Mitra Kooshki

PPARα ligands inhibit radiation-induced microglial inflammatory responses by negatively regulating NF-κB and AP-1 pathways

Whole-brain irradiation (WBI) can lead to cognitive impairment several months to years after irradiation. Studies on rodents have shown a rapid and sustained increase in activated microglia (brain macrophages) following brain irradiation, contributing to a chronic inflammatory response and a corresponding decrease in hippocampal neurogenesis. Thus, alleviating microglial activation following radiation represents a key strategy to minimize WBI-induced morbidity. We hypothesized that pretreatment with peroxisomal proliferator-activated receptor (PPAR)alpha agonists would ameliorate the proinflammatory responses seen in the microglia following in vitro radiation. Irradiating BV-2 cells (a murine microglial cell line) with single doses (2-10 Gy) of (137)Cs gamma-rays led to increases in (1) the gene expression of IL-1beta and TNFalpha, (2) Cox-2 protein levels, and (3) intracellular ROS generation. In addition, an increase in the DNA-binding activity of redox-regulated proinflammatory transcription factors AP-1 and NF-kappaB was observed. Pretreating BV-2 cells with the PPARalpha agonists GW7647 and Fenofibrate significantly inhibited the radiation-induced microglial proinflammatory response, in part, via decreasing (i) the nuclear translocation of the NF-kappaB p65 subunit and (ii) phosphorylation of the c-jun subunit of AP-1 in the nucleus. Taken together, these data support the hypothesis that activation of PPARalpha can modulate the radiation-induced microglial proinflammatory response.

The PPARα Agonist Fenofibrate Preserves Hippocampal Neurogenesis and Inhibits Microglial Activation After Whole-Brain Irradiation

PURPOSE Whole-brain irradiation (WBI) leads to cognitive impairment months to years after radiation. Numerous studies suggest that decreased hippocampal neurogenesis and microglial activation are involved in the pathogenesis of WBI-induced brain injury. The goal of this study was to investigate whether administration of the peroxisomal proliferator-activated receptor (PPAR) alpha agonist fenofibrate would prevent the detrimental effect of WBI on hippocampal neurogenesis. METHODS AND MATERIALS For this study, 129S1/SvImJ wild-type and PPARalpha knockout mice that were fed either regular or 0.2% wt/wt fenofibrate-containing chow received either sham irradiation or WBI (10-Gy single dose of (137)Cs gamma-rays). Mice were injected intraperitoneally with bromodeoxyuridine to label the surviving cells at 1 month after WBI, and the newborn neurons were counted at 2 months after WBI by use of bromodeoxyuridine/neuronal nuclei double immunofluorescence. Proliferation in the subgranular zone and microglial activation were measured at 1 week and 2 months after WBI by use of Ki-67 and CD68 immunohistochemistry, respectively. RESULTS Whole-brain irradiation led to a significant decrease in the number of newborn hippocampal neurons 2 months after it was performed. Fenofibrate prevented this decrease by promoting the survival of newborn cells in the dentate gyrus. In addition, fenofibrate treatment was associated with decreased microglial activation in the dentate gyrus after WBI. The neuroprotective effects of fenofibrate were abolished in the knockout mice, indicating a PPARalpha-dependent mechanism or mechanisms. CONCLUSIONS These data highlight a novel role for PPARalpha ligands in improving neurogenesis after WBI and offer the promise of improving the quality of life for brain cancer patients receiving radiotherapy.

Chronic Administration of the Angiotensin-Converting Enzyme Inhibitor, Ramipril, Prevents Fractionated Whole-Brain Irradiation-Induced Perirhinal Cortex-Dependent Cognitive Impairment

