The Role Of Hippocampal Glypican4 In Epilepsy

Epilepsy is one of the most common serious neurological disorders,affecting about 65 million people globally.In China,the overall prevalence of epilepsy was 2.89‰.Development of an epileptic seizure requires physiological changes that lead to both heightened neuronal excitability and abnormal synchronization of discharge within the affected neuronal network.Epilepsies result from long-lasting plastic changes in the brain affecting the expression of receptors and channels,and involve sprouting and reorganization of synapses,as well as reactive gliosis.Current theories of pathophysiology stress neuronal dysfunction and damage,and aberrant connections as relevant factors.Most antiepileptic drugs target neuronal mechanisms.However,nearly one-third of patients are refractory to available medications;and a deeper understanding of mechanisms may be required to conceive more effective therapies.Recent studies point to significant contribution by glia-especially astrocytes and microglia-in the pathophysiology of epilepsy.Astrocyte-secreted moleculespowerfully control the formation,maturation,function and elimination of synapses through various secreted and contact-mediated signals.In recent years,several families of astrocyte-secreted molecules have been identified,and these molecules have been shown to regulate several aspects of synaptic development and function in vitro and in vivo.The TSPs and hevin control the structural formation of excitatory synapses between CNS neurons,but these synapses are postsynaptically silent because they lack AMPA receptors.A biochemical approach has been used to identify a family of astrocyte-secreted molecules that are sufficient to induce the formation of functional excitatory synapses.This family is Glypicans.Glypicans,a family of heparan sulphate proteoglycans(HSPGs),are anchored to the extracellular side of the cell plasma mem-brane by glycosyl-phosphatidylinositol(GPI)linkages,which can be cleaved by endogenous phospholipases,thus releasing the protein.There are six family members in mammals(GPC1 to GPC6),all of which have a similar core-protein size of approx.Human GPC4 is a 556-amino acid protein,and shows 93.5%identity to mouse K-glypican.The gene of GPC4 has been mapped to Xq26,and its mRNA has been shown to be present in astrocytes.Studies suggest that GPC4 is involved in the regulation of Wnt signaling and astrocyte GPC4 plays a role in formation of excitatory synapses.Epileptic seizures occur as a result of episodic abnormal synchronous discharges in cerebral neuronal networks.Although a variety of non-conventional mechanisms may play a role in epileptic synchronization,cascading excitation within networks of synaptically connected excitatory glutamatergic neurons is a classical mechanism.As is the case throughout the central nervous system,fast synaptic excitation within and between brain regions relevant to epilepsy is mediated predominantly by AMPA receptors.By inhibiting glutamate-mediated excitation,AMPA receptor antagonists markedly reduce or abolish epileptiform activity in in vitro preparations and confer seizure protection in a broad range of animal seizure models.NMD A receptors may also contribute to epileptiform activity,but NMDA receptor blockade is not sufficient to eliminate epileptiform discharges.The demonstrated clinical efficacy of perampanel,a high-potency,orally active non-competitive AMPA receptor antagonist,supports the concept that AMPA receptors are critical to epileptic synchronization and the generation and spread of epileptic discharges in human epilepsy.In the developing central nervous system,astrocyte glypicans 4 promote formation of excitatory synapses via GluAl AMPA receptors.At the resting potential,NMDA receptors(NMDARs)barely pass any current in response to activation by glutamate.Astrocytes secrete glypicans to increase the surface levels and clustering of the GluAl subunit of the AMPAR and thereby to induce excitatory synapse functionality.The neuronal receptor for the glypicans has yet to be identified,but studies in Drosophila melanogaster indicate that the protein tyrosine phosphatase receptor leukocyte antigen-related receptor(LAR)could be involved.In order to investigate the role of Glypican 4 in epilepsy,we chose the amygdala kainic acid microinfusion model as the chronic epilepsy animal model of our experiment.8-10 weeks,22-25g,male C57BL/6J mice,were grouped randomly.Adult mice were infused into the right basolateral amygdala with KA(0.3 mg in 0.5 ml PBS)or vehicle(0.5 ml PBS).Forty minutes after onset of KA-induced SE,chloral hydrate(10 mg/kg,i.p.)was administered to suppress SE.Behaviors were continuously monitored(24 h/day)during 2 weeks after SE.Spontaneous recurrent seizures(SRSs)were identified by review of video files by two independent trained readers blinded to both genotype and treatment of mice.Behavioral seizures were classified according to a modification of the Racine scale for mice.Mice were killed by pentobarbital overdose and perfused with ice-cold saline to remove the intravascular blood components.The brains were microdissected over wet ice,to obtain the hippocampus and prefrontal cortex.Real-time qPCR measurement of GPC4 in hippocampus after SE showed the expression level of GPC4 mRNA was increased.And western blot showed the expression level of GPC4 protein in hippocampus was lower than the control after SE.But the levels of GPC4 in PFC were not significantly different as shown by qPCR and western blot from the control.Next we tested the expression level of GPC4 in CA1 and non-CA1 subfields.Real-time qPCR measurement showed GPC4 mRNA in CA1 subfields after SE was increased.And western blot showed GPC4 protein concentrations in CA1 subfields were lower after SE.There was no difference in qPCR and western blot measurement between the two groups of mice after SE.Then,adeno-associated virus(AAV)-GPC4 shRNAs were used to silence GPC4 in CA1 and CA3.There was no difference between the control mice and shRNA mice in 40 mg/kg PTZ model.But,in 50 mg/kg PTZ model,ratio of seizure(stage2 and stage3)and seizure stage were significantly lower in CA1 GPC4 knockdown mice than the control.And ratio of seizure(stage2)and seizure stage were lower in CA1 GPC4 knockdown mice by an i.p.injection of 50 mg/kg PTZ.According to our results,we can conclude that the expression level of GPC4 protein in hippocampus decreased in chronic epilepsy model.And knockdown of GPC4 prevented epilepsy-like behavior in the PTZ model.