Targeted disruption of Kv2.1-VAPA association provides neuroprotection against ischemic stroke in mice by declustering Kv2.1 channels.

Kv2.1 channels mediate cell death-enabling loss of cytosolic potassium in neurons following plasma membrane insertion at somatodendritic clusters. Overexpression of the carboxyl terminus (CT) of the cognate channel Kv2.2 is neuroprotective by disrupting Kv2.1 surface clusters. Here, we define a seven-amino acid declustering domain within Kv2.2 CT (DP-2) and demonstrate its neuroprotective efficacy in a murine ischemia-reperfusion model. TAT-DP-2, a membrane-permeable derivative, induces Kv2.1 surface cluster dispersal, prevents post-injurious pro-apoptotic potassium current enhancement, and is neuroprotective in vitro by disrupting the association of Kv2.1 with VAPA. TAT-DP-2 also induces Kv2.1 cluster dispersal in vivo in mice, reducing infarct size and improving long-term neurological function following stroke. We suggest that TAT-DP-2 induces Kv2.1 declustering by disrupting Kv2.1-VAPA association and scaffolding sites required for the membrane insertion of Kv2.1 channels following injury. We present the first evidence of targeted disruption of Kv2.1-VAPA association as a neuroprotective strategy following brain ischemia.

Molecular Neuroprotection Induced by Zinc-Dependent Expression of Hepatitis C-Derived Protein NS5A Targeting Kv2.1 Potassium Channels.

We present the design of an innovative molecular neuroprotective strategy and provide proof-of-concept for its implementation, relying on the injury-mediated activation of an ectopic gene construct. As oxidative injury leads to the intracellular liberation of zinc, we hypothesize that tapping onto the zinc-activated metal regulatory element (MRE) transcription factor 1 system to drive expression of the Kv2.1-targeted hepatitis C protein NS5A (hepatitis C nonstructural protein 5A) will provide neuroprotection by preventing cell death-enabling cellular potassium loss in rat cortical neurons in vitro. Indeed, using biochemical and morphologic assays, we demonstrate rapid expression of MRE-driven products in neurons. Further, we report that MRE-driven NS5A expression, induced by a slowly evolving excitotoxic stimulus, functionally blocks injurious, enhanced Kv2.1 potassium whole-cell currents and improves neuronal viability. We suggest this form of "on-demand" neuroprotection could provide the basis for a tenable therapeutic strategy to prevent neuronal cell death in neurodegeneration.

A Rat Model of Perinatal Seizures Provoked by Global Hypoxia.

Hypoxic-ischemic encephalopathy (HIE) refers to acute brain injury that results from perinatal asphyxia. HIE is a major cause of neonatal seizures, and outcomes can range from apparent recovery to severe cognitive impairment, cerebral palsy, and epilepsy. Acute partial seizures frequently aid in indicating the severity and localization of brain injury. However, evidence also suggests that the occurrence of seizures further increases the likelihood of epilepsy in later life regardless of the severity of the initial injury. Here, we describe a neonatal rat model of seizure-provoking mild hypoxia without overt brain injury that has been used to investigate potential epileptogenic effects of hypoxia-associated seizures alone on neonatal brain development. Clinically, HIE is defined by brain injury, and thus, this model is not intended to mimic clinical HIE. Rather, its utility is in providing a model to understand the dynamic and long-term regulation of brain function and how this can be perturbed by early life seizures that are provoked by a commonly encountered pathophysiological trigger. Additionally, the model allows the study of brain pathophysiology without the potential confound of variable neuroanatomical changes that are reactive to widespread cell death.

Disruption of KV2.1 somato-dendritic clusters prevents the apoptogenic increase of potassium currents.

As the predominant mediator of the delayed rectifier current, KV2.1 is an important regulator of neuronal excitability. KV2.1, however, also plays a well-established role in apoptotic cell death. Apoptogenic stimuli induce syntaxin-dependent trafficking of KV2.1, resulting in an augmented delayed rectifier current that acts as a conduit for K+ efflux required for pro-apoptotic protease/nuclease activation. Recent evidence suggests that KV2.1 somato-dendritic clusters regulate the formation of endoplasmic reticulum-plasma membrane junctions that function as scaffolding sites for plasma membrane trafficking of ion channels, including KV2.1. However, it is unknown whether KV2.1 somato-dendritic clusters are required for apoptogenic trafficking of KV2.1. By overexpression of a protein derived from the C-terminus of the cognate channel KV2.2 (KV2.2CT), we induced calcineurin-independent disruption of KV2.1 somato-dendritic clusters in rat cortical neurons, without altering the electrophysiological properties of the channel. We observed that KV2.2CT-expressing neurons are less susceptible to oxidative stress-induced cell death. Critically, expression of KV2.2CT effectively blocked the increased current density of the delayed rectifier current associated with oxidative injury, supporting a vital role of KV2.1-somato-dendritic clusters in apoptogenic increases in KV2.1-mediated currents.