We hypothesized that chronic administration of the angiotensin-converting enzyme inhibitor, ramipril, to young adult male rats would prevent/ameliorate fractionated whole-brain irradiation-induced perirhinal cortex-dependent cognitive impairment. Eighty 12–14-week-old young adult male Fischer 344 rats received either: (1) sham irradiation, (2) 40 Gy of fractionated whole-brain irradiation delivered as two 5 Gy fractions/week for 4 weeks, (3) sham irradiation plus continuous administration of 15 mg/L of ramipril in the drinking water starting 3 days before irradiation, or (4) fractionated whole-brain irradiation plus ramipril. Cognitive function was assessed using a perirhinal cortex-dependent version of the novel object recognition task 26 weeks after irradiation. Microglial activation was determined in the perirhinal cortex and the dentate gyrus of the hippocampus 28 weeks after irradiation using the ED1 antibody. Neurogenesis was assessed in the granular cell layer and subgranular zones of the dentate gyrus using a doublecortin antibody. Fractionated whole-brain irradiation led to: (1) a significant impairment in perirhinal cortex-dependent cognitive function, (2) a significant increase in activated microglia in the dentate gyrus but not in the perirhinal cortex, and (3) a significant decrease in neurogenesis. Continuous administration of ramipril before, during, and after irradiation prevented the fractionated whole-brain irradiation-induced changes in perirhinal cortex-dependent cognitive function, as well as in microglial activation in the dentate gyrus. Thus, as hypothesized, continuous administration of the angiotensin-converting enzyme inhibitor, ramipril, can prevent the fractionated whole-brain irradiation-induced impairment in perirhinal cortex-dependent cognitive function.

Decreasing peroxiredoxin II expression decreases glutathione, alters cell cycle distribution, and sensitizes glioma cells to ionizing radiation and H2O2

Glioblastomas are notorious for their resistance to ionizing radiation and chemotherapy. We hypothesize that this resistance to ionizing radiation is due, in part, to alterations in antioxidant enzymes. Here, we show that rat and human glioma cells overexpress the antioxidant enzyme peroxiredoxin II (Prx II). Glioma cells in which Prx II is decreased using shRNA exhibit increased hyperoxidation of the remaining cellular Prxs, suggesting that the redox environment is more oxidizing. Of interest, decreasing Prx II does not alter other antioxidant enzymes (i.e., catalase, GPx, Prx I, Prx III, CuZnSOD, and MnSOD). Analysis of the redox environment revealed that decreasing Prx II increased intracellular reactive oxygen species in 36B10 cells; extracellular levels of H(2)O(2) were also increased in both C6 and 36B10 cells. Treatment with H(2)O(2) led to a further elevation in intracellular reactive oxygen species in cells where Prx II was decreased. Decreasing Prx II expression in glioma cells also reduced clonogenic cell survival following exposure to ionizing radiation and H(2)O(2). Furthermore, lowering Prx II expression decreased intracellular glutathione and resulted in a significant decline in glutathione reductase activity, suggesting a possible mechanism for the observed increased sensitivity to oxidative insults. Additionally, decreasing Prx II expression increased cell cycle doubling times, with fewer cells distributed to S phase in C6 glioma cells and more cells redistributed to the most radiosensitive phase of the cell cycle, G2/M, in 36B10 glioma cells. These findings support the hypothesis that inhibiting Prx II sensitizes glioma cells to oxidative stress, presenting Prxs as potential therapeutic targets.

PPARδ prevents radiation-induced proinflammatory responses in microglia via transrepression of NF-κB and inhibition of the PKCα/MEK1/2/ERK1/2/AP-1 pathway

Partial or whole-brain irradiation is often required to treat both primary and metastatic brain cancer. Radiation-induced normal tissue injury, including progressive cognitive impairment, however, can significantly affect the well-being of the approximately 200,000 patients who receive these treatments each year in the United States. Although the exact mechanisms underlying radiation-induced late effects remain unclear, oxidative stress and inflammation are thought to play a critical role. Microglia are key mediators of neuroinflammation. Peroxisomal proliferator-activated receptor (PPAR) δ has been shown to be a potent regulator of anti-inflammatory responses. Thus, we hypothesized that PPARδ activation would modulate the radiation-induced inflammatory response in microglia. Incubating BV-2 murine microglial cells with the PPARδ agonist L-165041 prevented the radiation-induced increase in: (i) intracellular reactive oxygen species generation, (ii) Cox-2 and MCP-1 expression, and (iii) IL-1β and TNF-α message levels. This occurred, in part, through PPARδ-mediated modulation of stress-activated kinases and proinflammatory transcription factors. PPARδ inhibited NF-κB via transrepression by physically interacting with the p65 subunit and prevented activation of the PKCα/MEK1/2/ERK1/2/AP-1 pathway by inhibiting the radiation-induced increase in intracellular reactive oxygen species generation. These data support the hypothesis that PPARδ activation can modulate radiation-induced oxidative stress and inflammatory responses in microglia.

