Brandon Turner                           Receptors & Channels                           May 10, 2012                 Inves...
2and extrasynaptic (Chiu et al 2005), neuronal migration (Bozzi et al 2012), or inherent geneticfactors (Gurba et al 2012,...
3signaling in other regions of the brain associated with idiopathic generalized epilepsies. Clearly,developmental factors ...
4to epileptic phenotypes. As reviewed by Pavlov et al, 2012, “Reversal of GABA transport incertain subpopulations of neuro...
5that increased extra-cellular GABA concentrations are not enough to mitigate the hyper-excitability of hippocampal neuron...
6been shown by S. Joshi et al to lower neurosteroid sensitivity, which they attribute to changes inseizure susceptibility ...
7while collybistin binds preferentially to α2 only. They assert that the subunit and the scaffoldingproteins form a trimer...
8holds promise in understanding how spatial localization of inhibitory receptors may contribute toTLE.       Most studies ...
9Conclusion       Comprehending epilepsy from the molecular to the physiological level is a difficult gapto bridge, but th...
10mitigating seizure genesis at a behavioral level. Although working in the hippocampus todiscover the origin of TLE genes...
11                                          ReferencesBozzi Y, Casarosa S and Caleo M. Epilepsy as a neurodevelopmental di...
12Loup F, Wieser HG, Yonekawa Y, Aguzzi A and Fritschy JM. Selective alterations in GABA A      subtypes in human temporal...
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GABAergic Currents In Epilepsy

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A review article I created for a class in 2012. The paper attempts to overview the roles of GABA(A) receptors [Including pharmacology, mutations, and developmental disorders] in causing or alleviating Temporal Lobe Epilepsy (TLE).

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GABAergic Currents In Epilepsy

  1. 1. Brandon Turner Receptors & Channels May 10, 2012 Investigating the Roles of GABAergic Inhibitory Currents in EpilepsyIntroduction Epilepsy is one of the more common neurological disorders in humans and ischaracterized by the occurrence of repeated seizures. Seizures themselves result from an area ofthe brain exhibiting large amounts of depolarizing currents/over activity. For this reason, pastresearch has focused on an up-regulation of excitatory neurotransmission, such as AMPAreceptors, to be the cause of such over activity (Rogawski, 2011). However, treatment of seizureswith anti-convulsant that target excitability has proved to be less effective than desired. Morerecent research has discussed the role of inhibitory currents, notably those mediated by the γ-amino butyric acid (GABAA) type A receptor, which is responsible for the majority of the fastinhibitory signaling in the brain. The GABAA receptor exists as a heteropentamer usuallycomprised of two pairs of α and β subunits and a variable subunit (γ, δ, ρ, π, ε), each of whichmay have several subtypes (α [1-6], β [1-3], etc). With such subunit variability and differences ofsubunit expression levels in specific brain regions, the pharmacology of the GABAA receptor hasa high degree of variance. The receptor binds several allosteric potentiating ligands, such asbenzodiazepines (BZDs), which require the γ subunit, and neurosteroids which bind directly tothe α subunit. The involvement of such receptors in epilepsy is bound to be highly complex. Indeed,many factors can contribute to epilepsy via GABAergic currents, such as environmentalintoxicants like RDX (Williams et al 2011), developmental defects involving time of onset ofseizures (Briggs SW and Galanopoulou AS, 2011), regulation of GABA transport both, synaptic
  2. 2. 2and extrasynaptic (Chiu et al 2005), neuronal migration (Bozzi et al 2012), or inherent geneticfactors (Gurba et al 2012, Macdonald et al 2009). It has also been shown that GABA subunitcomposition changes during the course of seizures and epileptogenesis (Loup et al 2000).Despite these problems, neurosteroids and benzodiazepines are known to be effectiveanticonvulsants if administered with proper timing, yet some types of epilepsy, such as temporallobe epilepsy (TLE), remain resistant to such treatment. Clearly the role of GABAA in epilepsy isnot straightforward and requires additional investigation if a more conclusive understanding ofthe disease and its