Zn(2+) -induced Ca(2+) release via ryanodine receptors triggers calcineurin-dependent redistribution of cortical neuronal Kv2.1 K(+) channels.

KEY POINTS: Increases in intracellular Zn(2+) concentrations are an early, necessary signal for the modulation of Kv2.1 K(+) channel localization and physiological function. Intracellular Zn(2+) -mediated Kv2.1 channel modulation is dependent on calcineurin, a Ca(2+) -activated phosphatase. We show that intracellular Zn(2+) induces a significant increase in ryanodine receptor-dependent cytosolic Ca(2+) transients, which leads to a calcineurin-dependent redistribution of Kv2.1 channels from pre-existing membrane clusters to diffuse localization. As such, the link between Zn(2+) and Ca(2+) signalling in this Kv2.1 modulatory pathway is established. We observe that a sublethal ischaemic preconditioning insult also leads to Kv2.1 redistribution in a ryanodine receptor-dependent fashion. We suggest that Zn(2+) may be an early and ubiquitous signalling molecule mediating Ca(2+) release from the cortical endoplasmic reticulum via ryanodine receptor activation. ABSTRACT: Sublethal injurious stimuli in neurons induce transient increases in free intracellular Zn(2+) that are associated with regulating adaptive responses to subsequent lethal injury, including alterations in the function and localization of the delayed-rectifier potassium channel, Kv2.1. However, the link between intracellular Zn(2+) signalling and the observed changes in Kv2.1 remain undefined. In the present study, utilizing exogenous Zn(2+) treatment, along with a selective Zn(2+) ionophore, we show that transient elevations in intracellular Zn(2+) concentrations are sufficient to induce calcineurin-dependent Kv2.1 channel dispersal in rat cortical neurons in vitro, which is accompanied by a relatively small but significant hyperpolarizing shift in the voltage-gated activation kinetics of the channel. Critically, using a molecularly encoded calcium sensor, we found that the calcineurin-dependent changes in Kv2.1 probably occur as a result of Zn(2+) -induced cytosolic Ca(2+) release via activation of neuronal ryanodine receptors. Finally, we couple this mechanism with an established model for in vitro ischaemic preconditioning and show that Kv2.1 channel modulation in this process is also ryanodine receptor-sensitive. Our results strongly suggest that intracellular Zn(2+) -initiated signalling may represent an early and possibly widespread component of Ca(2+) -dependent processes in neurons.

Decreased A-currents in hippocampal dentate granule cells after seizure-inducing hypoxia in the immature rat.

PURPOSE: Cerebral hypoxia is a major cause of neonatal seizures, and can lead to epilepsy. Pathologic anatomic and physiologic changes in the dentate gyrus have been associated with epileptogenesis in many experimental models, as this region is widely believed to gate the propagation of limbic seizures. However, the consequences of hypoxia-induced seizures for the immature dentate gyrus have not been extensively examined. METHODS: Seizures were induced by global hypoxia (5-7% O2 for 15 min) in rat pups on postnatal day 10. Whole-cell voltage-clamp recordings were used to examine A-type potassium currents (IA ) in dentate granule cells in hippocampal slices obtained 1-17 days after hypoxia treatment. KEY FINDINGS: Seizure-inducing hypoxia resulted in decreased maximum IA amplitude in dentate granule cells recorded within the first week but not at later times after hypoxia treatment. The decreased IA amplitude was not associated with changes in the voltage-dependence of activation or inactivation removal, or in sensitivity to inhibition by 4-aminopyridine (4-AP). However, consistent with the role of IA in shaping firing patterns, we observed in the hypoxia group a significantly decreased latency to first spike with depolarizing current injection from hyperpolarized potentials. These differences were not associated with changes in resting membrane potential or input resistance, and were eliminated by application of 10 m 4-AP. SIGNIFICANCE: Given the role of IA to slow action potential firing, decreased IA could contribute to long-term hippocampal pathology after neonatal seizure-inducing hypoxia by increasing dentate granule cell excitability during a critical window of activity-dependent hippocampal maturation.

