Study Of The Role And Mechanism Of BDNF/ TRPC In The Development Of Focal Cortical Dysplasia

Focal cortical dysplasia(FCD), which is characterized by columnar and laminar disorganization with various cellular abnormalities, is increasingly recognized as one of the leading causes of medically intractable epilepsy in pediatric patients. Several cellular and molecular mechanisms underlying the pathogenesis and epileptogenesis of FCD have been proposed, including abnormal migration and maturation of neurons during cortical development and abnormalities of the neuronal circuitry in epileptic foci. Recent evidence has indicated that cells originating from the subventricular zone(SVZ) may be the source of the abnormal cells(e.g., DNs and BCs) within the cortical lesions of FCD patients. In addition, our previous study in a freezing lesions model indicated that SVZ-derived neural progenitors are involved in the formation of focal microgyria. However, the mechanisms underlying the abnormal migration of abnormal cells within FCD lesions have not been fully elucidated.The transient receptor potential canonical(TRPC) channels are Ca2+-permeable, nonselective cation channels that are expressed in a variety of multicellular organisms. Based on their amino acid sequences and functional similarities, the TRPC family can be divided into three groups: TRPC1/4/5, TRPC3/6/7, and TRPC2. They are widely expressed in the brain and play various physiological roles in the central nervous system. TRPC1 have an important role in the early development and proliferation of neurons. TRPC1 contribute to netrin-1/ BDNF-mediated growth cone guidance. Moreover, it has been shown that TRPC1 is involved in basic fibroblast growth factor(bFGF) regulated the proliferation of neural stem cells. TRPC1 could participate in the formation of excitatory postsynaptic potentials. Additionally, in TRPC1 knockout mice, epileptiform burst firing can be abolished by 74 % of lateral septal neurons. Interestingly, in TRPC1/TRPC4 double knockout mice, pilocarpine-induced seizures in the lateral septum and cornu ammonis 1 region of the hippocampus were greatly improved. TRPC5 also play a key role in neural proliferation and differentiation. Furthermore, TRPC5 may be involved in abnormal neuronal migration in some neurological disorders, such as FCD. Recent studies found that TRPC5 regulate dendrite morphogenesis of neurons. Additionally, in cultured hippocampal neurons, TRPC5 have an important role in axon formation through activating the CaMKK/Ca MKIγ cascade. Increased expression of TRPC5 have been reported in the pilocarpine model of temporal lobe epilepsy. Furthermore, in TRPC5 knockout(KO) mice, a significantly reduced seizures and the minimal seizure-induced neuronal cell death confirmed that TRPC5 channels participated in the long-term potentiation during epileptiform bursts. Accumulating evidence has demonstrated that TRPC6 plays a critical role in neuronal development. For instance, in a heteromeric assembly, TRPC6 has an essential role in brain-derived neurotrophic factor(BDNF)-mediated nerve growth cone guidance in conjunction with C3, suggesting that TRPC6 activation may be involved in neuronal migration. The development of neuronal circuits requires the establishment of proper neuronal morphology and synapse formation. Recent studies have demonstrated that TRPC6 promotes dendritic growth of hippocampal neurons both in culture and in the brain. In addition, TRPC6 is localized to both pre- and postsynaptic areas, and TRPC6 overexpression induces excitatory synapse formation. Thus, TRPC6 may play a key role in the establishment of neuronal circuits.Above all, BDNF and TRPC channels paticipated in the early development and proliferation of neurons, and have a novel function in the migration of neurons and in the formation of neural circuits. Additionly, increasing findings have defined that TRPC might participate in the processes of epilepsy. Therefore, we speculate that BDNF/TRPC channels may contribute various physiological functions in the pathogenesis and epileptogenesis of FCD.In order to elucidate the mechanisms underlying the role of BDNF/TRPC pathway in the pathogenesis and epileptogenesis of FCD, we designed following experiment: First, we identified the distribution of BDNF and TRPC channels in the cortical leisions from patients with focal cortical dysplasia(FCD) by means of Real time PCR, Western blot and immunohistochemistry. Second, we established WT-FCD,TRPC6-/--FCD and TRPC6+/+-FCD mouse models, respectively, and continuously observed the process of the migration of SVZ-derived NPCs labelled with eGFP. We investigate the potential role of TRPC6 involved in NPCs migration by guiding nerve growth cones. The results show as follows:Ⅰ. Expression and cellular distribution of BDNF and its receptor Trk B in FCD1. Real time PCR and western blot analysis showed BDNF mRNA and protein expression in total homogenates of normal control cortex(CTX); Immunohistochemistry showed that BDNF mainly distributed in pyramidal neurons in the grey matter and glial-like cells in the white matter.2. There was a statistically significant increase in BDNF m RNA and protein levels in the total homogenates from the FCDIa, FCDIIa and FCDIIb cases compared with the CTX. Importantly, the expression levels of BDNF mRNA and protein were significantly increased in the FCDIIa and FCDIIb cortical lesions compared with the FCDIa specimens. The immunostaining results demonstrated that in FCDIa, FCDIIa and FCDIIb cortical lesions, moderate to strong BDNF staining was observed in the neurons, including the microcolumns, DNs and BCs in the gray matter and the heterotopic neurons in the white matter. There was also moderate BDNF staining in glia-like cells. Increased TrkB protein levels were observed in FCD versus CTX tissues, however, there was no statistical differences in P75 protein levels between FCD and CTX.3. Above results suggesting that the binding of BDNF to TrkB, not P75, may support or enhance the survival of immature cells(DNs and BCs) and may participate the formation of DNs and BCs by guiding nerve growth cone. The immature cells(DNs and BCs) interact with cells that have reached maturity and have normal synaptic connections to produce seizures.