John F. Emery

Overexpression of mitochondrial Hsp70/Hsp75 in rat brain protects mitochondria, reduces oxidative stress, and protects from focal ischemia.

Mitochondria are known to be central to the cells response to ischemia, because of their role in energy generation, in free radical generation, and in the regulation of apoptosis. Heat shock protein 75 (Hsp75/Grp75/mortalin/TRAP1) is a member of the HSP70 chaperone family, which is targeted to mitochondria. Overexpression of Hsp75 was achieved in rat brain by DNA transfection, and expression was observed in both astrocytes and neurons. Rats were subjected to 100 mins middle cerebral artery occlusion followed by assessment of infarct volume, neurological score, mitochondrial function, and levels of oxidative stress at 24 h reperfusion. Overexpression of Hsp75 reduced infarct area from 44.6%±21.1% to 25.7%±12.1% and improved neurological outcome significantly. This was associated with improved mitochondrial function as shown by protection of complex IV activity, marked reduction of free radical generation detected by hydroethidine fluorescence, reduction of lipid peroxidation detected by 4-hydroxy-2-nonenol immunoreactivity, and increased preservation of ATP levels. This suggests that targeting mitochondria for protection may be a useful strategy to reduce ischemic brain injury.

Regulation of apoptotic and inflammatory cell signaling in cerebral ischemia - the complex roles of Heat Shock Protein 70

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 focuses on the heat shock protein 70 family and considers especially the functions of the inducible member, heat shock protein 72, in the setting of cerebral ischemia. In general, inhibiting apoptotic signaling at multiple points and up-regulating survival signaling, heat shock protein 70 has a net prosurvival effect. Heat shock protein 70 has both antiinflammatory and proinflammatory effects depending on the cell type, context, and intracellular or extracellular location. Intracellular effects are often antiinflammatory with inhibition of nuclear factor-&kgr;B signaling. Extracellular effects can lead to inflammatory cytokine production or induction of regulatory immune cells and reduced inflammation.

Overexpression of Mitochondrial Hsp70/Hsp75 Protects Astrocytes against Ischemic Injury in vitro

Mitochondrial heat shock protein 70 (mtHsp70/Hsp75/Grp75/mortalin/TRAP-1/PBP74) is an essential mitochondrial chaperone and a member of the heat shock protein 70 (HSP70) family. Although many studies have shown the protective properties of overexpression of the cytosolic inducible member of the HSP70 family, Hsp72, few studies have investigated the protective potential of Hsp75 against ischemic injury. Mitochondria are one of the primary targets of ischemic injury in astrocytes. In this study, we analyzed the effects of Hsp75 overexpression on cellular levels of reactive oxygen species (ROS), mitochondrial membrane potential, ATP levels, and viability during the ischemia-like conditions of oxygen-glucose deprivation (OGD) or glucose deprivation (GD) in primary astrocytic cultures. We show that Hsp75 overexpression decreases ROS production and preserves mitochondrial membrane potential during GD, and preserves ATP levels and cell viability during OGD. These findings indicate that Hsp75 can provide protection against ischemia-like in vitro injury and suggest that it should be further studied as a potential candidate for protection against ischemic injury.

Astrocyte targeted overexpression of Hsp72 or SOD2 reduces neuronal vulnerability to forebrain ischemia

