The Roles And Mechanisms Of Nogo-A In Malformations Of Cortical Development

Malformations of cortical development (MCD) is considered to be central nervoussystem disorders, which be common in children. In recent years, the diagnosis of MCDs wassignificantly increased due to the high-definition imaging technology continues to progress, Ithas been demonstrated that approximately40percent of adult patients and more than80percent of pediatric with intractable epilepsy are associated with MCD. Thus, MCDrepresents a well-recognized cause of intractable epilepsy and the pathogenesis of MCD hasbecame a central concern in the field of intractable epilepsy. Tuberous sclerosis complex(TSC) and focal cortical dysplasia type IIb (FCDIIb) are common MCDs in pharmacoresistantepilepsy patients. Although the clinical manifestations are not identical, several reports showthat TSC and FCDIIb share a number of similar histopathological and molecular alterations,including disorganized lamination and the presence of misshapen cells, such as dysmorphicneurons (DNs) and balloon cells (BCs) or giant cells (GCs; also referred to as TS cells),suggesting that a common pathogenic mechanism is involved in the pathogenesis of TSC andFCDIIb. The previous studies have shown misshapen cells may be generally recognized as theintrinsic pacemaker of epileptiform discharges in MCD. The clinical data also proved MCDpatients with misshapen cells have greater chance of seizures. Therefore, It is important toclarify the origin of misshapen cells to understanding the pathology development of MCD.Pathologically, misshapen cells has two characteristics,"misshapen" and "ectopy".Misshapen means abnormal cell morphology, axon development and point. Ectopic means theerror distribution of misshapen cells in cortex, resulting in cortex structural disorder anddeformity. The misshapen and ectopy were considered as the important reason of theexcessive excitatory neural network formation in MCD lesion. Until now, the pathogenesis ofmisshapen cells remains unclear. A more popular view is that FCD have been defined as a"neuronal migration disorder" and misshapen cells occur due to aberrant neuronal motilityduring brain development. Therefore, these molecular involved in regulating axon growth andNPCs migration become the focus. Nogo-A protein, an amino acid membrane protein, attractsour attention due to its characteristics of both regulating axon growth and cell migration. Nogo-A, the longest isoform of Nogo and a member of the reticulon family,is widelyinvolved in the development of central nervous system(CNS). Under physiological conditions,the high levels of Nogo-A expressed in embryonic and and located in oligodendrocytes anddeveloping neurons. It has2hydrophobic transmembrane domains separated by a66-amino-acid residue region that potently causes growth cone collapse and inhibition ofneurite outgrowth in vitro. This result reveals the important role played by the Nogo-A proteinin CNS diseases. More recently, Nogo-A has been implicated in regulating the migration ofolfactory ensheathing cells and NPCs in the embryonic mouse cortex via regulation of RhoAby NgR. In vivo, RhoA activation increased the amount of NPCs migrating to ectopic sites. Inaddition, the studies shown that Nogo-A protein may affect the distribution of selective ionchannel expression in the surface of neurons. However, no reports to date have investigatedthe relationship between Nogo-A signaling and NPC migration in SVZ. In view of the abovescientific research background, we propose the following scientific questions: what is thedetailed expression pattern of Nogo-A and it receptors in MCD? Whether does Nogo-Aexpressed in misshapen cell? What is the role of Nogo-A signal system in the pathogenesis ofMCD.To address these concerns: First,we identified the distribution of Nogo-A system inMCD from patients with medically intractable epilepsy by means of RT-PCR, western blotand immunohistochemistry. Second, we tested the expression and activation of signalingmolecule involved in Nogo-A pathway. Third, to reveal the role of Nogo-A in thepathogenesis of MCD, we have established a rat model of a microgyral malformation, andcontinuously observed the process of the migration and differentiation of SVZ-derived NPCslabelled with eGFP (named as"eGFP-FCFL"model).In addition, by using recombinantNogo-A protein and antagonists of Nogo-A, We investigate the potential role of Nogo-Ainvolved in NPCs migration,which is related with pathogenesis of misshapen in MCDs. Theresults show as following:Ⅰ. Expression and cellular distribution of Nogo-Aand NgR in MCD1．RT-PCR and western blot analysis showed Nogo-A and NgR mRNA and proteinexpression in total homogenates of normal control cortex (CTX); we observed that theexpression of Nogo-A and its receptor NgR were upregulated in TSC and FCDIIb corticallesions at the mRNA levels and Nogo-A protein levels were clearly upregulated in FCDIIb cortical lesions.2．The immunostaining results demonstrated that the high-level expression of protein,including Nogo-A and its receptors, was mainly localized within DNs, BCs and GCs. Theresults of double-labeling immunofluorescence showed: excluding a small part of BCs ofNogo-a IR, which show the glial cell lineage, the most of the misshapen cells of Nogo-A andNgR IR show the neurons lineage. These date suggested that the activation of Nogo-A signaltransduction pathways might be involved in the pathogenesis of cortical lesions in TSC andFCDIIb.3．We used the marginal cortex around the lesion as a control and found that theexpression of Nogo-A is not significantly elevated. Thus, epilepsy seizures in MCDs patientsmay not be a key factor for the upexpression of Nogo-A.