Karen Horsburgh

Synaptic NMDA receptor activity boosts intrinsic antioxidant defenses.

Intrinsic antioxidant defenses are important for neuronal longevity. We found that in rat neurons, synaptic activity, acting via NMDA receptor (NMDAR) signaling, boosted antioxidant defenses by making changes to the thioredoxin-peroxiredoxin (Prx) system. Synaptic activity enhanced thioredoxin activity, facilitated the reduction of overoxidized Prxs and promoted resistance to oxidative stress. Resistance was mediated by coordinated transcriptional changes; synaptic NMDAR activity inactivated a previously unknown Forkhead box O target gene, the thioredoxin inhibitor Txnip. Conversely, NMDAR blockade upregulated Txnip in vivo and in vitro, where it bound thioredoxin and promoted vulnerability to oxidative damage. Synaptic activity also upregulated the Prx reactivating genes Sesn2 (sestrin 2) and Srxn1 (sulfiredoxin), via C/EBPβ and AP-1, respectively. Mimicking these expression changes was sufficient to strengthen antioxidant defenses. Trans-synaptic stimulation of synaptic NMDARs was crucial for boosting antioxidant defenses; chronic bath activation of all (synaptic and extrasynaptic) NMDARs induced no antioxidative effects. Thus, synaptic NMDAR activity may influence the progression of pathological processes associated with oxidative damage.

Forebrain mineralocorticoid receptor overexpression enhances memory, reduces anxiety and attenuates neuronal loss in cerebral ischaemia

The nuclear mineralocorticoid receptor (MR), a high‐affinity receptor for glucocorticoids, is highly expressed in the hippocampus where it underpins cognitive, behavioural and neuroendocrine regulation. Increased neuronal MR expression occurs early in the response to cellular injury in vivo and in vitro and is associated with enhanced neuronal survival. To determine whether increased neuronal MR might be causal in protecting against ischaemic damage in vivo we generated a forebrain‐specific MR‐overexpressing transgenic mouse (MR‐Tg) under the control of the CamKII alpha promoter, and subjected mice to transient cerebral global ischaemia induced by bilateral common carotid artery occlusion for 20 min. We also separately assessed the effects of MR overexpression on hypothalamic–pituitary–adrenal (HPA) axis activity and cognitive and affective functions in noninjured animals. Our results showed that MR‐Tg mice had significantly reduced neuronal death following transient cerebral global ischaemia compared to wild‐type littermates. This effect was not associated with alterations in basal or poststress HPA axis function or in arterial blood pressure. MR‐Tg mice also demonstrated improved spatial memory retention, reduced anxiety and altered behavioural response to novelty. The induction of neuronal MR appears to offer a protective response which has potential therapeutic implications in cerebral ischaemia and cognitive and affective disorders.

The subtype of GluN2 C-terminal domain determines the response to excitotoxic insults.

Summary It is currently unclear whether the GluN2 subtype influences NMDA receptor (NMDAR) excitotoxicity. We report that the toxicity of NMDAR-mediated Ca2+ influx is differentially controlled by the cytoplasmic C-terminal domains of GluN2B (CTD2B) and GluN2A (CTD2A). Studying the effects of acute expression of GluN2A/2B-based chimeric subunits with reciprocal exchanges of their CTDs revealed that CTD2B enhances NMDAR toxicity, compared to CTD2A. Furthermore, the vulnerability of forebrain neurons in vitro and in vivo to NMDAR-dependent Ca2+ influx is lowered by replacing the CTD of GluN2B with that of GluN2A by targeted exon exchange in a mouse knockin model. Mechanistically, CTD2B exhibits stronger physical/functional coupling to the PSD-95-nNOS pathway, which suppresses protective CREB activation. Dependence of NMDAR excitotoxicity on the GluN2 CTD subtype can be overcome by inducing high levels of NMDAR activity. Thus, the identity (2A versus 2B) of the GluN2 CTD controls the toxicity dose-response to episodes of NMDAR activity.

Mild oxidative stress activates Nrf2 in astrocytes, which contributes to neuroprotective ischemic preconditioning

Haskew-Layton et al. (1) reported that subtoxic doses of H2O2 fails to activate nuclear factor erythroid 2-related factor (Nrf2) in astrocytes and triggers Nrf2-independent responses that protect cocultured neurons. Contrary to this, we show that mild oxidative insults, including subtoxic H2O2, strongly activate astrocytic Nrf2/antioxidant response element (ARE)-dependent gene expression, which, moreover, contributes to neuroprotective ischemic preconditioning.

