Shawei Chen

Involvement of de novo ceramide biosynthesis in tumor necrosis factor- α/cycloheximide-induced cerebral endothelial cell death

Cytokines, including tumor necrosis factor-α (TNF-α), may elicit cytotoxic response through the sphingomyelin-ceramide signal transduction pathway by activation of sphingomyelinases and the subsequent release of ceramide: the universal lipid second messenger. Treatment of bovine cerebral endothelial cells (BCECs) with TNF-α for 16 h followed by cycloheximide (CHX) for 6 h resulted in an increase in ceramide accumulation, DNA fragmentation, and cell death. Application of a cell permeable ceramide analogue C2 ceramide, but not the biologically inactive C2 dihydroceramide, also induced DNA laddering and BCEC death in a concentration- and time-dependent manner. TNF-α/CHX-mediated ceramide production apparently is not a result of sphingomyelin hydrolysis because sphingomyelin content does not decrease in this death paradigm. In addition, an acidic sphingomyelinase inhibitor, desipramine, had no effect on TNF-α/CHX-induced cell death. However, addition of fumonisin B1, a selective ceramide synthase inhibitor, attenuated TNF-α/CHX-induced intracellular ceramide elevation and BCEC death. Together, these findings suggest that ceramide plays at least a partial role in this paradigm of BCEC death. Our results show, for the first time, that ceramide derived from de novosynthesis is an alternative mechanism to sphingomyelin hydrolysis in the BCEC death process initiated by TNF-α/CHX.

Methylprednisolone inhibition of TNF-α expression and NF-kB activation after spinal cord injury in rats

Post-traumatic inflammatory reaction has been implicated in the secondary injury after SCI. TNF-alpha is a key inflammatory mediator, which plays a pathogenetic role in cell death in inflammatory disorders and traumatic brain injury. TNF-alpha exerts its effector actions, at least partially, through the activation of a pro-inflammatory transcription factor, NF-kB, which in turn upregulates such genes as iNOS, cytokines, adhesive molecules, and others. Consistent with a post-traumatic inflammatory reaction after SCI, we noted an increase in TNF-alpha expression by Western blotting (4.5-fold increase at 1 day after SCI, P<0.01) and immunohistochemistry in a rat SCI model. Post-traumatic TNF-alpha expression was accompanied by an increase in NF-kB binding activity in nuclear proteins isolated from the injured cord (3.9-fold increase, P<0.01). MP is the only drug proven effective in improving neurological function in patients with acute SCI. The mechanism of action of MP is not fully understood, but is thought to be related to its antioxidant effects. MP is also a potent anti-inflammatory agent, which has been recently shown to inhibit NF-kB binding activity. MP (30 mg/kg, i.v.) given immediately after SCI reduced TNF-alpha expression by 55% (P<0.01) and NF-kB binding activity. These findings suggest that post-traumatic inflammatory activity that is mediated by the TNF-alpha-NF-kB cascade can be suppressed by MP.

Amyloid-β peptide induces oligodendrocyte death by activating the neutral sphingomyelinase-ceramide pathway

Amyloid-β peptide (Aβ) accumulation in senile plaques, a pathological hallmark of Alzheimers disease (AD), has been implicated in neuronal degeneration. We have recently demonstrated that Aβ induced oligodendrocyte (OLG) apoptosis, suggesting a role in white matter pathology in AD. Here, we explore the molecular mechanisms involved in Aβ-induced OLG death, examining the potential role of ceramide, a known apoptogenic mediator. Both Aβ and ceramide induced OLG death. In addition, Aβ activated neutral sphingomyelinase (nSMase), but not acidic sphingomyelinase, resulting in increased ceramide generation. Blocking ceramide degradation with N-oleoyl-ethanolamine exacerbated Aβ cytotoxicity; and addition of bacterial sphingomyelinase (mimicking cellular nSMase activity) induced OLG death. Furthermore, nSMase inhibition by 3-O-methyl-sphingomyelin or by gene knockdown using antisense oligonucleotides attenuated Aβ-induced OLG death. Glutathione (GSH) precursors inhibited Aβ activation of nSMase and prevented OLG death, whereas GSH depletors increased nSMase activity and Aβ-induced death. These results suggest that Aβ induces OLG death by activating the nSMase–ceramide cascade via an oxidative mechanism.

