Rajendra Goswami

Interferon-γ-Oligodendrocyte Interactions in the Regulation of Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an animal model of the human demyelinating disorder multiple sclerosis (MS). The immune cytokine interferon-gamma (IFN-γ) is believed to participate in disease pathogenesis in both EAE and MS. In the present study, we examined the significance of IFN-γ-oligodendrocyte interactions in the course of EAE. For the purpose of our study, we used the previously described [proteolipid protein/suppressor of cytokine signaling 1 (PLP/SOCS1)] transgenic mouse line that displays suppressed oligodendrocyte responsiveness to IFN-γ. PLP/SOCS1 mice developed EAE with an accelerated onset associated with enhanced early inflammation and markedly increased oligodendrocyte apoptosis. Moreover, we found that IFN-γ pretreatment of mature oligodendrocytes in vitro had a protective effect against oxidative stress and the inhibition of proteasome activity and resulted in upregulation in expression of a number of chemokines, including CXCL10 (IP10), CCL2 (MCP-1), CCL3 (MCP-1α), and CCL5 (RANTES). These results suggest that IFN-γ-oligodendrocyte interactions are of significance to the clinical and pathological aspects of EAE. In addition, the present study suggests that oligodendrocytes are not simply targets of inflammatory injury but active participants of the neuroimmune network operating during the course of EAE.

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.

Oxidized phosphatidylcholine is a marker for neuroinflammation in multiple sclerosis brain

Multiple sclerosis (MS) is a common autoimmune neurodegenerative disease of unknown cause, which results in inflammation and plaques of demyelination in brain and eventual axonal degeneration. We report the novel presence of oxidized phosphatidylcholine [1‐palmitoyl‐2‐(5′‐oxo)valeryl‐sn‐glycero‐3‐phosphorylcholine (POVPC)], a lipid associated with inflammatory diseases such as atherosclerosis and lung disease, in the brain of MS patients. The OxPC epitope was detected by Western blotting with the E06 monoclonal antibody. E06‐positive lipid was present in the highest amounts in MS plaques, which also showed evidence of low‐molecular‐weight (15‐kDa) OxPC‐modified protein. E06 reactivity did not change with post‐mortem interval, and E06‐positive lipids were largely absent from control tissue. We then used a second monoclonal antibody (AB1‐2, which recognizes the E06/T15 idiotype and therefore detects the presence of antibody to OxPC) to show that MS brain samples were strongly positive for the 50‐kDa antibody heavy chain. We also showed that isoelectric focussing of the oligoclonal IgG characteristic of MS revealed some immunoglobulin bands that Western blotted with the AB1‐2 antibody. Spinal cords from mice induced to undergo experimental allergic encephalomyelitis (EAE) also showed strong AB1‐2 reactivity by both immunocytochemistry and Western blot analysis. We therefore conclude that we can detect both OxPC and 15‐kDa protein modified by OxPC and the antibody to the antibody to OxPC (antiidiotype) in pathological tissue and suggest that this could play a role in the progression of MS.

Ceramide in rafts (detergent-insoluble fraction) mediates cell death in neurotumor cell lines

Detergent‐resistant lipid microdomains (Rafts) were isolated from human oligodendroglioma (HOG), human neuroblastoma (LA‐N‐5), and immortalized dorsal root ganglion (F‐11) cell lines by sucrose‐density gradient ultracentrifugation and shown to be enriched in cholesterol, sphingomyelin, and ceramide. [3H]palmitate labeling allowed the Raft fraction to be easily identified as a sharp peak of 3H radioactivity in the 5–30% sucrose interphase. Treatment of [3H]palmitate‐labeled cells with staurosporine (to activate caspase 8 and induce apoptosis) or exogenous sphingomyelinase specifically increased the [3H]ceramide content of the Raft fraction. Depletion of cholesterol with β‐methylcyclodextran decreased Raft formation and partially blocked staurosporine‐induced apoptosis. Similarly, treatment of cells with Fumonisin B1 to inhibit de novo sphingolipid synthesis by 50% reduced the labeling of the Raft fraction and partially blocked staurosporine‐induced apoptosis. Staurosporine treatment activated neutral sphingomyelinase but had no effect on acid sphingomyelinase activity or on other lysosomal hydrolases, such as α‐L‐fucosidase. Most of the neutral sphingomyelinase activity is in the Raft fraction, suggesting that the conversion of sphingomyelin to ceramide in Rafts is an important event in neural cell apoptosis.

