Jianrong Li

Nitric Oxide Suppresses Apoptosis via Interrupting Caspase Activation and Mitochondrial Dysfunction in Cultured Hepatocytes

Nitric oxide (NO) is a potent inhibitor of apoptosis in many cell types, including hepatocytes. We and others have described NO-dependent decreases in caspase activity in cells undergoing apoptosis. However, previous work has not determined whether NO disrupts the proteolytic processing and thus the activation of pro-caspases. Here we report that NO suppresses proteolytic processing and activation of multiple pro-caspases in intact cells, including caspase-3 and caspase-8. We found that both exogenous NO as well as endogenously produced NO via adenoviral inducible NO synthase gene transfer protected hepatocytes from tumor necrosid factor (TNF) α plus actinomycin D (TNFα/ActD)-induced apoptosis. Affinity labeling with biotin-VAD-fmk of all active caspase species in TNFα-mediated apoptosis identified four newly labeled spots (activated caspases) present exclusively in TNFα/ActD-treated cells. Both NO and the caspase inhibitor, Ac-DEVD-CHO, prevented the appearance of the four newly labeled spots or active caspases. Immunoanalysis of affinity labeled caspases demonstrated that caspase-3 was the major effector caspase. Western blot analysis also identified the activation of caspase-8 in the TNFα/ActD-treated cells, and the activation was suppressed by NO. Furthermore, NO inhibited several other events associated with caspase activation in cells, including release of cytochrome c from mitochondria, decrease in mitochondrial transmembrane potential, and cleavage of poly(ADP-ribose) polymerase in TNFα/ActD-treated cells. These findings indicate the involvement of multiple caspases in TNFα-mediated apoptosis in hepatocytes and establish the capacity of NO to inhibit not only active caspases but also caspase activation.

Isolation and culture of rat and mouse oligodendrocyte precursor cells

The ability to isolate oligodendroglial precursor cells (OPCs) provides a powerful means to characterize their differentiation, properties and potential for myelin repair. Although much knowledge is available for isolation of OPCs from the rat central nervous system, preparation and maintenance of mouse OPCs has been until recently a challenge owing to difficulties in obtaining a sufficient quantity of purified OPCs. Here, we describe protocols to prepare highly enriched rat OPCs and nearly homogenous mouse OPCs. The mouse method generates predominantly OPCs from cortical neural progenitor cells as clonal aggregates called “oligospheres” by taking advantage of molecular genetic tools. Isolated OPCs can be further differentiated into oligodendrocytes. Collectively, we describe simple and efficient methods for the preparation and in vitro maintenance of enriched OPCs from rats and mice. Isolation and culture of a large, homogenous population of rodent OPCs should significantly facilitate studies on OPC lineage progression and their utility in myelin repair after injury.

Glutathione Peroxidase-Catalase Cooperativity Is Required for Resistance to Hydrogen Peroxide by Mature Rat Oligodendrocytes

Oxidative mechanisms of injury are important in many neurological disorders, including hypoxic-ischemic brain damage. Cerebral palsy after preterm birth is hypothesized to be caused by hypoxic-ischemic injury of developing oligodendrocytes (OLs). Here we examined the developmental sensitivity of OLs to exogenous hydrogen peroxide (H2O2) with stage-specific rat oligodendrocyte cultures. We found that H2O2 itself or that generated by glucose oxidase was more toxic to developing than to mature OLs. Mature OLs were able to degrade H2O2 faster than developing OLs, suggesting that higher antioxidant enzyme activity might be the basis for their resistance. Catalase expression and activity were relatively constant during oligodendrocyte maturation, whereas glutathione peroxidase (GPx) was upregulated with a twofold to threefold increase in its expression and activity. Thus, it appeared that the developmental change in resistance to H2O2 was caused by modulation of GPx but not by catalase expression. To test the relative roles of catalase and GPx in the setting of oxidative stress, we measured enzyme activity in cells exposed to H2O2 and found that H2O2 induced a decrease in catalase activity in developing but not in mature OLs. Inhibition of GPx by mercaptosuccinate led to an increase in the vulnerability of mature OLs to H2O2 as well as a reduction in catalase activity. Finally, H2O2-dependent inactivation of catalase in developing OLs was prevented by the GPx mimic ebselen. These data provide evidence for a key role for GPx-catalase cooperativity in the resistance of mature OLs to H2O2-induced cell death.

