Stress hyperglycemia exacerbates inflammatory brain injury after stroke.

Post-stroke hyperglycemia occurs in 30% - 60% of ischemic stroke patients as part of the systemic stress response, but neither clinical evidence nor pre-clinical studies indicate whether post-stroke hyperglycemia affects stroke outcome. Here we investigated this issue using a mouse model of permanent ischemia. Mice were maintained either normoglycemic or hyperglycemic during the interval of 17 - 48 hours after ischemia onset. Post-stroke hyperglycemia was found to increase infarct volume, blood-brain barrier disruption, and hemorrhage formation, and to impair motor recovery. Post-stroke hyperglycemia also increased superoxide formation by peri-infarct microglia/macrophages. In contrast, post-stroke hyperglycemia did not increase superoxide formation or exacerbate motor impairment in p47 phox-/- mice, which cannot form an active superoxide-producing NADPH oxidase-2 complex. These results suggest that hyperglycemia occurring hours-to-days after ischemia can increase oxidative stress in peri-infarct tissues by fueling NADPH oxidase activity in reactive microglia/macrophages, and by this mechanism contribute to worsened functional outcome.

α-Synuclein triggers cofilin pathology and dendritic spine impairment via a PrPC-CCR5 dependent pathway.

Cognitive dysfunction and dementia are critical symptoms of Lewy Body dementias (LBD). Specifically, alpha-synuclein (αSyn) accumulation in the hippocampus leading to synaptic dysfunction is linked to cognitive deficits in LBD. Here, we investigated the pathological impact of αSyn on hippocampal neurons. We report that either αSyn overexpression or αSyn pre-formed fibrils (PFFs) treatment triggers the formation of cofilin-actin rods, synapse disruptors, in cultured hippocampal neurons and in the hippocampus of synucleinopathy mouse models and of LBD patients. In vivo, cofilin pathology is present concomitantly with synaptic impairment and cognitive dysfunction. Rods generation prompted by αSyn involves the co-action of the cellular prion protein (PrPC) and the chemokine receptor 5 (CCR5). Importantly, we show that CCR5 inhibition, with a clinically relevant peptide antagonist, reverts dendritic spine impairment promoted by αSyn. Collectively, we detail the cellular and molecular mechanism through which αSyn disrupts hippocampal synaptic structure and we identify CCR5 as a novel therapeutic target to prevent synaptic impairment and cognitive dysfunction in LBD.

Cofilactin rod formation mediates inflammation-induced neurite degeneration.

Stroke, trauma, and neurodegenerative disorders cause loss of neurites (axons and dendrites) in addition to neuronal death. Neurite loss may result directly from a primary insult, secondary to parental neuron death, or secondary to a post-injury inflammatory response. Here, we use lipopolysaccharide and the alarmin S100ß to selectively evaluate neurite loss caused by the inflammatory response. Activation of microglia and infiltrating macrophages by these stimuli causes neurite loss that far exceeds neuronal death, both in vitro and in vivo. Neurite loss is accompanied by the formation of cofilactin rods and aggregates (CARs), which are polymers of cofilin-1 and actin induced by oxidative stress and other factors. Mice deficient in either cofilin-1 or the superoxide-generating enzyme NADPH oxidase-2 show reduced CAR formation, neurite loss, and motor impairment. The findings identify a mechanism by which inflammation leads to neurite loss via CAR formation and highlight the relevance of neurite loss to functional impairment.

Bioenergetic and excitotoxic determinants of cofilactin rod formation.

Cofilactin rods (CARs), which are 1:1 aggregates of cofilin-1 and actin, lead to neurite loss in ischemic stroke and other disorders. The biochemical pathways driving CAR formation are well-established, but how these pathways are engaged under ischemic conditions is less clear. Brain ischemia produces both ATP depletion and glutamate excitotoxicity, both of which have been shown to drive CAR formation in other settings. Here, we show that CARs are formed in cultured neurons exposed to ischemia-like conditions: oxygen-glucose deprivation (OGD), glutamate, or oxidative stress. Of these conditions, only OGD produced significant ATP depletion, showing that ATP depletion is not required for CAR formation. Moreover, the OGD-induced CAR formation was blocked by the glutamate receptor antagonists MK-801 and kynurenic acid; the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors GSK2795039 and apocynin; as well as an ROS scavenger. The findings identify a biochemical pathway leading from OGD to CAR formation in which the glutamate release induced by energy failure leads to activation of neuronal glutamate receptors, which in turn activates NADPH oxidase to generate oxidative stress and CARs.

