their agedmatched controls [43]. 3.2.2. Acid sphingomyelinases The main localization of A-SMase is in the acidic environment of lysosomes, although a secretory form of ASMase exists (for details refer to [14,16]). Mice defective in ASMase show selective degeneration of Purkinje cells and loss of the cerebellar motor coordination system at an early age [47] suggesting a role for SM metabolism in neuronal survival, and highlighting the selectivity among neuronal types. The differential subcellular localization of A- and N-SMase and the restricted movement of ceramide among intracellular compartments [48] determine the molecular targets that mediate ceramide actions and cellular fate. In neurons, compartmentalization is even more pronounced due to their polarized structure and to the enrichment of acidic endosomes or lysosomes in the cell bodies. In fact, we have demonstrated that distal axons are rich in N-SMase activity but contain almost no A-SMase activity, which is concentrated in cell bodies/proximal axons [49]. Consequently, in sympathetic neurons the axonal pool of ceramide, responsible for the inhibition of axonal growth, must be generated by a N-SMase. On the other hand, some evidence exists for the involvement of A-SMase in ceramide generation in other systems such as PC12 cells [50] and SK-N-BE neuroblastoma cells [51]. The preference of neurons versus cell lines for signaling through N-SMase has been related to the lack of functioning cell cycle and proliferation characteristic of postmitotic neurons [36]. 3.3. Mixed mechanisms in ceramide generation A contribution of both pathways in the generation of bioactive ceramide has been found in some cell systems. In neuroblastoma Neuro2a cells, ceramide generated in response to RA treatment derives from the de novo pathway (possibly by activation of SPT) as well as from activation of N-SMase [4]. Likewise, generation of ceramide from the de novo pathway and from SM protect from the cell death that naturally occurs in cultures of cerebellar Purkinje neurons [52]. 3.4. Role of p75NTR in ceramide generation The discovery that neurotrophins binding to the p75NTR stimulate SM hydrolysis with simultaneous elevation of

ceramide [53,54] highlighted the importance of ceramide in the regulation of death and survival in the nervous system. Neurotrophins bind to a member of the Trk family of receptors in addition to the p75NTR. All neurotrophins bind to p75NTR with similar affinity, however their affinity to the Trk receptors is more discriminative such that nerve growth factor (NGF) binds TrkA, brain-derived neurotrophic factor (BDNF) binds TrkB and NT3 binds preferentially TrkC but also TrkA but with much less affinity than NGF [55]. Activation of p75NTR leads to accumulation of ceramide from SM breakdown [54]. A direct correlation between the levels of p75NTR expression and the levels of ceramide has been reported for neuroblastoma cells [56] and hippocampal neurons [36]. In this last neuronal type only when p75NTR is expressed at high levels, ceramidemediated apoptosis occurs. In addition, a cross-talk between Trk and p75NTR signaling pathways exists so that simultaneous binding of neurotrophins to a member of the Trk family and p75NTR blocks the ability of p75NTR to activate SMase [53]. In PC12 cells the inhibitory crosstalk between Trk A and p75NTRdependent SM breakdown requires phosphatidylinositide-3kinase (PI3K) activation, and localizes to caveolae-related domains [50]. In neuroblastoma cells the negative regulation between TrkA and p75 is also mediated by PI3K, and involves protein kinase C (PKC) activation [57]. In sympathetic neurons, which express TrkA and p75NTR we found that, in addition to the negative cross-talk between TrkA and p75NTR, there is a dramatic down-regulation of p75NTR expression when TrkA is not active, which also limits ceramide production [2]. Therefore, in the absence of TrkA activation elevation of ceramide is limited, transient and insufficient to perform the main function of ceramide in sympathetic neurons, which is survival. The transient pool of ceramide generated from SM might, however, mediate other short-term cellular effects. In cultured embryonic hippocampal neurons, which express TrkB and TrkC but not TrkA generation of an active pool of ceramide from SM by the action of a N-SMase has been demonstrated upon treatment with NGF [35,36]. Importantly, this pool of ceramide is critical to promote neurite outgrowth or cell death, depending on the age of the neuronal cultures [35,36]. In cerebellar neurons, NGF-induced activation of N-SMase through p75NTR generates the pool of ceramide that mediates glutamate release [58]. In other cases ceramide generated from SM hydrolysis activated by p75NTR has been involved in cellular process without sufficient direct evidence. In subplate neurons, ceramide protects against apoptosis. Although a role for ceramide in survival is supported by the decrease in neuronal survival that takes place upon inhibition of de novo ceramide synthesis, direct indication of SMase activation downstream p75NTR is missing [59]. Moreover, since subplate neurons express TrkB and p75NTR but not TrkA, the SMase pathway would likely be activated by NGF, not by BDNF as the authors propose [59]. A very similar scenario was reported in subventricular zonederived interneurons. Based on the evidence that the ceramide synthesis inhibitor myriocin caused a decrease in dendrite development it was suggested that the p75NTR/ ceramide signaling pathway plays a role in dendritogenesis; however no

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activation of SMase has been demonstrated and, contrary to the notion of negative cross-talk between Trk and p75NTR, BDNF induced more pronounced activation of dendritogenesis than NGF although these cells express TrkB but not TrkA [60]. In conclusion, activation of SMase by p75NTR represents a major pathway of ceramide generation in cells of the nervous system, however a very rigorous examination of the origin of bioactive ceramide must be performed before assigning a central role to this pathway. 4. Bioactive sphingolipids: ceramide or its metabolites? Like ceramide, the ceramide metabolites SPh and S1P induce cell proliferation, differentiation and survival in several non-neuronal as well as neuronal systems, some of which are discussed below. In many cases the cellular responses to ceramide and its metabolites are different. Ceramide, but not SPh or S1P protects spinal motoneurons from apoptosis [61]. In hippocampal neurons exogenous ceramide but not SPh or S1P promotes survival at low concentration although all three sphingolipids trigger cell death at concentrations higher than 5 micromolar [62]. Ceramide can be rapidly converted to Sph and S1P (Fig. 1), hence it is important to understand whether it is ceramide or a metabolite, which exert the cellular effect. Ceramide provided to sensory neurons protects from apoptosis, however the actual lipid mediator seems to be SPh or S1P since in the presence of the ceramidase inhibitor Noleoyl-ethanolamine (NOE), ceramide induces apoptosis [63]. Likewise, in dopaminergic neurons inhibition of the conversion of ceramide into S1P by N-N′-dimethyl sphingosine (DMS) blocks the protection against apoptosis [64]. In other systems it is ceramide and not a metabolite, which has the cellular effect. NGF-induced apoptosis in hippocampal neurons is enhanced by treatment with the ceramidase inhibitor D-e-MAPP and, in the absence of NGF, increasing concentrations of D-e-MAPP results in ceramide increase and apoptosis indicating the involvement of ceramide itself, and not a downstream metabolite in inducing cell death [36]. Likewise we demonstrated that the ceramidase inhibitor NOE and the SPhK inhibitor DMS consistently increase ceramide-induced neuroprotection in sympathetic neurons and they are able to reduce apoptosis in the absence of exogenous ceramide. In addition, SPh and S1P are unable to protect from apoptosis neither they induced apoptosis of sympathetic neurons [2]. Others examples in which inhibition of exogenous ceramidase mimic and/or enhances ceramide effects are the induction of apoptosis by RA in neural stem cells [30], the apoptosis of oligodendrocytes induced by Aβ [44] and the induction of apoptosis in SH-SY5Y neuroblastoma cells by ceramide [65]. 5. Ceramide and its metabolites in survival and apoptosis of neurons and glia Neuronal survival is essential for the maintenance of the neuronal circuits that secure the proper functioning of the

