The Role Of Transient Receptor Potential Vanilloid Receptor 4 In Malformation Of Cortical Development

Epilepsy is a chronic disease which is characterized by excessive abnormal synchronized discharge and will result in transient brain dysfunction. The annual incidence of epilepsy is 3.5‰?3.7‰, however, there are 15%?25% epilepsy patients not sensitive to antiepileptic drugs and become medically intractable epilepsy. Malformation of cortical development?MCD? is one of important etiology of pharmacoresistant epilepsies. In China, it has been estimated that 57.6% intractable epilepsy patients who undergo surgery are caused by MCD.Moreover, pharmacoresistant childhood epilepsies caused by MCD are count 80%. Therefore,the research on pathogenesis and epileptogenesis will be helpful for treatment of medically intractable epilepsy.MCD includes a large spectrum of pathological change of disease, such as focal cortical dysplasia ?FCD?,tuberous sclerosis complex ?TSC?, lissencephaly, megalencephalon andheterotopias. According to classification system of FCD proposed by International league against epilepsy ?ILAE? in 2011, the FCD include isolated type and associated type. Isolated FCD can bepathologically differentiated into FCD ? and FCD ?. FCD ? is characterized by cortical architecturalabnormalities and microcolumn. FCD ? displays not only cortical dysplasia but also malformed cells ?MCs?, which include dysmorphic neurons ?DNs? and balloon cells ?BCs?.Most studies indicated that MCs may come from abnormal migration, mature and death of neural precursor cells located in ventricular zone during brain development.Electrophysilogical research demonstrated that DNs have hyperexcitability which may be pacemaker of epileptic lesions. However, BCs is electrophysiologically characterized by which could not generate action potential. The role of MCs in MCD needs to be elucidated.Transient Receptor Potential ?TRP? superfamily is the channel located in cellular or subcellular membrane, with a remarkable diversity of gating properties, selectivity and specific activation mechanisms. The TRP family includes TRPV, TRPC, TRPM, TRPA, TRPP and TRPML. TRPV4, a member of TRP superfamily, is the capsaicin receptor which can be activated by a variety of stimuli including warm temperatures, hypotonicity and endogenous lipids. Previous studies have detected that TRPV4 is widely expressed in central nervous system. Increasing evidences demonstrated that TRPV4 plays crucial roles in physiological processes such as synaptic plasticity. In addition, TRPV4 was also implicated in a variety of neurological disorders such as cerebral ischemia,neuropathic pain and seizure-Endogenous metabolites of arachidonic acid, which is the agonist of TRPV4, increase during the seizure.More importantly, activation of TRPV4 might contribute to the regulation of neurotransmitters release and subsequently disrupt neural excitabilities-inhibilities balance.To elucidate the role of TRPV4 in MCD, we detected expression and distribution of TRPV4 inresected MCD specimens by using western blot, immunohistochemistry and double immunofluorescence staining. In addition, we investigated the calcium influx regulated by TRPV4 in primary cultured cortical neurons. Moreover, we established MCD animal model and seizure model to reveal the role of TRPV4 in epileptogenesis in MCD, and patch clamp was employed to found effect of TRPV4 on synaptic transmission. The results are listed as following:?. Expression and distribution of TRPV4 in FCD s specimens1. Western blot analysis showed TRPV4 protein expression in CTX. Immunohistochemical study founded immunoreactivity was mainly located in neurons, glial cells and endothelial cells.2. Compared with CTX, there was a significant increase of TRPV4 protein expression in FCD specimen homogenate. Besides, the expression of TRPV4 was significantly higher in FCD ? than FCD I, but there has no significant difference between FCD ?a and FCD ?b.Immunoreactivity was mainly founded in microcolumns and malformed cells. Double-labelling experiments confirmed the co-localization of TRPV4 immunostaining with the neuronal