Chengbiao Lu

Activation of Mitochondrial ATP-Dependent Potassium Channels Protects Neurons against Ischemia-Induced Death by a Mechanism Involving Suppression of Bax Translocation and Cytochrome c Release

Neurons express a variety of plasma-membrane potassium channels that play important roles in regulating neuronal excitability and synaptic transmission, but also contain mitochondrial ATP-sensitive potassium channels, the functions of which are unknown. Studies of cardiac cells suggest that similar mitochondrial ATP-sensitive potassium channels are involved in the process of ischemic preconditioning, suggesting a role in regulating cell survival. The authors report that mice given diazoxide, an activator of mitochondrial ATP-sensitive potassium channels, exhibited a large (60% to 70%) decrease in cortical infarct size after permanent occlusion of the middle cerebral artery. Diazoxide decreases neuronal apoptosis and increases astrocyte survival and activation in the penumbral region of the ischemic cortex. The neuroprotective effect of diazoxide is abolished by 5-hydroxydecanoate, a selective antagonist of mitochondrial ATP-sensitive potassium channels. Studies of cultured hippocampal neurons reveal that diazoxide depolarizes mitochondria, prevents cytochrome c release, and protects cells against death induced by staurosporine and chemical hypoxia. Diazoxide increased the levels of Bcl2 and inhibited the association of Bax with mitochondria in neurons exposed to an apoptotic insult, suggesting that activation of mitochondrial ATP-sensitive potassium channels may stabilize mitochondrial function by differentially modulating proapoptotic and anti-apoptotic proteins. Collectively, the data suggest that mitochondrial ATP-sensitive potassium channels play a key role in modulating neuronal survival under ischemic conditions, and identify agents that activate mitochondrial ATP-sensitive potassium channels as potential therapeutics for stroke and related neurodegenerative conditions.

Membrane properties of rat embryonic multipotent neural stem cells

We have characterized several potential stem cell markers and defined the membrane properties of rat fetal (E10.5) neural stem cells (NSC) by immunocytochemistry, electrophysiology and microarray analysis. Immunocytochemical analysis demonstrates specificity of expression of Sox1, ABCG2/Bcrp1, and shows that nucleostemin labels both progenitor and stem cell populations. NSCs, like hematopoietic stem cells, express high levels of aldehyde dehydrogenase (ALDH) as assessed by Aldefluor labeling. Microarray analysis of 96 transporters and channels showed that Glucose transporter 1 (Glut1/Slc2a1) expression is unique to fetal NSCs or other differentiated cells. Electrophysiological examination showed that fetal NSCs respond to acetylcholine and its agonists, such as nicotine and muscarine. NSCs express low levels of tetrodotoxin (TTX) sensitive and insensitive sodium channels and calcium channels while expressing at least three kinds of potassium channels. We find that gap junction communication is mediated by connexin (Cx)43 and Cx45, and is essential for NSC survival and proliferation. Overall, our results show that fetal NSCs exhibit a unique signature that can be used to determine their location and assess their ability to respond to their environment.

Dimethyl Sulfoxide Suppresses NMDA- and AMPA-Induced Ion Currents and Calcium Influx and Protects against Excitotoxic Death in Hippocampal Neurons

Dimethyl sulfoxide (DMSO) is widely used in neuroscience research as a solvent for various pharmacological agents in both cell culture and in vivo studies and is also used in humans to treat musculoskeletal problems and pain. We now report that concentrations of DMSO to which neurons are typically exposed in experimental studies and in human patients (0.5-1.5%) inhibit glutamate responses in hippocampal neurons. DMSO suppresses, in a rapidly reversible manner, electrophysiological responses and calcium influx induced by glutamate, N-methyl-d-aspartate, and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate. Moreover, DMSO can prevent excitotoxic death of the neurons. These findings have important implications for the use of DMSO as a solvent in studies that involve glutamatergic neurotransmission. Our data also identify a mechanism that might explain clinical effects of DMSO on both peripheral and CNS neurons and suggest a potential use for DMSO in the treatment of excitotoxic neurodegenerative conditions.

Involvement of mitochondrial K+ release and cellular efflux in ischemic and apoptotic neuronal death.