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nervous system in the adult. However, it is well established that apoptosis is a normal feature in the development of the mammalian nervous system. During development neurons compete for limiting amounts of neurotrophins and approximately half of the neurons originally present in many parts of the central nervous system (CNS) and peripheral nervous system (PNS) undergo apoptosis. This massive loss of neurons is an important adaptive mechanism for establishing neuronal populations of the correct size and to guarantee the elimination of neurons that contact inappropriate targets [66,67]. Extensive apoptosis also occurs in glial cells during development and, at least for oligodendrocytes, a “competition for neurotrophin”-like mechanism has also been recognized [68]. Ultimately, the survival of neurons and glia depends on the balance of the actions of several factors and signals from neighboring cells. In addition to the death of postmitotic neurons, apoptosis also takes place in the proliferative zones of the brain both embryonically and postnatally. These are naïve cells that have not extended processes, and therefore a target-dependent mechanism cannot account for their death; instead apoptosis seems to be temporally and spatially coordinated with neuronal differentiation [69]. Although apoptosis is essential for tissue remodeling during development of the nervous system, inappropriate activation of apoptosis may contribute to neuronal loss in Alzheimer's and Parkinson's disease and other neurological disorders [70]. Sphingolipids, in particular ceramide, have demonstrated to regulate apoptosis, survival and differentiation in cells of the nervous system as well as in cell lines used as neuronal and glial models. Many neuronal models have been used to understand the molecular events involved in the regulation of neuronal apoptosis or survival by sphingolipids. Although these models offer a simple approach to obtain information of possible molecular mechanisms operating in a particular cellular process, the extrapolation to the physiological situation in vivo must be done with great caution as has been recently highlighted [71]. The role of ceramide as a regulator of neuronal apoptosis is complex since ceramide induces or protects from apoptosis depending on the experimental model considered. The determinant factor in the final outcome of ceramide actions in neurons is unknown. Some factors including the cellular type, the stage of cell development, the concentrations of ceramide used and the subcellular location of ceramide accumulation have been discussed [72]. The localization of neurons to the PNS or CNS does not seem to be crucial, neither is the localization of neurons to a specific area within the brain. Moreover, the mechanisms activated downstream of sphingolipid accumulation are also cell type-specific. A good example is the regulation of the signaling protein Akt. Our work demonstrated that in sympathetic neurons ceramide causes Akt activation [2], however in PC 12 cells (a model for sympathetic neurons) ceramide induces Akt dephosphorylation and inactivation [73]. In view of the cell-type specificity of the effects of sphingolipids I have decided to present the evidence available on the role of ceramide and ceramide metabolites on specific neuronal and glial types. In addition we present a table summarizing the cellular effects of ceramide and S1P in cells of neural origin (Table 1).

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5.1. PC12 cells The rat pheocromocytoma cell line PC12 represents the most widely used system to study the regulatory mechanisms of neuronal survival and apoptosis. PC12 cells have been used as models for sympathetic neurons since they differentiate extending neurites when provided with NGF [74,75], as well as models for dopaminergic neurons since they synthesize and take up dopamine [76]. Furthermore, many studies directed to understand cellular death after hypoxia, have been performed in this model system. Extensive evidence supports the notion that apoptosis induced by various insults in PC12 cells, including serum deprivation [77–79], hypoxia [80], serum and glucose deprivation [81], angiotensin II [82,83] and choline deficiency [84] is mediated by ceramide. Exogenously added short- and longchain ceramides also induce apoptosis of naïve PC12 cells [85] and apoptosis and neurite retraction in NGF-differentiated PC12 cells [76]. Addition of NGF or other neurotrophic factors at the time of treatment with short-chain ceramide dramatically decreases ceramide-induced apoptosis [86]. In many systems the ceramide metabolite S1P triggers cellular effects opposite to ceramide, which led to the proposal of the existence of a ceramide-S1P rheostat [87]. In PC12 cells NGF stimulates the formation of S1P by activating the enzyme SPhK, in a process that requires activation of the neurotrophin receptor TrkA [78]. NGF not only regulates SPhK 1 at the activity level but also increases SPhK 1 gene expression [88]. Accumulation of endogenous S1P secondary to NGF treatment (as well as addition of exogenous S1P) protects PC12 cells from apoptosis induced by serum withdrawal or by ceramide [77,78]. This evidence indicates that NGF acts as a regulator of the intracellular ration of ceramide to S1P in PC12 cells [78]. The observation that elevation of S1P cellular content is required for the neurotrophic-like factor prosaposin to prevent staurosporinand ceramide-induced cell death [89] emphasizes the significance of the intracellular balance between ceramide and S1P in this cell type. In contradiction with the notion that S1P is antiapoptotic, recent work indicated that exogenous S1P causes apoptosis of PC12 cells, although at concentrations higher than those used by Spiegel's group [90]. Differential expression of S1P receptors in PC12 cells might provide some insights for the contradictory findings, although it has been demonstrated that G protein-coupled receptor signaling does not mediate the antiapoptotic effect of S1P in PC12 [77]. By contrast, the effect of S1P on neurite outgrowth depends on S1P receptors. Conflicting evidence indicates that exogenous S1P induces rapid neurite retraction in differentiated PC12 [91] but enhances NGF-induced neuritogenesis in naïve PC 12 cells [78]. NGF causes translocation of SPhK from the cytoplasm to the plasma membrane and triggers preferential activation and internalization of the S1P receptor S1P1 over S1P2 [92]. S1P1 activation is linked to neurite extension while activation of S1P2 causes neurite retraction. Several molecular mechanisms activated by ceramide and S1P in PC12 cells have been reported. Ceramide leads to very

early increase of mitochondrial and cytosolic Ca2+ concentration [93], to the formation of reactive oxygen species (ROS) of mitochondrial origin followed by the translocation of NFκB to the nucleus [76,79], and to the activation of c-Jun aminoterminal kinase (JNK) [86] and caspases [85]. In spite of initial indications that the production of ROS is necessary for ceramide-induced apoptosis in PC12 cells to occur [76], later evidence suggests that the increase of cytosolic [Ca2+] and the production of ROS are not responsible for ceramide-induced apoptosis, but the increase in mitochondrial [Ca2+] is required [94]. This increase in mitochondrial [Ca2+] does not result from entry of extracellular or cytosolic Ca2+ but might be mediated by Ca2+ transfer from the ER to the mitochondria since closer interactions between these two organelles exist in ceramidetreated PC12 cells [93], as well as in mesencephalic neurons (see below) [95]. Ceramide-induced calcium transfer from ER to mitochondria is regulated by at least two concerted mechanisms. Ceramide induces cyclin-dependent kinase 5 (CDK5) activation, which mediates tau phosphorylation and dissociation from microtubules, facilitating organelle movement and redistribution [95]. However, CDK5 itself is not responsible for Ca2+ transfer, which requires the activation of caspase 8 and the cleavage of the caspase substrate Bid to cause cytochrome c release and apoptosis [95,96]. Supporting the role of caspase 8, overexpression of the caspase 8 inhibitor CrmA reduces TNFαinduced ceramide increase and apoptosis of PC12 cells [97]. Whether ceramide directly activates caspase 8 in neuronal cells is unknown but in T cell lines the sequence of events triggered by ceramide is caspase-2 and caspase-8 activation; Bid cleavage and translocation; mitochondrial damage; caspase-9 and-3 activation, and cell apoptosis [98]. Caspase 3 activation takes place in ceramide-treated PC12 cells, but the evidence suggests that caspase3 does not mediate apoptosis [96]. The significance of ceramide-induced NFκB translocation to the nucleus [76,79] is uncertain since there is strong indication that NFκB activation contributes to the survival (not death) of PC12 cells and sympathetic neurons [99–101], and that NFκB plays a physiological role in maintaining survival of CNS neurons [102]. The survival of NGF-dependent PC12 cells is determined by the functional interplay between NFκB and c-Jun that compete for binding to the transcriptional coactivator p300/ CBP [100]. c-Jun expression is regulated by several signaling pathways. Among them, the concerted activation of ERK 1/2 and JNK leads to c-Jun phosphorylation and increase of its own expression, which is important for neuritogenesis in PC12 cells [103]. NGF and other neurotrophic factors that block ceramideinduced apoptosis in PC12 cells do not alter JNK activity and moreover, they are efficient when provided after JNK activation suggesting that neurotrophic factors act downstream of JNK [86]. On the other hand, endogenous S1P promotes survival by inhibiting JNK [77]. However both S1P and NGF act independently of Akt and ERK 1/2 [77,86] indicating that the promotion of survival is due to inhibition of pro-apoptotic events rather than activation of classical pro-survival signals. Similarly, Salinas and co-workers have proposed that ceramideinduced apoptosis in PC12 cells is not due to activation of a proapoptotic pathway but depends on inhibition of the Akt survival

E.I. Posse de Chaves / Biochimica et Biophysica Acta 1758 (2006) 1995–2015

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Table 1 Effect of ceramide and ceramide metabolites in neurons and glial cells Cellular effect