The PPARδ agonist GW0742 inhibits neuroinflammation, but does not restore neurogenesis or prevent early delayed hippocampal-dependent cognitive impairment after whole-brain irradiation.

Brain tumor patients often develop cognitive impairment months to years after partial or fractionated whole-brain irradiation (WBI). Studies suggest that neuroinflammation and decreased hippocampal neurogenesis contribute to the pathogenesis of radiation-induced brain injury. In this study, we determined if the peroxisomal proliferator-activated receptor (PPAR) δ agonist GW0742 can prevent radiation-induced brain injury in C57Bl/6 wild-type (WT) and PPARδ knockout (KO) mice. Dietary GW0742 prevented the acute increase in IL-1β mRNA and ERK phosphorylation measured at 3h after a single 10-Gy dose of WBI; it also prevented the increase in the number of activated hippocampal microglia 1 week after WBI. In contrast, dietary GW074 failed to prevent the radiation-induced decrease in hippocampal neurogenesis determined 2 months after WBI in WT mice or to mitigate their hippocampal-dependent spatial memory impairment measured 3 months after WBI using the Barnes maze task. PPARδ KO mice exhibited defects including decreased numbers of astrocytes in the dentate gyrus/hilus of the hippocampus and a failure to exhibit a radiation-induced increase in activated hippocampal microglia. Interestingly, the number of astrocytes in the dentate gyrus/hilus was reduced in WT mice, but not in PPARδ KO mice 2 months after WBI. These results demonstrate that, although dietary GW0742 prevents the increase in inflammatory markers and hippocampal microglial activation in WT mice after WBI, it does not restore hippocampal neurogenesis or prevent early delayed hippocampal-dependent cognitive impairment after WBI. Thus, the exact relationship between radiation-induced neuroinflammation, neurogenesis, and cognitive impairment remains elusive.

Knocking Out Peroxisome Proliferator-Activated Receptor (PPAR) α Inhibits Radiation-Induced Apoptosis in the Mouse Kidney through Activation of NF-κB and Increased Expression of IAPs

Abstract Zhao, W., Iskandar, S., Kooshki, M., Sharpe, J. G., Payne, V. and Robbins, M. E. Knocking Out Peroxisome Proliferator-Activated Receptor (PPAR) α Inhibits Radiation-Induced Apoptosis in the Mouse Kidney through Activation of NF-κB and Increased Expression of IAPs. Radiat. Res. 167, 581–591 (2007). Peroxisome proliferator-activated receptor (PPAR) α, a member of the ligand-activated nuclear receptor superfamily, plays an important role in lipid metabolism and glucose homeostasis and is highly expressed in the kidney. The present studies were aimed at testing the hypothesis that PPARα knockout mice would exhibit decreased radiation-induced apoptosis due to exacerbated activation of NF-κB (NFKB) and expression of pro-survival factors. Thirty wild-type mice (29S1/SvImJ) and 30 PPARα knockout mice were irradiated with a single total-body dose 10 Gy of 137Cs γ rays; controls were sham-irradiated. Tissue samples were collected at 3, 6, 12, 24 and 48 h postirradiation. Apoptosis was quantified using immunohistochemical staining for apoptotic bodies and cleaved caspase 3. Radiation-induced apoptosis was observed in both mouse strains in a time-dependent manner. However, the level of apoptosis was significantly suppressed in PPARα knockout mice compared with wild-type mice at 6 h postirradiation (P < 0.05). This inhibition of radiation-induced apoptosis was associated with time-dependent increases in NF-κB DNA-binding activity, IκBα phosphorylation, and expression of other antiapoptosis factors in the PPARα knockout mouse kidneys but not in wild-type animals. These data support the hypothesis that the loss of PPARα expression leads to the suppression of radiation-induced apoptosis in the mouse kidney, mediated through activation of NF-κB and up-regulation of anti-apoptosis factors.