treatment are to be found. In this review, I will focus on changes inGABAergic inhibitory current due to developmental changes, GABA concentration variations inmultiple forms of epilepsy, and on receptor specific changes in temporal lobe epilepsy in thehopes of directing future research to develop anticonvulsants targeting these receptors that moreaccurately despite the inherent complexity of their role in epileptogenesis.Developmental Malformations in GABAergic Systems Developmental miscues can result in a host of neurological disorders that include manysubtypes of epilepsy. Some of these include genetic defects that decrease the migration ofGABAergic neurons to their proper positions, including interneurons of the cortex andhippocampus. Two of these factors, Dlx and Reelin, have been shown in knockout and mutationstudies to impair the migration of such inhibitory neurons to the cortex and proper layer in thehippocampus, respectively (Bozzi et al 2012), and have been implicated several forms ofepilepsy. Many other factors have been shown to induce epilepsy in model animals as well, allconcerning cellular circuits and positioning in the brain. Notably, Velisek et al have recentlyshown that haploinsufficiency of BRD2 (Bromodomain- containing gene 2) in mice contributesto lack of GABAergic neurons in the neocortex and striatum and a deficiency in GABAergic
  3. 3. 3signaling in other regions of the brain associated with idiopathic generalized epilepsies. Clearly,developmental factors and genetic regulation can play into developing epilepsy by removinginhibition in different brain regions. Other changes in normal neuronal function, most notably seizures at an early age, caninduce epilepsy in adult animal models. Depending on age, GABAA currents can be eitherdepolarizing (early age) or hyperpolarizing. Recently, this has been shown to be linked to theconcentration of Cl- within the cell, which affects GABAA subunit compositions (Succol et al2012). The authors used both electrophysiological and immunostaining to show that changes inintracellular chloride concentrations affected EGABA depending on the expression levels ofKCC2, a potassium/chloride cotransporter. Consistent with this data, it has been shown thatdisruption of KCC2 funciton can contribute to early life seizures and that these seizuresthemselves can alter GABAA subunit composition as well (Reviewed by Briggs SW,Galanopoulou AS, 2011). It may be that a rapid influx of chloride ions due to compensatoryinhibition during seizures may contribute to the changes in GABAA receptor subunitcomposition, or that changes in chloride trafficking may result in a reversion to immature-likeexpression and function of GABAA receptors, causing them to be depolarizing rather thaninhibitory. Further research into the temporal allocation of shifts in chloride gradients,transporter activity, and subunit composition during the ictal period of seizures may provideinsight into epileptogenesis.Changes in Extracellular and Synaptic GABA Concentrations Changes in GABA concentrations at the synapse due to disruptions in transport orsynthesis could contribute to altered levels of GABAA signaling, which could, in turn, contribute
  4. 4. 4to epileptic phenotypes. As reviewed by Pavlov et al, 2012, “Reversal of GABA transport incertain subpopulations of neurons or individual cells in epileptic tissue cannot be ruled out. Someinterneurons in chronic epilepsy may become metabolically more active, express more GAD andhave elevated intracellular GABA concentrations.” This speaks the idea that overexpression ofGAD (Glutamate Decarboxylase), which converts Glutamate to GABA, could raise intracellularGABA and reverse transportation, which is largely dependent on voltage and concentrationgradients (Pavlov et al, 2012). Consistent with this, impaired GABA uptake by GABAtransporter deficiency has been shown to cause tremors and ataxia in mice (Chiu et al 2005).Despite the possible negative roles of GAT impairment in epilepsy, it has been shown that GATimpairment functions to increase tonic inhibition in epileptic rats (Frahm et al, 2003) byincreasing available GABA to the extrasynaptic, high affinity and slow desensitizing receptors.In this sense, GABA transporters could serve as a possible drug target for anticonvulsants shouldallosteric up-regulation be possible. Much of the regulation of extrasynaptic GABA concentrations is dependent on thefunction