Mediator

Cell type

Inducer

Mechanism

Reference

Apoptosis

Ceramide

PC12 cells

C2-cer C2-cer C2-cer, C8-cer C2-cer Serum deprivation Serum/glucose deprivation NGF/cAMP withdrawal TNFα Angiotensin II Choline deficiency S1P Ceramide+NOE Suramin C2-ceramide > 5 micromolar NGF C2-ceramide; SMase S1P C2-ceramide; SMase; etoposide C2-ceramide; serum deprivation C2-ceramide C2-ceramide; serum withdrawal C2-ceramide; SMase C2-ceramide C2-ceramide; PDMP

JNK NFkB/ROS DAPK CDK5; tau dephosphorylation; Bid

[85,86] [76] [106] [95] [77,78]

JNK NFkB Caspase 8

[81] [79] [97] [82,83] [84] [90] [63] [29]

S1P Ceramide

Sensory neurons

Ceramide

Hippocampal neurons

S1P Ceramide

Ceramide

Ceramide

Survival

Cortical neurons

Mesencephalic neurons

Ceramide

Motoneurons

Ceramide

HN9, 10e

Ceramide Ceramide

PCC7-Mz1 cells Oligodendrocytes

Ceramide

Astrocytes

S1P Ceramide

PC12 cells Sympathetic neurons

S1P Ceramide Ceramide? S1P Ceramide S1P Differentiation/ neurite extension

Cerebellar granule cells

S1P S1P Ceramide

Sensory neurons Immature hippocampal neuron Purkinje cells Mesencephalic neurons Motoneurons Oligodendrocytes PC12 NGF-primed sensory neurons Hippocampal neurons Hippocampal neurons 1DIV

C2-ceramide > 1 micromolar C2-ceramide C2-ceramide C6-ceramide > 5 micromolar Serum deprivation; C2-ceramide RA NGF Aβ25-35 Palmitic acid C2-ceramide

Caspase 3 p38 NFkB; reentry to cell cycle

[62] [36] [114] [62,123]

JNK DAPK

[5,128,131,130] [129,134] JNK; Bax; Caspase 9; caspase 3 Akt and GSK3 dephosphorylation JNK/c-Jun; p38

[132] [142,146] [5] [143]

Caspase 9; caspase 3 p38; Akt inactivation; BAD; GSK3; cas 3

[144,147]

ROS; NFkB CDK5; tau dephosphorylation, Bid

[148] [149] [95]

Bax; [Ca2 + ]int increase; caspase 3

[150]

JNK JNK; DP5 Raf-1/Erk Cytochrome c release

[152,153] [30] [158] [163,44] [165] [167]

NGF C6-ceramide

SPhK activation/JNK inhibition

[78] [117]

C6-ceramide C6-ceramide; PPMP Ceramide

Inhibition of ROS and C-Jun TrkA and PP1 activation

[118] [2,119] [63]

C6-ceramide < 5 micromolar C6-ceramide; SM, SPh, S1P C2-ceramide < 1 micromolar SMase, low C6-ceramide NT3 NGF S1P C6-ceramide; SMase NGF

Inhibition of oxidative stress SPhK activation and translocation SphK/S1P, activation SphK/S1P, activation

[62] [141] [64] [150] [164] [5,77] [92] [121] [35,36] (continued on next page)

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Table 1 (continued) Cellular effect

Mediator

Cell type

Inducer

Neurite retraction/ Inhibition of axonal growth

Ceramide Ceramide Ceramide S1P Ceramide Ceramide Ceramide

Motoneurons Neuro 2A PC12 cells

SMase; low C6-ceramide C2-ceramide; SPh C2-cer S1P C6-ceramide; SMase; PPMP C2-ceramide C2-ceramide

Sympathetic neurons Cerebellar granule cells Cortical neurons

pathway [73]. Under their experimental conditions, pretreatment with ceramide for a short time blocks NGF-induced Akt activation by a mechanism that might involve activation of the serine/threonine protein phosphatase 2 (PP2A) a known target of ceramide. The likelihood of such a mechanism in neurons that respond to NGF should be carefully considered in view of the evidence of ceramide activation of the NGF receptor TrkA. Although pre-treatment of PC12 cells with ceramide for short periods of time as those used in Salina's studies might not have affected TrkA, it has been demonstrated that longer ceramide exposure dramatically increases NGF-induced TrkA activation and even causes TrkA phosphorylation in the absence of NGF in PC12 cells [104]. Our group demonstrated similar NGF-independent activation of TrkA by ceramide in sympathetic neurons (see below) [2]. Since Akt phosphorylation represents one of the main downstream effects of TrkA activation [55,105] the enhancement of TrkA signaling by ceramide will counteract ceramide-induced Akt dephosphorylation and the final cellular outcome will depend on the balance of both signals. Also required for ceramide-induced apoptosis of PC12 cells is the activation of the Ca2+/calmodulin-regulated, serine/ threonine kinase Death-Associated Protein-kinase (DAPK) [106]. DAPK is an actin filament-associated pro-apoptotic protein [107] with undeniable relevance in the nervous system. In rat brain, DAPK mRNA is present from embryonic day 13 (E13) throughout the proliferative and postmitotic regions within the cerebral cortex, hippocampus and cerebellar Purkinje cells; after birth DAPK expression is restricted mainly to the hippocampus [108,109]. DAPK has been implicated in ischemic injury [110,111] and in neuronal death in epilepsy [112,113]. The involvement of DAPK in ceramide-induced apoptosis in cultured primary neurons has also been reported (see below) [114]. In non-neuronal cells the mechanism underlying the apoptotic effect of DAPK involves inhibition of integrin-mediated cell adhesion and extracellular matrix signal transduction [115]. Although it seems unlikely that this mechanism would be restricted to a particular set of cells, direct demonstration in neurons is still pending. Other target identified for DAPK in neurons is the CaM-regulated protein kinase kinase (CaMKK), which has been proposed as a therapeutic target in neurodegenerative diseases [116]. 5.2. Sympathetic neurons Sympathetic neurons represent the prototype neurons of the PNS and have been used extensively to study the molecular

Mechanism

Inhibition of NGF uptake PP2A, tau dephosphorylation

Reference [150] [4] [76] [91] [49,120] [135] [142,146]

details of neuronal apoptosis since in culture they die in a synchronous manner when deprived of NGF. It has been long known that upon NGF withdrawal, addition of short-chain ceramide to the culture medium prevents programmed cell death [117] but only in more recent years the mechanisms behind the survival effect of ceramide have been deciphered. Our laboratory demonstrated that treatment of sympathetic neurons with C6-ceramide results in increase of endogenous long-chain ceramides and that ceramide generated from the “de novo” pathway is as effective as C6-ceramide in inhibiting apoptosis [2]. The generation of endogenous ceramides after treatment of cells with the short-chain ceramide analogues C2and C6-ceramide had been described previously for fibroblasts [118] and A549 human lung carcinoma cell line [119]. A proposed mechanism for the neuroprotective action of ceramide in sympathetic neurons includes the blockade of oxidative stress and c-jun induction that takes place early after NGF deprivation [120]. However, this cannot be the sole mechanism since C6ceramide is able to prevent apoptosis when provided to the neurons after ROS have been produced and c-jun has been activated [2]. We found that C6-Cer activates the neurotrophin receptor TrkA and its downstream effector Akt [2]. As mentioned above, activation of TrkA by ceramide had been previously demonstrated in PC12 cells [104]. However, since ceramide causes apoptosis in PC12 cells [77,78,85] the relevance for ceramide-induced TrkA in this neuron-like system is unknown. Our most recent work indicates that C6-ceramideinduced TrkA activation in the absence of NGF takes place in sphingolipid and cholesterol-rich microdomains (lipid rafts). C6-ceramide accumulates in lipid rafts, where TrkA resides, likely creating “ceramide-patches” that allow for increase local density of TrkA leading to dimerization and autophosphorylation1. TrkA activation, however, does not represent the only pro-survival mechanism elicited by ceramide. We demonstrated that protein phosphatase 1 (PP1) is activated in ceramide-treated sympathetic neurons and is required for ceramide-induced protection against apoptosis [121]. Although the direct targets of ceramide-activated PP1 in sympathetic neurons have not been completely identified, we showed that ceramide is able to prevent the hyperphosphorylation of retinoblastoma gene product (pRb) that takes place upon NGF deprivation, by a mechanism dependent on PP-1 activation. We proposed that reduced phosphorylation of pRb will abort the attempt to reenter the cell cycle after NGF withdrawal, which in neurons can

1

Posse de Chaves, in preparation.