Brief forebrain ischemia is a model of the delayed hippocampal neuronal loss seen in patients following cardiac arrest and resuscitation. Previous studies demonstrated that selective dysfunction of hippocampal CA1 subregion astrocytes occurs hours to days before delayed neuronal death. In this study we tested the strategy of directing protection to astrocytes to protect neighboring neurons from forebrain ischemia. Two well‐studied protective proteins, heat shock protein 72 (Hsp72) or superoxide dismutase 2 (SOD2), were genetically targeted for expression in astrocytes using the astrocyte‐specific human glial fibrillary acidic protein (GFAP) promoter. The expression constructs were injected stereotacticly immediately above the hippocampal CA1 region on one side of the rat brain two days prior to forebrain ischemia. Cell type specific expression was confirmed by double label immunohistochemistry. When the expression constructs were injected two days before transient forebrain ischemia, the loss of CA1 hippocampal neurons observed seven days later was significantly reduced on the injected side compared with controls. This neuroprotection was associated with significantly better preservation of astrocyte glutamate transporter‐1 immunoreactivity at 5‐h reperfusion and reduced oxidative stress. Improving the resistance of astrocytes to ischemic stress by targeting either the cytosolic or mitochondrial compartment was thus associated with preservation of CA1 neurons following forebrain ischemia. Targeting astrocytes is a promising strategy for neuronal preservation following cardiac arrest and resuscitation.

Overexpressing GRP78 influences Ca2+ handling and function of mitochondria in astrocytes after ischemia-like stress.

Ca(2+) transfer from endoplasmic reticulum (ER) to mitochondria at contact sites between the organelles can induce mitochondrial dysfunction and programmed cell death after stress. The ER-localized chaperone glucose-regulated protein 78kDa (GRP78/BiP) protects neurons against excitotoxicity and apoptosis. Here we show that overexpressing GRP78 protects astrocytes against ischemic injury, reduces net flux of Ca(2+) from ER to mitochondria, increases Ca(2+) uptake capacity in isolated mitochondria, reduces free radical production, and preserves respiratory activity and mitochondrial membrane potential after stress. We conclude that GRP78 influences ER-mitochondrial Ca(2+) crosstalk to maintain mitochondrial function and protect astrocytes from ischemic injury.

Inflammatory response of microglial BV-2 cells includes a glycolytic shift and is modulated by mitochondrial glucose-regulated protein 75/mortalin.

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.

Mitochondrial Protection Attenuates Inflammation-Induced Impairment of Neurogenesis In Vitro and In Vivo

The impairment of hippocampal neurogenesis has been linked to the pathogenesis of neurological disorders from chronic neurodegenerative disease to the progressive cognitive impairment of children who receive brain irradiation. Numerous studies provide evidence that inflammation downregulates neurogenesis, with multiple factors contributing to this impairment. Although mitochondria are one of the primary targets of inflammatory injury, the role of mitochondrial function in the modulation of neurogenesis remains relatively unstudied. In this study, we used neurosphere-derived cells to show that immature doublecortin (Dcx)-positive neurons are uniquely sensitive to mitochondrial inhibition, demonstrating rapid loss of mitochondrial potential and cell viability compared with glial cells and more mature neurons. Mitochondrial inhibition for 24 h produced no significant changes in astrocyte or oligodendrocyte viability, but reduced viability of mature neurons by 30%, and reduced survival of Dcx+ cells by 60%. We demonstrate that protection of mitochondrial function with mitochondrial metabolites or the mitochondrial chaperone mtHsp75/mortalin partially reverses the inflammation-associated impairment of neurogenesis in vitro and in irradiated mice in vivo. Our findings highlight mitochondrial mechanisms involved in neurogenesis and indicate mitochondria as a potential target for protective strategies to prevent the impairment of neurogenesis by inflammation.

Pupillary reflex dilation and skin temperature to assess sensory level during combined general and caudal anesthesia in children.

Background:  Regional anesthesia causes sympathetic blockade, vasodilation and higher skin temperature in anesthetized dermatomes. Measurement of skin temperature changes might provide a useful estimate of the level of caudal anesthesia in children. Pupillary reflex dilation (PRD) allows estimation of the sensory level during combined general/epidural anesthesia in adults, but has not been assessed in children. This study was designed to evaluate skin temperature and PRD as methods of estimating sensory level in children receiving combined general/caudal epidural anesthesia.

Quantitative characterization and analysis of the dynamic NF-κB response in microglia

BackgroundActivation 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.ResultsWe 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.ConclusionsWe 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.