of GABA transporters in astrocytes. GAT-3, which is primarily found in astrocytes,contributes substantially to extracellular GABA levels and can induce increased tonic inhibitionin the presensce of glutamate (Heja et al 2009). In vivo studies have also shown that uptake ofglutamate in hippocampal astrocytes is necessary for GABA release by GAT-2,3 and can convertexcess amounts of glutamate excitatory signaling to inhibition in an epileptic model (Heja et al2012). Due to increased variability of GABAA subunits during seizures, increased tonic GABAcan initiate a negative feedback loop and prevent the spread of excess excitation to other brainregions without the need of specifically designed drugs for the variant receptors. However, thismay not serve as an effective treatment for epilepsy, as past studies have demonstrated in vitro
  5. 5. 5that increased extra-cellular GABA concentrations are not enough to mitigate the hyper-excitability of hippocampal neurons in an epileptic model (Yeh et al 2005). Since extracellular GABA levels and tonic inhibition seem to be inefficient at preventingseizures, investigation of vesicular GABA transporters (VGAT) may provide insight intomechanisms for preventing seizures. It has recently been shown that epileptic neurons, asinduced by excess exposure to glutamate, express a truncated form of VGAT that accumulates atnon-synaptic sites (Gomes et al 2011). However, removal of VGAT from vesicles contributes toincreased synaptic GABA concentrations, suggesting that VGAT truncation may serve as amechanism to increase inhibition in their model of temporal lobe epilepsy rather than furtheringthe progression of the disease. Given that synaptic GABAA receptors desensitize more rapidlythan extrasynaptic receptors, it would seem that enhancing GABA at the synapse may also proveto be ineffective at alleviating epileptogenesis. Since the level of GABA transport appears to beproviding negative feedback in epileptic models, their dysfunction could contribute toepileptogenesis. However, due to their regulation by both voltage and concentration gradients,modifying these transporters would prove difficult at the least, suggesting that changes inreceptor expression and subunit concentration could provide more insight into understandingepileptogenesis and drug design.GABA Receptor Expression and Composition Prior to and Following Seizures Temporal lobe epilepsy (TLE) is among the most common forms of epilepsy in adultsand is mainly thought to arise from alterations in excitation/inhibition in the hippocampus (Joshiet al 2011). Those with the disease normally display a loss of both CA1 and CA3 pyramidalneurons and changes in receptor expression in dentate gyrus cells (DGCs). These changes have
  6. 6. 6been shown by S. Joshi et al to lower neurosteroid sensitivity, which they attribute to changes inseizure susceptibility of women dependent on the menstrual cycle (known to cause alterations inthe levels of endogenous neurosteroids in the brain). Indeed, recent studies have shown usingradiolabeled ligand binding that rats with temporal lobe epilepsy show decreased binding in thecerebral cortex and further show that this corresponds to a decrease in expression of α1, γ, δ,GABAB receptors, and GAD, the enzyme that converts glutamate to GABA (Mathew et al2012). Past studies have also shown that changes in CA1 and CA3 cells include a markeddecrease in α1 subunit expression and an increase in α2/3 expression (Loup et al 2000). Also,Rajasekaran et al had recently shown that the low affinity of neurosteroids for epileptic GABAAreceptors is likely due to lower incorporation of the δ subunit, which is down-regulated in TLE.How intracellular mechanisms contribute to subunit regulation is currently not well understood,but recent studies have shown that δ subunit incorporation is largely dependent on intracellular[Cl-]. Increased intracellular [Cl] in the presence of a KCC2 knockdown decreased the amount ofδ subunit incorporated into receptors, as shown by immunostaining, present at the membrane, aswell as increasing the incorporation of α3 subunits and decreasing the incorporation of α1subunits (Succol et al 2012). The authors focused on this shift of subunit incorporation primarilyto elucidate the shift of GABAA from depolarizing to hyperpolarizing during neuronaldevelopment, which they showed to be mediated by KCC2 up-regulation. However, this couldalso point to a method of altered subunit expression in epileptic animals, but whether the KCC2ion transporter is involved is not known. In addition to differential regulation of GABAA subunits during epilepsy, there is also aloss of synaptic GABAA receptors that may be due to changes in gephyrin and collybistinscaffolding. Past studies have shown that gephyrin preferentially binds to α2 and α3 subunits,