Gabapentin promotes inhibition by enhancing hyperpolarization-activated cation currents and spontaneous firing in hippocampal CA1 interneurons.

The H-current (I(H)) regulates membrane electrical activity in many excitable cells. The antiepileptic drug gabapentin (GBP) has been shown to increase I(H) in hippocampal area CA1 pyramidal neurons, and this has been proposed as an anticonvulsant mechanism of action. I(H) also regulates excitability in some types of hippocampal interneuron that provide synaptic inhibition to CA1 pyramidal neurons, suggesting that global pharmacological I(H) enhancement could have more complex effects on the local synaptic network. However, whether I(H) in CA1 interneurons is modulated by GBP has not been examined. In this study, we tested the effects of GBP on I(H) on hippocampal area CA1 stratum oriens non-pyramidal neurons, and on spontaneous inhibitory postsynaptic currents (sIPSCs) in CA1 pyramidal neurons in immature rat brain slices. GBP (100µM) increased I(H) in approximately 67% of interneurons that exhibited I(H), with no apparent effect on cell types that did not exhibit I(H). GBP also increased the frequency of spontaneous (but not miniature) inhibitory postsynaptic currents in pyramidal neurons without altering amplitudes or rise and decay times. These data indicate that I(H) in a subset of CA1 interneuron types can be increased by GBP, similarly to its effect on I(H) in pyramidal neurons, and further, that indirectly increased spontaneous inhibition of pyramidal neurons could contribute to its anticonvulsant effects.

Increased basal synaptic inhibition of hippocampal area CA1 pyramidal neurons by an antiepileptic drug that enhances I(H).

The hyperpolarization-activated cation current (I(H)) regulates the electrical activity of many excitable cells, but its precise function varies across cell types. The antiepileptic drug lamotrigine (LTG) was recently shown to enhance I(H) in hippocampal CA1 pyramidal neurons, showing a potential anticonvulsant mechanism, as I(H) can dampen dendrito-somatic propagation of excitatory postsynaptic potentials in these cells. However, I(H) is also expressed in many hippocampal interneurons that provide synaptic inhibition to CA1 pyramidal neurons, and thus, I(H) modulation may indirectly regulate the inhibitory control of principal cells by direct modulation of interneuron activity. Whether I(H) in hippocampal interneurons is sensitive to modulation by LTG, and the manner by which this may affect the synaptic inhibition of pyramidal cells has not been investigated. In this study, we examined the effects of LTG on I(H) and spontaneous firing of area CA1 stratum oriens interneurons, as well as on spontaneous inhibitory postsynaptic currents in CA1 pyramidal neurons in immature rat brain slices. LTG (100 microM) significantly increased I(H) in the majority of interneurons, and depolarized interneurons from rest, promoting spontaneous firing. LTG also caused an increase in the frequency of spontaneous (but not miniature) IPSCs in pyramidal neurons without significantly altering amplitudes or rise and decay times. These data indicate that I(H) in CA1 interneurons can be increased by LTG, similarly to I(H) in pyramidal neurons, that I(H) enhancement increases interneuron excitability, and that these effects are associated with increased basal synaptic inhibition of CA1 pyramidal neurons.

Persistently decreased basal synaptic inhibition of hippocampal CA1 pyramidal neurons after neonatal hypoxia-induced seizures.

Hypoxia is the most common cause of neonatal seizures and can lead to epilepsy, but the epileptogenic mechanisms are not yet understood. We have previously shown that hypoxia-induced seizures in the neonatal rat result in acutely decreased amplitudes and frequency of spontaneous and miniature inhibitory postsynaptic currents (sIPSCs and mIPSCs) in hippocampal CA1 pyramidal neurons. In the current study, we asked whether such changes persist for several days following hypoxia-induced seizures. Similar to the acute findings, we observed decreased frequency and amplitudes of sIPSCs and decreased mIPSC amplitudes in CA1 pyramidal neurons at 3-5 days after hypoxia. However, in contrast to the acute findings, we observed no differences between hypoxia-treated and control groups in mIPSC frequency. Additionally, by 7 days after hypoxia, sIPSC amplitudes in the hypoxia group had recovered to control levels, but sIPSC frequency remained decreased. These data indicate that the persistently decreased sIPSC frequency result from decreased firing of presynaptic inhibitory interneurons, with only transient possible changes in postsynaptic responses to GABA release.