Mild Fluid Percussion Injury in Mice Produces Evolving Selective Axonal Pathology and Cognitive Deficits Relevant to Human Brain Injury

Mild traumatic brain injury (TBI) accounts for up to 80% of clinical TBI and can result in cognitive impairment and white matter damage that may develop and persist over several years. Clinically relevant models of mild TBI for investigation of neurobiological changes and the development of therapeutic strategies are poorly developed. In this study we investigated the temporal profile of axonal and somal injury that may contribute to cognitive impairments in a mouse model of mild TBI. Neuronal perikaryal damage (hematoxylin and eosin and Fluoro-Jade C), myelin integrity (myelin basic protein and myelin-associated glycoprotein), and axonal damage (amyloid precursor protein), were evaluated by immunohistochemistry at 4 h, 24 h, 72 h, 4 weeks, and 6 weeks after mild lateral fluid percussion brain injury (0.9 atm; righting time 167 +/- 15 sec). At 3 weeks post-injury spatial reference learning and memory were tested in the Morris water maze (MWM). Levels of damage to neuronal cell bodies were comparable in the brain-injured and sham groups. Myelin integrity was minimally altered following injury. Clear alterations in axonal damage were observed at various time points after injury. Axonal damage was localized to the cingulum at 4 h post-injury. At 4 and 6 weeks post-injury, axonal damage was evident in the external capsule, and was seen at 6 weeks in the dorsal thalamic nuclei. At 3 weeks post-injury, injured mice showed an impaired ability to learn the water maze task, suggesting injury-induced alterations in search strategy learning. The evolving localization of axonal damage points to ongoing degeneration after injury that is concomitant with a deficit in learning.

Increased neuronal damage in apolipoprotein E-deficient mice following global ischaemia.

There is accumulating evidence that apolipoprotein E (apoE) plays a role in regulating the response to and outcome following brain injury. The present study compared the histological outcome and recovery following an episode of global ischaemia in apoE-deficient mice and wild-type littermates (12-week-old males, n = 8 per group). Transient global ischaemia was induced for a period of 17 min and the animals were allowed to recover for 72 h. Transient global ischaemia induced selective neuronal degeneration in several brain regions in wild-type mice. There was statistically significant increased ischaemic neuronal damage in apoE-deficient mice compared with wild-type mice in six of the seven regions examined (hippocampal regions CA1, CA3/CA4 and dentate gyrus; thalamus; cortex and caudate nucleus; P < 0.05). The data substantiate a role for apoE in modifying the response of the CNS to acute injury.

MRI is a sensitive marker of subtle white matter pathology in hypoperfused mice

White matter (WM) abnormalities, possibly resulting from hypoperfusion, are key features of the aging human brain. It is unclear, however, whether in vivo magnetic resonance imaging (MRI) approaches, such as diffusion tensor and magnetization transfer MRI are sufficiently sensitive to detect subtle alterations to WM integrity in mouse models developed to study the aging brain. We therefore investigated the use of diffusion tensor and magnetization transfer MRI to measure structural changes in 4 WM tracts following 1 month of moderate hypoperfusion, which results in diffuse WM pathology in C57Bl/6J mice. Following MRI, brains were processed for evaluation of white and gray matter pathology. Significant reductions in fractional anisotropy were observed in the corpus callosum (p = 0.001) and internal capsule (p = 0.016), and significant decreases in magnetization transfer ratio were observed in the corpus callosum (p = 0.023), fimbria (p = 0.032), internal capsule (p = 0.046) and optic tract (p = 0.047) following hypoperfusion. Hypoperfused mice demonstrated diffuse axonal and myelin pathology which was essentially absent in control mice. Both fractional anisotropy and magnetization transfer ratio correlate with markers of myelin integrity/degradation and not axonal pathology. The study demonstrates that in vivo MRI is a sensitive measure of diffuse, subtle WM changes in the murine brain.