Brain energy metabolism: A roadmap for future research.

Although we have learned much about how the brain fuels its functions over the last decades, there remains much still to discover in an organ that is so complex. This article lays out major gaps in our knowledge of interrelationships between brain metabolism and brain function, including biochemical, cellular, and subcellular aspects of functional metabolism and its imaging in adult brain, as well as during development, aging, and disease. The focus is on unknowns in metabolism of major brain substrates and associated transporters, the roles of insulin and of lipid droplets, the emerging role of metabolism in microglia, mysteries about the major brain cofactor and signaling molecule NAD+, as well as unsolved problems underlying brain metabolism in pathologies such as traumatic brain injury, epilepsy, and metabolic downregulation during hibernation. It describes our current level of understanding of these facets of brain energy metabolism as well as a roadmap for future research.

Use of Botulinum Toxin for Limb Immobilization for Rehabilitation in Rats with Experimental Stroke.

Motor rehabilitation strategies after unilateral stroke suggest that the immobilization of the healthy, unimpaired limb can promote the functional recovery of a paretic limb. In rodents, this has been modeled using casts, harnesses, and other means of restricting the use of the non-paretic forelimb in models of experimental stroke. Here, we evaluated an alternative approach, using botulinum toxin injections to limit the function of the non-paretic forelimb. Adult male rats were subjected to permanent ligation of the left distal middle cerebral artery, resulting in right forelimb paresis. The rats were then subjected to: (1) no treatment; (2) botulinum toxin injections 1 day post stroke; or (3) cast placement 5 days post stroke. Casts were removed after 5 weeks, while the botulinum toxin injection effectively immobilized subjects for approximately the same duration. Rats with bilateral forelimb impairment due to the stroke plus casting or botulinum injections were still able to feed and groom normally. Both immobilization groups showed modest recovery following the stroke compared to those that did not receive immobilization, but the casting approach led to unacceptable levels of animal stress. The botulinum toxin approach to limb immobilization had both advantages and disadvantages over traditional physical limb immobilization. The major advantage was that it was far less stress-inducing to the subject animals and appeared to be well tolerated. A disadvantage was that the paresis took roughly 10 weeks to fully resolve, and any degree of residual paresis could confound the interpretation of the behavioral assessments.

Neuronal Oxidative Stress Promotes α-Synuclein Aggregation In Vivo.

Both genetic and environmental factors increase risk for Parkinson's disease. Many of the known genetic factors influence α-synuclein aggregation or degradation, whereas most of the identified environmental factors produce oxidative stress. Studies using in vitro approaches have identified mechanisms by which oxidative stress can accelerate the formation of α-synuclein aggregates, but there is a paucity of evidence supporting the importance of these processes over extended time periods in brain. To assess this issue, we evaluated α-synuclein aggregates in brains of three transgenic mouse strains: hSyn mice, which overexpress human α-synuclein in neurons and spontaneously develop α-synuclein aggregates; EAAT3-/- mice, which exhibit a neuron-specific impairment in cysteine uptake and resultant neuron-selective chronic oxidative stress; and double-transgenic hSyn/EAAT3-/- mice. Aggregate formation was evaluated by quantitative immunohistochemistry for phosphoserine 129 α-synuclein and by an α-synuclein proximity ligation assay. Both methods showed that the double transgenic hSyn/EAAT3-/- mice exhibited a significantly higher α-synuclein aggregate density than littermate hSyn mice in each brain region examined. Negligible aggregate formation was observed in the EAAT3-/- mouse strain, suggesting a synergistic rather than additive interaction between the two genotypes. A similar pattern of results was observed in assessments of motor function: the pole test and rotarod test. Together, these observations indicate that chronic, low-grade neuronal oxidative stress promotes α-synuclein aggregate formation in vivo. This process may contribute to the mechanism by which environmentally induced oxidative stress contributes to α-synuclein pathology in idiopathic Parkinson's disease.