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lead to apoptosis [121]. It is important to highlight that contrary to evidence from PC12 cells, Akt, which is a known substrate of serine/threonine protein phosphatases is not dephosphorylated but instead Akt phosphorylation is increased in ceramidetreated sympathetic neurons. As discussed above, this might be explained by the balance of signals generated downstream of TrkA activation. An alternative explanation is that Akt might be a better substrate for PP2A than for PP1 since we have found that PP2A activity is very low in sympathetic neurons [121]. With respect to the role of ceramide in axonal growth and regeneration of sympathetic neurons we have shown that C6ceramide, bacterial SMase and ceramide accumulated intracellularly by inhibition of GlcCer synthesis, inhibit axonal growth of sympathetic neurons independently of cell death [49,122]. The ceramide pool that inhibits axonal growth is generated from SM in distal axons, but not in cell bodies. Analysis of endogenous SMase activities demonstrated that distal axons are rich in N-SMase activity but contain almost no A-SMase, which is concentrated in cell bodies/proximal axons. This is consistent with the notion that generation of ceramide from SM by N-SMAse in axons inhibits axonal growth [49]. The mechanism responsible for axonal growth inhibition involves reduction of NGF uptake and decrease of ERK activation in ceramide-treated neurons [2,49]. 5.3. DRG/sensory neurons Sensory neurons resemble sympathetic neurons in that they strongly depend on neurotrophins for survival and undergo apoptosis when deprived of trophic support. Neurons from the dorsal root ganglia (DRG) represent a more heterogeneous population with approximately one third of the neurons responding to NGF. When exposed to exogenous natural ceramides sensory neuron survival rate is increased both in the absence or presence of NGF [63]. Thus, sensory neurons respond to ceramide like sympathetic neurons, however, the actual lipid mediator in sensory neuron survival is not ceramide but a metabolite, possibly SPh or S1P. The ceramidase inhibitor NOE not only blocks survival but induces apoptosis, indicating that ceramide is indeed proapoptotic in DRG neurons [63]. Other work supporting the proapoptotic role of ceramide in DRG neurons demonstrated that treatment with the drug suramin, cause increase of endogenous ceramide in sensory neurons and leads to apoptosis [29]. The mechanism of ceramide-induced apoptosis involves translocation of NFκB to the nucleus and stimulation to re-entry the cell cycle as indicated by the increase in cyclin D1 [29]. NGF regulates neurite outgrowth and elongation in a subpopulation of sensory neurons, and this regulation is altered by S1P. If S1P is provided to DRG neurons together with NGF at the time of plating, it blocks NGF-induced neurite outgrowth; but S1P enhances NGF-induced neurite outgrowth when given after a few hours of exposure to NGF [92]. The differences in the effects elicited by S1P are explained by NGFmediated regulation of S1P receptors. NGF causes an increase in the expression of S1P1 and a decrease in the expression of

2003

S1P2, which promotes and inhibits neurite growth respectively [92]. 5.4. Hippocampal neurons Hippocampal neurons death is responsible for memory dysfunction and is characteristic of some neurodegenerative diseases such as AD. Several lines of evidence have recognized ceramide and its metabolites as important second messengers in hippocampal neuron biology. The effects of ceramide on hippocampal neuron survival depend on the developmental neuronal stage and the concentration of ceramide used. At concentrations below 5 micromolar exogenous ceramide either does not affect survival during the first day in culture [123] or increases cell viability [62]. In addition, ceramide concentrations lower than 1 micromolar protect hippocampal neurons from several insults such as excitotoxicity, FeSO4 and amyloid β-peptide (Aβ) [124]. At concentrations over 5 micromolar exogenous ceramide given to immature hippocampal neurons causes apoptosis [62,123]. In mature hippocampal neurons (after 6–7 DIV) even low concentrations of ceramide cause cell death [62]. Similarly, treatment with NGF during the first day in culture does not compromise neuronal viability but after 2 DIV NGF causes apoptosis [36]. This switch in the response to NGF correlates with the increase in expression of p75NTR and the increase in ceramide generation [36]. Some studies indicate that the mechanism of NGF-induced apoptosis might require mitogen-activated protein kinase (MAPK) activation, particularly JNK, although the intermediates between ceramide and JNK and the mechanisms downstream of JNK have not been identified [36]. Activation of the MAPK/JNK pathway in ceramide-treated hippocampal neurons, was not found in other studies [62], however further differences such as the lack of expression of p75NTR also existed in these, compared with Futerman's studies and might be explained by differences in the neuronal cultures. More recently, a pivotal role for DAPK in ceramide-induced apoptosis of hippocampal neurons was discovered [114]. Expression of DAPK is increased by ceramide in rat hippocampal neurons and neurons isolated from DAPK null mice are significantly more resistant to undergo apoptosis when challenged with ceramide analogs or high NGF concentrations [114]. Underscoring the physiological relevance of DAPK in hippocampal neuron apoptosis, DAPK mRNA expression becomes restricted to the hippocampus after birth [109]. Contrary to the situation in PC12 cells, in hippocampal neurons S1P does not have effects opposite to ceramide on survival. Indeed, at low concentrations exogenous S1P does not affect neuronal survival; but causes apoptosis at higher doses [62,125]. S1P-induced apoptosis is blocked by the Ca2+ chelator BAPTA-AM and by protein phosphatase inhibitors, and involves c-Fos-containing activator protein-1 (AP-1) complexes as transcription factors [125]. Unfortunately whether S1P itself or ceramide derived from S1P is responsible for the apoptosis has not been investigated. With respect to the role of sphingolipids in hippocampal neurite outgrowth, Futerman's group demonstrated, using the

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inhibitors FB1 and PDMP (Fig. 1), that during hippocampal axon development, ceramide must be converted to GlCer to maintain normal growth [123,126]. In addition, the first stage of differentiation, that is the formation of minor processes from lamellipodia, is independent of ceramide metabolism into GlCer and is stimulated by short-chain ceramide analogs and by generation of endogenous ceramide by SMase [123]. Moreover, ceramide participates in dendritic formation and elongation [62]. Although these findings are in apparent contradiction with our data from sympathetic neurons (see above), our studies were performed after 5–10 DIV and under our experimental conditions we only examined axonal extension but we could not evaluate neurite outgrowth, axonal branching or dendrite extension [122]. As mentioned above, one of the main pathways for ceramide generation in neurons is triggered by activation of the neurotrophin receptor p75NTR. Hippocampal neurons express p75NTR as well as TrkB and TrkC, but the expression of TrkA in these neurons is negligible at least up to 4 DIV [35,36]. Consequently, NGF is able to activate p75NTR without activating Trk, and to increase ceramide levels by activation of N-SMase. This, in turn, leads to significant activation of axonal growth during the first day in culture [35]. Other important effects of ceramide in hippocampal neurons include modulation of ion currents, neurotransmitter release and synaptic transmission [127,128]. These actions of ceramide have been recently reviewed in detail [129]. 5.5. Cerebellar neurons Significant number of studies addressed the role of ceramide in apoptosis of neurons of cerebellar origin. Cerebellar granule cells (CGC) represent the most abundant neurons of the CNS. Treatment of CGC in culture with exogenous short-chain ceramides or bacterial SMase causes apoptosis [5,130–133]. Serum/K+ deprivation or exposure of granule cells to the anticancer agent etoposide leads to accumulation of endogenous ceramide and CGN death [131]. Recent studies have revealed, at least in part, the mechanisms involved in ceramide-induced apoptosis of CGCs. Ceramide induces dephosphorylation of Akt and glycogen synthase kinase-3 (GSK3), possibly through the activation of a PP2A-like activity [134]. Multiple mechanisms might contribute to the promotion of apoptosis by GSK3. Among them, GSK3 is a natural activator of the mitogenactivated protein kinase kinase kinase (MEKK), which in turns triggers the JNK signaling pathway [135]. Consistent with this possibility, ceramide strongly stimulates JNK phosphorylation in CGCs [131]. Gene transcription activation downstream of JNK results in increase of BAX, decrease in mitochondrial membrane potential, release of cytochrome c from the mitochondria and activation of caspase-9 and caspase 3 [136]. These findings contradict earlier observations that a caspase 3 inhibitor was unable to block morphological alterations and cell death triggered by prolonged granule cell exposure to ceramide [132]. In addition to the activation of apoptosis, C2-cer induces rapid and transient activation of cell migration and inhibits

