Sherri L. Christian

Central nervous system regulation of mammalian hibernation: implications for metabolic suppression and ischemia tolerance

Torpor during hibernation defines the nadir of mammalian metabolism where whole animal rates of metabolism are decreased to as low as 2% of basal metabolic rate. This capacity to decrease profoundly the metabolic demand of organs and tissues has the potential to translate into novel therapies for the treatment of ischemia associated with stroke, cardiac arrest or trauma where delivery of oxygen and nutrients fails to meet demand. If metabolic demand could be arrested in a regulated way, cell and tissue injury could be attenuated. Metabolic suppression achieved during hibernation is regulated, in part, by the central nervous system through indirect and possibly direct means. In this study, we review recent evidence for mechanisms of central nervous system control of torpor in hibernating rodents including evidence of a permissive, hibernation protein complex, a role for A1 adenosine receptors, mu opiate receptors, glutamate and thyrotropin‐releasing hormone. Central sites for regulation of torpor include the hippocampus, hypothalamus and nuclei of the autonomic nervous system. In addition, we discuss evidence that hibernation phenotypes can be translated to non‐hibernating species by H2S and 3‐iodothyronamine with the caveat that the hypothermia, bradycardia, and metabolic suppression induced by these compounds may or may not be identical to mechanisms employed in true hibernation.

The B Cell Antigen Receptor Regulates the Transcriptional Activator β-Catenin Via Protein Kinase C-Mediated Inhibition of Glycogen Synthase Kinase-3

β-Catenin is a transcriptional activator that is regulated by glycogen synthase kinase-3 (GSK-3). GSK-3 is constitutively active in unstimulated cells where it phosphorylates β-catenin, targeting β-catenin for rapid degradation. Receptor-induced inhibition of GSK-3 allows β-catenin to accumulate in the cytoplasm and then translocate to the nucleus where it promotes the transcription of genes such as c-myc and cyclin D1. Wnt hormones, the best known regulators of β-catenin, inhibit GSK-3 via the Disheveled protein. However, GSK-3 is also inhibited when it is phosphorylated by Akt, a downstream target of phosphatidylinositol 3-kinase (PI3K). We have previously shown that B cell Ag receptor (BCR) signaling leads to activation of PI3K and Akt as well as inhibition of GSK-3. Therefore, we hypothesized that BCR engagement would induce the accumulation of β-catenin via a PI3K/Akt/GSK-3 pathway. We now show that BCR ligation causes an increase in the level of β-catenin in the nuclear fraction of B cells as well as an increase in β-catenin-dependent transcription. Direct inhibition of GSK-3 by LiCl also increased β-catenin levels in B cells. This suggests that GSK-3 keeps β-catenin levels low in unstimulated B cells and that BCR-induced inhibition of GSK-3 allows the accumulation of β-catenin. Surprisingly, we found that the BCR-induced phosphorylation of GSK-3 on its negative regulatory sites, as well as the subsequent up-regulation of β-catenin, was not mediated by Akt but by the phospholipase C-dependent activation of protein kinase C. Thus, the BCR regulates β-catenin levels via a phospholipase C/protein kinase C/GSK-3 pathway.

Persistent tolerance to oxygen and nutrient deprivation and N-methyl-D-aspartate in cultured hippocampal slices from hibernating Arctic ground squirrel

Hibernating Arctic ground squirrel (hAGS), Spermophilus parryii, survive profound decreases in cerebral perfusion during torpor and return to normal blood flow during intermittent rewarming periods without neurologic damage. Hibernating AGS tolerate traumatic brain injury in vivo, and acute hippocampal slices from hibernating animals tolerate oxygen and glucose deprivation. It remains unclear, however, if neuroprotection results from intrinsic tissue properties or from differences in response to acute trauma associated with slice preparation. The goal of this work was therefore to determine whether an intrinsic tissue tolerance persists in chronic culture of AGS hippocampal slices at 37 °C. A second goal was to address N-methyl-D-aspartate (NMDA) receptor involvement and channel arrest as potential mechanisms of intrinsic tissue tolerance. Baseline neuronal survival and tolerance to oxygen and nutrient deprivation (OND), an in vitro model of ischemia–reperfusion, were assessed in the CA1 region of hippocampal slices from juvenile, hAGS and interbout euthermic AGS (ibeAGS). Early in culture (insult onset at 3 h), slices from both hAGS and ibeAGS tolerate OND (4h deprivation followed by 20 h recovery) and 500 μmol/L NMDA plus 20 mmol/L KCl. Later in culture (insult onset at 24 h), tolerance persists in slices from hAGS but not in slices from ibeAGS. Ouabain (Na+K+ATPase inhibitor) administered 24 h in culture enhances survival of slices from hAGS (assessed 24h later). Thus, tolerance to OND in slices from hAGS is due to intrinsic tissue properties likely involving NMDA receptors and ion channel arrest.