Blast-induced axonal degeneration in the rat cerebellum in the absence of head movement.

Blast exposure can injure brain by multiple mechanisms, and injury attributable to direct effects of the blast wave itself have been difficult to distinguish from that caused by rapid head displacement and other secondary processes. To resolve this issue, we used a rat model of blast exposure in which head movement was either strictly prevented or permitted in the lateral plane. Blast was found to produce axonal injury even with strict prevention of head movement. This axonal injury was restricted to the cerebellum, with the exception of injury in visual tracts secondary to ocular trauma. The cerebellar axonal injury was increased in rats in which blast-induced head movement was permitted, but the pattern of injury was unchanged. These findings support the contentions that blast per se, independent of head movement, is sufficient to induce axonal injury, and that axons in cerebellar white matter are particularly vulnerable to direct blast-induced injury.

Glucose availability limits microglial nitric oxide production.

Metabolic intermediates influence inflammation not only through signaling effects, but also by fueling the production of pro-inflammatory molecules. Microglial production of nitric oxide (NO) requires the consumption of NADPH. NADPH consumed in this process is regenerated from NADP+ primarily through the hexose monophosphate shunt, which can utilize only glucose as a substrate. These factors predict that glucose availability can be rate-limiting for glial NO production. To test this prediction, cultured astrocytes and microglia were incubated with lipopolysaccharide and interferon-γ to promote expression of inducible nitric oxide synthase, and the rate of NO production was assessed at defined glucose concentrations. Increased NO production was detected only in cultures containing microglia. The NO production was markedly slowed at glucose concentrations below 0.5 mM, and comparably reduced by inhibition of the hexose monophosphate shunt with 6-aminonicotinamide. Reduced NO production caused by glucose deprivation was partly reversed by malate, which fuels NADPH production by malate dehydrogenase, and by NADPH itself. These findings highlight the role of the hexose monophosphate shunt in fueling NO synthesis and suggest that microglial NO production in the brain may be limited at sites of low glucose availability, such as abscesses or other compartmentalized infections.

α-synuclein aggregates induce c-Abl activation and dopaminergic neuronal loss by a feed-forward redox stress mechanism.

Oxidative stress and α-synuclein aggregation both drive neurodegeneration in Parkinson's disease, and the protein kinase c-Abl provides a potential amplifying link between these pathogenic factors. Suppressing interactions between these factors may thus be a viable therapeutic approach for this disorder. To evaluate this possibility, pre-formed α-synuclein fibrils (PFFs) were used to induce α-synuclein aggregation in neuronal cultures. Exposure to PFFs induced oxidative stress and c-Abl activation in wild-type neurons. By contrast, α-synuclein - deficient neurons, which cannot form α-synuclein aggregates, failed to exhibit either oxidative stress or c-Abl activation. N-acetyl cysteine, a thiol repletion agent that supports neuronal glutathione metabolism, suppressed the PFF - induced redox stress and c-Abl activation in the wild-type neurons, and likewise suppressed α-synuclein aggregation. Parallel findings were observed in mouse brain: PFF-induced α-synuclein aggregation in the substantia nigra was associated with redox stress, c-Abl activation, and dopaminergic neuronal loss, along with microglial activation and motor impairment, all of which were attenuated with oral N-acetyl cysteine. Similar results were obtained using AAV-mediated α-synuclein overexpression as an alternative means of driving α-synuclein aggregation in vivo. These findings show that α-synuclein aggregates induce c-Abl activation by a redox stress mechanism. c-Abl activation in turn promotes α-synuclein aggregation, in a feed-forward interaction. The capacity of N-acetyl cysteine to interrupt this interaction adds mechanistic support its consideration as a therapeutic in Parkinson's disease.

A tandem activity-based sensing and labeling strategy enables imaging of transcellular hydrogen peroxide signaling.