  7. 7. 7while collybistin binds preferentially to α2 only. They assert that the subunit and the scaffoldingproteins form a trimeric complex via co-immunoprecipitation and that disruption of thisinteraction may lead to epilepsy, as seen in a genetic mutation causing a change in the SH3domain of collybistin which is known to cause mental disability and seizures (Saiepour et al2010). More recent studies have also shown that gephyrin is able to bind to α1 subunits as well,also using immunoprecipitation (Mukherjee et al 2011). Since α1-3 containing receptors are theprimary receptors found at synaptic sites, changes in gephyrin binding, expression, orlocalization may contribute to loss of these receptors at the synapse and change thepharmacological properties of these sites during epilepsy. Congruent with this idea, other studieshave shown that induction of status epilepticus alters gephyrin and neuroligin-2 expresssion, butthese did not have an effect on GABAA clustering . Mechanisms underlying the lack ofcorrelation between the two remain unexplained, but other studies have shown that such arelationship does exist. Forstera et al had recently demonstrated that splicing of gephyrin exonsis altered in brain regions undergoing TLE or cellular stress which corresponded to a loss of α2subunit containing receptors at the synapse. It is possible that a decrease of gephyrin binding andthe reported up-regulation of α2 GABAA subunits could contribute to an unnatural clustering ofthese receptors at extra-synaptic sites, which normally contain α4 α6 subunits, specifically.However, Forstera et al report that no concurrent mutations in gephyrin are concurrent withtemporal lobe epilepsy in humans, which may indicate that collybistin-gephyrin clustering, alongwith their association with GABAA receptors is complex and requires additional study. Whetherthese changes in protein scaffolding are reflect potential feedback mechanisms to stopepileptogenesis or a method in which seizures are generated will require additional research and
  8. 8. 8holds promise in understanding how spatial localization of inhibitory receptors may contribute toTLE. Most studies have alluded to the involvement of hippocampal neurons in the pathogenesisof TLE, but, due to the aberrant shuffling of subunits and inherent neuro-plasticity thataccompanies seizures many forms of TLE have remained resistant to anti-convulsants. Aspreviously described, GABAA receptors in the hippocampus display lowered sensitivity to bothbenzodiazepines and neurosteroids, possibly due to increases in α4 containing receptors, whichare insensitive to BZDs, and a decrease in δ containing receptors, which have a high sensitivityto neurosteroids (Joshi et al 2011). A recent study has shown that other brain regions may beinvolved in the propagation of excitatory signaling from the hippocampus and are involved in thedevelopment of seizures. The thalamus, particularly the Thalamic Parafascicular Nucleus, hasbeen shown to be directly involved in the development of seizures (Langlois et al 2010).Specifically, they show in vivo that inhibition of NMDA receptors and/or the presence ofGABAA antagonists injected into the TPN was sufficient to prevent behavioral effects followingthe creation of an epileptic center in the hippocampus. In agreement with these findings, similardata was published using muscimol to activate GABAA receptors in the mediodorsal region ofthe thalamus and similar results were observed, most notably being the attenuation of seizureduration (Sloan et al 2011). Although changes in receptor signaling and expression may bepresent in the thalamus as well, it provides a novel target for preventing TLE behavioral effectsthat circumvents the complex problem of receptor subunit regulation, shuffling, or changes inligand concentrations. Until the generation of seizures is fully understood, the thalamus may bethe best option for treating patients with TLE and provides a novel target for specific drugs thatcould serve as anti-convulsants for TLE patients.
