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Overexpression of the inducible heat shock protein 70, Hsp72, has broadly cytoprotective effects and improves outcome following stroke. A full understanding of how Hsp72 protects cells against injury is elusive, though several distinct mechanisms are implicated. One mechanism is its anti-inflammatory effects. We study the effects of Hsp72 overexpression on activation of the transcription factor NF-κB in microglia combining experimentation and mathematical modeling, using TNFα to stimulate a microglial cell line stably overexpressing Hsp72. We find that Hsp72 overexpression reduces the amount of NF-κB DNA binding activity, activity of the upstream kinase IKK, and amount of IκBα inhibitor phosphorylated following TNFα application. Simulations evaluating several proposed mechanisms suggest that inhibition of IKK activation is an essential component of its regulatory activities. Unexpectedly we find that Hsp72 overexpression reduces the initial amount of the RelA/p65 NF-κB subunit in cells, contributing to the attenuated response. Neither mechanism in isolation, however, is sufficient to attenuate the response, providing evidence that Hsp72 relies upon multiple mechanisms to attenuate NF-κB activation. An additional observation from our study is that the induced expression of IκBα is altered significantly in Hsp72 expressing cells. While the mechanism responsible for this observation is not known, it points to yet another means by which Hsp72 may alter the NF-κB response. This study illustrates the multi-faceted nature of Hsp72 regulation of NF-κB activation in microglia and offers further clues to a novel mechanism by which Hsp72 may protect cells against injury.

Recent studies suggest a link between mitochondria and proinflammatory cytokine generation. We previously demonstrated that overexpression of mitochondrial chaperone glucose-regulated protein75 (Grp75/mortalin) protects mitochondria. In this study we investigated the modulation of the lipopolisaccharide (LPS)-induced inflammatory response of microglial BV-2 cells by Grp75. We demonstrate that LPS-induced activation promotes significant metabolic changes suppressing mitochondrial function and increasing glycolysis. Overexpression of Grp75 attenuates the LPS-induced oxidative and metabolic responses, and suppresses proinflammatory activation, which depends on both NF-κB activation and lactate. Thus overexpression of Grp75 provides a novel strategy to modulate proinflammatory cytokine production of relevance to inflammation-associated pathologies., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Background: Activation of the NF-κB transcription factor and its associated gene expression in microglia is a key component in the response to brain injury. Its activation is dynamic and is part of a network of biochemical species with multiple feedback regulatory mechanisms. Mathematical modeling, which has been instrumental for understanding the NF-κB response in other cell types, offers a valuable tool to investigate the regulation of NF-κB activation in microglia at a systems level., Results: We quantify the dynamic response of NF-κB activation and activation of the upstream kinase IKK using ELISA measurements of a microglial cell line following treatment with the pro-inflammatory cytokine TNFα. A new mathematical model is developed based on these data sets using a modular procedure that exploits the feedback structure of the network. We show that the new model requires previously unmodeled dynamics involved in the stimulus-induced degradation of the inhibitor IκBα in order to properly describe microglial NF-κB activation in a statistically consistent manner. This suggests a more prominent role for the ubiquitin-proteasome system in regulating the activation of NF-κB to inflammatory stimuli. We also find that the introduction of nonlinearities in the kinetics of IKK activation and inactivation is essential for proper characterization of transient IKK activity and corresponds to known biological mechanisms. Numerical analyses of the model highlight key regulators of the microglial NF-κB response, as well as those governing IKK activation. Results illustrate the dynamic regulatory mechanisms and the robust yet fragile nature of the negative feedback regulated network., Conclusions: We have developed a new mathematical model that incorporates previously unmodeled dynamics to characterize the dynamic response of the NF-κB signaling network in microglia. This model is the first of its kind for microglia and provides a tool for the quantitative, systems level study the dynamic cellular response to inflammatory stimuli.

Minocycline, a tetracycline antibiotic, is now known to protect cells via an anti-inflammatory mechanism. We further explored this effect using an in vitro model of ischemia-like injury to neurons. Coculturing neurons with microglia, the brain's resident immune cell, modestly increased cell death due to oxygen and glucose deprivation (OGD), compared to neurons alone. Treatment of cocultures with minocycline decreased cell death to a level significantly lower than that of neurons alone. Treatment of cocultures with minocycline or inhibitors of various immune mediators, also led to decreased cell death. Importantly, treatment of neuron cultures without added microglia with these same inhibitors of tissue plasminogen activator, matrix metalloproteinases, TNF-alpha and inducible nitric oxide synthase as well as minocycline also led to decreased cell death. Thus, anti-inflammatory treatments appear to be directly protective of neurons from in vitro ischemia.

Reactive gliosis is a hallmark of brain pathology and the injury response, yet the extent to which astrocytes proliferate, and whether this is central to astrogliosis is still controversial. We determined the fraction of mature astrocytes that proliferate in a mouse stroke model using unbiased stereology as a function of distance from the infarct edge. Cumulatively 11.1±1.2% of Aldh1l1+ astrocytes within 400 ¼m in the cortical penumbra incorporate BrdU in the first week following stroke, while the overall number of astrocytes does not change. The number of astrocytes proliferating fell sharply with distance with more than half of all proliferating astrocytes found within 100 ¼m of the edge of the infarct. Despite extensive cell proliferation primarily of microglia and neutrophils/monocytes in the week following stroke, few mature astrocytes re-enter cell cycle, and these are concentrated close to the infarct boundary. [ABSTRACT FROM AUTHOR]