Lucian Soane

Sulforaphane Protects Astrocytes Against Oxidative Stress and Delayed Death Caused by Oxygen and Glucose Deprivation

Oxidative stress is an important molecular mechanism of astrocyte injury and death following ischemia/reperfusion and may be an effective target of intervention. One therapeutic strategy for detoxifying the many different reactive oxygen and nitrogen species that are produced under these conditions is induction of the Phase II gene response by the use of chemicals or conditions that promote the translocation of the transcriptional activating factor NRF2 from the cytosol to the nucleus, where it binds to genomic antioxidant response elements. This study tested the hypothesis that pre‐ or post‐treatment of cultured cortical astrocytes with sulforaphane, an alkylating agent known to activate the NRF2 pathway of gene expression protects against death of astrocytes caused by transient exposure to O2 and glucose deprivation (OGD). Rat cortical astrocytes were exposed to 5 μM sulforaphane either 48 h prior to, or for 48 h after a 4‐h period of OGD. Both pre‐ and post‐treatments significantly reduced cell death at 48 h after OGD. Immunostaining for 8‐hydroxy‐2‐deoxyguanosine, a marker of DNA/RNA oxidation, was reduced at 4 h reoxygenation with sulforaphane pretreatment. Sulforaphane exposure was followed by an increase in cellular and nuclear NRF2 immunoreactivity. Moreover, sulforaphane also increased the mRNA, protein level, and enzyme activity of NAD(P)H/Quinone Oxidoreductase1, a known target of NRF2 transcriptional activation. We conclude that sulforaphane stimulates the NRF2 pathway of antioxidant gene expression in astrocytes and protects them from cell death in an in vitro model of ischemia/reperfusion.

C5b-9 Terminal Complement Complex Protects Oligodendrocytes from Death by Regulating Bad Through Phosphatidylinositol 3-Kinase/Akt Pathway

Apoptosis of oligodendrocytes is induced by serum growth factor deprivation. We showed that oligodendrocytes and progenitor cells respond to serum withdrawal by a rapid decline of Bcl-2 mRNA expression and caspase-3-dependent apoptotic death. Sublytic assembly of membrane-inserted terminal complement complexes consisting of C5b, C6, C7, C8, and C9 proteins (C5b-9) inhibits caspase-3 activation and apoptotic death of oligodendrocytes. In this study, we examined an involvement of the mitochondria in oligodendrocyte apoptosis and the role of C5b-9 on this process. Decreased phosphatidylinositol 3-kinase and Akt activities occurred in association with cytochrome c release and caspase-9 activation when cells were placed in defined medium. C5b-9 inhibited the mitochondrial pathway of apoptosis in oligodendrocytes, as shown by decreased cytochrome c release and inhibition of caspase-9 activation. Phosphatidylinositol 3-phosphate kinase and Akt activities were also induced by C5b-9, and the phosphatidylinositol 3-phosphate kinase inhibitor LY294002 reversed the protective effect of C5b-9. Phosphatidylinositol 3-phosphate kinase activity was also responsible for the phosphorylation of Bad at Ser112 and Ser136. This phosphorylation resulted in dissociation of Bad from the Bad/Bcl-xL complex in a Giα-dependent manner. The mitochondrial pathway of oligodendrocyte apoptosis is, therefore, inhibited by C5b-9 through post-translational regulation of Bad. This mechanism may be involved in the promotion of oligodendrocyte survival in inflammatory demyelinating disorders affecting the CNS.

Mechanisms of Impaired Mitochondrial Energy Metabolism in Acute and Chronic Neurodegenerative Disorders

Altered mitochondrial energy metabolism contributes to the pathophysiology of acute brain injury caused by ischemia, trauma, and neurotoxins and by chronic neurodegenerative disorders such as Parkinsons and Huntingtons diseases. Although much evidence supports that the electron transport chain dysfunction in these metabolic abnormalities has both genetic and intracellular environmental causes, alternative mechanisms are being explored. These include direct, reversible inhibition of cytochrome oxidase by nitric oxide, release of mitochondrial cytochrome c, oxidative inhibition of mitochondrial matrix dehydrogenases and adenine nucleotide transport, the availability of NAD for dehydrogenase reactions, respiratory uncoupling by activities such as that of the permeability transition pore, and altered mitochondrial structure and intracellular trafficking. This review focuses on the catabolism of neuronal NAD and the release of neuronal mitochondrial NAD as important contributors to metabolic dysfunction. In addition, the relationship between apoptotic signaling cascades and disruption of mitochondrial energy metabolism is considered in light of the fine balance between apoptotic and necrotic neural cell death.

