S. A. Dawson

Overexpression of Akt (protein kinase B) confers protection against apoptosis and prevents formation of ceramide in response to pro-apoptotic stimuli

An immortalized dorsal root ganglion cell line F‐11 exhibits many properties of spinal cord neurons and undergoes apoptosis in response to growth factor withdrawal and the exogenous addition of inhibitors of phosphatidylinositol‐3‐kinase (PI3K). To elucidate the mechanism of apoptosis we generated F‐11 clones which overexpressed either the p110 subunit of PI3K, a constitutively active form of protein kinase B/Akt (Myristoylated Akt), or a dominant‐negative form (c‐Akt). The first two constructs were protective against apoptosis induced by PI3K inhibitors such as wortmannin and LY294002. Caspase‐3 (CPP32) levels peaked at 4 hr to 6 hr in response to pro‐apoptotic drugs, and this increase was attenuated by 50% in F‐11 with constitutively active Akt. The Akt protection was confirmed by DNA fragmentation studies. Both neo‐transfected and the c‐Akt dominant‐negative transfected F‐11 cells showed increased ceramide formation (twofold) in response to staurosporine, wortmannin, or LY294002; whereas cells with a constitutively active Akt (Myr‐Akt) showed no increase in ceramide when treated with staurosporine, wortmannin, or LY294002. Ceramide was a more potent activator of CPP32 and an inducer of apoptosis when added as the native form (hydroxy‐ or nonhydroxy‐), rather than the more water‐soluble C2‐ceramide. Overexpression of PI3K (p110) and Akt protected cells against ceramide‐induced apoptosis, suggesting that Ceramide action is upstream of Akt in these cells and suggesting that Akt might be a target for inhibition by ceramide. Both staurosporine and C2‐ceramide activated the Jun kinase (JNK) cascade and C2‐ceramide increased caspase‐3 (CPP32) activity in cells expressing wild‐type c‐Jun, but not dominant‐negative (TAM‐67) c‐Jun. We suggest that this pathway is also involved in apoptosis, consistent with the idea that ceramide has multiple kinase and kinase‐modulating targets in the apoptotic pathway of neurons. J. Neurosci. Sci. 57:884–893, 1999.

Cyclic AMP Protects Against Staurosporine and Wortmannin‐Induced Apoptosis and Opioid‐Enhanced Apoptosis in Both Embryonic and Immortalized (F‐11κ7) Neurons

Abstract: The mechanism by which opiates affect fetal development is unknown, but one potential target is the programmed cell death (apoptosis) pathway of neurons. Apoptosis was induced in both primary neuronal cultures from embryonic day 7 cerebral hemispheres of chick brain (E7CH) and the F‐11κ7 cell line (an immortalized mouse neuroblastoma × dorsal root ganglion hybrid stably transfected to overexpress κ‐opioid receptors) by either staurosporine or the phosphatidylinositol 3‐kinase inhibitors wortmannin and LY294002. Cells pretreated with either the μ‐specific opioid agonist morphiceptin (E7CH) or the κ‐specific opioid agonist U69,593 (F‐11κ7) for 24 h showed increased apoptosis in response to staurosporine or wortmannin when compared with nonpretreated cells. The effects of morphiceptin and U69,593 were time‐ and dose‐dependent and antagonist‐reversible, suggesting that they were receptor‐mediated. Neither morphiceptin nor U69,593 by themselves had any measurable effect on cell viability or DNA fragmentation, and coaddition of opiates at the same time as staurosporine, wortmannin, or LY294002 did not enhance apoptosis. Time course studies indicated a maximal opioid effect at a time (16–24 h) when inhibition of adenylate cyclase had been maximal for many hours. Addition of dibutyryl cyclic AMP either before or at the time of opioid addition protected against apoptosis and reduced fragmentation to levels seen for staurosporine plus dibutyryl cyclic AMP alone. The specificity for cyclic AMP was confirmed by showing protection with the specific agonist Sp‐adenosine 3′,5′‐cyclic monophosphothioate and increased killing with the antagonist Rp‐adenosine 3′,5′‐cyclic monophosphothioate. We conclude that the opioid enhancement of apoptosis is based on the inhibition of adenylate cyclase and that the effect is time‐dependent.