Arctic ground squirrel (Spermophilus parryii) hippocampal neurons tolerate prolonged oxygen– glucose deprivation and maintain baseline ERK1/2 and JNK activation despite drastic ATP loss

Oxygen—glucose deprivation (OGD) initiates a cascade of intracellular responses that culminates in cell death in sensitive species. Neurons from Arctic ground squirrels (AGS), a hibernating species, tolerate OGD in vitro and global ischemia in vivo independent of temperature or torpor. Regulation of energy stores and activation of mitogen-activated protein kinase (MAPK) signaling pathways can regulate neuronal survival. We used acute hippocampal slices to investigate the role of ATP stores and extracellular signal-regulated kinase (ERK)1/2 and Jun NH2-terminal kinase (JNK) MAPKs in promoting survival. Acute hippocampal slices from AGS tolerated 30 mins of OGD and showed a small but significant increase in cell death with 2 h OGD at 37 C. This tolerance is independent of hibernation state or season. Neurons from AGS survive OGD despite rapid ATP depletion by 3 mins in interbout euthermic AGS and 10 mins in hibernating AGS. Oxygen—glucose deprivation does not induce JNK activation in AGS and baseline ERK1/2 and JNK activation is maintained even after drastic depletion of ATP. Surprisingly, inhibition of ERK1/2 or JNK during OGD had no effect on survival, whereas inhibition of JNK increased cell death during normoxia. Thus, protective mechanisms promoting tolerance to OGD by AGS are downstream from ATP loss and are independent of hibernation state or season.

Decreased NR1 Phosphorylation and Decreased NMDAR Function in Hibernating Arctic Ground Squirrels

Heterothermic mammals such as ground squirrels tolerate ischemia and N‐methyl‐D‐aspartate (NMDA) better than homeothermic mammals such as rats both in vivo and in vitro, and this tolerance is enhanced in the hibernating state. However, the cellular mechanisms underlying this tolerance remain unclear. NMDA receptors (NMDAR) play a key role in excitotoxicity. The purpose of the current study was therefore to test the hypothesis that NMDAR are down‐regulated in hibernating Arctic ground squirrels (hAGS; Spermophilus parryii). To address this hypothesis, we used Western blot analysis to investigate NMDAR phosphorylation, an activator of NMDAR function, and internalization in naïve hippocampal tissue from hAGS, interbout euthermic AGS (ibeAGS), and rats. Furthermore, we used fura‐2 calcium imaging to examine NMDAR function in cultured hippocampal slices from hAGS, ibeAGS, and rats. We report that phosphorylation of the NMDAR1 (NR1) subunit is decreased in hippocampal tissue from hAGS and that the NMDAR component of Glu‐induced increase in [Ca2+]i is decreased in hippocampal slices from hAGS. Moreover, the fraction of NR1 in the functional membrane pool in AGS is less than that in rats.

Activated Ras/MEK Inhibits the Antiviral Response of Alpha Interferon by Reducing STAT2 Levels

ABSTRACT The ability of interferon (IFN) to induce the expression of antiviral genes, and therefore suppress viral infection, is dependent on the activity of cellular suppressors. The Ras/MEK pathway is one of these cellular suppressors, since the activation of Ras/MEK permits viral replication in the presence of alpha IFN (IFN-α). Here, we have investigated the mechanism by which activated Ras/MEK inhibits the IFN-α response. We found that the induction of antiviral proteins in response to IFN-α was impaired in Ras-transformed NIH 3T3 (RasV12) cells. The inhibition of the Ras/MEK pathway restored the IFN-mediated induction of antiviral genes, indicating that activated Ras interrupts the IFN pathway upstream of antiviral gene transcription. Indeed, the IFN-induced phosphorylation of signal transducer and activator of transcription 1 (STAT1) and STAT2 was inhibited in RasV12 cells compared to that of vector control cells. In addition, we found that the total amount of STAT2 was reduced in RasV12 cells. To determine if the impaired IFN-α response can be rescued by restoring the overall level of STAT2, we overexpressed STAT2 in RasV12 cells. The IFN-α-induced phosphorylation of STAT1 and STAT2, as well as the expression of antiviral protein, were restored, and IFN-induced antiviral protection was partially restored. Moreover, we demonstrated that the downregulation of STAT2 levels by Ras/MEK was mediated at the transcriptional level. Thus, the activation of the Ras/MEK pathway reduces the amount of STAT2 available for propagating the IFN signal, resulting in the impairment of the IFN-α-induced antiviral response.

