The Involvement Of SVZ-derived Neural Precursor Cells In The Epileptogenicity Of Malformations Of Cortical Development

Malformations of cortical development (MCDs) are increasingly recognized as important causes of medically intractable epilepsy in pediatric patients. With the continuous development of clinical diagnostic techniques, particularly the emergence of high-resolution magnetic resonance imaging, the diagnosis rate of MCDs is increasing, the latest data showed that about 40% of refractory epilepsy is caused by the MCDs. Therefore, the pathogenic mechanism of MCDs become a major concern.Clinical classification of MCDs is more complex, according to the latest classification, focal cortical dysplasia (FCD) are the common types of MCDs. A large number of studies confirmed that FCD is abnormal neuronal migration diseases (NMDs). During the late stage of cortical development, a variety of exogenous stimuli cause the abnormal migration of newborn neurons resulted in FCD sequently. In addition, clinical studies have also indicated that the misshapen cells, such as balloon cells (BCs), within the cortical lesions of FCD are in some degree of positive correlation with the severity of seizure. Recent studies have demonstrated that BCs are derived in part from the neural precursor cells (NPCs) in subventricular zone (SVZ). Accordingly, we speculate that the abnormal migration of SVZ-derived NPCs is closely related to the epileptogenicity of MCDs. Therefore, to observe the the migration and differentiation of SVZ-derived cells in FCD lesions and to study the roles and neurochemical features of misshapen cells within the cortical dysplastic lesions of MCDs will help people better understand and clarify the mechanisms of epileptogenicity of MCDs.In this study, we have established a rat model of a microgyral malformation, and continuously observed the process of the migration and differentiation of SVZ-derived NPCs labelled with CM-DiI (named as"DiI-FCD"model). In addition, by using the patch-clamp recording techniques in the brain slice, we investigated the electrophysiological characteristics of the SVZ-NPCs- derived pyramidal neurons in the microgyral and paramicrogyral regions. Furthermore, the alterations of neurochemical features in the microgyral/ paramicrogyral of the DiI-FCD model and the cortical lesions of MCDs have also been studied in the present study. The main results are as follows:1. Experimental study of the the involvement of SVZ-derived neural precursor cells in the epileptogenicity of malformations of cortical development1.1 Combined use of neonatally cortical freez-lesion and CM-DiI intracerebroventricular injection methods, we successfully established a"DiI labeled SVZ NPCs- focal cortical dysplasia"animal model (DiI-FCD model). The results showed that DiI-positive (DiI+) cells, which form a migratory stream from SVZ to FCD cortical lesion (SFMS), could be continuously observed from P10 to P90.1.2 By using double immunofluorescence techniques, the molecular phenotype of the DiI(+) were further identified. The results showed that the DiI(+) cells in the SFMS of P10 rats expressed Nestin, DCX and OTX1/2, which confirmed that these cells are from SVZ-derived NPCs. Further studies revealed that most of the DiI(+) cells in SFMS differentiate into glial cells, which probably in the formation of local gliosis. However, part of the DiI (+) cells differentiate into neurons, and with the surrounding cells to form a synapse.1.3 Electrophysiological studies showed that: (1) The intrinsic membrane properties of DiI(+) neurons in the microgyral and paramicrogyral regions suggest that these cells are more immature, but higher excitability, than the control neurons from age-matched animals. (2) The depolarization and repolarization process of the DiI(+) neurons shows faster than those of normal neurons, and the absolute value of the AHP amplitude increased significantly in DiI(+) cells. (3) During the P10 phase, the amplitude of spontaneous EPSC (sEPSC) in the DiI(+) neurons are higher than normal neurons; however, during the other stages (P30-P90), there is no significant difference between each group. In addition, the sEPSC frequency in the early years to adulthood are significant higher than normal cortical neurons. (4) Compared with normal cortical neurons, the amplitude of evoked-EPSC (eEPSC) in DiI (+) neurons increased slightly, but did not achieve statistical significance. Moreover, the 10-90% rise time and decay time has increased compared with normal neurons. Interestingly, by analysising the paired-plus eEPSC, the data shows an impairment of feedback inhibition in DiI(+) neurons.2. The molecular phenotype of SVZ-derived neural precursor cells