Linking kindling to increased glutamate release in the dentate gyrus of the hippocampus through the STXBP5/tomosyn-1 gene.

INTRODUCTION: In kindling, repeated electrical stimulation of certain brain areas causes progressive and permanent intensification of epileptiform activity resulting in generalized seizures. We focused on the role(s) of glutamate and a negative regulator of glutamate release, STXBP5/tomosyn-1, in kindling. METHODS: Stimulating electrodes were implanted in the amygdala and progression to two successive Racine stage 5 seizures was measured in wild-type and STXBP5/tomosyn-1-/- (Tom-/-) animals. Glutamate release measurements were performed in distinct brain regions using a glutamate-selective microelectrode array (MEA). RESULTS: Naïve Tom-/- mice had significant increases in KCl-evoked glutamate release compared to naïve wild type as measured by MEA of presynaptic release in the hippocampal dentate gyrus (DG). Kindling progression was considerably accelerated in Tom-/- mice, requiring fewer stimuli to reach a fully kindled state. Following full kindling, MEA measurements of both kindled Tom+/+ and Tom-/- mice showed significant increases in KCl-evoked and spontaneous glutamate release in the DG, indicating a correlation with the fully kindled state independent of genotype. Resting glutamate levels in all hippocampal subregions were significantly lower in the kindled Tom-/- mice, suggesting possible changes in basal control of glutamate circuitry in the kindled Tom-/- mice. CONCLUSIONS: Our studies demonstrate that increased glutamate release in the hippocampal DG correlates with acceleration of the kindling process. Although STXBP5/tomosyn-1 loss increased evoked glutamate release in naïve animals contributing to their prokindling phenotype, the kindling process can override any attenuating effect of STXBP5/tomosyn-1. Loss of this "braking" effect of STXBP5/tomosyn-1 on kindling progression may set in motion an alternative but ultimately equally ineffective compensatory response, detected here as reduced basal glutamate release.

Kindling-induced asymmetric accumulation of hippocampal 7S SNARE complexes correlates with enhanced glutamate release.

PURPOSE: To correlate kindling-associated alterations of the neurotransmitter secretory machinery, glutamate release in the trisynaptic hippocampal excitatory pathway, and the behavioral evolution of kindling-induced epileptogenesis. METHOD: Neurotransmitter release requires the fusion of vesicle and plasma membranes; it is initiated by formation of a stable, ternary complex (7SC) of SNARE [soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptor] proteins. Quantitative Western blotting was used to monitor levels of 7SC and SNARE regulators [NSF, SV2 (synaptic vesicle protein 2)] in hippocampal synaptosomes from amygdala-kindled animals. Hippocampal synaptic glutamate release was measured in vivo with a unique microelectrode array (MEA) that uses glutamate oxidase to catalyze the breakdown of glutamate into a reporter molecule. KEY FINDINGS: Ipsilateral hippocampal accumulation of 7SC developed with onset of amygdalar kindling, but became permanent only in animals stimulated to at least Racine stage 3; the ratio peaked and did not increase with more than two consecutive stage 5 seizures. Chronic 7SC asymmetry was seen in entorhinal cortex and the hippocampal formation, particularly in dentate gyrus (DG) and CA1, but not in the other brain areas examined. There was a strong correlation between asymmetric 7SC accumulation and increased total hippocampal SV2. Following a 30-day latent period, amplitudes of spontaneous synaptic glutamate release were enhanced in ipsilateral DG and reduced in ipsilateral CA3 of kindled animals; increased volleys of synaptic glutamate activity were seen in ipsilateral CA1. SIGNIFICANCE: Amygdalar kindling is associated with chronic changes in the flow of glutamate signaling in the excitatory trisynaptic pathway and with early but permanent changes in the mechanics of vesicular release in ipsilateral hippocampal formation.

Levetiracetam prevents kindling-induced asymmetric accumulation of hippocampal 7S SNARE complexes.

PURPOSE: Understanding the molecular mechanisms underlying epilepsy is crucial to designing novel therapeutic regimens. This report focuses on alterations in the secretory machinery responsible for neurotransmitter (NT) release. Soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptor (SNARE) complexes mediate the fusion of synaptic vesicle and active zone membranes, thus mediating NT secretion. SNARE regulators control where and when SNARE complexes are formed. Previous studies showed an asymmetric accumulation of 7S SNARE complexes (7SC) in the ipsilateral hippocampus of kindled animals. The present studies probe the persistence of 7SC accumulation and the effect of the anticonvulsant, levetiracetam (LEV), on 7SC and SNARE regulators. METHOD: Quantitative Western blotting was used to monitor levels of 7SC and SNARE regulators in hippocampal synaptosomes from kindled animals both before and after LEV treatment. RESULTS: The asymmetric accumulation of 7SC is present 1-year postamygdalar kindling. The synaptic vesicle protein, synaptic vesicle protein 2 (SV2), a primary LEV-binding protein, and the SNARE regulator Tomosyn increase, whereas NSF decreases in association with this accumulation. Treatment with LEV prevented kindling-induced accumulation of SV2, but did not affect the transient increase of Tomosyn or the long-term decrease NSF. LEV treatment retarded the electrical and behavioral concomitants of amygdalar kindling coincident with a decrease in accumulation of 7SC. CONCLUSIONS: The ipsilateral hippocampal accumulation of SNARE complexes is an altered molecular process associated with kindling that appears permanent. Kindling epileptogenesis alters synaptosomal levels of the SNARE regulators: NSF, SV2, and Tomosyn. Concomitant treatment with LEV reverses the kindling-induced 7SC accumulation and increase of SV2.

