The Roles And Mechanisms Of Acid-sensing Ion Channel1a In The Pathogenesis Of Focal Cortical Dysplasia

Malformations of cortical development (MCD), which is characterized by sporadicarchitectural and cytoarchitectural malformations of the cerebral cortex, is recognized as themajor cause of chronic medically intractable epilepsy. It has been estimated that25%–40%of pharmacoresistant childhood epilepsies are caused by MCD and that75%of patientswith MCD will have epilepsy in their lives.MCD is a general term for a series of abnormal cortical development disorders, whichincludes focal cortical dysplasia (FCD), tuberous sclerosis complex (TSC),Hemimegalencephaly, Polymicrogyria and Sturge-Weber syndrome. According to theliterature, FCD is the most common cause of intractable epilepsy, susceptible to surgicaltreatment in China. In2011, a new three-tiered system was proposed by InternationalLeague Against Epilepsy, which includes both isolated and associated FCD variants.Isolated forms of FCD can be histopathologically distinguished into type I and type II. FCDtype I is characterized by cortical dyslamination and the presence of hypertrophic neurons(HNs). FCD type II displays additional cytological abnormalities, including dysmorphicneurons (DNs) and balloon cells (BCs). HNs demonstrate a pyramidal morphology with acentral nucleus. DNs were defined as neurons with large nucleolated nuclei, abnormal somasize and orientation and abundant Nissl substances. BCs were identified as morphologicallyabnormal cells with a thin membrane, eosinophilic cytoplasm and one or more eccentricallylocated nuclei.Most previous studies focused on the source and function of malformed cells (MCs).MCs might be a malformation due to abnormal migration, mature and cell death duringembryonic brain development. Electrophysiological studies demonstrated DNs couldsustain epileptic activity due to their hyperexcitability, however BCs are unable to generateaction potential. Despite of all the findings on MCs, the exact mechanism of FCD has notyet been elucidated. Acid-sensing ion channels (ASICs) represent proton-gated members of thedegenerin/epithelial Na+channel family, which are able to flux Na+and Ca2+. In mammals,four ASICs genes encode six different ASICs subunits (ASIC1a, ASIC1b, ASIC2a, ASIC2b,ASIC3, ASIC4). ASIC1a, which is encoded by the ACCN2gene, is widely expressed incentral and peripheral nervous system. Increasing evidences have demonstrated that ASIC1aplays crucial roles in physiological processes such as synaptic plasticity, learning-memoryand fear conditioning. In addition, ASIC1a was also implicated in a variety of neurologicaldisorders such as cerebral ischemia, multiple sclerosis and epilepsy. Down-regulation ofASIC1a mRNA level has been reported in the hippocampal CA1-2region of temporal lobeepilepsy (TLE) rat model. More importantly, both in vivo and in vitroexperiments demonstrated that ASIC1a shortened seizure duration and prevented seizureprogression, suggesting that ASIC1a could terminate seizures. Intriguingly, blockade ofASICs by amiloride delayed the onset of limbic seizures and the occurrence of statusepilepticus. To our knowledge, no reports to date have investigated the roles of ASIC1a inFCD. In our preliminary experiment, we detected decreased ASIC1a mRNA and proteinexpression in FCD compare with normal cortex (CTX). However, the detailed expressionpattern of ASIC1a, and its functional role in the pathogenesis and epileptogenesis of FCDare unclear.To address these concerns: First, we identified the distribution of ASIC1a in MCDspecimens from patients with intractable epilepsy. Second, to clarify the role of ASIC1a inthe epileptogenesis of MCD, a double-hit MCD rat model was established. We investigatedthe effect of ASICs blocker (Amiloride) on the latency, duration and times of seizure.Third, by primary neuron culture, we investigated the effect of ASIC1a in the neuronapoptosis. Fourth, to investigate the expression and distribution of IL-2signaling system,which might adjust the expression and function of ASIC1a.I．Expression of AISC1a in human FCD specimens.1．Real-time PCR and western blot analysis showed ASIC1a mRNA and proteinexpression in total homogenates of CTX. Immunohistochemical study revealedimmunoreactivity was mainly observed in neurons and gial-like cells.2．There was a statistically significant decrease of ASIC1a mRNA and proteinexpression in dysplastic cortices of FCD, in comparison with CTX. Moreover, the expression of ASIC1a was significantly lower in FCD type II than FCD type I.Immunohistochemistry results indicated that the overall immunoreactivity of the ASIC1astaining was diminished in FCD specimens compared to the CTX samples. In FCD, ASIC1aimmunoreactivity was mainly observed in reactive astrocytes and a minority of malformedcells, including hypertrophic neurons, dysmorphic neurons and balloon cells. Confocalfluresence analysis showed:①co-localization of ASIC1a with the neuronal marker andastrocytic marker in normal-appearing neurons and reactive astrocytes respectively;②mostMCs expressing ASIC1a were co-labeled with neuronal rather than astrocytic markers,indicating a neuronal lineage.3．