Insult-provoked transformation of neuronal networks into epileptic ones involves multiple mechanisms. Intervention studies have identified both dysregulated inflammatory pathways and NRSF-mediated repression of crucial neuronal genes as contributors to epileptogenesis. However, it remains unclear how epilepsy-provoking insults (e.g., prolonged seizures) induce both inflammation and NRSF, and whether common mechanisms exist. We examined miR-124 as a candidate dual regulator of NRSF- and inflammatory-pathways. Status epilepticus (SE) led to reduced miR-124 expression via SIRT1, and in turn MiR-124 repression, via C/EBPα, upregulated NRSF. We tested whether augmenting miR-124 after SE would abort epileptogenesis by preventing inflammation and NRSF upregulation. SE-sustaining animals developed epilepsy but supplementing miR-124 did not modify epileptogenesis. Examining this result further, we found that synthetic miR-124 effectively blocked NRSF upregulation and rescued NRSF target genes, but also augmented microglia activation and inflammatory cytokines. Thus, miR-124 attenuates epileptogenesis via NRSF while promoting epilepsy via inflammation.

Key pointsr Mutations in the Ca v 3.2 T-type Ca 2+ channel were found in patients with idiopathic generalized epilepsies, yet the mechanisms by which these mutations increase neuronal excitability and susceptibility to seizures remain to be determined.r Using electrophysiological and transfection methods, we validate in cultured hippocampal neurons the hypothesis that an epilepsy mutation increases neuronal excitability.r Mutations in the I-II loop of the channel increase trafficking to the plasma membrane without altering trafficking into dendrites. Mutations also enhance dendritic arborization.r Additionally, we provide the first evidence that Ca v 3.2 can signal to Ca 2+ -regulated transcription factors, which are known to play important roles in neuronal development and gene expression.Abstract T-type calcium channels play essential roles in regulating neuronal excitability and network oscillations in the brain. Mutations in the gene encoding Ca v 3.2 T-type Ca 2+ channels, CACNA1H, have been found in association with various forms of idiopathic generalized epilepsy. We and others have found that these mutations may influence neuronal excitability either by altering the biophysical properties of the channels or by increasing their surface expression. The goals of the present study were to investigate the excitability of neurons expressing Ca v 3.2 with the epilepsy mutation, C456S, and to elucidate the mechanisms by which it influences neuronal properties. We found that expression of the recombinant C456S channels substantially increased the excitability of cultured neurons by increasing the spontaneous firing rate and reducing the threshold for rebound burst firing. Additionally, we found that molecular determinants in the I-II loop (the region in which most childhood absence epilepsy-associated mutations are found) substantially increase the surface expression of T-channels but do not alter the relative distribution of channels into dendrites of cultured hippocampal neurons. Finally, we discovered that expression of C456S channels promoted dendritic growth and arborization. These effects were reversed to normal by either the absence epilepsy drug ethosuximide or a novel T-channel blocker, TTA-P2. As Ca 2+ -regulated transcription factors also increase dendritic development, we tested a transactivator trap assay and found that the C456S variant can induce changes in gene transcription. Taken together, our findings suggest that gain-of-function mutations in Ca v 3.2 T-type Ca 2+ channels increase seizure susceptibility by directly altering neuronal electrical properties and indirectly by changing gene expression.V.-S. Eckle, A. Shcheglovitov and I. Vitko are co-first authors and contributed equally to the paper.

Objective To develop a constitutively active K+ leak channel using TREK-1 (TWIK-related potassium channel 1; TREK-M) that is resistant to compensatory down-regulation by second messenger cascades, and to validate the ability of TREK-M to silence hyperactive neurons using cultured hippocampal neurons. To test if adenoassociated viral (AAV) delivery of TREK-M could reduce the duration of status epilepticus and reduce neuronal death induced by lithium-pilocarpine administration. Methods Molecular cloning techniques were used to engineer novel vectors to deliver TREK–M via plasmids, lentivirus, and AAV using a cytomegalovirus (CMV)-enhanced GABRA4 promoter. Electrophysiology was used to characterize the activity and regulation of TREK–M in human embryonic kidney (HEK-293) cells, and the ability to reduce spontaneous activity in cultured hippocampal neurons. Adult male rats were injected bilaterally with self-complementary AAV particles composed of serotype 5 capsid into the hippocampus and entorhinal cortex. Lithium-pilocarpine was used to induce status epilepticus. Seizures were monitored using continuous video–electroencephalography (EEG) monitoring. Neuronal death was measured using Fluoro-Jade C staining of para-formaldehyde-fixed brain slices. Results TREK-M inhibited neuronal firing by hyperpolarizing the resting membrane potential and decreasing input resistance. AAV delivery of TREK-M decreased the duration of status epilepticus by 50%. Concomitantly it reduced neuronal death in areas targeted by the AAV injection. Significance These findings demonstrate that TREK-M can silence hyperexcitable neurons in the brain of epileptic rats and treat acute seizures. This study paves the way for an alternative gene therapy treatment of status epilepticus, and provides the rationale for studies of AAV-TREK-M’s effect on spontaneous seizures in chronic models of temporal lobe epilepsy.