Asymmetric accumulation of hippocampal 7S SNARE complexes occurs regardless of kindling paradigm.

Modifications of neurotransmission may contribute to the synchronization of neuronal networks that are a hallmark of epileptic seizures. In this study we examine the synaptosomal proteins involved in neurotransmitter release to determine if alterations in their interactions correlate with the chronic epileptic state. Using quantitative western blotting, we measured the levels of 7S SNARE complexes and SNARE effectors in the effected hippocampi from animals that were electrically kindled through stimulation from one of three different foci. All three kindling paradigms, amygdalar, entorhinal, and septal, were associated with an accumulation of 7S SNARE complexes in the ipsilateral hippocampus, measured 1 month after completion of kindling. Of the eight SNARE effectors examined (alpha-SNAP, NSF, SV2A/B, Munc18a/nSec1, Munc13-1, Complexins 1 and 2, and synaptotagmin I), there was a statistically significant bihemispheric increase of hippocampal SV2 and decrease of NSF upon kindling; neither by itself would be expected to account for the asymmetry of SNARE complex distribution. These data suggest that an ipsilateral hippocampal accumulation of SNARE complexes is a permanent alteration of kindling-induced epilepsy, regardless of stimulation pathway. The significance of these findings toward a molecular understanding of epilepsy will be discussed.

Hypersensitivity of pulmonary chemoreflex induced by poly-L-lysine: role of cationic charge.

This study was carried out to investigate the role of cationic charge in the hypersensitivity of pulmonary C-fibers induced by airway exposure to synthetic cationic protein poly-L-lysine (PLL) in anesthetized rats. Inhalation of PLL aerosol induced a distinctly irregular breathing pattern, and significantly enhanced the pulmonary chemoreflex responses to capsaicin. However, after the cationic charges were completely removed from PLL by succinylation, the succinylated PLL no longer produced any change in either the baseline breathing pattern or the reflex responses to capsaicin. In addition, the effects of PLL were also abolished after premixing it with a polyanion, poly-L-glutamic or poly-L-aspartic acid, before delivery. In sharp contrast, when delivered within 5 min after the PLL aerosol, these two polyanions were completely ineffective in reversing the effects of PLL. Electrophysiological recording of the afferent activity of single pulmonary C-fibers further supported our conclusion that the cationic charge carried by this protein is primarily responsible for generating the stimulatory and sensitizing effects of PLL on these afferents.

Cross-talk between tuberin, calmodulin, and estrogen signaling pathways.

Lymphangioleiomyomatosis (LAM) is a rare disease that occurs primarily in women and has been linked to both estrogen-mediated signaling events and mutations associated with the tuberous sclerosis complex 2 gene product tuberin. These two observations fostered the hypothesis that tuberin's impact on estrogen-mediated signaling might be through a direct interaction with the intracellular receptor for estrogen, estrogen receptor alpha (ERalpha). In the study presented here, tuberin was shown to co-immunoprecipitate and directly bind ERalpha through a domain localized within the carboxyl 73 amino acids of tuberin. This domain had previously been shown to serve as a binding domain for the intracellular calcium signaling molecule calmodulin (CaM). Competition binding studies identified a potential competitive relationship for binding of tuberin by ERalpha and CaM. Additionally, tuberin-ERalpha interactions were found to be modulated by the presence of tuberin's predominant intracellular binding partner hamartin, suggesting that tuberin-hamartin interactions negatively impact the ability of tuberin to modulate ERalpha-mediated gene transcription events. Cumulatively, data presented here support the hypothesis that interactions between tuberin, ERalpha, and CaM may play a critical role in the pathology of LAM disease.

Studies of calmodulin-dependent regulation.

Methods are presented for purifying bovine testes calmodulin and the calmodulin-regulated plasma-membrane calcium ATPase from human erythrocytes by calcium dependent affinity chromatography. The assay of CaM Kinase II using a synthetic peptide substrate is also described.

A calmodulin binding site in the tuberous sclerosis 2 gene product is essential for regulation of transcription events and is altered by mutations linked to tuberous sclerosis and lymphangioleiomyomatosis.

Mutations in the tuberous sclerosis 2 (TSC2) gene product have been genetically linked to the pathology of both tuberous sclerosis (TSC) and the gender-specific lung disease, lymphangioleiomyomatosis (LAM). Both diseases are classified as disorders of cellular migration, proliferation, and differentiation. Earlier studies from our laboratory (1) linked TSC2 with steroid/nuclear receptor signaling. Studies presented here provide evidence for calmodulin (CaM) signaling in the propagation of this TSC2 activity. Far Western screening of a lambda phage human brain cDNA library to identify interacting proteins for the TSC2 gene product (tuberin) yielded multiple clones encoding human CaM. Direct binding with 32P-labeled tuberin demonstrated Ca2+-dependent binding to CaM-Sepharose which was lost upon deletion of the C-terminal 72 residues. The sequence (1740)WIARLRHIKRLRQRIC(1755) was identified as one capable of forming a basic amphipathic helix indicative of CaM binding domains in known calmodulin binding proteins. Studies with a synthetic peptide of this sequence demonstrated very tight Ca2+-dependent binding to CaM as judged by tryptophan fluorescence perturbation studies and phosphodiesterase activation by CaM. Deletion mutagenesis studies further suggested that this CaM binding domain is required for tuberin modulation of steroid receptor function and that mutations in this region may be involved in the pathology of TSC and LAM.