  9. 9. 9Conclusion Comprehending epilepsy from the molecular to the physiological level is a difficult gapto bridge, but the numerous studies already done have shown many changes at both levels, bothbefore and after the occurrence of seizures. The myriad of developmental factors that couldcontribute to the onset of epilepsy are not well understood beyond their number, yet thenumerous in ways in which they affect the nervous system, as per neuronal development andmigration or receptor/transporter mutations that cause aberrant signaling in mature neurons. Thissignificantly narrows the mechanisms in which developmental disorders can contribute toepileptogenesis, but correcting neuronal migration in the brain is unlikely, while finding methodsthat can target malformed regions and restore them to their normal inhibitory function by anti-convulsant drugs is much more likely. GABA transporters, however, seem to serve a role asincreasing GABAergic current in epileptic areas of the brain and their selective modulation byallosteric drugs would also prove arduous due to their voltage sensitivity and dependence onintra/extra cellular GABA concentrations. It is possible that an increase in GAD activity may inneurons or astrocytes may be able to shunt the concentration of GABA to favor increasedexportation, but it has been shown that increased ambient GABA at the synapse or in extra-synaptic regions have been ineffective at preventing seizures. A more novel target would beselective modulation of GABAA receptors to increase tonic and phasic inhibition, but the regionsaffected by seizure generation display irregular GABAA subunit composition and pharmacology,further complicating the effect of anti-convulsants. Also, TLE is markedly insensitive to anti-convulsant drugs, perhaps due to this or the inability of such drugs to act in the epileptic centers.Yet new research suggests that inhibiting the progression of seizures to other areas of the brainvia increased inhibition or decreased excitation in the thalamus may be an effective way of
  10. 10. 10mitigating seizure genesis at a behavioral level. Although working in the hippocampus todiscover the origin of TLE genesis, preventing the spread of seizures from here by increasedinhibition in the thalamus may be the most effective direction to work with until the exactmechanism of the marked increase in excitability of hippocampal neurons is discovered andmore specific drugs can be developed.
  11. 11. 11 ReferencesBozzi Y, Casarosa S and Caleo M. Epilepsy as a neurodevelopmental disorder (2012). Front. Psych. 3(19): DOI: 10/3389/fpsyt.2012.00019.Briggs SW and Galanopoulou AS. Altered GABA signaling in early life epilepsies (2011). Neural Plasticity. 2011: DOI: 10.1155/2011/527605Chiu CS, Brickley S, Jensen K. Southwell A, Mckinney S, Cull-Candy S, Mody I and Lester HA. GABA transporter deficiency causes tremor, ataxia, nervousness, and increased GABA-induced tonic conductance in cerebellum (2005). Journ. Neurosci. 25(12): 3234 – 3245.Forstera B, Belaidi AA, Juttner R, Bernert C, Tsokos M, Lehmann TN, Horn P, Dehnicke C, Schwarz G and Meir JC. Irregular RNA splicing curtails postsynaptic gephyrin in the cornu ammonis of patients with epilepsy. Brain. 133:3778-3794.Frahm C, Stief F, Zuschratter W, Draguh A. Unaltered control of extracellular GABA- concentration through GAT-1 in the hippocampus of rats after pilocarpine-induced status epilepticus (2003). Epilepsy Res. 52: 243-252Gomes JR, Lobo AC, Melo CV, Inacio AR, Takano J, Iwata N, Saido TC, de Almeida LP, Wieloch T and Duarte CB. Cleavage of the vesicular GABA transporter under excitotoxic conditions is followed by accumulation of the truncated transporter in nonsynaptic sites (2011). 