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.

Acid sphingomyelinase and inhibition by phosphate ion: role of inhibition by phosphatidyl‐myo‐inositol 3,4,5‐triphosphate in oligodendrocyte cell signaling

There is ample evidence that both acid (ASMase) and neutral (NSMase) sphingomyelinases play a role in cell death so inhibitors of either enzyme could have significant value as protectors against neurodegeneration. We used a fluorogenic sphingomyelinase substrate, 6‐hexadecanoylamino‐4‐methylumbelliferyl‐phosphorylcholine, and a [14C]choline‐labeled sphingomyelin substrate to screen large numbers of phosphocompounds for inhibition of ASMase in extracts of human oligodendroglioma cells (HOG) and neonatal rat oligodendrocytes. Non‐competitive inhibition was observed with inorganic phosphate and AMP, which was a more potent inhibitor of ASMase than cyclic AMP, ADP or ATP. However, other nucleotide phosphates, sugar phosphates, nucleotide sugars and glycerol phosphate did not inhibit ASMase. Our key finding was that phosphatidyl‐myo‐inositol 3,4,5‐triphosphate [PtdIns (3,4,5)P3] was a much more potent inhibitor of ASMase than lysophosphatidic acid or phosphatidyl‐myo‐inositol 4,5‐diphosphate [PtdIns(4,5)P2]. When PtdIns(3,4,5)P3 was added to cultured cells we observed 50% inhibition of ASMase but no inhibition of other lysosomal hydrolases. After transfection of HOG cells with the tumor supressor phosphatase and tensin homolog protein (PTEN), which hydrolyses PtdIns(3,4,5)P3 to PtdIns(4,5)P2, we observed a two‐fold increase in ASMase activity. Furthermore, the phosphatidylinositol‐3‐kinase inhibitor wortmannin (which reduces PtdIns(3,4,5)P3 levels) also resulted in activation of ASMase. We propose that the small amount of ASMase activity associated with detergent‐resistant cell membranes (Rafts) is regulated by PtdIns(3,4,5)P3 and is most likely involved in receptor clustering and capping.

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.

Ceramide regulation of the tumor suppressor phosphatase PTEN in rafts isolated from neurotumor cell lines.

The neutral sphingolipid ceramide has been implicated in the apoptotic death of cells by a number of different mechanisms, including activation of protein kinase B (Akt) phosphatase. Here we present evidence that ceramide recruits the tumor suppressor PTEN (phosphatase and tensin homolog deleted from chromosome 10) into membrane microdomains (rafts), where it could act to reduce the levels of polyphosphoinositides necessary for the activation of Akt. A PTEN construct with a red‐fluorescent protein (RFP) tag was overexpressed in both a human cell line derived from oligodendroglioma (HOG) and a rat pheochromocytoma cell line (PC12) by means of an inducible promoter system (Tet‐Off). Induction of PTEN by removal of doxycycline enhanced both capsase‐3 and cell death with staurosporine, wortmannin, or C2‐ceramide, whereas antisense PTEN had the reverse effect. Overexpression of PTEN also increased acid sphingomyelinase (ASMase) activity. PTEN normally has a generalized (cytosolic/membrane) distribution, but treatment with C2‐ceramide translocated a fraction of the PTEN to the plasma membrane, showing a plasma membrane distribution similar to that observed for a prenylated green‐fluorescent (GFP) construct. PTEN was then shown to translocate to the detergent‐resistant membrane microdomain fraction (raft) of the plasma membrane. The colocalization of sphingomyelinases, ceramide, polyphosphoinositides, and PTEN in the raft fraction further suggests that the association of these lipids is critical for regulating cell death.