Molecular Cloning and Characterization of RGC-32, a Novel Gene Induced by Complement Activation in Oligodendrocytes

Sublytic complement activation on oligodendrocytes (OLG) down-regulates expression of myelin genes and induces cell cycle in culture. Differential display (DD) was used to search for new genes whose expression is altered in response to complement and that may be involved in cell cycle activation. DD bands showing either increased or decreased mRNA expression in response to complement were identified and designated ResponseGenes to Complement (RGC) 1–32.RGC-1 is identical with heat shock protein 105,RGC-2 with poly(ADP-ribose) polymerase, andRGC-10 with IP-10. A new gene, RGC-32, that encodes a protein of 137 amino acids was cloned. RGC-32 has no homology with other known proteins, and contains no motif that would indicate its function. In OLG, the mRNA expression was increased by complement activation and by terminal complement complex assembly. RGC-32 protein was localized in the cytoplasm and co-immunoprecipitated with cdc2 kinase. Overexpression of RGC-32 increased DNA synthesis in OLGxC6 glioma cell hybrids. These results suggest thatRGC-32 may play a role in cell cycle activation.

The Potential Role of Mitochondria in Pediatric Traumatic Brain Injury

Mitochondria play a central role in cerebral energy metabolism, intracellular calcium homeostasis and reactive oxygen species generation and detoxification. Following traumatic brain injury (TBI), the degree of mitochondrial injury or dysfunction can be an important determinant of cell survival or death. Literature would suggest that brain mitochondria from the developing brain are very different from those from mature animals. Therefore, aspects of developmental differences in the mitochondrial response to TBI can make the immature brain more vulnerable to traumatic injury. This review will focus on four main areas of secondary injury after pediatric TBI, including excitotoxicity, oxidative stress, alterations in energy metabolism and cell death pathways. Specifically, we will describe what is known about developmental differences in mitochondrial function in these areas, in both the normal, physiologic state and the pathologic state after pediatric TBI. The ability to identify and target aspects of mitochondrial dysfunction could lead to novel neuroprotective therapies for infants and children after severe TBI.

Sulforaphane protects immature hippocampal neurons against death caused by exposure to hemin or to oxygen and glucose deprivation.

Oxidative stress is a mediator of cell death following cerebral ischemia/reperfusion and heme toxicity, which can be an important pathogenic factor in acute brain injury. Induced expression of phase II detoxification enzymes through activation of the antioxidant response element (ARE)/Nrf2 pathway has emerged as a promising approach for neuroprotection. Little is known, however, about the neuroprotective potential of this strategy against injury in immature brain cells. In this study, we tested the hypothesis that sulforaphane (SFP), a naturally occurring isothiocyanate that is also a known activator of the ARE/Nrf2 antioxidant pathway, can protect immature neurons from oxidative stress‐induced death. The hypothesis was tested with primary mouse hippocampal neurons exposed to either O2 and glucose deprivation (OGD) or hemin. Treatment of immature neurons with SFP immediately after the OGD during reoxygenation was effective in protecting immature neurons from delayed cell death. Exposure of immature hippocampal neurons to hemin induced significant cell death, and both pre‐ and cotreatment with SFP were remarkably effective in blocking cytotoxicity. RT‐PCR analysis indicated that several Nrf2‐dependent cytoprotective genes, including NAD(P)H quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO1), and glutamate‐cysteine ligase modifier subunit (GCLM), which is involved in glutathione biosynthesis, were up‐regulated following SFP treatment both in control neurons and following exposure to OGD and hemin. These results indicate that SFP activates the ARE/Nrf2 pathway of antioxidant defense and protects immature neurons from death caused by stress paradigms relevant to those associated with ischemic and traumatic injury to the immature brain.

Tyrosine phosphorylation and activation of Janus kinase 1 and STAT3 by sublytic C5b-9 complement complex in aortic endothelial cells.

The pathway involving Janus kinase (JAK) and signal transducers and activators of transcription (STATs) plays an important role in differentiation and proliferation of cells initiated by receptor activation. In the present study we identified the JAK and STAT proteins activated by C5b-9 in human aortic endothelial cells (AEC). JAK1 but not JAK2 was tyrosine phosphorylated in response to sublytic C5b-9. STAT3 was rapidly tyrosine phosphorylated also by C5b-9. Pertussis toxin inhibited the C5b-9 induced JAK1 activation. However, phosphorylation of STAT3 was not inhibited by Pertussis toxin, although C5b-9 induced a time-dependent nuclear translocation of STAT3. These observations indicated that JAK1 is phosphorylated by C5b-9 through activation of trimeric G proteins of the Gi/Go family. Raf-1 and ERK1 were also activated by C5b-9 in human AEC in a G protein dependent manner. Therefore, JAK1 activity may be involved in activation of Raf-1 and ERK1 via G proteins activated by C5b-9. This study demonstrates the ability of membrane-inserted C5b-9 to activate JAK1 and STAT3 proteins, thus defining new signalling pathway by which C5b-9 may regulate gene activation.