We measured and manipulated intracellular potassium (K+) fluxes in cultured hippocampal neurons in an effort to understand the involvement of K+ in neuronal death under conditions of ischemia and exposure to apoptotic stimuli. Measurements of the intracellular K+ concentration using the fluorescent probe 1,3‐benzenedicarboxylic acid, 4,4′‐[1,4,10,13‐tetraoxa‐7,16‐diazacyclooctadecane‐7,16‐diylbis(5‐methoxy‐6,2‐benzofurandiyl)]bis‐, tetrakis [(acetyloxy) methyl] ester (PBFI) revealed that exposure of neurons to cyanide (chemical hypoxia), glutamate (excitotoxic insult) or staurosporine (apoptotic stimulus) results in efflux of K+ and cell death. Treatment of neurons with 5‐hydroxydecanoate (5HD), an inhibitor of mitochondrial K+ channels, reduced K+ fluxes in neurons exposed to each insult and increased the resistance of the cells to death. K+ efflux was attenuated, levels of oxyradicals were decreased, mitochondrial membrane potential was stabilized and release of cytochrome c from mitochondria was attenuated in neurons treated with 5HD. K+ was rapidly released into the cytosol from mitochondria when neurons were exposed to the K+ channel opener, diazoxide, or to the mitochondrial uncoupler, carbonyl cyanide 4(trifluoromethoxy)phenylhydrazone (FCCP), demonstrating that the intramitochondrial K+ concentration is greater than the cytosolic K+ concentration. The release of K+ from mitochondria was followed by efflux through plasma membrane K+ channels. In vivo studies showed that 5HD reduces ischemic brain damage without affecting cerebral blood flow in a mouse model of focal ischemic stroke. These findings suggest that intracellular K+ fluxes play a key role in modulating neuronal oxyradical production and cell survival under ischemic conditions, and that agents that modify K+ fluxes may have therapeutic benefit in stroke and related neurodegenerative conditions.

Direct cleavage of AMPA receptor subunit GluR1 and suppression of AMPA currents by caspase-3

Cysteine proteases of the caspase family play central roles in excecuting the cell death process in neurons during development of the nervous system and in neurodegenerative disorders. Recent findings suggest that caspases may also play roles in modulating neuronal plasticity in the absence of cell death. We previously reported that caspases can be activated in dendrites and synapses in response to activation of glutamate receptors. In the present study we demonstrate that the GluR1 subunit of the AMPA subtype of glutamate receptor is directly cleaved by caspase-3, and provide evidence that the cleavage of this subunit modulates neuronal excitability in ways that suggest important roles for caspases in regulating synaptic plasticity and cell survival. Whole-cell patch-clamp recordings in cultured rat hippocampal neurons showed that caspase activation in response to apoptotic stimuli selectively decreases AMPA channel activity without decreasing NMDA channel activity. Perfusion of neurons with recombinant caspase-3 resulted in a decreased AMPA current, demonstrating that caspase-3 activity is sufficient to suppress neuronal responses to glutamate. Exposure of radiolabeled GluR1 to recombinant caspase-3 resulted in cleavage of GluR1, demonstrating that this glutamate receptor protein is a direct substrate of this caspase. Our findings suggest roles for caspases in the modulation of neuronal excitability in physiological settings, and also identify a mechanism whereby caspases ensure that neurons die by apoptosis rather than excitotoxic necrosis in developmental and pathological settings.

Telomerase protects developing neurons against DNA damage-induced cell death

In mitotic cells, telomerase adds repeats of a DNA sequence (TTAGGG) to the ends of chromosomes (telomeres) thereby maintaining their length and preventing cellular senescence. We recently reported that the catalytic subunit of telomerase (TERT) is expressed in neuronal progenitor cells and in early postmitotic neurons in the developing rodent brain. We now report that TERT can protect cultured PC12 cells and embryonic hippocampal neurons against death induced by DNA damage. Overexpression of TERT in PC12 cells increases their resistance to the topoisomerase inhibitors camptothecin and etoposide. Hippocampal neurons in which TERT levels are decreased using antisense technology exhibit increased vulnerability to the DNA-damaging agents. Emerging findings suggest that DNA damage may trigger the death of neurons during brain development and in neurodegenerative disorders. Our data therefore suggest roles for TERT in modulating such cell deaths.

