Xin Ouyang

Pivotal role for neuronal Toll-like receptors in ischemic brain injury and functional deficits.

The innate immune system senses the invasion of pathogenic microorganisms and tissue injury through Toll-like receptors (TLR), a mechanism thought to be limited to immune cells. We now report that neurons express several TLRs, and that the levels of TLR2 and -4 are increased in neurons in response to IFN-γ stimulation and energy deprivation. Neurons from both TLR2 knockout and -4 mutant mice were protected against energy deprivation-induced cell death, which was associated with decreased activation of a proapoptotic signaling cascade involving jun N-terminal kinase and the transcription factor AP-1. TLR2 and -4 expression was increased in cerebral cortical neurons in response to ischemia/reperfusion injury, and the amount of brain damage and neurological deficits caused by a stroke were significantly less in mice deficient in TLR2 or -4 compared with WT control mice. Our findings establish a proapoptotic signaling pathway for TLR2 and -4 in neurons that may render them vulnerable to ischemic death.

Gamma secretase–mediated Notch signaling worsens brain damage and functional outcome in ischemic stroke

Mice transgenic for antisense Notch and normal mice treated with inhibitors of the Notch-activating enzyme γ-secretase showed reduced damage to brain cells and improved functional outcome in a model of focal ischemic stroke. Notch endangers neurons by modulating pathways that increase their vulnerability to apoptosis, and by activating microglial cells and stimulating the infiltration of proinflammatory leukocytes. These findings suggest that Notch signaling may be a therapeutic target for treatment of stroke and related neurodegenerative conditions.

Intravenous immunoglobulin (IVIG) protects the brain against experimental stroke by preventing complement-mediated neuronal cell death

Stroke is among the three leading causes of death worldwide and the most frequent cause of permanent disability. Brain ischemia induces an inflammatory response involving activated complement fragments. Here we show that i.v. Ig (IVIG) treatment, which scavenges complement fragments, protects brain cells against the deleterious effects of experimental ischemia and reperfusion (I/R) and prevents I/R-induced mortality in mice. Animals administered IVIG either 30 min before ischemia or after 3 h of reperfusion exhibited a 50–60% reduction of brain infarct size and a 2- to 3-fold improvement of the functional outcome. Even a single low dose of IVIG given after stroke was effective. IVIG was protective in the nonreperfusion model of murine stroke as well and did not exert any peripheral effects. Human IgG as well as intrinsic murine C3 levels were significantly higher in the infarcted brain region compared with the noninjured side, and their physical association was demonstrated by immuno-coprecipitation. C5-deficient mice were significantly protected from I/R injury compared with their wild-type littermates. Exposure of cultured neurons to oxygen/glucose deprivation resulted in increased levels of C3 associated with activation of caspase 3, a marker of apoptosis; both signals were attenuated with IVIG treatment. Our data suggest a major role for complement-mediated cell death in ischemic brain injury and the prospect of using IVIG in relatively low doses as an interventional therapy for stroke.

Involvement of PGC-1α in the formation and maintenance of neuronal dendritic spines

The formation, maintenance and reorganization of synapses are critical for brain development and the responses of neuronal circuits to environmental challenges. Here we describe a novel role for peroxisome proliferator-activated receptor γ co-activator 1α, a master regulator of mitochondrial biogenesis, in the formation and maintenance of dendritic spines in hippocampal neurons. In cultured hippocampal neurons, proliferator-activated receptor γ co-activator 1α overexpression increases dendritic spines and enhances the molecular differentiation of synapses, whereas knockdown of proliferator-activated receptor γ co-activator 1α inhibits spinogenesis and synaptogenesis. Proliferator-activated receptor γ co-activator 1α knockdown also reduces the density of dendritic spines in hippocampal dentate granule neurons in vivo. We further show that brain-derived neurotrophic factor stimulates proliferator-activated receptor γ co-activator-1α-dependent mitochondrial biogenesis by activating extracellular signal-regulated kinases and cyclic AMP response element-binding protein. Proliferator-activated receptor γ co-activator-1α knockdown inhibits brain-derived neurotrophic factor-induced dendritic spine formation without affecting expression and activation of the brain-derived neurotrophic factor receptor tyrosine receptor kinase B. Our findings suggest that proliferator-activated receptor γ co-activator-1α and mitochondrial biogenesis have important roles in the formation and maintenance of hippocampal dendritic spines and synapses.