Calcium influx through voltage-gated Ca 2+ channels constitutes one main source of intracellular Ca 2+ in neurons. Biophysical, pharmacological and molecular analyses have revealed the presence of two families of voltage-gated Ca 2+ channels (reviewed by Catterall, 1998). The family of low voltage-activated Ca 2+ channels (also known as T-type channels because of their tiny conductance and transient opening) generates fast-inactivating currents at relatively hyperpolarized potentials. The family of high voltageactivated (HVA) Ca 2+ channels requires stronger depolarizations for opening and can be further divided into several subfamilies including L, N, P, and Q-type Ca 2+ channels. Presently, there is little understanding about the cellular mechanisms that regulate low voltage-activated and HVA Ca 2+ channel expression during neuronal development and differentiation.The functional expression of T-type Ca 2+ channels is developmentally regulated in chicken nodose neurons (Pachuau and Martin-Caraballo 2007a). T-type Ca 2+ currents are restricted to a few nodose neurons between embryonic day 7 (E7) and E10 but are present in 60% of nodose neurons by E17. Although the functional expression of T-type Ca 2+ channels occurs late in development, transcripts of the T-type Ca 2+ channel pore-forming a1H subunit are already present by E7. T-type Ca 2+ channel expression can be evoked in vitro by culture of E7 nodose neurons with a heart-derived extract suggesting that extrinsic factors present in the culture medium regulate the functional expression of T-type Ca 2+ channels in chicken nodose neurons. Chicken ciliary These authors contributed equally to this study.Abbreviations used: BDNF, brain-derived neurotrophic factor; CNTF, ciliary neurotrophic factor; E7, embryonic day 7; EMEM, Eagle's minimal essential medium; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3P-dehydrogenase; GPI, glycosyl-phosphatidylinositol; HVA, high voltage activated; JAK, Janus tyrosine kinase; LIF, leukemia inhibitory factor; LIFRb, LIF receptor b; MAP, mitogenactivated protein; SH2, src homology 2; SOCS, suppressors of cytokine signaling; STAT, signal transducer and transducer of transcription. AbstractCulture of chicken nodose neurons with CNTF but not BDNF causes a significant increase in T-type Ca 2+ channel expression. CNTF-induced channel expression requires 12 h stimulation to reach maximal expression and is not affected by inhibition of protein synthesis, suggesting the involvement of a post-translational mechanism. In this study, we have investigated the biochemical mechanism responsible for the CNTFdependent stimulation of T-type channel expression in nodose neurons. Stimulation of nodose neurons with CNTF evoked a considerable increase in signal transducer and activator of transcription (STAT3) and extracellular signal-regulated kinase (ERK) phosphorylation. CNTF-evoked ERK phosphorylation was transient whereas BDNF-evoked activation of ERK was sustained. Pre-treatment of nodose neurons with the Janus tyrosine kina...

Neuropoietic cytokines such as ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) stimulate the functional expression of T-type Ca(2+) channels in developing sensory neurons. However, the molecular and cellular mechanisms involved in the cytokine-evoked membrane expression of T-type Ca(2+) channels are not fully understood. In this study we investigated the role of LIF in promoting the trafficking of T-type Ca(2+) channels in a heterologous expression system. Our results demonstrate that transfection of HEK-293 cells with the rat green fluorescent protein (GFP)-tagged T-type Ca(2+) channel α(1H)-subunit resulted in the generation of transient Ca(2+) currents. Overnight treatment of α(1H)-GFP-transfected cells with LIF caused a significant increase in the functional expression of T-type Ca(2+) channels as indicated by changes in current density. LIF also evoked a significant increase in membrane fluorescence compared with untreated cells. Disruption of the Golgi apparatus with brefeldin A inhibited the stimulatory effect of LIF, indicating that protein trafficking regulates the functional expression of T-type Ca(2+) channels. Trafficking of α(1H)-GFP was also disrupted by cotransfection of HEK-293 cells with the dominant-negative form of ADP-ribosylation factor (ARF)1 but not ARF6, suggesting that ARF1 regulates the LIF-evoked membrane trafficking of α(1H)-GFP subunits. Trafficking of T-type Ca(2+) channels required transient activation of the JAK and ERK signaling pathways since stimulation of HEK-293 cells with LIF evoked a considerable increase in the phosphorylation of the downstream JAK targets STAT3 and ERK. Pretreatment of HEK-293 cells with the JAK inhibitor P6 or the ERK inhibitor U0126 blocked ERK phosphorylation. Both P6 and U0126 also inhibited the stimulatory effect of LIF on T-type Ca(2+) channel expression. These findings demonstrate that cytokines like LIF promote the trafficking of T-type Ca(2+) channels.