Shinghua Ding

Enhanced Astrocytic Ca2+ Signals Contribute to Neuronal Excitotoxicity after Status Epilepticus

Status epilepticus (SE), an unremitting seizure, is known to cause a variety of traumatic responses including delayed neuronal death and later cognitive decline. Although excitotoxicity has been implicated in this delayed process, the cellular mechanisms are unclear. Because our previous brain slice studies have shown that chemically induced epileptiform activity can lead to elevated astrocytic Ca2+ signaling and because these signals are able to induce the release of the excitotoxic transmitter glutamate from these glia, we asked whether astrocytes are activated during status epilepticus and whether they contribute to delayed neuronal death in vivo. Using two-photon microscopy in vivo, we show that status epilepticus enhances astrocytic Ca2+ signals for 3 d and that the period of elevated glial Ca2+ signaling is correlated with the period of delayed neuronal death. To ask whether astrocytes contribute to delayed neuronal death, we first administered antagonists which inhibit gliotransmission: MPEP [2-methyl-6-(phenylethynyl)pyridine], a metabotropic glutamate receptor 5 antagonist that blocks astrocytic Ca2+ signals in vivo, and ifenprodil, an NMDA receptor antagonist that reduces the actions of glial-derived glutamate. Administration of these antagonists after SE provided significant neuronal protection raising the potential for a glial contribution to neuronal death. To test this glial hypothesis directly, we loaded Ca2+ chelators selectively into astrocytes after status epilepticus. We demonstrate that the selective attenuation of glial Ca2+ signals leads to neuronal protection. These observations support neurotoxic roles for astrocytic gliotransmission in pathological conditions and identify this process as a novel therapeutic target.

Photothrombosis ischemia stimulates a sustained astrocytic Ca2+ signaling in vivo

Although there is significant information concerning the consequences of cerebral ischemia on neuronal function, relatively little is known about functional responses of astrocytes, the predominant glial‐cell type in the central nervous system. In this study, we asked whether focal ischemia would impact astrocytic Ca2+ signaling, a characteristic form of excitability in this cell type. In vivo Ca2+ imaging of cortical astrocytes was performed using two‐photon (2‐P) microscopy during the acute phase of photothrombosis‐induced ischemia initiated by green light illumination of circulating Rose Bengal. Although whisker evoked potentials were reduced by over 90% within minutes of photothrombosis, astrocytes in the ischemic core remained structurally intact for a few hours. In vivo Ca2+ imaging showed that an increase in transient Ca2+ signals in astrocytes within 20 min of ischemia. These Ca2+ signals were synchronized and propagated as waves amongst the glial network. Pharmacological manipulations demonstrated that these Ca2+ signals were dependent on activation of metabotropic glutamate receptor 5 (mGluR5) and metabotropic γ‐aminobutyric acid receptor (GABABR) but not by P2 purinergic receptor or A1 adenosine receptor. Selective inhibition of Ca2+ in astrocytes with BAPTA significantly reduced the infarct volume, demonstrating that the enhanced astrocytic Ca2+ signal contributes to neuronal damage presumably through Ca2+‐dependent release of glial glutamate. Because astrocytes offer multiple functions in close communication with neurons and vasculature, the ischemia‐induced increase in astrocytic Ca2+ signaling may represent an initial attempt for these cells to communicate with neurons or provide feed back regulation to the vasculature.

Investigating the Putative Glycine Hinge in Shaker Potassium Channel

The crystal structure of an open potassium channel reveals a kink in the inner helix that lines the pore (Jiang, Y.X., A. Lee, J.Y. Chen, M. Cadene, B.T. Chait, and R. MacKinnon. 2002. Nature 417:523–526). The putative hinge point is a highly conserved glycine residue. We examined the role of the homologous residue (Gly466) in the S6 transmembrane segment of Shaker potassium channels. The nonfunctional alanine mutant G466A will assemble, albeit poorly, with wild-type (WT) subunits, suppressing functional expression. To test if this glycine residue is critical for activation gating, we did a glycine scan along the S6 segment in the background of G466A. Although all of these double mutants lack the higher-level glycosylation that is characteristic of mature Shaker channels, one (G466A/V467G) is able to generate voltage-dependent potassium current. Surface biotinylation shows that functional and nonfunctional constructs containing G466A express at comparable levels in the plasma membrane. Compared with WT channels, the shifted-glycine mutant has impairments in voltage-dependent channel opening, including a right-shifted activation curve and a decreased rate of activation. The double mutant has relatively normal open-channel properties, except for a decreased affinity for intracellular blockers, a consequence of the loss of the side chain of Val467. Control experiments with the double mutants M440A/G466A and G466A/V467A suggest that the flexibility provided by Gly466 is more important for channel function than its small size. Our results support roles for Gly466 both in biogenesis of the channel and as a hinge in activation gating.