Ⅱ. Expression and cellular distribution of TRPC channels in FCD1. Real time PCR and western blot analysis showed TRPC channels(C1,C5 and C6) m RNA and protein expression in total homogenates of normal control cortex(CTX); Immunohistochemistry showed that BDNF mainly distributed in pyramidal neurons in the grey matter and glial-like cells in the white matter.2. There was a statistically significant increase in TRPC channels(C1,C5 and C6) m RNA and protein levels in the total homogenates from the FCDIa, FCDIIa and FCDIIb cases compared with the CTX. Importantly, the expression levels of TRPC6 mRNA and protein were significantly increased in the FCDIIa and FCDIIb cortical lesions compared with the FCDIa specimens. The immunostaining results demonstrated that in FCDIa, FCDIIa and FCDIIb cortical lesions, moderate to strong TRPC channels(C1,C5 and C6) staining was observed in the neurons, including the microcolumns, DNs and BCs in the gray matter and the heterotopic neurons in the white matter. There was also moderate TRPC channels(C1,C5 and C6) staining in glia-like cells. Immunofluorescence experiments further confirmed that TRPC(C1,C5 and C6) co-localized with glutamate and GABA in the DNs and BCs. Most of the TRPC6-positive neurons were glutamatergic neurons, and a few were GABAergic neurons. However, in CTX tissues, there was no obvious difference in the numbers of glutamatergic and GABAergic neurons3. Above results suggesting that TRPC channels may parcitipate in the epileptogenesis of FCD. TRPC channels might induce the disrupt in the excitatory/inhibitory balance of neuronal circuits in the epileptic zone, resulting in the seizure activity. TRPC channels colocalized with GFAP in astrocyte cells, particularly in reactive astrocytes suggesting TRPC channels may might be involved in the process of astrocyte induced epilepsy in FCD. BDNF/TRPC6 pathway may be involved in the formation of heterotopic neurons, DNs and BCs by guiding nerve growth cones and may support or enhance the survival of immature cells(DNs and BCs) that interact with cells that have reached maturity and have normal synaptic connections to produce seizures. The highly expressed TRPC6 in FCD patients promotes dendritic growth and the development of dendritic spines and excitatory synapses via the CaMKIV-CREB pathway, resulting in dendritic abnormalities and abnormally excitable circuits. These results suggest that the overexpression of BDNF and TRPC6 and the activation of the TRPC6 signal transduction pathway in FCD lesions may contribute to the pathogenesis and epileptogenesis of FCD.Ⅲ. TRPC6 regulate the migration of SVZ neural precursor cell(NPCs)1. In this study, through combined use of neonatally cortical freeze-lesion, e GFP intracerebroventricular injection and lenti-virus TRPC6 RNAi transfection methods,we successfully established WT-FCD,TRPC6-/--FCD and TRPC6+/+-FCD mouse models, respectively, and continuously observed the process of the migration of SVZ-derived NPCs labelled with eGFP.2. At P5, there was no migration of eGFP-positive(eGFP +) cells in WT-FCD and TRPC6-/--FCD mouse models. However, in TRPC6+/+-FCD mouse models, a migratory stream from SVZ to FCD cortical lesion(SFMS) could be continuously observed. At P10, we found a few e GFP-positive(eGFP +) cells migrated from SVZ to cortical leisions in WT-FCD mouse models. Sporadic eGFP-positive(eGFP +) cells could be observed in TRPC6-/--FCD mouse models and direction of migration was disordered. However, in TRPC6+/+-FCD mouse models, we found a thick migratory stream from SVZ to FCD cortical lesion(SFMS).3. The intervention in vivo study results indicated that increased expression of TRPC6 could promote the migration of SVZ NPCs and the underlying mechanism of TRPC6 mediate the migration of SVZ-NPCs might through guiding the direction of nerve growth corn of SVZ-NPCs.In summary, firstly, the expression of BDNF and its recepter TrkB were higher in FCD versus CTX samples, especially distributed in the microcolumns, DNs and BCs in the gray matter and the heterotopic neurons in the white matter, suggesting that the binding of BDNF to Trk B, not P75, may support or enhance the survival of immature cells(DNs and BCs) and may participate the formation of DNs and BCs by guiding nerve growth cone. The immature cells(DNs and BCs) interact with cells that have reached maturity and have normal synaptic connections to produce seizures. Secondly, the expression of TRPC channels were higher in FCD versus CTX samples, especially distributed in misshappen cells, suggesting that TRPC channels might induce the disrupt in the excitatory/inhibitory balance of neuronal circuits in the epileptic zone, resulting in the seizure activity. BDNF/TRPC pathway may be involved in the formation of heterotopic neurons, DNs and BCs by guiding nerve growth cones and may support or enhance the survival of immature cells(DNs and BCs) that interact with cells that have reached maturity and have normal synaptic connections to produce seizures. The highly expressed TRPC6 in FCD patients promotes dendritic growth and the development of dendritic spines and excitatory synapses via the CaMKIV-CREB pathway, resulting in dendritic abnormalities and abnormally excitable circuits. Thirdly, increased expression of TRPC6 could promote the migration of SVZ NPCs and the underlying mechanism of TRPC6 mediate the migration of SVZ-NPCs might through guiding the direction of nerve growth corn of SVZ-NPCs.