The oligodendrocyte-specific G-protein coupled receptor GPR17 is a cell-intrinsic timer of myelination

The basic helix-loop-helix transcription factor Olig1 promotes oligodendrocyte maturation and is required for myelin repair. We characterized an Olig1-regulated G protein–coupled receptor, GPR17, whose function is to oppose the action of Olig1. Gpr17 was restricted to oligodendrocyte lineage cells, but was downregulated during the peak period of myelination and in adulthood. Transgenic mice with sustained Gpr17 expression in oligodendrocytes exhibited stereotypic features of myelinating disorders in the CNS. Gpr17 overexpression inhibited oligodendrocyte differentiation and maturation both in vivo and in vitro. Conversely, Gpr17 knockout mice showed early onset of oligodendrocyte myelination. The opposing action of Gpr17 on oligodendrocyte maturation reflects, at least partially, upregulation and nuclear translocation of the potent oligodendrocyte differentiation inhibitors ID2/4. Collectively, these findings suggest that GPR17 orchestrates the transition between immature and myelinating oligodendrocytes via an ID protein–mediated negative regulation and may serve as a potential therapeutic target for CNS myelin repair.

Peroxynitrite-Induced Neuronal Apoptosis Is Mediated by Intracellular Zinc Release and 12-Lipoxygenase Activation

Peroxynitrite toxicity is a major cause of neuronal injury in stroke and neurodegenerative disorders. The mechanisms underlying the neurotoxicity induced by peroxynitrite are still unclear. In this study, we observed that TPEN [N,N,N′,N′-tetrakis (2-pyridylmethyl)ethylenediamine], a zinc chelator, protected against neurotoxicity induced by exogenous as well as endogenous (coadministration of NMDA and a nitric oxide donor, diethylenetriamine NONOate) peroxynitrite. Two different approaches to detecting intracellular zinc release demonstrated the liberation of zinc from intracellular stores by peroxynitrite. In addition, we found that peroxynitrite toxicity was blocked by inhibitors of 12-lipoxygenase (12-LOX), p38 mitogen-activated protein kinase (MAPK), and caspase-3 and was associated with mitochondrial membrane depolarization. Inhibition of 12-LOX blocked the activation of p38 MAPK and caspase-3. Zinc itself induced the activation of 12-LOX, generation of reactive oxygen species (ROS), and activation of p38 MAPK and caspase-3. These data suggest a cell death pathway triggered by peroxynitrite in which intracellular zinc release leads to activation of 12-LOX, ROS accumulation, p38 activation, and caspase-3 activation. Therefore, therapies aimed at maintaining intracellular zinc homeostasis or blocking activation of 12-LOX may provide a novel avenue for the treatment of inflammation, stroke, and neurodegenerative diseases in which the formation of peroxynitrite is thought to be one of the important causes of cell death.

Nitric oxide-induced cell death in developing oligodendrocytes is associated with mitochondrial dysfunction and apoptosis-inducing factor translocation

Reactive nitrogen species are thought to be involved in both hypoxic‐ischemic and cytokine‐induced brain injury, including periventricular leukomalacia (PVL), the major pathological substrate of cerebral palsy in premature infants. PVL appears to be the result of perinatal inflammatory events and hypoxic‐ischemic injury to the cerebral white matter. The chronic disturbance of myelination resulting from PVL suggests that developing oligodendrocytes (OLs) are involved in its pathogenesis. We hypothesized that nitric oxide (NO) could participate in the pathogenesis of PVL through a toxic effect on developing OLs. Using primary cultures of highly enriched OLs we found that NO is toxic to developing OLs (O4+, O1–, MBP–), with an EC50 value of 236u2003±u2003125u2003µm of DETANOnoate. Peroxynitrite formation does not appear to be involved in NO toxicity in developing OLs, as determined by the failure of peroxynitrite scavengers as well as superoxide dismutase overexpression to prevent NO‐induced toxicity. Similarly, several pathways involving PARP, excitotoxicity, guanylyl cyclase and caspase activation were not related to NO toxicity to developing OLs. NO toxicity to OLs resulted in ATP depletion and loss of mitochondrial membrane potential (ΔΨ) in developing OLs. Apoptosis‐inducing factor (AIF) has been shown to be involved in caspase‐independent cell death, and we found that AIF translocated from mitochondria into the nucleus upon NO exposure. In conclusion, we suggest that the vulnerability of developing OLs to NO involves mitochondrial dysfunction and translocation of AIF from mitochondria to nuclei.

Tumor necrosis factor alpha mediates lipopolysaccharide-induced microglial toxicity to developing oligodendrocytes when astrocytes are present.

