ag-490 and Hypoxia

The cytokine erythropoietin (Epo) initiates adaptive cellular responses to both moderate environmental challenges and tissue damaging insults in various non-hematopoietic mammalian tissues including the nervous system. Neuroprotective and neuroregenerative functions of Epo in mammals are mediated through receptor-associated Janus kinase 2 and intracellular signaling cascades that modify the transcription of Epo-regulated genes. Signal transducers and activators of transcription (STAT) and phosphoinositol-3-kinase (PI3K) represent key components of two important Epo-induced transduction pathways. Our previous study on insects revealed neuroprotective and regenerative functions of recombinant human Epo (rhEpo) similar to those in mammalian nervous tissues. Here we demonstrate that rhEpo effectively rescues primary cultured locust brain neurons from apoptotic cell death induced by hypoxia or the chemical compound H-7. The Janus kinase inhibitor AG-490 and the STAT inhibitor sc-355797 abolished protective effects of rhEpo on locust brain neurons. In contrast, inhibition of PI3K with LY294002 had no effect on rhEpo-mediated neuroprotection. The results indicate that rhEpo mediates the protection of locust brain neurons through interference with apoptotic pathways by the activation of a Janus kinase-associated receptor and STAT transcription factor(s). The involvement of similar transduction pathways in mammals and insects for the mediation of neuroprotection and support of neural regeneration by Epo indicates that an Epo/Epo receptor-like signaling system with high structural and functional similarity exists in both groups of animals. Epo-like signaling involved in tissue protection appears to be an ancient beneficial function shared by vertebrates and invertebrates.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Apoptosis; Brain; Cell Survival; Cells, Cultured; Chromones; Enzyme Inhibitors; Erythropoietin; Grasshoppers; Humans; Hypoxia; Insect Proteins; Janus Kinase 2; Morpholines; Neurons; Neuroprotective Agents; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Signal Transduction; STAT Transcription Factors; Tyrphostins

The cellular and molecular mechanisms that govern the response of the perinatal brain to injury remain largely unexplored. We investigated the role of white matter astrocytes in a rodent model of diffuse white matter injury produced by exposing neonatal mice to chronic hypoxia-a paradigm that mimics brain injury in premature infants. We demonstrate the absence of reactive gliosis in the immature white matter following chronic hypoxia, as determined by astrocyte proliferation index and glial fibrillary acidic protein levels. Instead, Nestin expression in astrocytes is transiently increased, and the glial-specific glutamate transporters glutamate-aspartate transporter (GLAST) and glutamate transporter 1 (GLT-1) are reduced. Finally, we demonstrate that Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling-which is important in both astrocyte development and response to injury-is reduced in the white matter following hypoxia, as well as in primary astrocytes exposed to hypoxia in vitro. Hypoxia and JAK/STAT inhibition reduce glutamate transporter expression in astrocytes, but unlike hypoxia JAK/STAT inhibition downregulates GLAST expression without affecting GLT-1, as demonstrated in vitro by treatment with JAK inhibitor I and in vivo by treatment with the JAK/STAT inhibitor AG490 [(E)-2-cyano-3-(3,4-dihydrophenyl)-N-(phenylmethyl)-2-propenamide]. Our findings (1) demonstrate specific changes in astrocyte function after perinatal hypoxia, which might contribute to the particular pathogenesis of perinatal white matter injury, (2) provide evidence that at least part of these changes result from a disturbance of the JAK/STAT pathway by hypoxia, and (3) identify JAK/STAT signaling as a potential therapeutic target to restore normal GLAST expression and uptake of glutamate after perinatal brain injury.

Topics: Age Factors; Amino Acid Transport System X-AG; Animals; Animals, Newborn; Aspartic Acid; Astrocytes; Bromodeoxyuridine; Cell Count; Cells, Cultured; Disease Models, Animal; Enzyme Inhibitors; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Gliosis; Green Fluorescent Proteins; Hypoxia; Intermediate Filament Proteins; Janus Kinases; Male; Mice; Mice, Transgenic; Nerve Tissue Proteins; Nestin; Signal Transduction; STAT Transcription Factors; Tritium; Tyrphostins

Cerebral ischemia and reperfusion increase superoxide anions (O(2)(*-)) in brain mitochondria. Manganese superoxide dismutase (Mn-SOD; SOD2), a primary mitochondrial antioxidant enzyme, scavenges superoxide radicals and its overexpression provides neuroprotection. However, the regulatory mechanism of Mn-SOD expression during cerebral ischemia and reperfusion is still unclear. In this study, we identified the signal transducer and activator of transcription 3 (STAT3) as a transcription factor of the mouse Mn-SOD gene, and elucidated the mechanism of O(2)(*-) overproduction after transient focal cerebral ischemia (tFCI). We found that Mn-SOD expression is significantly reduced by reperfusion in the cerebral ischemic brain. We also found that activated STAT3 is usually recruited into the mouse Mn-SOD promoter and upregulates transcription of the mouse Mn-SOD gene in the normal brain. However, at early postreperfusion periods after tFCI, STAT3 was rapidly downregulated, and its recruitment into the Mn-SOD promoter was completely blocked. In addition, transcriptional activity of the mouse Mn-SOD gene was significantly reduced by STAT3 inhibition in primary cortical neurons. Moreover, we found that STAT3 deactivated by reperfusion induces accumulation of O(2)(*-) in mitochondria. The loss of STAT3 activity induced neuronal cell death by reducing Mn-SOD expression. Using SOD2-/+ heterozygous knock-out mice, we found that Mn-SOD is a direct target of STAT3 in reperfusion-induced neuronal cell death. Our study demonstrates that STAT3 is a novel transcription factor of the mouse Mn-SOD gene and plays a crucial role as a neuroprotectant in regulating levels of reactive oxygen species in the mouse brain.

Topics: Animals; Brain; Brain Infarction; Brain Ischemia; Cells, Cultured; Chromatin Immunoprecipitation; Cytochromes c; Disease Models, Animal; Electrophoretic Mobility Shift Assay; Embryo, Mammalian; Glucose; Humans; Hypoxia; Interleukin-6; Male; Mice; Mice, Knockout; Neurons; Neuroprotective Agents; Reperfusion; RNA, Small Interfering; STAT3 Transcription Factor; Superoxide Dismutase; Time Factors; Transfection; Tyrphostins; Up-Regulation