iNOS and nitrotyrosine expression after spinal cord injury

Secondary tissue damage after spinal cord injury (SCI) may be due to inflammatory mediators. After SCI, the nuclear factor-kappaB (NF-kappaB) transcription factor can activate many pro-inflammatory genes, one of which is inducible nitric oxide synthase (iNOS). iNOS catalyzes the synthesis of nitric oxide (NO), a key inflammatory mediator, which in turn reacts with superoxide to generate peroxynitrite. Peroxynitrite is a strong oxidant that can damage cellular enzymes, membranes, and subcellular organelles through the nitration of tyrosine residues on proteins. The presence of nitrotyrosine (NT) is an indirect chemical indicator of toxic NO and peroxynitrite-induced cellular damage. Using a New York University (NYU) impactor to induce SCI in adult rats, we examined the temporal and cellular expression of iNOS and NT. We observed a progressive increase in iNOS expression in the injured cord starting at day 1 with maximal expression occurring at day 7, as determined by Western blot analysis. iNOS expression corresponded temporally to an increase in iNOS enzyme activity after SCI. In parallel with the progressive increase in iNOS activity, NT expression also increased with time after SCI. The iNOS and NT immunoreactivity was localized in neurons, astrocytes, endothelial cells and ependymal cells at the epicenter and adjacent to the region of spinal cord impact and injury. Results from the present study suggest that increased iNOS and peroxynitrite anion, as reflected by the progressive accumulation of NT in the injured impacted spinal cord, may contribute to the secondary injury process after SCI.

Amyloid β Peptide–Induced Cerebral Endothelial Cell Death Involves Mitochondrial Dysfunction and Caspase Activation

Amyloid β peptide (Aβ), a 39 to 43 amino acid fragment of the β-amyloid precursor protein (βAPP), forms insoluble fibrillar accumulation in neurofibrillary tangles and vascular plaques. Aβ has been implicated in neuronal and vascular degeneration in brain regions susceptible to plaque formation because of its cytotoxic effect on neurons and endothelial cells (ECs). The authors used a murine cerebral endothelial cell (CEC) line and primary cultures of bovine CECs to explore the cytotoxic mechanism of Aβ. Aβ 1–40 and Aβ 25–35 peptides caused cell death in a dose-dependent and time-dependent manner. Exposure to either Aβ 25–35 or Aβ 1–40 at 10 μmol/L for 48 hours caused at least 40% cell death. Cerebral endothelial cell death was characterized by nuclear condensation, mitochondrial dysfunction, and nuclear and mitochondrial DNA damage. Aβ 25–35 activated both caspase-8 and caspase-3 in murine CECs. zVAD-fmk, a broad-spectrum caspase inhibitor, prevented Aβ 25–35-induced increase in caspase-3 activity and CEC death. N-acetyl-cysteine, an antioxidant, also prevented Aβ-induced cell death. Together, these findings indicate that Aβ-mediated CEC death is an apoptotic process that is characterized by increased oxidative stress, caspase activation, mitochondrial dysfunction, and nuclear and mitochondrial DNA damage.

Matrix metalloproteinase-9 and spontaneous hemorrhage in an animal model of cerebral amyloid angiopathy.

We examined the potential role of the extra‐cellular matrix‐degrading enzyme, matrix metalloproteinase‐9 (MMP‐9), in the pathogenesis of cerebral amyloid angiopathy (CAA)‐induced spontaneous hemorrhage. The amyloid‐beta peptide (Aβ) induced the synthesis, release and activation of MMP‐9 in murine cerebral endothelial cells, resulting in increased extracellular matrix degradation. Furthermore, extensive MMP‐9 immunoreactivity was observed in CAA‐vessels with evidence of microhemorrhage in aged APPsw transgenic mice, but not detected in aged wild type or young APPsw mice. These results suggest that increased vascular MMP‐9 expression, stimulated by Aβ, may play a role in the pathogenesis of spontaneous intracerebral hemorrhage in patients with CAA. Ann Neurol 2003;54:000–000

Methylprednisolone Protects Oligodendrocytes But Not Neurons after Spinal Cord Injury

Methylprednisolone (MP) is used to treat a variety of neurological disorders involving white matter injury, including multiple sclerosis, acute disseminated encephalomyelitis, and spinal cord injury (SCI). Although its mechanism of action has been attributed to anti-inflammatory or antioxidant properties, we examined the possibility that MP may have direct neuroprotective activities. Neurons and oligodendrocytes treated with AMPA or staurosporine died within 24 h after treatment. MP attenuated oligodendrocyte death in a dose-dependent manner; however, neurons were not rescued by the same doses of MP. This protective effect was reversed by the glucocorticoid receptor (GR) antagonist (11, 17)-11-[4-(dimethylamino)phenyl]-17-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one (RU486) and small interfering RNA directed against GR, suggesting a receptor-dependent mechanism. MP reversed AMPA-induced decreases in the expression of anti-apoptotic Bcl-xL, caspase-3 activation, and DNA laddering, suggesting anti-apoptotic activity in oligodendrocytes. To examine whether MP demonstrated this selective protection in vivo, neuronal and oligodendrocyte survival was assessed in rats subjected to spinal cord injury (SCI); groups of rats were treated with or without MP in the presence or absence of RU486. Eight days after SCI, MP significantly increased oligodendrocytes (CC-1-immunoreactive cells) after SCI, but neuronal (neuronal-specific nuclear protein-immunoreactive cells) number remained unchanged; RU486 reversed this protective effect. MP also inhibited SCI-induced decreases in Bcl-xL and caspase-3 activation. Consistent with these findings, the volume of demyelination, assessed by Luxol fast blue staining, was attenuated by MP and reversed by RU486. These results suggest that MP selectively inhibits oligodendrocyte but not neuronal cell death via a receptor-mediated action and may be a mechanism for its limited protective effect after SCI.

