INVESTIGATIONS OF THE STRUCTURE-FUNCTION RELATIONSHIP IN KAINATE RECEPTORS USING FÖRSTER RESONANCE ENERGY TRANSFER

Kainate receptors belong to the family of ion channels known as the ionotropic glutamate receptors. Ionotropic glutamate receptors mediate the majority of excitatory synaptic transmission, modulate the release of presynaptic glutamate, and facilitate dendrite formation. Kainate receptors are unique among the ionotropic glutamate receptors in being modulated by sodium ions. They have also been implicated in the development of higher learning and epilepsy. In recent years a wealth of structural data has become available for the AMPA and NMDA classes; however, the structural characterization of kainate receptors has been limited. The work in this dissertation utilizes luminescence resonance energy transfer (LRET) and single-molecule Förster Resonance Energy Transfer (smFRET) in order to address this gap in the knowledge. We have characterized the structural arrangement and dynamics of the homomeric (GluK2) receptors and identified structural changes involved in the functional modulation by ions and auxiliary proteins. Additionally, we have characterized the arrangement and dynamics of the heteromeric (GluK2/GluK5). These data will build a foundation for the full biophysical characterization of kainate receptors; and contribute to the development of subunit-specific modulatory compounds to be used for disease therapies, and for more detailed characterization of brain function at the molecular level

Quantifying AAV (hM3Dq) transfection in neocortical cells as a guide to DREADDs control of trauma-induced epileptogenesis

Le néocortex déclenche des activités paroxystiques suite aux lésions cérébrales traumatiques. Avec des blessures cérébrales pénétrantes, la déafférentation s'accompagne d'une longue période silencieuse du cortex atteint, et d'une augmentation des périodes d'hyperpolarisation du néocortex entourant la lésion. L'activité synchrone du réseau neuronal après une période de latence conduirait à l'épileptogenèse. Des tentatives de modifier la nature des crises ont été effectuées à l'aide de modèles animaux. L'une des plus prometteuse étant la chimiogenèse par l'injection des vecteurs viraux afin d'anéantir la réponse de certains récepteurs couplés aux protéines G aux ligands habituels, alors qu'ils réagissent aux médicaments désirés, comme N-oxyde de clozapine. Ces récepteurs appelés « DREADDs » exclusivement activés par ces médicaments ont été étudiés pour réduire le nombre et la sévérité des crises. Selon les résultats non-publiés de notre laboratoire, l'utilisation d'un DREADD excitateur près de «undercut» serait antiépileptogène. Nous pensons qu'une excitation ciblée pourrait optimiser l'effet antiépileptogène. L'excitation est directement liée aux neurones transduits. Nous postulons qu'une transduction optimale des neurones pourrait être atteinte par un dosage optimisé du virus injecté, tant au titre viral qu'au volume injecté. Pour prouver notre hypothèse nous avons utilisé trois différentes titrations de AAV2/8 et injecté différents volumes de ces titrations aux souris. Le volume de transfection corticale et le nombre des neurones transduits ont été quantifiés. Avec la titration E11gc/ml aucune transfection n'a été observée. Avec la titration E12gc/ml une corrélation quasi-linéaire a été observée entre le volume viral injecté, et le volume cortical transfecté, ainsi que le nombre de neurones transduits. Avec la titration E13gc/ml, une meilleure corrélation a été observée à la transduction neuronale qu'à la transfection corticale, par rapport au volume viral injecté. En conclusion, la titration E12gc/ml paraît être un meilleur choix pour nos futures études, la fiabilité de la titration E13gc/ml n'ayant pas été démontrée.The neocortex is the origin of paroxysmal activities that occur after traumatic brain injuries. In penetrating brain injuries, deafferentation causes long silent periods in affected cortex and increased hyperpolarization period in neocortical tissue around the injury. The synchronous neural network activity after a latent period may lead to epileptogenesis. Some attempts to alter seizures were done using animal models. One of the most promising involves chemogenetic tools via AAV viral vector injection to make some G protein-coupled receptors unresponsive to their natural ligands, and activated by the desired drug, such as clozapine-N-oxide. These designer receptors exclusively activated by designer drugs (DREADDs) have been studied in reducing the numbers and severity of seizures. According to unpublished works in our lab, using excitatory DREADDs in the vicinity of undercut was antiepileptogenic. We believe there could be an optimal level of excitation for yielding an optimal antiepileptogenic response. This excitation is in direct relation with neurons transfected. We hypothesize that the optimal neuronal transduction might be achieved with optimal dosage of virus delivered, in terms of viral titration and the volume of virus injected. To test this, we used three different titrations of AAV2/8 and we injected different volumes of these titrations in adult mice. Cortical transfection volume and number of neuronal transductions were estimated. With E11gc/ml titration, no transfection was visible. With E12gc/ml titration, an almost linear correlation was observed between the volume of virus injected and the number of neurons transduced and the cortical volume of transfection. With E13gc/ml titration the correlation between the injected AAV volume and the number of neuronal transductions was still good but there was a poor correlation between AAV volume and transfection volume. We concluded that E12gc/ml titration was a more reliable option for our further studies. The reliability of E13gc/ml titration needs to be proven