neurite outgrowth in immature CGC [137]. These effects have been attributed to disorganization of the microfilament network and to decreased phosphorylation of the microtubule-associated protein tau by a mechanism mediated by caspases and PP2A [137]. Based on: 1) the effects of ceramide on CGC in culture; 2) the evidence that mice overexpressing TNF-α present strong impairment of cerebellum development and 3) the fact that ceramide mediates many of the cellular effects of TNF-α; Falluel-Morel and collaborators proposed that ceramideinduced apoptosis plays a role in cerebellum development [136,137]. Supporting this notion Sonino and colleagues demonstrated in cerebellum a progressive increase in ceramide content in sphingolipid-enriched membrane domains from the time of neuronal differentiation and neuritogenesis to the latest stage of development, a time at which massive age-induced apoptotic death takes place [138]. A second, important neuronal type present in the cerebellum is Purkinje cells. Cellular levels of ceramide and other sphingolipids are critical for survival and differentiation of Purkinje cells. Inhibition of ceramide synthesis and depletion of sphingolipids result in Purkinje cell apoptosis, which is reverted by administration of C6-ceramide, SM, SPh or S1P [52]. These studies in vitro suggest that ceramide and related metabolites have a protective role against apoptosis in Purkinje cells. Conflicting with the notion that ceramide is a survival mediator in Purkinje cells, studies in animals in which the putative lysosomal ceramidase activator saposin D was deleted, demonstrated that accumulation of ceramide in the cerebellum is accompanied by selective degeneration and death of Purkinje cells [139]. This evidence favors the notion that a metabolite of ceramide such as S1P is the possible survival mediator in Purkinje cells. Although the observation that selective degeneration of Purkinje cells occurs in ASMase-deficient mice supports a role of SM-derived ceramide in neuronal survival [47], these findings could also be explained by decreased production of S1P. Interestingly, the loss of Purkinje cells in ASMase-deficient mice does not occur at random but it rather takes place in a very well-ordered array of stripes in which Purkinje cells expressing the protein zebrin II are more resistant to death [140]. Strikingly, the stripped pattern of zebrin II expression correspond to the expression of SPhK [141]. Neurons expressing SPhK might be able to synthesize more efficiently S1P from a reduced pool of ceramide, and S1P might be the true antiapoptotic mediator. Further research should determine the levels of sphingolipids (ceramide, SM, S1P) in the surviving Purkinje cells in order to be able to identify the bona fide survival mechanism. Taking in consideration the high expression of S1P receptors in cerebellar neurons, particularly Purkinje cells, and the secretion of S1P by cerebellar neurons [142] further studies in this area are granted. With respect to the role of ceramide in Purkinje cell differentiation, Furuya and colleagues showed that sphingolipid synthesis is required for proper dendrite differentiation of Purkinje cells in mixed cultures [52,142]. More specifically, the loss of ceramide and/or SM was identified as responsible for dendritic abnormalities. Interestingly, CGCs

E.I. Posse de Chaves / Biochimica et Biophysica Acta 1758 (2006) 1995–2015

present in the cultures were unaffected by the decrease in sphingolipids. 5.6. Cortical neurons Cortical neurons respond to cellular ceramide elevation in a manner similar to CGCs. Treated with C2-cer or exogenous SMase cultured cortical neurons undergo apoptosis [2,144,145]. The molecular mechanisms activated by ceramide in cortical neurons are also very close to those activated in cerebellar neurons. A rapid dephosphorylation of Akt, likely mediated by PP2A, takes place in the first hour of ceramide treatment [146,147]. Akt dephosphorylation is required for ceramide-induced apoptosis since activation of Akt by insulin growth factor-1 prevented neuronal death [147]. Akt dephosphorylation is followed by dephosphorylation of the proapoptotic regulators BAD, Forkhead family transcription factors and GSK3, which after 2–6h cause mitochondrial depolarization and permeabilization, and activation of the intrinsic cascade pathway [146]. Although the targets downstream of GSK3 have not been directly identified, activation and nuclear translocation of JNK, activation of p38 and phosphorylation of c-Jun have been demonstrated in ceramide-treated cortical neurons [144,148]. The requirement of p38 activation has been confirmed, however JNK activation upon ceramide treatment was not found by others [149]. The involvement of ERK in ceramide-induced death in cortical neurons was also investigated with contradicting conclusions. Two research groups found that ceramide causes significant decreased of ERK 1/2 phosphorylation [144,149]. However, inhibition of Erk 1/2 phosphorylation by the MEK inhibitor PD98059 in the absence of ceramide did not cause apoptosis, from which Willaume et al. concluded that ERK 1/2 are not mediators of cortical neurons apoptosis [144]. Stoica and colleagues followed a similar strategy but used the MEK1/2 inhibitor U0126 for cotreatments with ceramide and found that U0126 is able to reduce ceramide-induced apoptosis concluding that ERK1/2 are involved in the apoptotic pathway [149]. If the decrease in Erk 1/2 phosphorylation induced by ceramide is required for ceramide-induced apoptosis it is very difficult to understand how an inhibitor that has the same effect than ceramide on Erk phosphorylation can prevent ceramide-induced cell death. Extensive evidence indicates that ceramide-induced apoptosis of cortical neurons is accompanied by caspase 3 and caspase 9 activation [144–146,149]. Indeed, by using caspase inhibitors Movsesyan and collaborators demonstrated the requirement of caspase 3 and caspase 9 but not caspase 8 activation in ceramide-induced apoptosis of cortical neurons [144]. The inability of caspase 3 inhibitor to prevent apoptosis of these neurons in previous work by Willaime et al. might be explained by the concentration of inhibitor used [144]. With respect to neurite development and extension, treatment of cortical neurons with ceramide causes rapid neurite retraction and loss of dendritic MAP2 immunoreactivity [144,148], however it is unclear if these effects are secondary to neuronal death or are due to independent actions of ceramide.

2005

5.7. Mesencephalic neurons Mesencephalic dopaminergic neurons represent the major cell population that degenerates in patients with Parkinson's disease (PD). Morphological studies on postmortem brain from patients with PD indicate that these neurons die by apoptosis. Almost a decade ago the hypothesis that the SM cycle could mediate neurodegeneration in PD [76] promoted studies aimed to understand the role of ceramide in apoptosis of mesencephalic neurons. The SM hypothesis of neuronal death was based on evidence indicating that 1) dopaminergic neurons that degenerate in PD express TNF-α receptors; 2) TNF-α immunoreactive glial cells localize in proximity to these neurons in PD patients and 3) ceramide is an important second messenger in TNF-α signaling. Classical experiments using short chain ceramides indicated that, as in hippocampal neurons, ceramide has dual effects in cultured mesencephalic neurons, depending on the concentration. Short-chain ceramide at concentrations less than 1 micromolar protects cultured mesencephalic neurons against glutamate cytotoxicity by conversion to its metabolite S1P [64]. Over micromolar concentrations exogenous ceramide induces apoptosis [150]. The apoptotic process involves generation of ROS and NFkB translocation to the nucleus [151]. As in PC12 cells, ceramide induces an increase in CDK5 activity, which leads to tau phosphorylation causing the clustering of ER and mitochondria and enhancing t-Bid-mediated Ca2+ transfer [95]. Tau phosphorylation can be catalyzed by GSK3 as well, however in contrast to CGC [134] and cortical neurons [144] ceramide does not activate GSK3 in mesencephalic neurons [95]. Moreover, although GSK3 is activated in CGCs, tau phosphorylation is not increased but is reduced in this neuronal type [137], highlighting once again, the cell specificity in ceramide effects. 5.8. Motoneurons The effects of ceramide in embryonic spinal motoneurons are similar than in hippocampal neurons. Low concentrations of C6-ceramide or treatment with bacterial SMase prevent the natural death that occurs in vitro and improves axonal elongation. Conversely, higher C6-ceramide concentrations lead motoneurons to apoptosis [152]. Ceramide prevents motoneuron apoptosis by inhibition of oxidative stress [61]. 5.9. Neuroblastoma cells Neuroblastoma cells represent good models to study the role of sphingolipids in neuronal apoptosis in the proliferative areas of the nervous system. In addition, understanding the mechanisms that mediate apoptosis in neuroblastoma cells is important to design strategies for the prevention of apoptosis in immortalized neuronal stem cells and progenitor cells, which have therapeutic potential in the treatment of neurodegenerative disorders. In general, ceramide causes differentiation and/or apoptosis in neuroblastoma cells,