Reactive oxygen species (ROS) like hydrogen peroxide (H2O2) are transient species that have broad actions in signaling and stress, but spatioanatomical understanding of their biology remains insufficient. Here, we report a tandem activity-based sensing and labeling strategy for H2O2 imaging that enables capture and permanent recording of localized H2O2 fluxes. Peroxy Green-1 Fluoromethyl (PG1-FM) is a diffusible small-molecule probe that senses H2O2 by a boronate oxidation reaction to trigger dual release and covalent labeling of a fluorescent product, thus preserving spatial information on local H2O2 changes. This unique reagent enables visualization of transcellular redox signaling in a microglia-neuron coculture cell model, where selective activation of microglia for ROS production increases H2O2 in nearby neurons. In addition to identifying ROS-mediated cell-to-cell communication, this work provides a starting point for the design of chemical probes that can achieve high spatial fidelity by combining activity-based sensing and labeling strategies.

Superoxide and Non-ionotropic Signaling in Neuronal Excitotoxicity.

Excitotoxicity is classically attributed to Ca2+ influx through NMDA receptors (NMDAr), leading to production of nitric oxide by neuronal nitric oxide synthase and superoxide by mitochondria, which react to form highly cytotoxic peroxynitrite. More recent observations warrant revision of the classic view and help to explain some otherwise puzzling aspects of excitotoxic cell injury. Studies using pharmacological and genetic approaches show that superoxide produced by NMDAr activation originates primarily from NADPH oxidase rather than from mitochondria. As NADPH oxidase is localized to the plasma membrane, this also provides an explanation for the extracellular release of superoxide and cell-to-cell "spread" of excitotoxic injury observed in vitro and in vivo. The signaling pathway linking NMDAr to NADPH oxidase involves Ca2+ influx, phosphoinositol-3-kinase, and protein kinase Cζ, and interventions at any of these steps can prevent superoxide production and excitotoxic injury. Ca2+ influx specifically through NMDAr is normally required to induce excitotoxicity, through a mechanism presumed to involve privileged Ca2+ access to local signaling domains. However, experiments using selective blockade of the NMDAr ion channel and artificial reconstitution of Ca2+ by other routes indicate that the special effects of NMDAr activation are attributable instead to concurrent non-ionotropic NMDAr signaling by agonist binding to NMDAr. The non-ionotropic signaling driving NADPH oxidase activation is mediated in part by phosphoinositol-3-kinase binding to the C-terminal domain of GluN2B receptor subunits. These more recently identified aspects of excitotoxicity expand our appreciation of the complexity of excitotoxic processes and suggest novel approaches for limiting neuronal injury.

A thermodynamic function of glycogen in brain and muscle.

Brain and muscle glycogen are generally thought to function as local glucose reserves, for use during transient mismatches between glucose supply and demand. However, quantitative measures show that glucose supply is likely never rate-limiting for energy metabolism in either brain or muscle under physiological conditions. These tissues nevertheless do utilize glycogen during increased energy demand, despite the availability of free glucose, and despite the ATP cost of cycling glucose through glycogen polymer. This seemingly wasteful process can be explained by considering the effect of glycogenolysis on the amount of energy obtained from ATP (ΔG'ATP). The amount of energy obtained from ATP is reduced by elevations in inorganic phosphate (Pi). Glycogen utilization sequesters Pi in the glycogen phosphorylase reaction and in downstream phosphorylated glycolytic intermediates, thereby buffering Pi elevations and maximizing energy yield at sites of rapid ATP consumption. This thermodynamic effect of glycogen may be particularly important in the narrow, spatially constrained astrocyte processes that ensheath neuronal synapses and in cells such as astrocytes and myocytes that release Pi from phosphocreatine during energy demand. The thermodynamic effect may also explain glycolytic super-compensation in brain when glycogen is not available, and aspects of exercise physiology in muscle glycogen phosphorylase deficiency (McArdle disease).

Technical and Comparative Aspects of Brain Glycogen Metabolism.