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.

Selective and biphasic effect of the membrane lipid peroxidation product 4-hydroxy-2,3-nonenal on N-methyl-D-aspartate channels

Increased oxyradical production and membrane lipid peroxidation occur in neurons under physiological conditions and in neurodegenerative disorders. Lipid peroxidation can alter synaptic plasticity and may increase the vulnerability of neurons to excitotoxicity, but the underlying mechanisms are unknown. We report that 4‐hydroxy‐2,3‐nonenal (4HN), an aldehyde product of lipid peroxidation, exerts a biphasic effect on NMDA‐induced current in cultured rat hippocampal neurons with current being increased during the first 2 h and decreased after 6 h. Similarly, 4HN causes an early increase and a delayed decrease in NMDA‐induced elevation of intracellular Ca2+ levels. In contrast, 4HN affects neither the ion current nor the Ca2+ response to α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA). The initial enhancement of NMDA‐induced current is associated with increased phosphorylation of the NR1 receptor subunit, whereas the delayed suppression of current is associated with cellular ATP depletion and mitochondrial membrane depolarization. Cell death induced by 4HN is attenuated by an NMDA receptor antagonist, but not by an AMPA receptor antagonist. A secreted form of amyloid precursor protein, previously shown to protect neurons against oxidative and excitotoxic insults, prevented each of the effects of 4HN including the early and late changes in NMDA current, delayed ATP depletion, and cell death. These findings show that the membrane lipid peroxidation product 4HN can modulate NMDA channel activity, suggesting a role for this aldehyde in physiological and pathophysiological responses of neurons to oxidative stress.

A link between maze learning and hippocampal expression of neuroleukin and its receptor gp78.

Neuroleukin (NLK) is a multifunctional protein involved in neuronal growth and survival, cell motility and differentiation, and glucose metabolism. We report herein that hippocampal expression of NLK and its receptor gp78 is associated with maze learning in rats. First, mRNA levels of NLK and gp78 were significantly increased in hippocampi of male Fischer‐344 rats following training in the Stone T‐maze and the Morris water maze. Second, a parallel increase was found in hippocampal NLK and gp78 proteins after maze learning. Third, NLK and gp78 mRNA and protein expression in hippocampus was reduced in a group of aged rats that showed more errors during the acquisition of the Stone maze task as compared with young rats. Finally, application of recombinant NLK to hippocampal neurons significantly enhanced glutamate‐induced ion currents, functional molecular changes that have been correlated with learning in vivo. Taken together, our results identify a novel association of hippocampal expression of NLK and its receptor gp78 with rat maze learning. Interaction of NLK with gp78 and subsequent signaling may strengthen synaptic mechanisms underlying learning and memory formation.

Phenformin Suppresses Calcium Responses to Glutamate and Protects Hippocampal Neurons against Excitotoxicity

Phenformin is a biguanide compound that can modulate glucose metabolism and promote weight loss and is therefore used to treat patients with type-2 diabetes. While phenformin may indirectly affect neurons by changing peripheral energy metabolism, the possibility that it directly affects neurons has not been examined. We now report that phenformin suppresses responses of hippocampal neurons to glutamate and decreases their vulnerability to excitotoxicity. Pretreatment of embryonic rat hippocampal cell cultures with phenformin protected neurons against glutamate-induced death, which was correlated with reduced calcium responses to glutamate. Immunoblot analyses showed that levels of the N-methyl-d-aspartate (NMDA) subunits NR1 and NR2A were significantly decreased in neurons exposed to phenformin, whereas levels of the AMPA receptor subunit GluR1 were unchanged. Whole-cell patch clamp analyses revealed that NMDA-induced currents were decreased, and AMPA-induced currents were unchanged in neurons pretreated with phenformin. Our data demonstrate that phenformin can protect neurons against excitotoxicity by differentially modulating levels of NMDA receptor subunits in a manner that decreases glutamate-induced calcium influx. These findings show that phenformin can modulate neuronal responses to glutamate, and suggest possible use of phenformin and related compounds in the prevention and/or treatment of neurodegenerative conditions.