in the malformations of cortical development2.1 Using western blot method, we further studied the expression of NMDA/ AMPA receptor subunits in the microgyral and paramicrogyral regions compared with the control cortex from sham-operated group. The results show that the GluR2 and GluR3 proteins increased in DiI-FCD group in each stage of P10-P90, and NR1 and NR2A were upregulated from P30 to P90. Except the P60 stage, NR2B protein levels are upregulated in the DiI-FCD group. In addition, we also found that IL-6, IL-6R and gp130 proteins in DiI-FCD model was significantly upregulated, extracellular high concentration of IL-6 stimulation can cause DiI (+) neuron spike frequency increased significantly.2.2 According to the data about the upregulation of the IL-6 and its receptors in the microgyral and paramicrogyral regions of DiI-FCD model, we futher studied the expression of IL-6 system in the cortical lesions of TSC and FCDIIb. The results demonstrated that upregulation of mRNA and increased protein levels of IL-6 and its receptors, including the IL-6 receptor (IL-6R) and gp130, were observed in both TSC and FCDIIb lesions. Immunohistochemical analyses indicated that IL-6 and IL-6R were strongly expressed in misshapen cells such as dysmorphic neurons, TS cells and balloon cells (BCs), while gp130 was expressed in almost all cells following a diffuse distribution pattern. Co-localization assays further revealed that most of the IL-6/IL-6R-positive misshapen cells were co-labeled with neuronal markers rather than astrocytic markers. This finding suggests that these cells have a neuronal lineage, with the exception of the IL-6/IL-6R-positive BCs in FCDIIb, the most of which have an astrocytic lineage. Additionally, the protein levels of JAK2 and phosphorylated STAT3 were strongly increased, suggesting the involvement of the gp130/JAK2/STAT3 pathway in IL-6 signal transduction in both TSC and FCDIIb. Soluble IL-6R, but not soluble gp130, was significantly increased in both TSC and FCDIIb lesions relative to CTX, indicating the activation of this trans-signaling pathway in TSC and FCDIIb lesions. These results suggest that overexpression within the IL-6 system and activation of IL-6 signal transduction pathway in TSC and FCDIIb cortical lesions may contribute to intrinsic and increased epileptogenicity.Conclusion:1. Using DiI-FCD model, we successfully observe the migration process of SVZ-derived NPCs to the microgyral/ paramicrogyral regions. The data further confirm the hypothesis that the SVZ-derived NPCs are involved in the formation of the epileptogenic foci of FCD.2. In the microgyral/paramicrogyral regions, the majority of the SVZ-derived NPCs differeniate into glial cells and neurons, and to format synaptic connections with the surround cells,suggesting the possibility of these cells to influence the local neural circuit.3. Electrophysiological studies indicated that the SVZ-NPCs-derived neurons in the the microgyral/paramicrogyral regions showed high excitability and recevied more excitatory input, while the feedback suppression to the presynaptic cells reduced. These data suggested that the high excitability of the SVZ-NPCs-derived neurons would result in the hyperexcitability of the local neural circuit, and consequently trigger the kindling of the epileptiform discharge.4. The alterations of the expression of the NMDA/AMPA receptor subunits suggested the imbalance of the excitatory receptors in the microgyral/paramicrogyral regions, which further supported our electrophysiological finding above.5. The clinical study demonstrated a strong association between overproduction and overexpression in the IL-6 system and MCDs. Our morphological study further indicated the cell-specific expression patterns of IL-6 and its receptors, which were mainly distributed in the DNs, GNs, BCs and TS cells. This finding suggests that these misshapen cells are the potential source of IL-6 in MCDs cortical lesions. Additionally, we showed that the gp130/JAK2/STAT3 signal-transduction pathway might be involved in the response of these cortical lesions to locally high concentrations of IL-6. Our data do not provide insight into the mechanism(s) of IL-6 that underlie the epileptogenic properties of TSC tubers and FCDIIb cortical lesions. However, it is likely that either the strong expression or cell-specific distribution of IL-6 and its receptors, or the activation of IL-6 signal transduction in these cortical lesions, contributes to the intrinsic and high epileptogenicity of MCDs.In summary, a wide range of evidence show that the SVZ–NPCs-derived ectopic neurons in the epileptogenic lesions are one of the possible epileptogenic factors for the epileptogenicity of MCDs.