Microdialysis studies of purine and monoamine release from the central nervous system in vivo

The effects of kainic acid and potassium on the release of endogenous adenosine and its metabolites, inosine, hypoxanthine and xanthine, from the rat hippocampus have been studied by in vivo microdialysis. In the hippocampus of rats anaesthetised with urethane the concentration of extracellular adenosine was estimated to be 0.8muM during the first two hrs after insertion of the dialysis probe. Kainic acid (0.1-25mM) in the perfusate evoked a concentration-dependent release of adenosine with an EC50 of 0.94mM. A 5min pulse of ImM kainic acid in the perfusate, during a sampling period of one hour, increased the 20mul dialysate levels from 3.68 +/- 0.21 to 7.66 +/- 0.82 pmol (mean +/- sem). A second stimulation (S2) 3hrs (hours) after the first stimulation (SI) also induced adenosine release. The S2/S1 ratio was 0.46 +/- 0.02. Kainate- evoked release of adenosine was shown to involve the production of action potentials since TTX (tetrodotoxin) significantly reduced the S2/S1 ratio by 53.85%. The release was reduced by incorporation into the perfusate of CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), a non-NMDA (N-methyl-D-aspartate) receptor antagonist, but not by NMDA receptor blockers, (+)-MK-801 (dizocilpine) or (+/-)-AP-5 ((+/-)-2-amino-5-phosphonopentanoic acid), indicating a non-NMDA receptor mediated process. The kappa agonist, U50 488H (trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneactemide methanesulphonate hydrochloride), significantly reduced the S2/S1 ratio by 55.77%. Release was reduced significantly by 44.23% by ascorbic acid (an antioxidant), 48.08% by glutathione (a scavenger of hydroperoxides) and 71.15% by oxypurinol (a xanthine oxidase inhibitor) indicating the involvement of free radicals in kainate-evoked adenosine release. Neither the adenosine A1 receptor antagonist CPT (8-cyclopentyl-1,3-dimethylxanthine) nor the A1 receptor agonist R-PIA (R(-) N6-(2-phenylisopropyl)adenosine) affected kainate-evoked release of adenosine. This indicates that activation of A1 receptors, by endogenous adenosine or an agonist, does not inhibit kainate-evoked release of adenosine. The present results indicate that kainate-evoked release of adenosine may be mediated by non-NMDA receptor activation, possibly requiring the propagation of action potentials and free radical production

Action of philanthotoxin on ion channels of arthropod muscle

Calcium ions play an important role in many signalling pathways involved in normal cell metabolism. Pertrebations of normal Ca++ signalling may also play a pivotal role in the initiation of cell death. In these studies I have examined the influx of 45Ca++ into the extensor tibiae muscle of the locust (Schistocerca gregaria ). 45Ca++ entry could be stimulated by the addition of glutamate receptor-agonists or by activation of voltage activated calcium channels. L-glutamate, L-quisqualate and NMDA stimulated the influx of 45Ca++ while L-aspartate had only a small effect. DL-ibotenate, kainate, AMPA and glycine had no effect on 45Ca++ uptake (all agonists were tested at concentrations up to (100μM). Glycine (1μM) enhanced the 45Ca++ entry induced by NMDA and L-glutamate. Only the glycine potentiation of L-glu-stimulated responses was abolished in the presence of Mg++ (2mM) or AP5 (10μM) whereas the NMDA-stimulated response was completely abolished by these agents. These finding suggests that in the presence of glycine, L-glutamate may activate NMDA receptors and that in the absence of glycine L-glu-stimulated 45Ca++ entry occurs via activation of the qGluR. Depolarisation of the extensor tibiae muscles (50mM KCl) stimulated 45Ca++ influx by activation of voltage-sensitive calcium channels. Philanthotoxin-343 (0.1μM) had no effect on depolarisation activated calcium entry, however, nifedipine (1μM) an L-type calcium channel antagonist inhibited this Ca++ influx. Nifedipine did not inhibit L-glu-stimulated Ca++ entry suggesting that in these muscles L-type Ca++ channels are not involved in the Ca++ influx pathway following G1uR activation. Philanthotoxin-433 (PhTX-433) and many of its synthetic analogues are potent inhibitors of locust GluR. In the future these analogues may prove as useful potential neuroprotective agents or as novel pesticides. Over 100 analogues of PhTX-433 have been synthesized with changes made in the four regions of the structure, the thermospermine moiety, the tyrosyl moiety, the butyryl moiety and the terminal amino moiety. The effects of different concentrations (10-4M to 10-14M) of synthetic analogues of PhTX-433 (PhTX-343, PhTX-343-Arg, PhTX-4) were investigated in the 45Ca++ influx assay using locust extensor tibiae muscle. PhTX-343-Arg was more potent (IC50= - 7x10-9) than PhTX- 343 (IC50= - 10-8M) or PhTX-4 in blocking 45Ca H uptake. These findings were further supported by electrophysiological studies. The interaction of these synthetic analogues of philanthotoxin with GluR of locust muscle were further investigated by examining the effect of these compounds on evoked excitatory post synaptic potentials. In recent years control of ticks have been very important issue because of the social and economical damage they cause. Neuromuscular transmission is a main target site for the chemical control of many pests. Philanthotoxin and its analogues block the glutamate receptors which are involved in arthropod neuromuscular transmission and thus may prove useful as novel pesticides. The action of synthetic analogues of philanthotoxin (C7PhTX-343, DNP12-, PhTX-343 and PhTX-343) were examined on evoked excitatory postsynaptic potential in tick coxal muscle. These compounds all antagonized the evoked EPSP. C7PhTX-343 and DNP12-PhTX-343 exhibited same potency (IC50 = 10-8M) and both were more potent than PhTX-343 (IC50 ='2X10-5M). In recent years Xenopus oocyte has taken over a new role as a test tube for the study of the biogenesis, functional architecture and modulation of plasma membrane protein. Attempts were made to express mRNA from embryonic tissue of tick and locust leg muscle in to Xenopus oocyte for pharmacological studies. Xenopus oocytes failed to translate RNA faithfully and efficiently from these sources. Rat brain RNA injected oocytes used as control, expressed routinely