We found decreased protein expression of parvalbumin (PV), calbindin (CB) andcalretinin (CR) in dysplastic cortices of FCD compared with CTX. Moreover, western blotanalysis showed the expression of PV, CB and CR was significantly lower in FCD type IIthan FCD type I.II．The modulatory effects of ASIC1a on seizure pattern in MCD rats model.1．We established a “double-hit” MCD rat model, which induced by in utero irradiationplus pentylenetetrazol (PTZ) injection. Immunostaining results showed there was adecreased ASIC1a expression in irradiation group, PTZ group and irradiation+PTZ groupcompared with CTX. The ASIC1a expression was significantly lower in irradiation+PTZgroup compared with irradiation group and PTZ group. Moreover, ASIC1a level wassignificantly lower in PTZ group compared with irradiation group.2．We injected amiloride, a non-specific ASIC blocker,90minutes before PTZinjection. The results of electroencephalograph (EEG) showed:①In the normal rats, we didnot detect any significantly difference in seizure latency, seizure duration and seizure times.②In the irradiated rats, with amiloride they have longer latency and reduced seizureduration after PTZ injection. However, we did not find any significantly difference inseizure times.III．The effect of ASIC1a on acidosis-induced apoptosis in cortical neurons.1．Primary cultured cortical neurons were treated with different concentration acidicculture medium for15,30and60min respectively. The lactate dehydrogenase (LDH) assay showed that acidic solution induced a dose-and time-dependent cytotoxic effect on corticalneurons. After treating with Amiloride or Psalmotoxin (PcTX-1), no significant differencewas found in acidic solution group, compared with control group.2．Blockage of ASIC1a significantly decreased the number of TUNEL-positive corticalneurons after exposing to acidic solution.40%and30%reduction of apoptotic rate weredetected in Amiloride and PcTX-1treating respectively.3． Western blot analysis showed the expression of caspase-3, which is a keyexecutioner in apoptosis. Amiloride and PcTX-1treating could down-regulate theacidosis-induced caspase-3activation.IV．Expression and cellular distribution of IL-2system in human FCD specimens.1．There was a statistically significant increase of IL-2and its receptors mRNA andprotein levels in dysplastic cortices of FCD, in comparison with CTX. Moreover, theexpression of IL-2and IL-2Rs was significantly higher in FCD type II than FCD type I. Insitu hybridization and immunohistochemistry results indicated that IL-2and IL-2Rs werestrongly expressed in hypertrophic neurons and neuronal microcolumns in FCD type I, andhighly expressed in malformed neurons in FCD type II. Confocal immunofluorescenceanalysis showed:①co-localization of IL-2/IL-2Rs with the neuronal marker and astrocyticmarker in normal-appearing neurons and reactive astrocytes respectively;②T cells, but notmicroglia (HLA-DR-positive) within the dysplastic cortex showed positive immunostainingof IL-2and IL-2Rs;○3co-localization of IL-2/IL-2Rs with NeuN, but not with theastrocytic marker GFAP in MCs.○4co-localization of IL-2/IL-2Rs immunostaining withvimentin in MCs.2．In comparison with CTX, western blot analysis showed the protein levels of Januskinase1(JAK1), JAK3and p-STAT5, which are important downstream factors in the IL-2signaling pathway, were significantly increased in FCD lesions. Moreover, Elisa resultsshowed soluble IL-2R (sIL-2R) was decreased in FCD specimens compared with CTX.These findings suggested the JAK-STAT pathway and sIL-2R involved in the pathogenesisof MCD.In the present study, we detected a significantly decrease of ASIC1a mRNA andprotein expression in FCD specimens. ASIC1a immunoreactivity was mainly observed in reactive astrocytes and a minority of MCs, including HNs, DNs and BCs. Subsequently, wedetected the decreased expression of ASIC1a in MCD rats. Treating with Amiloride,irradiated rats have longer latency and reduced seizure duration after PTZ injection.Moreover, blockage of ASIC1a significantly decreased the LDH amount, the number ofTUNEL-positive cortical neurons and caspase-3activation. In addition, we detectedincreased expression of IL-2/IL-2Rs/JAK-STAT pathway, which could potentially affect thefunction of ASIC1a. Altogether, these findings illuminate the ASIC1a involves in thepathogenesis of FCD.