The Role Of Matrix Metalloproteinase9in Malformations Of Cortical Development

Malformations of cortical development (MCDs), which are characterized by abnormalcytoarchitecture of the cerebral cortex, are important causes of drug-resistant epilepsy inchildren. Two common subtypes of MCDs, focal cortical dysplasia (FCD) and tuberoussclerosis complex (TSC), have many similar histological features. The pathogenesis of FCD isthought to be mainly caused by embryonic developmental insults that result in the formationof dysplastic lesions with abnormal neuronal proliferation, migration and differentiation.Among the three types of FCD, type IIb (FCDIIb) results in the most typical histologicalchanges and has a younger age of seizure onset, a shorter epilepsy duration, and an increasedseizure frequency compared with the other variants. TSC is an autosomal dominantmultisystem neurocutaneous syndrome that results from mutations of the TSC1or TSC2genes. Seizures occur in more than70to80%of the patients with TSC and are oftenrefractory to treatment. Despite the different etiologies of FCDIIb and TSC, they do share anumber of characteristic cellular and histological abnormalities as well as common clinicalfeatures, which suggest that they could have common pathomechanisms of epileptogenesis.Moreover, human genetic studies linked the TSC1gene to the development of FCDIIb.Recent evidence suggests that seizures originate within the dysplastic cortical lesions inFCDIIb and the cortical tubers in TSC. However, understanding epilepsy in FCDIIb and TSCremains a challenge.Until now, most of our interest has focused on the role of neurotransmitter receptors andion channels in the epileptogenesis of MCDs; little attention has been paid to the potentialrole of extracellular proteinases. Matrix metalloproteinases (MMPs) are a large family ofstructurally related zinc-dependent secreted or cellular membrane-bound proteinases. MMPsare essential for various normal biological processes such as embryonic development,angiogenesis and tissue remodeling. In the central nervous system (CNS), they are involved inseveral pathologies, including tumorigenesis, inflammation, ischemia, trauma and epilepsy.MMP9, one of the best characterized members in MMP family, is synthesized and secreted into the extracellular space as the inactive zymogen pro-MMP9, then converted to the activeform, act-MMP9. Recent studies have suggested that MMP9plays important roles in corticaldevelopment, synaptic plasticity, learning and memory. As epileptogenic plasticity involvesextensive nervous tissue remodeling, a series of experiments with animal models haveindicated that MMP9is intimately involved in epileptogenesis. However, little is known aboutthe role of MMP9in MCDs.To address the above concerns: First, we identified the expression and distribution ofMMP9in epileptic lesions of FCDIIb and TSC patients by means of RT-PCR, westernblotting and immunohistochemistry. Second, we tested the expression, activity anddistribution of MMP9in a FCD rat model. Third, to reveal the role of MMP9in thepathogenesis of MCDs, we further investigated the effect of MMP9on the migration ofSVZ-derived neural precursor cells (NPCs) to the microgyrus. The main results are asfollows:1．Expression and cellular distribution of MMP9in cortical lesions of patiens withMCDs1.1RT-PCR analysis showed the expression of MMP9mRNA in total homogenates ofFCDIIb and TSC lesions was dramatically higher than that in CTX samples.1.2Western blotting analysis indicated that the pro-MMP9protein levels weresignificantly higher in the epilepsy groups (including FCDIIb, TSC and TLE) than in the CTXsamples. However, only in FCDIIb and TSC, the act-MMP9protein levels were significantlyelevated compared with CTX.1.3As described in the previous studies, weak to moderate MMP9IR was observed inneurons and endothelial cells, and MMP9IR-positive glial cells were occasionally seen in theGM and WM of the CTX tissues1.4A strong MMP9IR was observed in the FCDIIb cortical lesions. The intensity scoresindicated a higher expression of MMP9in the FCDIIb samples compared with the CTXsamples. Moreover, the MMP9IR showed a specific cellular distribution pattern: i.e. in allFCDIIb tissues (n=9) there was a strong staining of MMP9in80%±6.5%of the BCs (n=352)and in89±3.3%of the DNs (n=573) and83±4.7%of the HNs (n=420), and also glial cells.Double labeling experiments confirmed that the MMP9IR was colocalized with vimentin (amarker of less mature glial cells commonly expressed by BCs)and NF200(a neuronalmarker). Further immunofluorescent studies demonstrated that most of the MMP9+glial cells were theGFAP+astrocytes and not the microglia.1.5A strong MMP9IR was present in the TSC tubers (n=14), which was similar to thatin the FCDIIb lesions. The intensity scores of MMP9IR in the TSC tubers were dramaticallyhigher than those in the CTX samples. For the cellular MMP9IR, there was a moderate tostrong IR in77%±5.3%of the GCs (n=491) and in85%±3.6%of the DNs (n=627). Inagreement with the abnormal differentiation of the GCs, our double labeling experimentsindicated that some of MMP9+GCs were vimentin+, and that some are NF200+. Furtherimmunofluorescent studies revealed that, as in the FCDIIb lesions, most MMP9+glial cells inthe TSC tubers were the GFAP+reactive astrocytes and not microglia.2．Expression, activity and cellular distribution of MMP9in FCD rat model2.1Western blotting analysis indicated that the MMP9protein levels were significantlyhigher in the FCD groups than in the control rats at postnatal10(P10) and postnatal20(P20).2.2Zymography assay indicated that the activity of MMP9was dramatically higher inmicrogyrus of FCD rats than in the control cortices at P10and P20.2.3Immunohistochemical studies suggested a stronger MMP9IR was observed inmicrogyrus of FCD rats than in the control cortices at P10. Further immunofluorescent studiesdemonstrated NeuN+neurons are the major sources of MMP9. MMP9IR-positive GFAP+glial cells were occasionally seen in the microgyrus.2.4Similar to the observation of P10, a stronger MMP9IR was observed in microgyrusof FCD rats than in the control cortices. Double labeling experiments indicated that MMP9colabelled with NeuN, and not GFAP.3．Effect of MMP9on the migration of SVZ-derived NPCs to the microgyrus3.1We successfully transfected the Ad-eGFP into the cells in the SVZ, and observed theSFMS as our previous research.3.2Immunofluorescent studies demonstrated the eGFP+cells colabelled with nestin (aneural stem cell marker) and DCX (a cytoskeletal protein expressed by the migrating NPCsfrom SVZ), suggesting that the eGFP+cells in the SFMS were derived from SVZ.3.3Inhibition of MMP9by FN-439significantly attenuated the number of eGFP+cellsin the SFMS at P10and P20.In summary, in the present study, we found an obvious increase of MMP9mRNA and protein expression in MCDs specimens compared with the CTX samples. The IHC studiesdetected MMP9at characteristically high levels in misshapen cells (e.g. DNs, HNs, GCs andBCs), which may be the intrinsic "pacemakers" that initiate and drive the epileptiform activityin MCDs. Further, we established a rat model of microgyral malformation and confirmed thatthe protein levels and activity of MMP9were dramatically higher in microgyrus than in thecontrol cortices at P10and P20. Consistent with our previous researches, we observed themigration of eGFP+SVZ-derived NPCs to the microgyrus, and inhibition of MMP9significantly attenuated the number of eGFP+cells in the SFMS at P10and P20. Takentogether, our findings suggest that MMP9may be linked with the pathogenesis of MCDs.