Extension of cerebral hypoperfusion and ischaemic pathology beyond MCA territory after intraluminal filament occlusion in C57Bl/6J mice

Rodent models of focal cerebral ischaemia are critical for understanding pathophysiological concepts in human stroke. The availability of genetically modified mice has prompted the adaptation of the intraluminal filament occlusion model of focal ischaemia for use in mice. In the present study, we investigated the effects of increasing duration of intraluminal occlusion on the extent and distribution of ischaemic pathology and local cerebral blood flow (LCBF) in C57Bl/6J mice, the most common background mouse strain. Volumetric assessment of ischaemic damage was performed after 15, 30 or 60 min occlusion followed by 24 h reperfusion. LCBF was measured after 15 and 60 min occlusion using quantitative 14C-iodoantipyrine autoradiography. The extent and distribution of ischaemic damage was highly sensitive to increasing occlusion duration. Recruitment of tissue outside MCA territory produced a steep increase in the volume of damage with increasing occlusion duration: 15 min (9+/-2 mm3); 30 min (56+/-6 mm3); 60 min (69+/-2 mm3). Significant increases in the severity of cerebral hypoperfusion were observed after 60 min compared to 15 min occlusion within and outside MCA territory, e.g. caudate nucleus (9+/-6 ml per 100 g per min at 60 min vs. 33 ml per 100 g per min at 15 min) and hippocampus (16+/-14 ml per 100 g per min at 60 min vs. 61+/-16 ml per 100 g per min at 15 min). MABP remained stable for 25 min after occlusion onset and declined thereafter. The integrity of the circle of Willis was examined by carbon black perfusion of the vasculature. A complete circle of Willis was present in only one of 10 mice. These results demonstrate that intraluminal filament occlusion in C57Bl/6J mice leads to an occlusion duration-dependent increase in severity of cerebral hypoperfusion and extension of ischaemic pathology beyond MCA territory.

Neuroprotection after Transient Global Cerebral Ischemia in Wlds Mutant Mice

The Wlds mouse mutant demonstrates a remarkable phenotype of delayed axonal and synaptic degeneration after nerve lesion. In this study, the authors tested the hypothesis that expression of Wld protein is neuroprotective in an in vivo mouse model of global cerebral ischemia. This model is associated with selective neuronal degeneration in specific brain regions such as the caudate nucleus and CA2 hippocampal pyramidal cell layer. The extent of neuronal damage was quantified in Wlds compared to wild-type mice after an identical episode of global cerebral ischemia. The results demonstrated a significant and marked reduction in the extent of neuronal damage in Wlds as compared to wild-type C57Bl/6 mice. In the caudate nucleus, Wld expression significantly reduced the percentage of ischemic neuronal damage after global ischemia (Wlds, 27.7 ± 16.8%; wild-type mice, 58.7 ± 32.3%; P = 0.036). Similarly, in the CA2 pyramidal cell layer, there was a significant reduction of neuronal damage in the Wlds mice as compared to wild-type mice after ischemia (Wlds, 17.7 ± 23.0%; wild-type mice, 41.9 ± 28.0%; P < 0.023). Thus, these results clearly demonstrate that the Wld gene confers substantial neuroprotection after cerebral ischemia, and suggest a new role to that previously described for Wlds.

Rapid Disruption of Axon–Glial Integrity in Response to Mild Cerebral Hypoperfusion

Myelinated axons have a distinct protein architecture essential for action potential propagation, neuronal communication, and maintaining cognitive function. Damage to myelinated axons, associated with cerebral hypoperfusion, contributes to age-related cognitive decline. We sought to determine early alterations in the protein architecture of myelinated axons and potential mechanisms after hypoperfusion. Using a mouse model of hypoperfusion, we assessed changes in proteins critical to the maintenance of paranodes, nodes of Ranvier, axon–glial integrity, axons, and myelin by confocal laser scanning microscopy. As early as 3 d after hypoperfusion, the paranodal septate-like junctions were damaged. This was marked by a progressive reduction of paranodal Neurofascin signal and a loss of septate-like junctions. Concurrent with paranodal disruption, there was a significant increase in nodal length, identified by Nav1.6 staining, with hypoperfusion. Disruption of axon–glial integrity was also determined after hypoperfusion by changes in the spatial distribution of myelin-associated glycoprotein staining. These nodal/paranodal changes were more pronounced after 1 month of hypoperfusion. In contrast, the nodal anchoring proteins AnkyrinG and Neurofascin 186 were unchanged and there were no overt changes in axonal and myelin integrity with hypoperfusion. A microarray analysis of white matter samples indicated that there were significant alterations in 129 genes. Subsequent analysis indicated alterations in biological pathways, including inflammatory responses, cytokine-cytokine receptor interactions, blood vessel development, and cell proliferation processes. Our results demonstrate that hypoperfusion leads to a rapid disruption of key proteins critical to the stability of the axon–glial connection that is mediated by a diversity of molecular events.