31(12): 4622 – 4635.Gurba KN, Hernandez CC, Hu N and Macdonald RL. The GABRB3 mutation, G32R, associated with childhood absence epilepsy alters α1β3γ2L GABAA receptor expression and channel gating (2012). Journ. Biol. Chem. 287, 12083-12097.Heja L, Barabas P, Nyitrai G, Kekesi KA, Lasztoczi B, Toke O, Tarkanyi G, Madsen K, Schousboe A, Dobolyi A, Palkovits M and Kardos J. Glutamate uptake triggers transporter-mediated GABA release from astrocytes (2009). PLoS ONE. 4(9): e7153. DOI:10.1371/jounal.pone.0007153.Heja L, Nyitrai G, Kekesi O, Dobolyi A, Szabo P, Fiath R, Ulbert I, Pal-Szenthe B, Palkovits M and Kardos J. Astrocytes convert network excitation to tonic inhibition of neurons (2012). BMC Biology. 10(26): http://www.biomedcentral.com/1741-7007/10/26.Jackson J, Chugh D, Nilsson P, Wood J, Carlstrom K, Lindvall O and Ekdahl CT. Altered synaptic properties during integration of adult-born hippocampal neurons following a seiaure insult (2012). PLoS One. 7(4). E35557.Joshi S, Rajasekaran K, Kapur J. GABAergic transmission in temporal lobe epilepsy: The role of neurosteroids (2011). Exp Neurol. Doi:10.1016/j.expneurol.2011.10.028Langlois M, Polack PO, Bernard H, David O, Charpier S, Depaulis A, and Deransart C. Involvement of the Thalamic Parafascicular Nucleus in Mesial Temporal Lobe Epilepsy (2010). Journ. Neurosci. 300(49):16523-16535
  12. 12. 12Loup F, Wieser HG, Yonekawa Y, Aguzzi A and Fritschy JM. Selective alterations in GABA A subtypes in human temporal lobe epilepsy (2000). Journ. Neurosci. 20(14): 5401 – 5419.Mathew J, Balakrishnan S, Antony S, Abraham PM and Paulose CS. Decreased GABA receptor in the cerebral cortex of epileptic rats: effect of Bacopa monnieri and Bacoside-A (2012). Journal of Biomedical Science. 19:25. http://www.jbiomedsci.com/content/19/1/25.Mukherjee J, Kretschmannova K, Gouzer G, Maric HM, Ramsden S, Tretter V, Harvey K, Davies PA, Triller A, Schindelin H and Moss SJ. The residence time of GABAARs at inhibitory synapses is determined by direct binding of the receptor α1 subunit to gephyrin (2011). Journ. Neurosci. 32(41):14677-14687.Pavlov I & Walker MC. Tonic GABAA receptor-mediated signaling in temporal lobe epilepsy (2012). Neuropharm. Doi:10.1016/j.neuropharm.2012.04.003Rajasekaran K, Joshi S, Sun C, Mtchedlishvilli Z and Kapur J. Receptors with low affinity for neurosteroids and GABA contribute to tonic inhibition of granule cells in epileptic animals (2010). Neurobio. Disease. 40: 490-501.Rogawski MA. Revisiting AMPA receptors as an antiepileptic drug target (2011). Epilep. Curr. 11(2): 56-63.Saiepour L, Fuchs C, Patrizi A, Sassoe-Pognetto M, Harvey RJ and Harvey K. Complex role of collybistin and gephyrin in GABAA receptor clustering (2010). Journ. Biol. Chem. 285(38): 29623-29631.Sloan DM, Zhang DX, and Bertram EH III. Increased GABAergic inhibition in the midline thalamus affects signaling and seizure spread in the hippocampus-prefrontal cortex pathway (2011). Epilepsia. 52(3):523-530.Succol F, Fiumelli H, Benfanati F, Cancedda L and Barberis A. Intracellular chloride concentration influences the GABAA receptor subunit composition (2012). Nature Comm. 3:738. DOI: 10.1038/ncomms1744.Velisek L, Shang E, Velskova J, Chachau T, Macchiarulo S, Maglakelidze G, Wolgemuth DJ and Greenberg DA. GABAergic neuron defecit as an idiopathic generalized epilepsy mechanism: The role of BRD2 haploinsufficiency in juvenile myoclonic epilepsy (2011). PLoS One. 6(8): E23656.Williams LR, Aroniadou-Anderjaska V, Qashu F, Finne H, Pidoplichko V, Bannon DI and Braga MFM. RDX binds to the GABAA receptor-convulsant site and blocks GABAA receptor- mediated currents in the amygdale: A mechanism for RDX-induced seizures. Envir. Health. Persp. 119(3): 357 – 363.Yeh JH, Jeng CJ, Chen YW, Lin HM, Wu YS and Tang CY. Selective enhancement of tonic inhibition by increasing ambient GABA is insufficient to suppress excitotoxicity in hippocampal neurons (2005). Biochem. Biophys. Res. Comm. 338: 1417-1425.

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