Amyloid-β peptide enhances tumor necrosis factor-α-induced iNOS through neutral sphingomyelinase/ceramide pathway in oligodendrocytes

Although accumulating evidence demonstrates that white matter degeneration contributes to pathology in Alzheimers disease (AD), the underlying mechanisms are unknown. In order to study the roles of the amyloid‐β peptide in inducing oxidative stress damage in white matter of AD, we investigated the effects of amyloid‐β peptide 25–35 (Aβ) on proinflammatory cytokine tumor necrosis factor‐α (TNF‐α)‐induced inducible nitric oxide synthase (iNOS) in cultured oligodendrocytes (OLGs). Although Aβ 25–35 by itself had little effect on iNOS mRNA, protein, and nitrite production, it enhanced TNF‐α‐induced iNOS expression and nitrite generation in OLGs. Aβ, TNF‐α, or the combination of both, increased neutral sphingomyelinase (nSMase) activity, but not acidic sphingomyelinase (aSMase) activity, leading to ceramide accumulation. Cell permeable C2‐ceramide enhanced TNF‐α‐induced iNOS expression and nitrite generation. Moreover, the specific nSMase inhibitor, 3‐O‐methyl‐sphingomyelin (3‐OMS), inhibited iNOS expression and nitrite production induced by TNF‐α or by the combination of TNF‐α and Aβ. Overexpression of a truncated mutant of nSMase with a dominant negative function inhibited iNOS mRNA production. 3‐OMS also inhibited nuclear factor κB (NF‐κB) binding activity induced by TNF‐α or by the combination of TNF‐α and Aβ. These results suggest that neutral sphingomyelinase/ceramide pathway is required but may not be sufficient for iNOS expression induced by TNF‐α and the combination of TNF‐α and Aβ.

Matrix metalloproteinase-9 in cerebral-amyloid-angiopathy-related hemorrhage

Spontaneous intracerebral hemorrhage (ICH) is one of the most recognized complications of cerebral amyloid angiopathy (CAA), but little is known about the molecular pathogenesis of this life-threatening complication. In this review, we present preliminary evidence which suggests that the extracellular-matrix-degrading protease, matrix metalloproteinase-9 (MMP-9), may play a role in the development of spontaneous ICH resulting from CAA. The amyloid-beta peptide (Abeta) induced the synthesis, cellular release, and activation of MMP-9 in murine cerebral endothelial cells (CECs), resulting in increased extracellular matrix (ECM) degradation. Furthermore, in a mouse model of CAA (APPsw transgenic mice), MMP-9 immunoreactivity was observed in amyloid-laden cerebral vessels in aged APPsw mice but not in young APPsw or aged wild-type mice. More extensive MMP-9 immunostaining was present in amyloid-laden vessels with evidence of microhemorrhage. These results suggest that increased vascular MMP-9 expression, stimulated by Abeta, may play a role in the pathogenesis of spontaneous intracerebral hemorrhage (ICH) in patients with CAA.

Amyloid beta peptide increases DP5 expression via activation of neutral sphingomyelinase and JNK in oligodendrocytes

There is growing recognition that white matter pathology is a common feature in Alzheimers disease. We have previously reported that the amyloid beta peptide (Aβ) induces apoptosis in oligodendrocytes (OLG), via activation of neutral sphingomyelinase (nSMase) and resultant generation of ceramide. In the current study, we report that both Aβ and ceramide increased expression of the proapoptotic protein DP5/Hrk (DP5), and release of cytochrome C from mitochondria to cytoplasm in OLGs. We provide evidence that the Jun N‐terminal kinase (JNK) signaling pathway mediates Aβ‐ and ceramide‐induced apoptosis: Both Aβ and ceramide activated JNK phosphorylation, and subsequent AP‐1 DNA binding activity; JNK siRNA decreased AP‐1 DNA binding, DP5 expression and reduced cell death. Furthermore, inhibition of nSMase attenuated Aβ‐induced JNK phosphorylation, AP‐1 DNA binding activity, DP5 expression, and cytochrome C release. Collectively, these results suggest that Aβ‐induced apoptosis involves the sequential activation of nSMase with ceramide generation, JNK activation, AP‐1 DNA binding, and DP5 expression.