marker NeuN or NF200 in microcolumnsDNs and BCs. However, TRPV4 and GFAP were co-localized in most glial cells and some BCs. TRPV4 and Glutamate, GABA and GAD67also co-locatedin DNs, BCs and microcolumns. These results suggested that TRPV4 may play important role in FCD.3. Furthermore, the upstream factor of TRPV4 PKC and PKA are expression in FCD and CTX. However, PKA has no significant difference between CTX and FCD while PKC up-regulated in FCD specimens. These results indicated that PKC but not PKA may regulate TRPV4 in FCD specimens.?. Expression and distribution of TRPV4 in TSC specimens1. We investigated the expression patterns of TRPV4 in cortical tubers of TSC compared with CTX. We found that TRPV4 was clearly up-regulated in cortical tubers at the protein levels. Immunostaining indicated that TRPV4 was specially distributed in abnormal cells,including dysplastic neurons ?DNs? and giant cells ?GCs?. In addition, double immunofluorescent staining revealed that TRPV4 was localized on NF200-positive neurons and GFAP-positive reactive astrocytes. Moreover, TRPV4 co-localized with both glutamatergic and GABAergic neurons.2. Furthermore, protein levels of protein kinase C ?PKC?, but not protein kinase A ?PKA?,the important upstream factors of the TRPV4, were significantly increased in cortical tubers.?. TRPV4 activation affected calcium influx of cortical neurons1. To provide a functional assessment of TRPV4 in the cortex, rat-derived cortical neurons were used in a calcium imaging system. We took advantage of the TRPV4 agonist,4aPDD, to demonstrate the specific activation of TRPV4. In cortical neurons, Ca²⁺ influx was stimulated by 4aPDD in a dose-dependent manner. We also demonstrated that 4aPDD addition leads to the activation of calcium influx rather than facilitating its release from intracellular calcium stores. Overall, these results demonstrated that TRPV4 serves as a route of Ca²⁺ influx into the cortical neurons in response to pharmacological stimuli.2. We next determined whether these signalling cascades are involved in the control of[Ca²⁺]i elevation by affecting TRPV4 activity in cortical neurons. Stimulation of PKC greatly potentiated 4aPDD-mediated elevations in [Ca²⁺]i. Moreover, we repeated the treatment with PMA in the presence of the TRPV4 inhibitor HC067047. The amplitudes of the 4?PDD-mediated [Ca²⁺]i responses were significantly decreased. However, PKA signaling cascade didn't have similar effect. Overall,we conclude that the [Cau+]i response to pharmacological stimuli may be regulated by the stimulation of a PKC signalling cascade and that this regulation occurs in a TRPV4-dependent manner.?. TRPV4 activation in epileptogenesis of MCD1. We established MCD model, which induced by MAM injection during pregnancy.Western blot and immunohistochemistry analysis revealed that TRPV4 expression has no difference between MCD model and control animals. These results indicated that TRPV4 may not be involved in pathogenesis of MCD;2. Then, we established status epilepticus ?SE? model induced by intraperitoneal injection of pilocarpine. Western blot and immunohistochemistry analysis revealed that TRPV4 expression was upregulated in SE model. These results indicated that TRPV4 may be participated in epileptogenesis of MCD;3. Finally, by using patch clamp, we demonstrated that activation of TRPV4 increase both sEPSC and mEPSC frequency in SE model. According to these results, we hypothesized that TRPV4 activation enhanced development of seizure recurrent.In summary,this study revealed that:1. TRPV4 and its upstream factors PKC are upregulated in MCD specimens and mainly located in reactive astrocytes or malformed cells;2. TRPV4 served as a route of Ca²⁺ influx into the cortical neurons in response to pharmacological activation. PKC but not PKA could regulate this function of TRPV4.3. Compared with control animal,the expression of TRPV4 didn't have significant difference in MCD model but it upregulated in SE model. Moreover, activation of TRPV4 could increase frequency of mEPSC and sEPSC in SE model.4. In conclusion, TRPV4 may play important role in epileptogenesis of MCD.