Mitochondrial Uncoupling Protein-4 Regulates Calcium Homeostasis and Sensitivity to Store Depletion-induced Apoptosis in Neural Cells

An increase in the cytoplasmic-free Ca2+ concentration mediates cellular responses to environmental signals that influence a range of processes, including gene expression, motility, secretion of hormones and neurotransmitters, changes in energy metabolism, and apoptosis. Mitochondria play important roles in cellular Ca2+ homeostasis and signaling, but the roles of specific mitochondrial proteins in these processes are unknown. Uncoupling proteins (UCPs) are a family of proteins located in the inner mitochondrial membrane that can dissociate oxidative phosphorylation from respiration, thereby promoting heat production and decreasing oxyradical production. Here we show that UCP4, a neuronal UCP, influences store-operated Ca2+ entry, a process in which depletion of endoplasmic reticulum Ca2+ stores triggers Ca2+ influx through plasma membrane “store-operated” channels. PC12 neural cells expressing human UCP4 exhibit reduced Ca2+ entry in response to thapsigargin-induced endoplasmic reticulum Ca2+ store depletion. The elevations of cytoplasmic and intramitochondrial Ca2+ concentrations and mitochondrial oxidative stress induced by thapsigargin were attenuated in cells expressing UCP4. The stabilization of Ca2+ homeostasis and preservation of mitochondrial function by UCP4 was correlated with reduced mitochondrial reactive oxygen species generation, oxidative stress, and Gadd153 up-regulation and increased resistance of the cells to death. Reduced Ca2+-dependent cytosolic phospholipase A2 activation and oxidative metabolism of arachidonic acid also contributed to the stabilization of mitochondrial function in cells expressing human UCP4. These findings demonstrate that UCP4 can regulate cellular Ca2+ homeostasis, suggesting that UCPs may play roles in modulating Ca2+ signaling in physiological and pathological conditions.

Telomere protection mechanisms change during neurogenesis and neuronal maturation: Newly generated neurons are hypersensitive to telomere and DNA damage

Telomeres are DNA–protein complexes at the ends of eukaryotic chromosomes that play an important role in maintaining the integrity of the genome. In proliferative stem cells and cancer cells, telomere length is maintained by telomerase, and telomere structure and functions are regulated by telomere-associated proteins. We find that telomerase levels are high in embryonic cortical neural progenitor cells (NPCs) and low in newly generated neurons (NGNs) and mature neurons (MNs). In contrast, telomere repeat-binding factor 2 (TRF2) expression is undetectable in early brain development in vivo and in cultured NPCs and is expressed at progressively higher levels as NPCs cease proliferation and differentiate into postmitotic neurons. The telomere-disrupting agent telomestatin induces a DNA damage response and apoptosis in NGNs (which have low levels of TRF2 and telomerase), whereas NPCs (which have high levels of telomerase) and MNs (which have high levels of TRF2) are resistant to telomere damage. Overexpression of TRF2 in NGNs protects them against death induced by telomestatin and other DNA-damaging agents. Knockdown of TRF2 expression in MNs and knock-out of telomerase reverse transcriptase in NPCs increased their sensitivity to telomere- and DNA-damaging agents but did not affect the vulnerability of NGNs. These findings suggest that TRF2 and telomerase function as distinct telomere protection mechanisms during the processes of neurogenesis and neuronal maturation and that hypersensitivity of NGNs to telomere damage results from relative deficiencies of both telomerase and TRF2.

Neuroprotective actions of a histidine analogue in models of ischemic stroke.

Histidine is a naturally occurring amino acid with antioxidant properties, which is present in low amounts in tissues throughout the body. We recently synthesized and characterized histidine analogues related to the natural dipeptide carnosine, which selectively scavenge the toxic lipid peroxidation product 4‐hydroxynonenal (HNE). We now report that the histidine analogue histidyl hydrazide is effective in reducing brain damage and improving functional outcome in a mouse model of focal ischemic stroke when administered intravenously at a dose of 20 mg/kg, either 30 min before or 60 min and 3 h after the onset of middle cerebral artery occlusion. The histidine analogue also protected cultured rat primary neurons against death induced by HNE, chemical hypoxia, glucose deprivation, and combined oxygen and glucose deprivation. The histidine analogue prevented neuronal apoptosis as indicated by decreased production of cleaved caspase‐3 protein. These findings suggest a therapeutic potential for HNE‐scavenging histidine analogues in the treatment of stroke and related neurodegenerative conditions.