Chronic exposure to κ-opioids enhances the susceptibility of immortalized neurons (F-11κ7) to apoptosis-inducing drugs by a mechanism that may involve ceramide

Abstract: Chronic exposure of embryonic brain to opioids leads to microcephaly and developmental abnormalities. An immortalized mouse neuroblastoma × dorsal root ganglion hybrid cell line stably transfected to overexpress κ‐opioid receptors (F‐11κ7) showed complete loss of κ‐receptor binding to [3H]U69,593 after exposure to the κ‐agonist U69,593 for 24 h. U69,593 had no measurable effect on cell viability as determined by either cell viability or DNA fragmentation assays. However, when cell death (apoptosis) was induced by either staurosporine or the phosphatidylinositol 3‐kinase inhibitors wortmannin and LY294002, cells pretreated with U69,593 for 24 h showed increased apoptosis compared with untreated cells. Thus, staurosporine (50 nM), wortmannin (4 µM), and LY294002 (30 µM) treatment for 24 h induced a 50% loss of cell viability and DNA fragmentation in 24 h. U69,593 pretreatment produced the same killing at lower concentrations, namely, 20 nM staurosporine, 2 µM wortmannin, and 14 µM LY294002, respectively. The effects of U69,593 were time‐, dose‐, and naloxone‐reversible, suggesting that they are receptor‐mediated. However, coaddition of U69,593 at the same time as staurosporine, wortmannin, or LY294002 did not enhance apoptosis. All three drugs that induced apoptosis were found to increase the level of ceramide, and pretreatment with U69,593 further increased the rate of formation of ceramide, a lipid that induces apoptosis in cells. We propose that chronic exposure to κ‐receptor agonists promotes increased vulnerability of neurons to apoptosis.

Differential regulation of ceramide in lipid-rich microdomains (rafts): antagonistic role of palmitoyl:protein thioesterase and neutral sphingomyelinase 2.

Cell differentiation and myelination involve a fine balance between stasis and programmed cell death, yet the genes that regulate this have not been clearly defined. We therefore studied two key gene products involved in oligodendrocyte plasma membrane lipid metabolism and their antagonistic role in ceramide‐mediated cell death signaling. Overexpression of palmitoyl:protein thioesterase (PPT1; verified by Western blot of the V5‐tagged protein and increased enzyme activity) resulted in decreased ceramide in the detergent‐resistant microdomain (DRM, or raft) relative to cholesterol and sphingomyelin (SM). This PPT1 overexpression also resulted in protection against cell death induced by either staurosporine or C2‐ceramide. In contrast, overexpression of neutral sphingomyelinase 2 (NSMase2; verified by Western blot of the FLAG‐tagged protein and increased enzyme activity) resulted in increased membrane NSMase and increased ceramide in rafts relative to cholesterol and SM. The difference in SM and ceramide turnover was quantitated by [3H]palmitate pulse‐chase labeling. Furthermore, when NBD‐SM was added to cells, it was hydrolyzed by NSMase‐transfected cells at more than twofold the rate in untransfected cells. NSMase2 overexpression enhanced cell death induced by staurosporine or C2‐ceramide, in contrast to the protective effect of PPT1 overexpression. The presence of a fraction of both PPT1 and NSMase2 in rafts together with their substrates (palmitoylated proteins and SM, respectively) suggests a mechanism for dynamic palmitoylation/depalmitoylation of certain proteins in controlling cell death via NSMase activation.

Expression of the receptor for advanced glycation end products in oligodendrocytes in response to oxidative stress.

Demyelination is a common result of oxidative stress in the nervous system, and we report here that the response of oligodendrocytes to oxidative stress involves the receptor for advanced glycation end products (RAGE). RAGE has not previously been reported in neonatal rat oligodendrocytes (NRO), but, by using primers specific for rat RAGE, we were able to show expression of messenger RNA (mRNA) for RAGE in NRO, and a 55‐kDa protein was detected by Western blotting with antibodies to RAGE. Neonatal rat oligodendrocytes stained strongly for RAGE, suggesting membrane localization of RAGE. Addition of low concentrations of hydrogen peroxide (100 μM) initiated 55‐kDa RAGE shedding from the cell membrane and the appearance of “soluble” 45‐kDa RAGE in the culture medium, followed by restoration of RAGE expression to normal levels. Increasing hydrogen peroxide concentration (>200 μM) resulted in no restoration of RAGE, and the cells underwent apoptosis and necrosis. We further confirmed the observation in a human oligodendroglioma‐derived (HOG) cell line. Both the antioxidant N‐acetyl‐L‐cysteine and the broad‐spectrum metalloproteases inhibitor TAPI0 were able partially to inhibit shedding of RAGE, suggesting involvement of metalloproteases in cleavage to produce soluble RAGE. The level of 55‐kDa RAGE in autopsy brain of patients undergoing neurodegeneration with accompanying inflammation [multiple sclerosis and neuronal ceroid‐lipofuscinosis (Battens disease)] was much lower than that in age‐matched controls, suggesting that shedding of RAGE might occur as reactive oxygen species accumulate in brain cells and be part of the process of neurodegeneration.