Pathogen inducible reporting in transgenic tobacco using a GFP construct

A model sentinel plant (phytosensor) capable of signaling pathogen attack in the field was obtained by transformation with a construct containing the green fluorescent protein (GFP) as a reporter gene, driven by the promoter of gn1 , a tobacco b-1,3glucanase gene. This is a first step towards the creation of sentinel plants for early disease diagnostics with an integral real-time, fluorescent-based reporting mechanism. The gn1 promoter is responsive to pathogens and salicylic acid (SA), which is synthesized by the plant during systemic acquired resistance (SAR). Transgenic plants were sprayed with benzo-(1,2,3)-thiadiazole-7-carbothioic acid S -methyl ester (BTH), an SA analogue, at different stages of development. The presence of GFP transcript in transgenic plants after the induction with 5 mM BTH was determined. GFP was detected as early as 48 h after induction using Western blot analysis, but the fluorescence could not be robustly detected spectrally. Plants younger than 8 weeks did not express detectable levels of GFP. We show systemic induction of gn1 /mgfp5-er by BTH in non-treated plant tissues. Time course of induction of gn1 /mgfp5-er by BTH in transgenic plants showed that induction and GFP accumulation was slow and maximum, GFP accumulation was found between 6 and 12 days after treatment. gn1 /mgfp5-er expression was also induced by inoculating plant leaves with the pathogen Plectosporium tabacinum . # 2003 Elsevier Science Ireland Ltd. All rights reserved.

Angiotensin-(1-7) prevents radiation-induced inflammation in rat primary astrocytes through regulation of MAP kinase signaling.

About 500,000 new cancer patients will develop brain metastases in 2013. The primary treatment modality for these patients is partial or whole brain irradiation which leads to a progressive, irreversible cognitive impairment. Although the exact mechanisms behind this radiation-induced brain injury are unknown, neuroinflammation in glial populations is hypothesized to play a role. Blockers of the renin-angiotensin system (RAS) prevent radiation-induced cognitive impairment and modulate radiation-induced neuroinflammation. Recent studies suggest that RAS blockers may reduce inflammation by increasing endogenous concentrations of the anti-inflammatory heptapeptide angiotensin-(1-7) [Ang-(1-7)]. Ang-(1-7) binds to the AT(1-7) receptor and inhibits MAP kinase activity to prevent inflammation. This study describes the inflammatory response to radiation in astrocytes characterized by radiation-induced increases in (i) IL-1β and IL-6 gene expression; (ii) COX-2 and GFAP immunoreactivity; (iii) activation of AP-1 and NF-κB transcription factors; and (iv) PKCα, MEK, and ERK (MAP kinase) activation. Treatment with U-0126, a MEK inhibitor, demonstrates that this radiation-induced inflammation in astrocytes is mediated through the MAP kinase pathway. Ang-(1-7) inhibits radiation-induced inflammation, increases in PKCα, and MAP kinase pathway activation (phosphorylation of MEK and ERK). Additionally Ang-(1-7) treatment leads to an increase in dual specificity phosphatase 1 (DUSP1). Furthermore, treatment with sodium vanadate (Na3VO4), a phosphatase inhibitor, blocks Ang-(1-7) inhibition of radiation-induced inflammation and MAP kinase activation, suggesting that Ang-(1-7) alters phosphatase activity to inhibit radiation-induced inflammation. These data suggest that RAS blockers inhibit radiation-induced inflammation and prevent radiation-induced cognitive impairment not only by reducing Ang II but also by increasing Ang-(1-7) levels.

Laser-Induced Fluorescence Imaging and Spectroscopy of GFP Transgenic Plants

Green fluorescent protein (GFP) and other fluorescent protein bioreporters can be used to monitor transgenes in plants. GFP is a valuable marker for transgene presence and expression, but remote sensing instrumentation for stand-off detection has lagged behind fluorescent protein marker biotechnology. However, both biology and photonics are needed for the monitoring technology to be fully realized. In this paper, we describe laser-induced fluorescence imaging and laser-induced fluorescence spectroscopy of GFP-transgenic plants in ambient light towards the application of remote sensing of transgenic plants producing GFP.