Mitochondrial Superoxide Production Negatively Regulates Neural Progenitor Proliferation and Cerebral Cortical Development

Although high amounts of reactive oxygen species (ROS) can damage cells, ROS can also play roles as second messengers, regulating diverse cellular processes. Here, we report that embryonic mouse cerebral cortical neural progenitor cells (NPCs) exhibit intermittent spontaneous bursts of mitochondrial superoxide (SO) generation (mitochondrial SO flashes) that require transient opening of membrane permeability transition pores (mPTP). This quantal SO production negatively regulates NPC self‐renewal. Mitochondrial SO scavengers and mPTP inhibitors reduce SO flash frequency and enhance NPC proliferation, whereas prolonged mPTP opening and SO generation increase SO flash incidence and decrease NPC proliferation. The inhibition of NPC proliferation by mitochondrial SO involves suppression of extracellular signal‐regulated kinases. Moreover, mice lacking SOD2 (SOD2−/− mice) exhibit significantly fewer proliferative NPCs and differentiated neurons in the embryonic cerebral cortex at midgestation compared with wild‐type littermates. Cultured SOD2−/− NPCs exhibit a significant increase in SO flash frequency and reduced NPC proliferation. Taken together, our findings suggest that mitochondrial SO flashes negatively regulate NPC self‐renewal in the developing cerebral cortex. STEM CELLS2012;30:2535–2547

Evidence that caspase‐1 is a negative regulator of AMPA receptor‐mediated long‐term potentiation at hippocampal synapses

Best known for their pivotal role in a form of programmed cell death called apoptosis, caspases may also function in more subtle physiological processes. Caspases are present in synapses and dendrites of neurons where they can be activated in response to glutamate receptor stimulation and calcium influx. Here we tested the hypothesis that caspase‐1 plays a role in modulating long‐term potentiation (LTP) at hippocampal synapses. We provide evidence that caspase‐1 plays a role in regulating α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor‐mediated calcium influx and synaptic plasticity in the hippocampus. LTP of excitatory postsynaptic potentials at CA1 synapses was significantly enhanced when hippocampal slices were treated with either a pan‐caspase inhibitor or a selective inhibitor of caspase‐1, but not by an inhibitor of caspase‐6. Inhibition of caspase‐1 significantly enhanced the AMPA current‐mediated component of LTP without affecting the N‐methyl‐d‐aspartate current‐mediated component. Calcium responses to AMPA were enhanced in hippocampal neurons treated with a caspase‐1 inhibitor suggesting that caspase‐1 normally functions to reduce AMPA receptor‐mediated calcium influx. These findings suggest that, by selectively reducing AMPA currents and calcium influx, caspase‐1 functions as a negative regulator of LTP at hippocampal synapses.

Permeability transition pore-mediated mitochondrial superoxide flashes mediate an early inhibitory effect of amyloid beta1−42 on neural progenitor cell proliferation

Cellular damage by reactive oxygen species and altered neurogenesis are implicated in the etiology of AD and the pathogenic actions of amyloid β-peptide (Aβ); the underlying mechanisms and the early oxidative intracellular events triggered by Aβ are not established. In the present study, we found that mouse embryonic cortical neural progenitor cells exhibit intermittent spontaneous mitochondrial superoxide (SO) flashes that require transient opening of mitochondrial permeability transition pores (mPTPs). The incidence of mitochondria SO flash activity in neural progenitor cells (NPCs) increased during the first 6-24 hours of exposure to aggregating amyloid β-peptide (Aβ1-42), indicating an increase in transient mPTP opening. Subsequently, the SO flash frequency progressively decreased and ceased between 48 and 72 hours of exposure to Aβ1-42, during which time global cellular reactive oxygen species increased, mitochondrial membrane potential decreased, cytochrome C was released from mitochondria and the cells degenerated. Inhibition of mPTPs and selective reduction in mitochondrial SO flashes significantly ameliorated the negative effects of Aβ1-42 on NPC proliferation and survival. Our findings suggest that mPTP-mediated bursts of mitochondrial SO production is a relatively early and pivotal event in the adverse effects of Aβ1-42 on NPCs. If Aβ inhibits NPC proliferation in the brains of AD patients by a similar mechanism, then interventions that inhibit mPTP-mediated superoxide flashes would be expected to protect NPCs against the adverse effects of Aβ.