Transfection of 2,6 and 2,3-sialyltransferase genes and GlcNAc-transferase genes into human glioma cell line U-373 MG affects glycoconjugate expression and enhances cell death

Human glioma cell line U‐373 MG expresses CMP‐NeuAc : Galβ1,3GlcNAc α2,3‐sialyltransferase [EC No. 2.4.99.6] (α2,3ST), UDP‐GlcNAc : β‐d‐mannoside β1,6‐N‐acetylglucosaminyltransferase V [EC 2.4.1.155] (GnT‐V) and UDP‐GlcNAc3: β‐d‐mannoside β1,4‐N‐acetylglucosaminyltransferase III [EC 2.4.1.144] (GnT‐III) but not CMP‐NeuAc : Galβ1,4GlcNAc α2,6‐sialyltransferase [EC 2.4.99.1] (α2,6ST) under normal culture conditions. We have previously shown that transfection of the α2,6ST gene into U‐373 cells replaced α2,3‐linked sialic acids with α2,6 sialic acids, resulting in a marked inhibition of glioma cell invasivity and a significant reduction in adhesivity. We now show that U‐373 cells, which are typically highly resistant to cell death induced by chemotherapeutic agents (< 10% death in 18 h), become more sensitive to apoptosis following overexpression of these four glycoprotein glycosyltransferases. U‐373 cell viability showed a three‐fold decrease (from 20 to 60% cell death) following treatment with staurosporine, C2‐ceramide or etoposide, when either α2,6ST and GnT‐V genes were stably overexpressed. Even glycosyltransferases typically raised in cancer cells, such as α2,3ST and GnT‐III, were able to decrease viability two‐fold (from 20 to 40% cell death) following stable overexpression. The increased susceptibility of glycosyltransferase‐transfected U‐373 cells to pro‐apoptotic drugs was associated with increased ceramide levels in Rafts, increased caspase‐3 activity and increased DNA fragmentation. In contrast, the same glycosyltransferase overexpression protected U‐373 cells against a different class of apoptotic drugs, namely the phosphatidylinositol 3‐kinase inhibitor LY294002. Thus altered surface protein glycosylation of a human glioblastoma cell line can lead to lowered resistance to chemotherapeutic agents.

Anti-tumor promoting effects of palmitoyl: protein thioesterase inhibitors against a human neurotumor cell line

Inhibiting the depalmitoylation of proteins disrupts cell survival signaling in tumor cells and leads to increased cell death. We chemically synthesized a non-hydrolyzable analog of the palmitoyl-cysteine thioester linkage (AcG-alpha-ketoamido-palmitoyl diamino propionate-VKIKK) (DAPKA) and showed that it inhibits palmitoyl:protein thioesterase (PPT1) in an in vitro assay using a specific fluorescent-based (4-methylumbelliferyl-beta-gluco-6-thiopalmitate) assay. We then showed that it killed cultured tumor cells and enhanced the killing of neurotumor cells by chemotherapeutic drugs such as etoposide and adriamycin. Overexpression of PPT1 protected against apoptosis induced by etoposide and the ketoamide and the inhibitory effect of the two was additive.

Multiple polyphosphoinositide pathways regulate apoptotic signalling in a dorsal root ganglion derived cell line