Regulation of KCNQ Genes as a Mechanism Underlying Epileptogenesis

Epilepsy can develop in response to a brain insult, such as an initial seizure, stroke or traumatic brain injury. This insult induces a variety of cellular and molecular changes, observed in the clinic as a latent period, leading to a state of chronic spontaneous seizures. KCNQ/Kv7 channels are voltage-gated potassium channels which regulate neuronal excitability and protect against hyperexcitability through the Kv7 current. The transcriptional expression of KCNQ/Kv7 channels has previously been shown to be regulated by the transcription factors REST/NRSF, Sp1 and NFAT in peripheral neurons, but the presence of these mechanisms in the brain has not been examined. In this project, organotypic hippocampal slice cultures were used to investigate changes in Kcnq/Kv7 expression changes in epileptic conditions and in response to REST modulation. Adenovirus was used to deliver REST overexpression or a dominant negative REST to the slice cultures, with a novel delivery technique enabling up to 41% infection of total cells, including neurons and microglia, without observable toxicity. REST modulation had no effect on Kcnq2/3 expression, but inhibition of HDACs, which are recruited by REST to repress its target genes, caused de-repression of Kcnq2, suggesting Kcnq2 is regulated by HDACs. The chemoconvulsants kainate and 4-aminopyridine both caused a large upregulation of the epileptic marker BDNF. Kainate exposure caused a downregulation of Kcnq2, associated with a reduction of the Kv7.2 protein it encodes, specific to the CA1 hippocampal region. Expression patterns suggest that in contrast to REST’s known direct repression of BDNF, it may indirectly contribute to Bdnf upregulation following seizure through its pro-epileptic effects. Furthermore, Bdnf levels were correlated with Kcnq2 and Kcnq3, and Bdnf appears to help to maintain Kcnq2/3 expression in epileptic conditions. The downregulation of Kcnq2 after kainate exposure may contribute to epileptogenesis and could provide an area for therapeutic targeting

The Photooxidation of Domoic Acid

Domoic acid (DA) is a naturally occurring cyanotoxin, which upon ingestion, is responsible for amnesic shellfish poisoning (ASP) in both humans and animals. Produced by the marine diatom, Pseudonitzschia, DA is accumulated by a number of marine organisms including shellfish, clams and mussels which upon consumption can lead to headaches, nausea and seizures. Possessing a variety of functional groups the structure of DA contains three carboxyl groups, a pyrrole ring and a potent conjugated diene region allowing for binding to glutamate receptors in the dorsal hippocampus of the brain causing the described detrimental effects. Although limitations have been placed regarding the amount of DA that may be contained in seafood no limitations have been placed on the amount present in drinking water. Natural degradation of the toxin may occur through reactive oxygen species such as the hydroxyl radical and singlet oxygen at the conjugated diene region. In this work the photooxidation of DA via singlet oxygen has been studied using sorbic acid as a model compound. The three major reaction pathways observed during the photooxdiation process for both acids include 2 + 4 cycloaddition to produce endoperoxides , 2 + 2 reaction to afford aldehydes and ketones or an ene reaction to generate hydroperoxides. Under similar reaction conditions for SA and DA, the endoperoxide has been seen to be the major product for photoxidation of SA while the hydroperoxide has been seen to be the dominant product during photooxidation of DA