Development of a bispecific monoclonal antibody as a universal immunoprobe for detecting biotinylated macromolecules

Bispecific monoclonal antibody (BsMab) combining two different antigen binding sites, anti-biotin and anti-HRPO paratopes, could be used as a universal immunoprobe for detecting all biotinylated macromolecules. First, a mouse hybridoma cell line secreting monospecific anti-biotin Mab was generated and characterized. Second, a quadroma cell line which could continuously secrete bsMab (anti-biotin x anti-HRPO) was developed by a nonselective microelectrofusion method. The supernatant containing bsMab was collected from tissue culture medium and purified with two affinity columns. This bsMab has comparable avidity to commercial streptavidin-HRPO when tested against biotinylated macromolecules. Compared to streptavidin, this bsMab can bind the enzyme and thus eliminate the need for chemical conjugation. This bsMab can be used as a promising immunoprobe for detecting many macromolecules bearing biotin markers, such as protein, phage, liposome and DNA in different bioassay systems.

Distribution of NMDA receptor subunit NR1 in arctic ground squirrel central nervous system.

Hibernation is a natural model of neuroprotection and adult synaptic plasticity. NMDA receptors (NMDAR), which play key roles in excitotoxicity and synaptic plasticity, have not been characterized in a hibernating species. Tolerance to excitotoxicity and cognitive enhancement in Arctic ground squirrels (AGS, Spermophilus parryii) suggests that NMDAR expression may decrease in hibernation and increase upon arousal. NMDAR consist of at least one NMDAR1 (NR1) subunit, which is required for receptor function. Localization of NR1 reflects localization of the majority, if not all, NMDAR complexes. The purpose of this study, therefore, was to characterize the distribution of NR1 subunits in AGS central nervous system using immunohistochemistry. In addition, we compare NR1 expression in hippocampus of hibernating AGS (hAGS) and inter-bout euthermic AGS (ibeAGS) and assess changes in cell somata size using NR1 stained sections in three hippocampal sub-regions (CA1, CA3, and dentate gyrus). For the first time, we report that immunoreactivity of anti-NR1 is widely distributed throughout the central nervous system in AGS and is similar to other species. No differences exist in the expression and distribution of NR1 in hAGS and ibeAGS. However, we report a significant decrease in size of hippocampal CA1 and dentate gyrus NR1-expressing neuronal somata during hibernation torpor.

Activation and phosphatidylinositol 3-kinase-dependent phosphorylation of protein kinase C-epsilon by the B cell antigen receptor

Protein kinase C (PKC) enzymes play an important role in B cell antigen receptor (BCR) signaling, linking the BCR to the activation of mitogen-activated protein kinases as well as the NF-kappa B, and AP-1 transcription factors. There are eleven different PKC isoforms, each of which is likely to have a unique set of substrates and hence a unique role in signal transduction. Although PKC-alpha, PKC-beta, PKC-delta, and PKC-zeta have been shown to be targets of BCR signaling, the full spectrum of PKC enzymes that are activated by the BCR remains to be determined. In this report, we show that PKC-epsilon is a target of BCR signaling. We found that PKC-epsilon is highly expressed in B cells and that BCR engagement causes PKC-epsilon to translocate from the cytosol to cellular membranes. This presumably reflects the binding of PKC-epsilon to its membrane-associated lipid activator, diacylglycerol. We also found that BCR engagement resulted in the phosphatidylinositol 3-kinase-dependent phosphorylation of PKC-epsilon. This modification may promote the full activation of PKC-epsilon. Activation of PKC-epsilon could be a key event in BCR signaling since PKC-epsilon has been strongly linked to cell survival and proliferation in other cell types.