Sublytic C5b-9-stimulated Schwann cell survival through PI 3-kinase-mediated phosphorylation of BAD

Sublytic C5b‐9 induces cell cycle activation, proliferation, and rescue from apoptosis in Schwann cells. The signaling pathways for C5b‐9‐mediated rescue were investigated. Following serum withdrawal, DNA fragmentation, detected by TUNEL and FACS analysis, was 56.7% ± 7.3 and 91.9% ± 2.4 in cultured sciatic nerve Schwann cells from 6‐day‐old rats after 18 h and 24 h, respectively. Apoptosis was confirmed by inhibition of DNA fragmentation in a dose‐dependent manner by DMQD‐CHO, a caspase‐3 inhibitor. Treatment with sublytic C5b‐9 generated with purified components (C5*9) or Ab+C7‐depleted serum (C7dHS)+C7 rescued 89% and 86% of Schwann cells, respectively, as compared with cells treated with C5*6, C8, C9, or Ab+C7dHS. Sublytic C5b‐9 increased Schwann cell PI‐3 kinase and Akt activity maximally at 5 min 3.14 ± 0.5‐fold and 3.56 ± 0.4‐fold, respectively, over controls. ERK‐1 activity was maximally stimulated 2.98‐fold at 15 min. Inhibition of PI‐3 kinase by LY294002 abrogated the C5b‐9‐mediated Schwann cell rescue from apoptosis, while inhibition of ERK‐1 with PD098,059 did not. PI‐3 kinase‐Akt pathway activation by C5b‐9 induced, within 15 min, a 6.34 ± 1.2‐fold increase in BAD phosphorylation at Ser 136, but not at Ser 112. Downstream Bcl‐xL protein was increased 2.61‐fold ± 0.34‐fold by 18 h and 3.9‐fold ± 0.84‐fold by 24 h over controls. LY294002 prevented both BAD phosphorylation at Ser 136 and Bcl‐xL protein induction, while PD098,059 did not. Our data indicated that sublytic C5b‐9 rescued Schwann cell from apoptosis via activation of PI‐3 kinase‐Akt, BAD phosphorylation on Ser 136 and increased expression of Bcl‐xL. Sublytic C5b‐9 detected on Schwann cell in vivo during inflammatory neuropathy may facilitate survival of Schwann cell capable of remyelination. GLIA 36:58–67, 2001.

TAT-mediated endocytotic delivery of the loop deletion Bcl-2 protein protects neurons against cell death

Protein delivery mediated by protein transduction domains (PTD) such as the HIV‐1 TAT‐PTD has emerged as a promising approach for neuroprotection. The objective of this study was to generate and evaluate the neuroprotective potential of TAT fusion proteins using constructs based on Bcl‐2 anti‐death family proteins. A TAT‐Bcl‐2 construct with the loop domain deleted (TAT‐Bcl‐2Δloop) was tested for its ability to transduce neuronal cells and to promote survival. The potential mechanism of TAT‐mediated protein internalization in neural cells was also investigated. The purified TAT‐Bcl‐2Δloop binds to neural cell and rat brain mitochondria, and transduces cultured neural cell lines and primary cortical neurons when used at nm concentrations. Effective internalization of TAT‐Bcl‐2Δloop occurs at 37°C but not at 4°C, consistent with an endocytotic process. Both cell association and internalization require interaction of TAT‐Bcl‐2Δloop with cell surface heparan sulfate proteoglycans. TAT‐mediated protein delivery in neuronal cells occurs through a lipid raft‐dependent endocytotic process, inhibited by the cholesterol‐sequestering agent nystatin. Transducible loop deleted Bcl‐2 increases the survival of cortical neurons following trophic factor withdrawal and also rescues neural cell lines from staurosporine‐induced death. These results support the concept of using protein transduction of Bcl‐2 constructs for neuroprotection.

Postnatal Developmental Regulation of Bcl-2 Family Proteins in Brain Mitochondria

Although it has been long recognized that the relative balance of pro‐ and antiapoptotic Bcl‐2 proteins is critical in determining the susceptibility to apoptotic death, only a few studies have examined the level of these proteins specifically at mitochondria during postnatal brain development. In this study, we examined the age‐dependent regulation of Bcl‐2 family proteins using rat brain mitochondria isolated at various postnatal ages and from the adult. The results indicate that a general down‐regulation of most of the proapoptotic Bcl‐2 proteins present in mitochondria occurs during postnatal brain development. The multidomain proapoptotic Bax, Bak, and Bok are all expressed at high levels in mitochondria early postnatally but decline in the adult. Multiple BH3‐only proteins, including direct activators (Bid, Bim, and Puma) and the derepressor BH3‐only protein Bad, are also present in immature brain mitochondria and are down‐regulated in the adult brain. Antiapoptotic Bcl‐2 family members are differentially regulated, with a shift from high Bcl‐2 expression in immature mitochondria to predominant Bcl‐xL expression in the adult. These results support the concept that developmental differences in upstream regulators of the mitochondrial apoptotic pathway are responsible for the increased susceptibility of cells in the immature brain to apoptosis following injury.