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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., Author Summary Inducing heat shock or overexpressing certain heat shock proteins (HSPs) is known to protect against brain injury, such as that resulting from stroke. Understanding the mechanisms underlying protection at the cellular and molecular level is a subject of intense research, as such knowledge may prove beneficial in designing future therapies. Regulation of the activation of the key inflammatory transcription factor Nuclear Factor κB (NF-κB) is believed to be one critical mechanism. However how its activation is altered by Hsp72 remains unresolved. Here we examine NF-κB signaling in microglia cells overexpressing Hsp72, combining experimentation and mathematical modeling. We show that Hsp72 affects signaling using at least two essential and distinct mechanisms: attenuation of upstream kinase (IKK) activity and reduction of steady state NF-κB protein levels. We provide numerical evidence suggesting that neither mechanism in isolation is sufficient to account for the observed signaling. Furthermore, our observations suggest an intriguing additional level of regulation of gene expression and protein synthesis of the IκBα inhibitor, which opens interesting new avenues of research. These results provide novel insight into the mechanisms by which Hsp72 may regulate inflammation and protect brain cells from injury.

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., PLoS Computational Biology, 10 (2), ISSN:1553-734X, ISSN:1553-7358

Astrocytes have been shown to protect neurons and increase their survival in many pathological settings. Manipulating astrocyte functions is thus an important strategy to enhance neuronal survival and improve outcome following cerebral ischemia. Increasing evidence supports the involvement of microRNAs (miRNA), some of them being astrocyte-enriched, in the regulation of cerebral ischemia. This mini review will focus on several recently reported astrocyte-enriched miRNAs (miR-181 and miR-29 families and miR-146a), their validated targets, regional expression and effects on outcome after cerebral ischemia.

The already established and widely used intravenous application of recombinant tissue plasminogen activator as a re-opening strategy for acute vessel occlusion in ischemic stroke was recently added by mechanical thrombectomy, representing a fundamental progress in evidence-based medicine to improve the patient’s outcome. This has been paralleled by a swift increase in our understanding of pathomechanisms underlying many neurovascular diseases and most prevalent forms of dementia. Taken together, these current advances offer the potential to overcome almost two decades of marginally successful translational research on stroke and dementia, thereby spurring the entire field of translational neuroscience. Moreover, they may also pave the way for the renaissance of classical neuroprotective paradigms.\ud \ud This review reports and summarizes some of the most interesting and promising recent achievements in neurovascular and dementia research. It highlights sessions from the 9th International Symposium on Neuroprotection and Neurorepair that have been discussed from April 19th to 22nd in Leipzig, Germany. To acknowledge the emerging culture of interdisciplinary collaboration and research, special emphasis is given on translational stories ranging from fundamental research on neurode- and -regeneration to late stage translational or early stage clinical investigations.

It is commonly believed that sustained elevations in the mitochondrial matrix Ca2+ concentration are a major feature of the intracellular cascade of lethal events during cerebral ischemia. The physical association between the endoplasmic reticulum (ER) and mitochondria, known as the mitochondria-associated ER membrane (MAM), enables highly efficient transmission of Ca2+ from the ER to mitochondria under both physiological and pathological conditions. Molecular chaperones are well known for their protective effects during cerebral ischemia. It has been demonstrated recently that many molecular chaperones coexist with MAM and regulate the MAM and thus Ca2+ concentration inside mitochondria. Here, we review recent research on cerebral ischemia and MAM, with a focus on molecular chaperones and ER-mitochondrial calcium transfer.

Stroke causes brain injury with activation of an inflammatory response that can contribute to injury. We tested the hypothesis that the anti-inflammatory cytokine interleukin-4 (IL-4) reduces injury after stroke using IL-4 knockout (KO) adult male mice.IL-4 KO and wild-type mice were subjected to transient middle cerebral artery occlusion. Outcome was assessed by triphenyltetrazolium chloride staining for infarct volume, neuroscore and spontaneous activity for behavioral outcome, and immunostaining and stereological counting for cellular response.Infarction volume at 24 hours was significantly larger in IL-4 KO mice, neurological score was significantly worse, and spontaneous activity was reduced compared with wild-type mice. Increased macrophage/microglial infiltration, increased numbers of myeloperoxidase-positive cells, and increased Th1/Th2 ratio were observed in the infarct core in IL-4 KO mice. Reduced astrocyte activation was observed in the cortical penumbra in IL-4 KO mice. Recombinant IL-4 administered intracerebroventricularly before middle cerebral artery occlusion significantly reduced infarct volume, improved neurological score, reduced macrophages/microglia, and lowered the Th1/Th2 ratio in IL-4 KO mice, but not in wild-type.Loss of IL-4 signaling in KO mice was associated with worse outcome, and this was reversed by giving exogenous IL-4. Worsened outcome was associated with increased inflammation in the core, which was reversed in IL-4 KO but not significantly changed in wild-type mice by exogenous IL-4. This is consistent with IL-4 signaling leading to reduced inflammation in the core and a possible beneficial role for activated astrocytes in the penumbra.

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., BMC Bioinformatics, 12, ISSN:1471-2105

Although heat shock proteins have been studied for decades, new intracellular and extracellular functions in a variety of diseases continue to be discovered. Heat shock proteins function within networks of interacting proteins; they can alter cellular physiology rapidly in response to stress without requiring new protein synthesis. This review will focus on the heat shock protein 70 family and consider especially the functions of the inducible member, heat shock protein 72, in the setting of cerebral ischemia. In general, inhibiting apoptotic signalling at multiple points and upregulating survival signaling, heat shock protein 70 has a net pro-survival effect. Heat shock protein 70 has both anti-inflammatory and pro-inflammatory effects depending on the cell type, context, and intracellular or extracellular location. Intracellular effects are often anti-inflammatory with inhibition of Nuclear Factor κB signaling. Extracellular effects can lead to inflammatory cytokine production or induction of regulatory immune cells and reduced inflammation.