Prolonged exposure of cortical neurons to oligomeric amyloid-β impairs NMDA receptor function via NADPH oxidase-mediated ROS production: protective effect of green tea (–)-epigallocatechin-3-gallate

Excessive production of Aβ (amyloid β-peptide) has been shown to play an important role in the pathogenesis of AD (Alzheimers disease). Although not yet well understood, aggregation of Aβ is known to cause toxicity to neurons. Our recent study demonstrated the ability for oligomeric Aβ to stimulate the production of ROS (reactive oxygen species) in neurons through an NMDA (N-methyl-d-aspartate)-dependent pathway. However, whether prolonged exposure of neurons to aggregated Aβ is associated with impairment of NMDA receptor function has not been extensively investigated. In the present study, we show that prolonged exposure of primary cortical neurons to Aβ oligomers caused mitochondrial dysfunction, an attenuation of NMDA receptor-mediated Ca2+ influx and inhibition of NMDA-induced AA (arachidonic acid) release. Mitochondrial dysfunction and the decrease in NMDA receptor activity due to oligomeric Aβ are associated with an increase in ROS production. Gp91ds-tat, a specific peptide inhibitor of NADPH oxidase, and Mn(III)-tetrakis(4-benzoic acid)-porphyrin chloride, an ROS scavenger, effectively abrogated Aβ-induced ROS production. Furthermore, Aβ-induced mitochondrial dysfunction, impairment of NMDA Ca2+ influx and ROS production were prevented by pre-treatment of neurons with EGCG [(−)-epigallocatechin-3-gallate], a major polyphenolic component of green tea. Taken together, these results support a role for NADPH oxidase-mediated ROS production in the cytotoxic effects of Aβ, and demonstrate the therapeutic potential of EGCG and other dietary polyphenols in delaying onset or retarding the progression of AD.

Neuronal protective role of PBEF in a mouse model of cerebral ischemia.

Pre-B-cell colony-enhancing factor (PBEF) (also known as nicotinamide phosphoribosyltransferase) is a rate-limiting enzyme in the salvage pathway for mammalian biosynthesis of nicotinamide adenine dinucleotide (NAD+). By synthesizing NAD+, PBEF functions to maintain an energy supply that has critical roles in cell survival. Cerebral ischemia is a major neural disorder with a high percentage of mortality and disability. Ischemia leads to energy depletion and eventually neuronal death and brain damage. This study investigated the role of PBEF in cerebral ischemia using a photothrombosis mouse model. Using immunostaining, we initially determined that PBEF is highly expressed in neurons, but not in glial cells in the mouse brain. To study the role of PBEF in ischemia in vivo, we used PBEF knockout heterozygous (Pbef+/−) mice. We showed that these mice have lower PBEF expression and NAD+ level than do wild-type (WT) mice. When subjected to photothrombosis, Pbef+/− mice have significantly larger infarct volume than do age-matched WT mice at 24 hours after ischemia. Higher density of degenerating neurons was detected in the penumbra of Pbef+/− mice than in WT mice using Fluoro-Jade B staining. Our study shows that PBEF has a neuronal protective role in cerebral ischemia presumably through enhanced energy metabolism.

Inactivation of P2X2 purinoceptors by divalent cations

1 P2X2 channels are activated by extracellular ATP. Despite being commonly described as non‐desensitizing, P2X2 receptors do desensitize or inactivate. In the unspliced, 472 amino acid isoform of the P2X2 receptor, inactivation required membrane disruption and the presence of extracellular Ca2+. 2 The ability to inactivate whole‐cell currents developed slowly after breaking in. In contrast, currents from excised patches exhibited rapid (∼100 ms) inactivation with a dependence on extracellular Ca2+, ATP and voltage. 3 The inactivation rate increased with the fourth power of [Ca2+] suggesting that the functional channel may be a tetramer. Ca2+ had both a higher affinity and a larger Hill coefficient for inactivation than Mg2+, Ba2+ or Mn2+. Trivalent cations at concentrations up to the solubility product of ATP had no effect. The change in apparent co‐operativity with ionic species suggests the presence of experimentally unresolved ligand‐insensitive kinetic steps. 4 Based on the weak voltage dependence of inactivation and the lack of effect of intracellular Ca2+ buffers, the Ca2+‐binding sites are probably located near the extracellular surface of the membrane. 5 The recovery from inactivation was slow, with a time constant of ∼7 min. 6 Ca2+‐sensitive inactivation only appeared when the membrane was disrupted in some manner. Treatment with actin and microtubule reagents did not induce inactivation, suggesting that an intact cytoskeleton is not necessary. 7 Inactivation rates observed in different patch configurations suggest that the induction of Ca2+‐dependent inactivation was due to the loss of a diffusible cofactor located in the membrane or the cytoplasm.