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however not all neuroblastoma cells respond equally to ceramide elevation. Some examples follow. The embryonic hippocampal cell line HN9.10e has been extensively used as an experimental model for hippocampal neurons. These cells are a somatic fusion product of hippocampal neurons from embryonic day 18 C57BL/6 mice and N18TG2 neuroblastoma cells. They exhibit morphological and cytoskeletal features which are typical of their neuronal parent but which are not expressed by the neuroblastoma parent [153]. Study of apoptosis in undifferentiated HN9.10e provides information of cellular processes that occurs during embryogenesis. Serum deprivation of undifferentiated HN9.10e cells causes accumulation of intracellular ceramide and apoptosis. The apoptotic process is also triggered by short-chain ceramide analogs [154]. The cell death mechanisms include translocation of the proapoptotic Bcl-2 family member Bax to the mitochondria and release of cytochrome c without mitochondria depolarization or swelling [154,155]. A later increase in intracellular [Ca2+] takes place, however, contrary to the case of PC12 cells, mitochondrial [Ca2+] does not change. Caspase 3 is activated many hours after cytochrome c release [154]. Recently, it has been reported that the early elevation of ceramide that follows serum deprivation is the result of the change in the balance between SMase and SM synthase activity of nuclear origin [156]. In neuroblastoma neuro 2A cells differentiated with RA there is a rapid and sustained increase in ceramide and a decrease in SM [4]. Direct application of natural or short chain ceramides or SPh also induces neurite differentiation [4], being the effect of SPh, strictly related to its conversion to ceramide [157]. RA also causes differentiation of PCC7-Mz1 cells into neurons, astroglial cells and fibroblasts while a fraction of RAtreated cultures dies by apoptosis. Although RA causes ceramide elevation, in this case by activation of the enzyme SPT [30], ceramide does not promote differentiation but induces apoptosis since treatment with FB1 reduces RA-induced cell death but not neurite formation [30]. The differential response of PCC7-Mz1 and Neuro2A cells to ceramide-induced differentiation was interpreted as depending on the degree of commitment that the cell already has at the time of ceramide increase [30]. While Neuro2a cells have already committed to a neuronal differentiation, PCC7-Mz1 cells still conserve the potential to differentiate in all cell types derived from the neuroectoderm. On the other hand treatment of undifferentiated SK-N-BE neuroblastoma cells with TNF-α causes ceramide elevation and increased differentiation, while TNF-α induces apoptosis in RA-differentiated SK-N-BE cells [51]. Short chain ceramides also induce apoptosis in SH-SY5Y neuroblastoma cells [65,145], SKN-SH cells [158] and hypothalamic GT1-7 cells [159], while addition of S1P induces SH-SY5Y cells proliferation [65]. 5.10. Glial cells 5.10.1. Oligodendrocytes Chao and collaborators demonstrated that ceramide elevation causes selective apoptosis in mature oligodendrocytes without

affecting astrocytes and oligodendrocyte precursors [160,161]. The mechanism of apoptosis involves activation of JNK [160] and p38α [162]. Ceramide originates from the hydrolysis of SM that occurs upon binding of NGF to p75NTR expressed in oligodendrocytes. Retroviral expression of Trk A in mature oligodendrocytes inhibits NGF-induced p75 NTR -dependent apoptosis, blocks JNK stimulation, and instead, induces ERK 1/2 activation [163]. Interestingly, although under physiological conditions in vivo oligodendrocytes do not express p75NTR, upregulation of p75NTR messenger RNA and protein were demonstrated in oligodendrocytes and microglia/macrophages in multiple sclerosis plaques [164], suggesting a role for p75NTR (and possibly ceramide) in cell death in this disease. Likewise, ceramide might also play a role in Aß-induced oligodendrocyte death since inhibition of SMase is able to block the apoptotic pathway activated by Aß that involves JNK and the proapototic protein DP5 [44,165]. Direct evidence of ceramide accumulation and/or SMase activation in Aβ-treated oligodendrocytes however has not yet been presented. As in other cell types SPh and S1P exert effects different than ceramide in oligodendrocytes. SPh causes non-apoptotic cell death and S1P does not affect survival. S1P activates JNK/p38 although it causes activation of Erk 1/2 [162] and is able to induce phosphorylation of cyclic AMP-response element binding protein (CREB) downstream of Erk 1/2 [166]. CREB activation and survival support is also elicited by treatment of oligodendrocytes with the neurotrophin NT3. The survival mechanism activated by NT3 is similar to the mechanisms discussed for PC12 cells treated with NGF; exposure of oligodendrocytes to NT3 causes translocation of SPhK to the plasma membrane and SPhK activation with the consequent increase in S1P [166]. 5.10.2. Astrocytes As mentioned above, some studies indicated that the survival of astrocytes in a mixed culture with oligodendrocytes is not affected by ceramide [160]. On the other hand, other reports showed that treatment of cortical astrocytes with palmitate [167] or C2-ceramide [168,169] causes apoptosis, which is mediated by an increase in ceramide generated de novo. Activation of Raf-1/Erk [167] and release of mitochondrial cytochrome c [169] are involved in ceramide-induced apoptosis. One well-characterized role of ceramide in astrocytes is in proliferation. Astrocyte proliferation is essential during development of the nervous system and is the main process activated during astrogliosis in response to ischemia, brain injury and in neurodegenerative diseases. In quiescent primary astrocytes induction of proliferation with basic Fibroblast Growth Factor (bFGF) causes a rapid and transient decrease in ceramide content, and exogenous ceramide counteracts the proliferating actions of bFGF by decreasing Erk 1/2 activation [170]. The decrease in ceramide levels upon bFGF treatment is due to stimulation of SM synthase [171]. Exogenously added S1P also causes astrocyte proliferation in the absence of bFGF by a mechanism mediated by S1P receptors [172]. Importantly, bFGF induce astrocytes to release S1P but not SphK. Moreover, ceramide, which has antiproliferative effects in astrocytes

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causes a decrease in S1P release [172]. These results are consistent with the presence of the S1P/ceramide “rheostat” in astrocytes. 6. Ceramide and ceramide metabolites in neurodegenerative diseases The role of sphingolipids in the development and progression of several neurological diseases, in particular lipid storage diseases, is well recognized. In addition, in the past few years increasing evidence has pointed out the relevance of simpler sphingolipids like ceramide in other neurological dysfunctions, particularly dementias. 6.1. Alzheimer’s disease Alzheimer's disease (AD) is a devastating progressive neurodegenerative disorder with characteristic clinical and pathological features. Since age is a major risk factor for AD, the incidence of this disease is rising as people continue to live longer, especially in developed countries. The number of individuals who have AD is projected to almost double by 2025 in North America underscoring the importance of understanding the molecular mechanisms that lead to the disease development. Accumulation of Aβ leads to the progression of characteristic morphological features of AD namely intraneuronal neurofibrillary tangles, extracellular amyloid plaques (neuritic or senile plaques), and cerebrovascular angiopathies [173,174]. A link between AD and lipid homeostasis has been provided initially by the finding that the E4 allele of apolipoprotein E (apoE) associates with a higher risk of developing both late familial [175,176] and sporadic AD [177] as well as certain early-onset forms of the disease [178]. Later studies have