It has been known for over 50 years that brain has significant glycogen stores, but the physiological function of this energy reserve remains uncertain. This uncertainty stems in part from several technical challenges inherent in the study of brain glycogen metabolism, and may also stem from some conceptual limitations. Factors presenting technical challenges include low glycogen content in brain, non-homogenous labeling of glycogen by radiotracers, rapid glycogenolysis during postmortem tissue handling, and effects of the stress response on brain glycogen turnover. Here, we briefly review aspects of glycogen structure and metabolism that bear on these technical challenges, and discuss ways these can be overcome. We also highlight physiological aspects of glycogen metabolism that limit the conditions under which glycogen metabolism can be useful or advantageous over glucose metabolism. Comparisons with glycogen metabolism in skeletal muscle provide an additional perspective on potential functions of glycogen in brain.

Methodological considerations for studies of brain glycogen.

Glycogen stores in the brain have been recognized for decades, but the underlying physiological function of this energy reserve remains elusive. This uncertainty stems in part from several technical challenges inherent in the study of brain glycogen metabolism. These include low glycogen content in the brain, non-homogeneous labeling of glycogen by radiotracers, rapid glycogenolysis during postmortem tissue handling, and effects of the stress response on brain glycogen turnover. Here we briefly review the aspects of the glycogen structure and metabolism that bear on these technical challenges and present ways they can be addressed.

Cofilin-actin rod formation in experimental stroke is attenuated by therapeutic hypothermia and overexpression of the inducible 70 kD inducible heat shock protein (Hsp70).

BACKGROUND AND PURPOSE: Cofilin-actin rods are covalently linked aggregates of cofilin-1 and actin. Under ischemic conditions, these rods have been observed in neuronal dendrites and axons and may contribute to the loss of these processes. Hypothermia (Hypo) and the 70 kD inducible heat shock protein (Hsp70) are both known to improve outcomes after stroke, but the mechanisms are uncertain. Here, we evaluated the effect of these factors on cofilin-actin rod formation in a mouse model of stroke. MATERIALS AND METHODS: Mice were subjected to distal middle cerebral artery occlusion (dMCAO) and treated with Hypo using a paradigm previously shown to be neuroprotective. We similarly studied mice that overexpressed transgenic (Tg) or were deficient knockout (Ko) in the inducible 70 kDa heat shock protein (Hsp70), also previously shown to be protective by our group and others. Cofilin-actin rod formation was assessed by histological analysis at 4 and 24 h after dMCAO. Its expression was analyzed in three different regions, namely, infarct core (the center of the infarct), middle cerebral artery (MCA) borderzone (the edge of the brain regions supplied by the MCA), and the ischemic borderzone (border of ischemic lesion). Ischemic lesion size and neurological deficits were also assessed. RESULTS: Both Hypo-treated and Hsp70 Tg mice had smaller lesion sizes and improved neurological outcomes, whereas Hsp70 Ko mice had larger lesion sizes and worsened neurological outcomes. Cofilin-actin rods were increased after stroke, but were reduced by therapeutic Hypo and in Hsp70 Tg mice. In contrast, cofilin-actin rods were increased in ischemic brains of Hsp70 Ko mice. CONCLUSIONS: Cofilin-actin rod formation was suppressed under the conditions of neuroprotection and increased under circumstances where outcome was worsened. This suggests that cofilin-actin rods may act to participate in or exacerbate ischemic pathology and warrants further study as a potential therapeutic target.

Excitotoxic superoxide production and neuronal death require both ionotropic and non-ionotropic NMDA receptor signaling.

NMDA-type glutamate receptors (NMDAR) trigger superoxide production by neuronal NADPH oxidase-2 (NOX2), which if sustained leads to cell death. This process involves Ca2+ influx through NMDAR channels. By contrast, comparable Ca2+ influx by other routes does not induce NOX2 activation or cell death. This contrast has been attributed to site-specific effects of Ca2+ flux through NMDAR. Here we show instead that it stems from non-ionotropic signaling by NMDAR GluN2B subunits. To evaluate non-ionotropic effects, mouse cortical neurons were treated with NMDA together with 7-chlorokynurenate, L-689,560, or MK-801, which block Ca2+ influx through NMDAR channels but not NMDA binding. NMDA-induced superoxide formation was prevented by the channel blockers, restored by concurrent Ca2+ influx through ionomycin or voltage-gated calcium channels, and not induced by the Ca2+ influx in the absence of NMDAR ligand binding. Neurons expressing either GluN2B subunits or chimeric GluN2A/GluN2B C-terminus subunits exhibited NMDA-induced superoxide production, whereas neurons expressing chimeric GluN2B/GluN2A C-terminus subunits did not. Neuronal NOX2 activation requires phosphoinositide 3-kinase (PI3K), and NMDA binding to NMDAR increased PI3K association with NMDA GluN2B subunits independent of Ca2+ influx. These findings identify a non-ionotropic signaling pathway that links NMDAR to NOX2 activation through the C-terminus domain of GluN2B.