Reactive microglia and astrocytes are present in lesions of white matter disorders, such as periventricular leukomalacia and multiple sclerosis. However, it is not clear whether they are actively involved in the pathogenesis of these disorders. Previous studies demonstrated that microglia, but not astrocytes, are required for lipopolysaccharide (LPS)-induced selective killing of developing oligodendrocytes (preOLs) and that the toxicity is mediated by microglia-derived peroxynitrite. Here we report that, when astrocytes are present, the LPS-induced, microglia-dependent toxicity to preOLs is no longer mediated by peroxynitrite but instead by a mechanism dependent on tumor necrosis factor-α (TNFα) signaling. Blocking peroxynitrite formation with nitric oxide synthase (NOS) inhibitors or a decomposition catalyst did not prevent LPS-induced loss of preOLs in mixed glial cultures. PreOLs were highly vulnerable to peroxynitrite; however, the presence of astrocytes prevented the toxicity. Whereas LPS failed to kill preOLs in cocultures of microglia and preOLs deficient in inducible NOS (iNOS) or gp91phox, the catalytic subunit of the superoxide-generating NADPH oxidase, LPS caused a similar degree of preOL death in mixed glial cultures of wild-type, iNOS−/−, and gp91phox−/− mice. TNFα neutralizing antibody inhibited LPS toxicity, and addition of TNFα induced selective preOL injury in mixed glial cultures. Furthermore, disrupting the genes encoding TNFα or its receptors TNFR1/2 completely abolished the deleterious effect of LPS. Our results reveal that TNFα signaling, rather than peroxynitrite, is essential in LPS-triggered preOL death in an environment containing all major glial cell types and underscore the importance of intercellular communication in determining the mechanism underlying inflammatory preOL death.

Microfluidic compartmentalized co-culture platform for CNS axon myelination research

This paper presents a circular microfluidic compartmentalized co-culture platform that can be used for central nervous system (CNS) axon myelination research. The microfluidic platform is composed of a soma compartment and an axon/glia compartment connected through arrays of axon-guiding microchannels. Myelin-producing glia, oligodendrocytes (OLs), placed in the axon/glia compartment, interact with only axons but not with neuronal somata confined to the soma compartment, reminiscent to in vivo situation where many axon fibres are myelinated by OLs at distance away from neuronal cell bodies. Primary forebrain neurons from embryonic day 16–18 rats were cultured inside the soma compartment for two weeks to allow them to mature and form extensive axon networks. OL progenitors, isolated from postnatal day 1-2 rat brains, were then added to the axon/glia compartment and co-cultured with neurons for an additional two weeks. The microdevice showed fluidic isolation between the two compartments and successfully isolated neuronal cell bodies and dendrites from axons growing through the arrays of axon-guiding microchannels into the axon/glia compartment. The circular co-culture device developed here showed excellent cell loading characteristics where significant numbers of cells were positioned near the axon-guiding microchannels. This significantly increased the probability of axons crossing these microchannels as demonstrated by the more than 51 % of the area of the axon/glia compartment covered with axons two weeks after cell seeding. OL progenitors co-cultured with axons inside the axon/glia compartment successfully differentiated into mature OLs. These results indicate that this device can be used as an excellent in vitro co-culture platform for studying localized axon-glia interaction and signalling.

The anti-apoptotic actions of nitric oxide in hepatocytes.

Nitric oxide (NO) has anti-apoptotic actions in hepatocytes in vitro and in vivo. The protection is mediated via the S-nitrosylation of procaspases as well as the active caspase enzymes. NO stimulation of the cGMP/protein kinase G pathway also appears to contribute to the protective effect of NO. Here we review the evidence for the direct inhibition of NO with the apoptotic signaling pathways in hepatocytes.

12-Lipoxygenase plays a key role in cell death caused by glutathione depletion and arachidonic acid in rat oligodendrocytes

Oxidative injury to premyelinating oligodendrocytes (preOLs) in developing white matter has been implicated in the pathogenesis of periventricular leukomalacia, the lesion underlying most cases of cerebral palsy in premature infants. In this study, we investigated the pathways of OL death induced by intracellular glutathione (GSH) depletion. We found that the lipoxygenase (LOX) inhibitors AA‐861 and BMD‐122 (N‐benzyl‐N‐hydroxy‐5‐phenylpentamide; BHPP), but not the cyclooxygenase (COX) inhibitor indomethacin, fully protected the cells from GSH depletion caused by cystine deprivation. Arachidonic acid (AA), the substrate for 12‐LOX, potentiated the toxicity of mild cystine deprivation and at higher concentration was itself toxic. This toxicity was also blocked by 12‐LOX inhibitors. Consistent with a role for 12‐LOX in the cell death pathway, 12‐LOX activity increased following cystine deprivation in OLs. Blocking 12‐LOX with AA‐861 effectively inhibited the accumulation of reactive oxygen species (ROS) induced by cystine deprivation. These data suggest that, in OLs, intracellular GSH depletion leads to activation of 12‐LOX, ROS accumulation and cell death. Mature OLs were more resistant than preOLs to cystine deprivation. The difference in sensitivity was not due to a difference in 12‐LOX activity but rather appeared to be related to the presence of stronger antioxidant defense mechanisms in mature OLs. These results suggest that 12‐LOX activation plays a key role in oxidative stress‐induced OL death.