Ⅱ. The expression and activation of signaling molecule involved in Nogo-ApathwayA novel Nogo-A-specific domain that binds to NgR has been defined. Nogo-A binds toand activates NgR, which is complexed with the transmembrane proteins LINGO-1and p75or TROY, and then acts as an inhibitory signal transducing subunit. The activation of theNgR/LINGO-1/P75or TROY complex of the neurotrophin receptor leads to the activation ofthe RhoA and RhoA kinase pathway. Moreover, antibodies blocking NgR have shown thesame motility-enhancing effect, supporting a direct role for surface Nogo-A in cell migration.Therefore, LINGO-1, P75, TROY and other downstream factors involved in the RhoA kinasepathway were also investigated.1．Western blot analyses revealed that the protein levels of LINGO-1and TROY weresignificantly increased in TSC tubers and FCDIIb cortical lesions compared with CTX tissues.The P75protein level in TSC tubers and FCDIIb cortical lesions remained unchanged, andRhoA expression was slightly higher in TSC tubers and FCDIIb cortical lesions2．The immunostaining results demonstrated that the high-level expression of protein,including LINGO-1,TORY and RhoA, was mainly localized within DNs,BCs and GCs.These data suggest that the NgR/LINGO-1/TROY signal transduction pathway may beinvolved in the response of these cortical lesions to the high concentrations of Nogo-A.Ⅲ. The similarity analysis of expression and cellular distribution between Nav1.1,Nav1.3and Nogo-A in MCD. The aim of this study was to investigate the protein expression and cellular distributionof Nav1.1and Nav1.3in FCDIIb cortical lesions.1．Western blot analysis demonstrated no significant changes were observed in theexpression of Nav1.1, whereas a notable increase in Nav1.3expression was found in FCDIIblesion tissue when compared with CTX tissue.2．Immunohistochemistry analyses results demonstrated Nav1.3immunoreactivity waswidely present in FCDIIb lesion tissue, specifically, high levels of Nav1.3immunoreactiveproteins were located mainly in cytomegalic neurons of different sizes and shapes. Theexpression pattern of Nav1.3is similar with Nogo-A. Our correlation studies have suggestedthat the expression of Nav1.3is inversely correlated with the expression of Nogo-A in FCDIIblesion tissue.Ⅳ. Nogo-Aregulate the migration of SVZ neural precursor cell (NPCs)In this study, we have established a rat model of a microgyral malformation, andcontinuously observed the space-time distribution of Nogo-A protein during different periodsof the rat brain development. By using recombinant Nogo-A protein and antagonists ofNogo-A, we investigate the potential role of Nogo-A involved in NPCs migration, which isrelated with pathogenesis of misshapen in MCDs.1．Combined use of neonatally cortical freez-lesion and eGFP intracerebroventricularinjection methods, we successfully established a "eGFP labeled SVZ NPCs-focal corticaldysplasia"animal model (eGFP-FCFL model). First, the model is accord with the behaviorcharacteristics of FCD model animal which be report by previous study; in addition, themorphology and neuropathology studies showed that the most common type of freezinginjury recreated a microgyric four-layer cortex comprising,not normal six-layer cortex,neurons disorganized and columnar polarity disappears. Moreover, occasionally, somepyknosis of neurons and abnormal morphology neurons were found. These characteristics areconsistent with previous literature reports. The establishment of the FCFL model is suitablefor our further experiments and provide a basis for the follow-up study.2．eGFP-positive (eGFP+) cells, which form a migratory stream from SVZ to FCDcortical lesion (SFMS), could be continuously observed from P10to P75. By using doubleimmunofluorescence techniques, the molecular phenotype of the eGFP (+) were furtheridentified. The results showed that the eGFP (+) cells in the SFMS of P10rats expressed PSA-NACM, CRMP4, DCX, which confirmed that these cells are from SVZ-derived NPCs.3．We observed the expression of Nogo-A protein in different developmental stages ofthe cortical areas in model group and control group. These data showed the expression ofNogo-A protein was decreased in the cerebral cortex with the development of the rat brain.However, intriguingly, the expression of Nogo-A was significantly increased in model grouprelative to those of control group at P10,P20,P30. Further immunohistochemical study foundthat the Nogo-A protein mainly located in the cell membrane of neurons in normal cortex ofcontrol group. However, in the model group, the Nogo-A IR of cytoplasmic, membrane andaxons were observed and is strong than those in control group. These results provided amorphological basis for the role of Nogo-A involved the pathogenesis mechanisms of MCD.4．The intervention study results showed that the number of eGFP(+) cells migrating tothe microgyral malformation region decreased via increasing the expression of Nogo-A andSMFS showed a linear distribution.. On the contrary, the number of eGFP (+) cells migratingto the microgyral malformation region increased via using the antagonist of Nogo-A and themigration of NPCs became more diffuse. This found suggest the role of Nogo-A in regulatingthe migration of SVZ NPCs.In summary, in the present study, we found an obvious increase of Nogo-A and itreceptors mRNA and protein expression in MCD specimens compared with the controlsample. The high-level expression of protein, including Nogo-A and NgR, was mainlylocalized within misshapen cells (DNs, GNs, GCs and BCs). Subsequently, we observed theactivation of signaling molecule LINGO-1, TROY, and RhoA not P75in TSC and FCDIIblesions, which suggested the ternary complex of NgR/TROY/LINGO-1may be the primarysignaling pathway involved in the pathogenesis of MCD, not NgR/P75/LINGO-1. Moreover,our found showed upregulated expression of Nav1.3may contribute to the increasedexcitability of cortical neuron circuits in FCDIIb lesion tissue. In addition, abnormalexpression of Nogo-A may be involved in the pathogenesis of misshapen cells by regulatingthe migration of SVZ NPCs. Altogether, our studies has highlighted the role of Nogo-Asignaling pathway in pathogenesis of MCD, but the specific mechanism have yet to be morein-depth study.