The polyphosphoinositides play important roles in transmembrane signalling but are also involved in anchoring cell surface proteins, organellar transport, cytoskeleton organization, and cell survival. The polyphosphoinositides synthesized by phosphatidylinositol‐3 kinase (PI‐3K), (Ptd(3,4)InsP2, and PtdIns(3,4,5)P3), appear to play a critical role in cell survival by membrane recruitment and activation of Akt kinase. Inhibitors of PI3K, wortmannin, and LY294002, induced a time‐dependent activation of caspase‐3 (CPP32), with a peak at 6 hr, leading to subsequent cell death by apoptosis in a dorsal root ganglion cell line (F‐11). Lowering cyclic AMP (cAMP) levels enhanced both caspase‐3 activation and cell death induced by PI3K inhibitors, whereas a nonhydrolyzable cAMP analog (Bt2cAMP), lowered CPP32 and was protective We stably transfected the F‐11 cells with the constitutively active p110 catalytic subunit of PI‐3 kinase and observed resistance to both caspase‐3 (CPP32) activation and subsequent apoptosis induced by either wortmannin or LY294002. Treatment of F‐11 cells with bradykinin (BK) stimulated the hydrolysis of a different polyphosphoinositide, PtdIns(4,5)P2, and enhanced both wortmannin‐induced caspase‐3 (CPP32) activation and subsequent apoptosis. PtdIns(4,5)P2 is also a precursor of the anti‐apoptotic PtdIns(3,4,5) P3 and lowering cAMP levels with opioid agonists for 30 min enhanced both the hydrolysis of PtdIns(4,5) P2 and cellular apoptosis. The enhancement was opioid dose‐dependent and opioid antagonist (naloxone)‐ reversible and was also seen following 24‐hr exposure to opioids such as U69,593 and Dala2, Dleu5 enkephalin (DADLE). However, unlike the bradykinin stimulation of PtdIns(4,5)P2 hydrolysis following activation of phospholipase C, the opioid‐enhanced hydrolysis was independent of external Ca2+ and was blocked by pertussis toxin, suggesting a different mechanism involving GI, Go, or βγ‐subunits. In summary, both the receptor‐mediated lowering of cAMP levels and the hydrolysis of 4,5‐polyphosphoinositides have no direct effect on caspase‐3 activity or apoptosis but do exacerbate the activation of caspase‐3‐like activity and subsequent cell death by apoptosis induced by inhibitors of 3‐polyphosphoinositide synthesis. We suggest that multiple polyphosphoinositide pathways are involved in the regulation of apoptosis. J. Neurosci. Res. 59:136–144, 2000

Oxidized phosphatidylcholine formation and action in oligodendrocytes.

Reactive oxygen species play a major role in neurodegeneration. Increasing concentrations of peroxide induce neural cell death through activation of pro‐apoptotic pathways. We now report that hydrogen peroxide generated sn‐2 oxidized phosphatidylcholine (OxPC) in neonatal rat oligodendrocytes and that synthetic OxPC [1‐palmitoyl‐2‐(5′‐oxo)valeryl‐sn‐glycero‐3 phosphorylcholine, POVPC] also induced apoptosis in neonatal rat oligodendrocytes. POVPC activated caspases 3 and 8, and neutral sphingomyelinase (NSMase) but not acid sphingomyelinase. Downstream pro‐apoptotic pathways activated by POVPC treatment included the Jun N‐terminal kinase proapoptotic cascade and the degradation of phospho‐Akt. Activation of NSMase occurred within 1 h, was blocked by inhibitors of caspase 8, increased mainly C18 and C24:1 ceramides, and appeared to be concentrated in detergent‐resistant microdomains (Rafts). We concluded that OxPC initially activated NSMase and converted sphingomyelin into ceramide to mediate a series of downstream pro‐apoptotic events in oligodendrocytes.

Differential regulation of sphingomyelin synthesis and catabolism in oligodendrocytes and neurons

Neurons (both primary cultures of 3‐day rat hippocampal neurons and embryonic chick neurons) rapidly converted exogenous NBD‐sphingomyelin (SM) to NBD‐Cer but only slowly converted NBD‐Cer to NBD‐SM. This was confirmed by demonstrating low in vitro sphingomyelin synthase (SMS) and high sphingomyelinase (SMase) activity in neurons. Similar results were observed in a human neuroblastoma cell line (LA‐N‐5). In contrast, primary cultures of 3‐day‐old rat oligodendrocytes only slowly converted NBD‐SM to NBD‐Cer but rapidly converted NBD‐Cer to NBD‐SM. This difference was confirmed by high in vitro SMS and low SMase activity in neonatal rat oligodendrocytes. Similar results were observed in a human oligodendroglioma cell line. Mass‐Spectrometric analyses confirmed that neurons had a low SM/Cer ratio of (1.5 : 1) whereas oligodendroglia had a high SM/Cer ratio (9 : 1). Differences were also confirmed by [3H]palmitate‐labeling of ceramide, which was higher in neurons compared with oligodendrocytes. Stable transfection of human oligodendroglioma cells with neutral SMase, which enhanced the conversion of NBD‐SM to NBD‐Cer and increased cell death, whereas transfection with SMS1 or SMS2 enhanced conversion of NBD‐Cer to NBD‐SM and was somewhat protective against cell death. Thus, SMS rather than SMases may be more important for sphingolipid homeostasis in oligodendrocytes, whereas the reverse may be true for neurons.