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demonstrated significant alterations of several lipids (e.g., ceramide, gangliosides, cholesterol and sulfatide) in AD. Extensive evidence supports an important role of cholesterol in the development and possibly progression of AD (reviewed by [179,180]. The role of sphingolipids is also emerging. Ceramide elevation in the brain is evident at an early stage in AD patients [181,182]. This substantial elevation of ceramide might result from degradation of sulfatide, which is reduced in brain grey and white matter and in cerebrospinal fluid of patients with AD [181,183]. Indeed, in AD brains the profile of molecular species of ceramide correlates more closely with sulfatide molecular species than with SM molecular species [181]. Sulfatide mass content seem to be modulated by apoE [184,185] but the reasons for the pronounced deficiency of sulfatide in AD brain are still unknown. Nevertheless, sulfatide deficiency has been suggested as a potentially useful biomarker of AD [184]. On the other hand, studies by Mattson and collaborators suggested that the increase of ceramide in AD may result, at least in part, from membrane-associated oxidative stress and might be accompanied by a decrease in SM [182]. 6.1.1. Modulation of APP cleavage and Aβ production by lipids Aβ is a peptide derived from intramembrane sequential cleavage of the amyloid precursor protein (APP) by β-site APP cleaving enzyme 1 (BACE1) and γ-secretase (Fig. 2) [186]. The amyloidogenic pathway of APP clevage occurs normally in all cells rendering two main Aβ peptides of 40 and 42 aminoacids respectively (Aβ40 and Aβ42). Under physiological conditions however, APP is predominantly cleaved by α-secretase, resulting in the secretion of soluble APP (sAPPα) and precluding excessive Aβ formation. The proteolytic processing of APP takes place predominantly in post-Golgi secretory and endocytic compartments and at the plasma membrane [187,188]. The amyloidogenic processing of APP seems to

Fig. 2. Amyloidogenic and non-amyloidogenic processing of APP. The transmembrane protein APP is cleaved by α-secretases in cholesterol- and sphingolipid-poor domains and by β-secretase (BACE) in lipid rafts domains generating the correspondent C-terminal fragments (CTFs) C83 and C99 and the large ectomains sAPPα and sAPPβ respectively. α-cleavage of APP precludes the formation of Aß. γ-secretase cleavage of C83 results in the release of P3. Aβ is the result of γ-secretase activity of. In both pathways γ-secretase releases APP intracellular domain (AICD), which may be involved in nuclear signaling.

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occurs primarily in lipid rafts, whereas the non-amyloidogenic cleavage takes place in non-raft regions of the membranes [189]. The involvement of rafts in Aβ production is supported by evidence that BACE1 [190,191], γ-secretase [192–195] and APP [190,196,197] localize at least in part, to lipid rafts. Sphingolipids and cholesterol are major components of lipid rafts and changes in cellular sphingolipids or cholesterol levels regulate Aβ generation. The correlation between cellular cholesterol and Aβ production is still controversial. Several studies indicated that cholesterol depletion leads to decreased Aβ production [190,198,199] and stimulation of the nonamyloidogenic cleavage pathway [200]. Conversely, at least one report showed increased Aβ generation upon moderate reduction of cellular cholesterol levels [201]. These discrepancies have been reconciled in a model that takes in consideration the impact of different levels of cholesterol on the structure of lipid rafts [202]. Similarly, the precise role of sphingolipids in APP processing and Aβ production is contentious. The discrepancy in the data obtained by different research groups might result from the use of different approaches to regulate sphingolipid levels. In cultured cells expressing APP, exogenous addition of C6ceramide or N-SMase increases Aß generation by regulating βcleavage through BACE1 stabilization but without affecting γcleavage of APP [203]. Elevated Aß secretion is accompanied by an increase in α- and β-C-terminal fragments (CTF) production but with no change in APP expression or maturation. Accordingly, treatment with FB1 causes reduction of ceramide levels and a decrease in Aβ as well as in α- and β-CTF generation [203]. Interestingly the authors found that the reduction of SM that results from treatment with N-SMase or treatment with FB1 cause similar decrease in cholesterol clustering into cholesterol-rich domains, however only when decreased cholesterol in lipid rafts is accompanied by reduced ceramide levels (FB1 treatment) it resulted in decreased Aß generation. Conversely, decreased cholesterol in lipid rafts in the presence of elevated ceramide (treatment with nSMase) leads to increased Aß production. Although these results suggest that ceramide could be creating “lipid raft-like patches” where BACE1 might localize, the authors claim that, at list C6ceramide, does not affect the overall distribution of BACE among microdomains. Instead they attribute their results to a more direct effect of ceramide on BACE1 activity by posttranslational stabilization of BACE1 [203]. More recently, the pool of ceramide involved in BACE1 stabilization has been suggested to derive from p75NTR-activated N-SMase, although direct activation of N-SMase has not been measured in those studies [204]. Work by Sawamura and collaborators demonstrated that reduction of cellular sphingolipid levels by inhibition of SPT (treatment with myriocin or use of a mutant defective in the LCB1 subunit of palmitoyltransferase) results in increased secretion of sAPPα but not sAPPβ, and increase production of Aβ42 [205]. Since with this experimental approach the levels of all sphingolipids, including ceramide are reduced, their results contradict the findings of Puglielli et al. [203]. Although some

of the discrepancies could be attributed to the accumulation of sphinganine (and PKC inhibition) in cells treated with FB1 [205], the inconsistency in Aβ production remains unexplained. In addition, cells treated with the GlcCer synthase inhibitor PDMP showed reduced secretion of both sAPP and Aβ [206]. The authors inferred that reduced Aβ secretion depends on the decrease of glycosphingolipids and not on ceramide accumulation since exogenous C6-ceramide caused no change in sAPP secretion in their model. They interpreted that decreased generation of Aβ upon cellular glycosphingolipid depletion might result from reduced access of BACE1 to APP in the endocytic compartments due to reduction of APP maturation in the secretory pathway, and reduction of APP movement to the plasma membrane. This study [206] therefore suggests that glycosphingolipids might be implicated in the forward transport of APP in the secretory pathway. In spite of the great effort dedicated to understand the role of lipids in the regulation of Aβ production during disease, much less attention was paid to the physiological functions of APP and Aβ. New exciting evidence suggests that APP processing and Aβ production regulates cholesterol and SM metabolism [207]. Aβ42, at physiological concentrations, directly activates N-SMase and causes a decrease in SM levels, while Aβ40 reduces de novo cholesterol synthesis by inhibiting the key enzyme of the biosynthetic pathway, hydroxy-methyl glutaryl coenzyme-A reductase (HMG-CoA reductase). Interestingly, this work suggests that production of ROS that had previously been involved in many of the effects of Aβ (see below) is not responsible for Aβ-induced N-SMase activation at lower (physiological) levels of Aβ [207]. 6.1.2. Ceramide as a second-messenger in the cytotoxic effects of Aβ Several lines of evidence linked Aß with neuronal death [208]. Aβ causes death of neurons by mechanisms dependent and independent of caspases [209–211]. We have recently demonstrated that in neurons exposed to Aβ exclusively in the axons neurons die by a process of caspase-mediated apoptosis, which is secondary to caspase-independent axonal degeneration [212]. The molecular events that lead to Aβ-induced neuronal degeneration and death are under debate. Some studies suggest that a direct effect of Aβ on neurons is required. Interaction of Aβ with RAGE [213], α-7AChR [214], and p75NTR [215] has been linked to AD. Other studies support the notion that the effect of Aβ is indirect, activating microglia and causing inflammation [216]. There is sufficient evidence that inflammatory mechanisms induced by TNF-α and other cytokines are involved in AD. In addition, induction of membrane-associated oxidative stress has been identified as a key mechanism in Aβinduced toxicity. Previous studies demonstrated that Aβinduced cell death is mediated by a mechanism involving lipid peroxidation and oxidative stress [217,218] and that there is cross-talk between the oxidation-stress system and the SM/ Cer pathway. Ceramide has also been identified as a possible second messenger in Aβ-induced death. Evidence from different