Reactive astrocytes and therapeutic potential in focal ischemic stroke

Astrocytes are specialized and the most abundant cell type in the central nervous system (CNS). They play important roles in the physiology of the brain. Astrocytes are also critically involved in many CNS disorders including focal ischemic stroke, the leading cause of brain injury and death in patients. One of the prominent pathological features of a focal ischemic stroke is reactive astrogliosis and glial scar formation. Reactive astrogliosis is accompanied with changes in morphology, proliferation, and gene expression in the reactive astrocytes. This study provides an overview of the most recent advances in astrocytic Ca(2+) signaling, spatial, and temporal dynamics of the morphology and proliferation of reactive astrocytes as well as signaling pathways involved in the reactive astrogliosis after ischemic stroke based on results from experimental studies performed in various animal models. This review also discusses the therapeutic potential of reactive astrocytes in focal ischemic stroke. As reactive astrocytes exhibit high plasticity, we suggest that modulation of local reactive astrocytes is a promising strategy for cell-based stroke therapy.

Ion permeation and block of P2X(2) purinoceptors: single channel recordings.

Abstract. P2X2 purinoceptors are cation-selective channels activated by ATP and its analogues. Using single channel measurements we studied the channels selectivity for the alkali metal ions and organic monovalent cations NMDG+, Tris+, TMA+, and TEA+. The selectivity sequence for currents carried by alkali metal ions is: K+ > Rb+ > Cs+ > Na+ > Li+, which is Eisenman sequence IV. This is different from the mobility sequence of the ions in free solution suggesting there is weak interaction between the ions and the channel interior. The relative conductance for alkali ions increases linearly in relation to the Stokes radius. The organic ions NMDG+, Tris+, TMA+ and TEA+ were virtually impermeant. The divalent ions (Mn2+, Mg2+, Ca2+ and Ba2+) induced a fast block visible as a reduction in amplitude of the unitary currents. Using a single-site binding model, the divalent ions exhibited an equilibrium affinity sequence of Mn2+ > Mg2+ > Ca2+ > Ba2+.

Tail end of the s6 segment: role in permeation in shaker potassium channels.

The permeation pathway in voltage-gated potassium channels has narrow constrictions at both the extracellular and intracellular ends. These constrictions might limit the flux of cations from one side of the membrane to the other. The extracellular constriction is the selectivity filter, whereas the intracellular bundle crossing is proposed to act as the activation gate that opens in response to a depolarization. This four-helix bundle crossing is composed of S6 transmembrane segments, one contributed by each subunit. Here, we explore the cytoplasmic extension of the S6 transmembrane segment of Shaker potassium channels, just downstream from the bundle crossing. We substituted cysteine for each residue from N482 to T489 and determined the amplitudes of single channel currents and maximum open probability (P o,max) at depolarized voltages using nonstationary noise analysis. One mutant, F484C, significantly reduces P o,max, whereas Y483C, F484C, and most notably Y485C, reduce single channel conductance (γ). Mutations of residue Y485 have no effect on the Rb+/K+ selectivity, suggesting a local effect on γ rather than an allosteric effect on the selectivity filter. Y485 mutations also reduce pore block by tetrabutylammonium, apparently by increasing the energy barrier for blocker movement through the open activation gate. Replacing Rb+ ions for K+ ions reduces the amplitude of single channel currents and makes γ insensitive to mutations of Y485. These results suggest that Rb+ ions increase an extracellular energy barrier, presumably at the selectivity filter, thus making it rate limiting for flux of permeant ions. These results indicate that S6T residues have an influence on the conformation of the open activation gate, reflected in both the stability of the open state and the energy barriers it presents to ions.

Evidence for non-independent gating of P2X2 receptors expressed in Xenopus oocytes

BackgroundP2X2 receptor is an ATP-activated ion channel which is widely expressed in the nervous system, and mediates synaptic transmission.ResultsWe recorded currents of P2X2 receptors expressed in Xenopus oocytes from outside-out patches and have found that currents recorded from patches containing a single or multiple P2X2 channels differ in a manner suggesting positive cooperativity. First, the currents from multichannel patches exhibit simultaneous transitions more frequently than predicted from the activity of independent channels. Second, the mean open lifetime at the current level of a single channel in a multichannel burst is about six times longer than the open time of currents from single channel patches, a trend opposite to what is expected of independent channels. These results indicate that the channels have positive cooperativity and that the longer opening is due to a slower closing rate. Third, from kinetic analysis the likelihood of the cooperative model is significantly larger than that of the independent model. Fourth, the open channel noise of currents from patches containing multiple channels is less than half that from a single channel, which is consistent with the channel properties being different when they are active in groups.ConclusionTaken together, our results suggest that P2X2 receptors are non-independent, but interact with positive cooperativity.