Fibrin-targeting immunotherapy protects against neuroinflammation and neurodegeneration.

Activation of innate immunity and deposition of blood-derived fibrin in the central nervous system (CNS) occur in autoimmune and neurodegenerative diseases, including multiple sclerosis (MS) and Alzheimer's disease (AD). However, the mechanisms that link disruption of the blood-brain barrier (BBB) to neurodegeneration are poorly understood, and exploration of fibrin as a therapeutic target has been limited by its beneficial clotting functions. Here we report the generation of monoclonal antibody 5B8, targeted against the cryptic fibrin epitope γ377-395, to selectively inhibit fibrin-induced inflammation and oxidative stress without interfering with clotting. 5B8 suppressed fibrin-induced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and the expression of proinflammatory genes. In animal models of MS and AD, 5B8 entered the CNS and bound to parenchymal fibrin, and its therapeutic administration reduced the activation of innate immunity and neurodegeneration. Thus, fibrin-targeting immunotherapy inhibited autoimmunity- and amyloid-driven neurotoxicity and might have clinical benefit without globally suppressing innate immunity or interfering with coagulation in diverse neurological diseases.

Cofilin-actin rod formation in neuronal processes after brain ischemia.

Functional impairment after brain ischemia results in part from loss of neuronal spines and dendrites, independent of neuronal death. Cofilin-actin rods are covalently linked aggregates of cofilin-1 and actin that form in neuronal processes (neurites) under conditions of ATP depletion and oxidative stress, and which cause neurite degeneration if not disassembled. ATP depletion and oxidative stress occur with differing severity, duration, and time course in different ischemic conditions. Here we evaluated four mouse models of brain ischemia to define the conditions that drive formation of cofilin-actin rods. Three of the models provide early reperfusion: transient middle cerebral artery occlusion (MCAo), transient bilateral common carotid artery occlusion (CCAo), and cardiac arrest / cardiopulmonary resuscitation (CA/CPR). Early reperfusion restores ATP generating capacity, but also induces oxidative stress. The fourth model, photothrombotic cortical infarction, does not provide reperfusion. Cofilin-actin rods were formed in each of these models, but with differing patterns. Where acute reperfusion occurred, rod formation was maximal within 4 hours after reperfusion. Where infarction occurred, rods continued to form for at least 24 hours after ischemic onset, and extended into the adjacent non-ischemic tissue. Interventions that limit cofilin-actin rod formation may help to preserve integrity of neuronal processes in permanent ischemia.

Low-frequency cortical activity is a neuromodulatory target that tracks recovery after stroke.

Recent work has highlighted the importance of transient low-frequency oscillatory (LFO; <4 Hz) activity in the healthy primary motor cortex during skilled upper-limb tasks. These brief bouts of oscillatory activity may establish the timing or sequencing of motor actions. Here, we show that LFOs track motor recovery post-stroke and can be a physiological target for neuromodulation. In rodents, we found that reach-related LFOs, as measured in both the local field potential and the related spiking activity, were diminished after stroke and that spontaneous recovery was closely correlated with their restoration in the perilesional cortex. Sensorimotor LFOs were also diminished in a human subject with chronic disability after stroke in contrast to two non-stroke subjects who demonstrated robust LFOs. Therapeutic delivery of electrical stimulation time-locked to the expected onset of LFOs was found to significantly improve skilled reaching in stroke animals. Together, our results suggest that restoration or modulation of cortical oscillatory dynamics is important for the recovery of upper-limb function and that they may serve as a novel target for clinical neuromodulation.