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studies indicated that treatment of cultured cells with Aβ [38,39,44,45,182] or intracerebral administration of Aβ to rats [40] causes ceramide elevation and that the apoptotic effects of Aβ are mimicked by exogenous short-chain ceramides [38,42,45]. The origin of the ceramide pool generated upon Aβ treatment is still unclear. Aβ-induced activation of N-SMase has been demonstrated in human primary neurons, oligodendrocytes and cerebral endothelial cells [38,44,45]. Unfortunately, the Aβ peptide used for most of the studies in oligodendrocytes and endothelial cells was Aβ25–35, which does not exist in vivo. Although Aβ25–35 and Aβ40 showed similar potency in the induction of apoptosis [219,220], this cannot be interpreted as an indication that the two peptides cause apoptosis by the same mechanisms. Hence, direct demonstration of N-SMase activation by the relevant peptide Aβ42 or Aβ40 in oligodendrocytes is still missing. This is particularly relevant in view of evidence discussed above showing that Aβ42 but not Aβ40 activates N-SMase at physiological concentrations [207]. Other studies in vivo implicating N-SMase in the development of AD suffer of poor identification of the Aβ peptide used [40]. In addition, studies in hippocampal neurons that suggest that SM is the source of ceramide generation were performed with the relevant peptide Aβ42 but did not examine SMase activity [182]. Moreover, in this last work, data derived from experiments using the SPT inhibitor myriocin are difficult to interpret. Although the authors claim that hippocampal neurons were depleted of SM upon treatment with myriocin, the actual levels of SM were only slightly decreased; and ceramide levels were unaffected or conversely, Cer C24:0 was significantly increased in myoricin-treated neurons compared to untreated neurons. Nevertheless, myriocin was able to prevent the increase in ceramide triggered by Aβ and partially protected neurons from Aβ-induced apoptosis, which suggests that under those experimental conditions the pool of ceramide involved in Aβ actions derives more likely from de novo pathway of ceramide synthesis [182]. The mechanisms by which Aβ causes N-SMase activation and ceramide accumulation seem to be redox-sensitive and to entail activation of NADPH oxidase and/or regulation of glutathione metabolism [38,44]. In support for the involvement of oxidative stress, in hippocampal neurons treated with Aβ, ceramide elevation is accompanied by an increase in the peroxidation product 4-hydroxynonenal (HNE) and is prevented by the antioxidant α-tocopherol [182]. Unexpectedly though, treatment with myriocin completely abolishes the increase in 4HNE induced by Aβ, which would suggest that lipid peroxidation is secondary to Aβ-induced ceramide elevation or alternatively that myriocin has unrelated, nonspecific effects in this system. In conclusion, there is growing and exciting evidence that a reciprocal regulation between lipids (sphingolipids and cholesterol) and Aβ exists. Sphingolipids have emerged as significant regulators of Aβ production; and alteration of sphingolipid metabolism might be associated with the development of sporadic AD. More studies in vivo are required to understand

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the molecular details of the role of sphingolipids in Aß generation and toxicity. 6.2. HIV-associated dementia In the late stages of acquired immunodeficiency syndrome (AIDS) 20–30% of the patients present severe neurological disabilities identified as Human Immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) [221]. Histopathology of brains from HAD patients demonstrate gliosis, abnormalities of dendritic processes and neuronal apoptosis [222]. The exact mechanisms by which the virus causes apoptosis are still unknown. HIV-1 infects susceptible cells by fusion of the viral membrane with the cell plasma membrane. This process is mediated by the interaction of the HIV-1 envelope glycoprotein gp120 with CD4 and the co-receptors CXC chemokine receptor 4 and 5 (CXCR4 and CCR5) present on the host cell surface [223]. Several lines of evidence indicate that these interactions occur at distinct domains on the target-cell membrane [224] and that disruption of domain structure by cholesterol depletion causes inhibition of HIV infection [225]. Recent evidence indicates that the HIV coat protein gp120, which has been implicated in the pathogenesis of HAD, causes neuronal apoptosis. gp120 induces the activation of SMase (primarily N-SMase) and generation of ceramide. SMase activation is triggered by coupling of gp120 to the CXCR4 and the induction of NADPH oxidase-mediated production of superoxide radicals [37]. Consistently, recent finding by Haughey and colleagues have shown that overproduction of ceramide may be involved in neuronal death in HAD patients [226]. Sphingolipid deregulation is more pronounced in HAD patients with an apoE4 genotype, who have worse prognoses [227], however the link between apoE genotype and the altered sphingolipid metabolism is unclear and deserves further study. Contrary to the notion that increased ceramide is detrimental for HIV-related disease it was demonstrated that elevation of ceramide by pharmacological manipulation of the ceramide synthetic pathway, by treatment with SMase, or by direct addition of long-chain ceramides makes cells resistant to HIV infection due to inhibition of membrane fusion [228]. As a consequence the biosynthetic pathway of ceramide has been proposed as a possible novel target for HIV treatment. Further studies are required to reconcile the pros and cons of ceramide increase in HAD. 7. Conclusions In summary, it is clear that simple sphingolipids like ceramide and S1P play pivotal roles in life and death of cells from the nervous system. The cellular fate is intrinsic to the neuronal type and a same molecular target can be regulated by the same sphingolipid with opposing results. Nevertheless some general mechanisms exist in the action of ceramide and its metabolites in different neuronal types. In particular, the mechanisms involved in the induction of neuronal apoptosis are shared by many neuronal types. On the other hand, more

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specific molecular pathways are activated in systems in which sphingolipids induce cell survival. Although the reasons for differential mechanisms to be activated in certain neuronal types and not in others are still obscure, it is obvious that we cannot attribute “a priori” a certain cellular outcome to ceramide or S1P treatment. Acknowledgments Studies in our laboratory are supported by grants from the Canadian Institutes of Health Research (CIHR), Alzheimer Society Canada, The Alzheimer Association (USA) and an establishment grant from the Alberta Heritage Foundation for Medical Research (AHFMR). References [1] Y.A. Hannun, L.M. Obeid, The Ceramide-centric universe of lipidmediated cell regulation: stress encounters of the lipid kind, J. Biol. Chem. 277 (2002) 25847–25850. [2] M.S. Song, E.I. Posse de Chaves, Inhibition of rat sympathetic neuron apoptosis by ceramide. Role of p75NTR in ceramide generation, Neuropharmacology 45 (2003) 1130–1150. [3] P.W. Wertz, D.T. Downing, Ceramides of pig epidermis: structure determination, J. Lipid Res. 24 (1983) 759–765. [4] L. Riboni, A. Prinetti, R. Bassi, A. Caminiti, G. Tettamanti, A mediator role of ceramide in the regulation of neuroblastoma Neuro2a cell differentiation, J. Biol. Chem. 270 (1995) 26868–26875. [5] R.E. Toman, V. Movsesyan, S.K. Murthy, S. Milstien, S. Spiegel, A.I. Faden, Ceramide-induced cell death in primary neuronal cultures: upregulation of ceramide levels during neuronal apoptosis, J. Neurosci. Res. 68 (2002) 323–330. [6] Y.A. Hannun, Functions of ceramide in coordinating cellular responses to stress, Science 274 (1996) 1855–1859. [7] J.M. Holopainen, M.I. Angelova, P.K. Kinnunen, Vectorial budding of vesicles by asymmetrical enzymatic formation of ceramide in giant liposomes, Biophys. J. 78 (2000) 830–838. [8] R. Bose, M. Verheij, A. Haimovitz-Friedman, K. Scotto, Z. Fuks, R. Kolesnick, Ceramide synthase mediates daunorubicin-induced apoptosis: an alternative mechanism for generating death signals, Cell 82 (1995) 405–414. [9] G.S. Dbaibo, W. El-Assaad, A. Krikorian, B. Liu, K. Diab, N.Z. Idriss, M. El-Sabban, T.A. Driscoll, D.K. Perry, Y.A. Hannun, Ceramide generation by two distinct pathways in tumor necrosis factor alpha-induced cell death, FEBS Lett. 503 (2001) 7–12. [10] D.K. Perry, The role of de novo ceramide synthesis in chemotherapyinduced apoptosis, Ann. N. Y. Acad. Sci. 905 (2000) 91–96. [11] A. Senchenkov, D.A. Litvak, M.C. Cabot, Targeting Ceramide Metabolism—A Strategy for Overcoming Drug Resistance, J. Natl. Cancer Inst. 93 (2001) 347–357. [12] Y.A. Hannun, C. Luberto, K.M. Argraves, Enzymes of sphingolipid metabolism: from modular to integrative signaling, Biochemistry 40 (2001) 4893–4903. [13] E. Wang, W.P. Norred, C.W. Bacon, R.T. Riley, A.H. Merrill Jr., Inhibition of sphingolipid biosynthesis by fumonisins. Implications for diseases associated with Fusarium moniliforme, J. Biol. Chem. 266 (1991) 14486–14490. [14] F.M. Goni, A. Alonso, Sphingomyelinases: enzymology and membrane activity, FEBS Lett. 531 (2002) 38–46. [15] T. Levade, J.P. Jaffrezou, Signalling sphingomyelinases: which, where, how and why? Biochim. Biophys. Acta 1438 (1999) 1–17. [16] N. Marchesini, Y.A. Hannun, Acid and neutral sphingomyelinases: roles and mechanisms of regulation, Biochem. Cell. Biol. 82 (2004) 27–44. [17] H. Birbes, S.E. Bawab, L.M. Obeid, Y.A. Hannun, Mitochondria and

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