Dysmorphic neurons express markers of inhibitory glycinergic signaling in focal cortical dysplasia IIb.

Astrocytic and microglial phenotypes in focal cortical dysplasia.

Focal cortical dysplasia (FCD) IIb and tuberous sclerosis complex (TSC) are common causes of drug-resistant epilepsy in children. However, the etiologies related to the development of FCD IIb and TSC are not fully understood. α-synuclein (α-syn) is a member of synucleins family that plays crucial roles in modulating synaptic transmission in central nervous system. Here, we explored the expression profiles and potential pathogenic functions of α-syn in cortical lesions of epileptic patients with FCD IIb and TSC. Surgical specimens from epileptic patients with FCD IIb and TSC, as well as FCD rats generated by in utero X-ray-radiation were adopted in this study and studied with immunohistochemistry, immunofluorescence, western blotting, and co-immunoprecipitation etc. molecular biological techniques. Our results showed that α-syn expression was reduced in FCD IIb and TSC lesions. Specifically, α-syn protein was intensely expressed in dysplastic neurons (DNs) and balloon cells (BCs) in FCD IIb lesions, whereas was barely detected in DNs and giant cells (GCs) of TSC lesions. Additionally, p-α-syn, the aggregated form of α-syn, was detected in DNs, BCs, GCs, and glia-like cells of FCD IIb and TSC lesions. We previous showed that the function of N-methyl-D-aspartate receptor (NMDAR) was enhanced in FCD rats generated by X-ray-radiation. Here, we found the interaction between α-syn and NMDAR subunits NMDAR2A, NMDAR2B were augmented in cortical lesions of FCD patients and FCD rats. These results suggested a potential role of α-syn in the pathogenesis of FCD IIb and TSC by interfering with NMDAR.

Abnormal maturation and differentiation of neocortical neurons in epileptogenic cortical malformation: Unique distribution of layer-specific marker cells of focal cortical dysplasia and hemimegalencephaly

Transmantle dysplasia is a rare type of focal cortical dysplasia (FCD) characterized by expansion of the cortex from the deep white matter to the surface and in which there is a FCD IIA or IIB pathologic pattern. To characterize possible mechanisms underlying this regional disorder of radial migrating cells, we studied the expression patterns of neocortical layer-specific markers using immunohistochemistry in surgical specimens from 5 FCD IIA and 4 FCD IIB cases in children. All neuronal cells expressed the mature neuron marker MAP2/2B but not the microglia markers Iba-1 and CD68. Some layer-specific markers showed distinct expression patterns. TBR1-positive, SATB2-positive, and FOXP1-positive cells were diffusely distributed in the cortex and/or the white matter. TBR1-positive and FOXP1-positive cells were generally more numerous in FCD IIB than in FCD IIA and were mostly in the cortical molecular and upper layers. FOXP1-, FOXP2-, and CUTL1-positive cells also expressed the immature neuron marker, Nestin/PROX1, whereas TBR1-, CTIP2-, and SATB2-positive cells only expressed MAP2/2B. These data highlight differences between FCD IIB and FCD IIA with more cells having the immature marker in upper layer markers in the former. By analyzing layer-specific marker expression patterns, we identified apparent neuronal maturation differences between FCD IIA and FCD IIB in cases of transmantle dysplasia.

Focal cortical dysplasia (FCD) is a brain malformation that causes medically refractory epilepsy. FCD is classified into three categories based on structural and cellular abnormalities, with FCD type II being the most common and characterized by disrupted organization of the cortex and abnormal neuronal development. In this study, we employed cell-type deconvolution and single-cell signatures to analyze bulk RNA-seq from multiple transcriptomic studies, aiming to characterize the cellular composition of brain lesions in patients with FCD IIa and IIb subtypes. Our deconvolution analyses revealed specific cellular changes in FCD IIb, including neuronal loss and an increase in reactive astrocytes (astrogliosis) when compared to FCD IIa. Astrogliosis in FCD IIb was further supported by a gene signature analysis and histologically confirmed by glial fibrillary acidic protein (GFAP) immunostaining. Overall, our findings demonstrate that FCD II subtypes exhibit differential neuronal and glial compositions, with astrogliosis emerging as a hallmark of FCD IIb. These observations, validated in independent patient cohorts and confirmed using immunohistochemistry, offer novel insights into the involvement of glial cells in FCD type II pathophysiology and may contribute to the development of targeted therapies for this condition.

Tuberous sclerosis complex (TSC) and focal cortical dysplasia (FCD) type IIb are the predominant causes of drug-refractory epilepsy in children. Dysmorphic neurons (DNs), giant cells (GCs), and balloon cells (BCs) are the most typical pathogenic profiles in cortical lesions of TSC and FCD IIb patients. However, mechanisms underlying the pathological processes of TSC and FCD IIb remain obscure. The Plexin-B2-Sema4C signalling pathway plays critical roles in neuronal morphogenesis and corticogenesis during the development of the central nervous system. However, the role of the Plexin-B2 system in the pathogenic process of TSC and FCD IIb has not been identified. In the present study, we investigated the expression and cell distribution characteristics of Plexin-B2 and Sema4C in TSC and FCD IIb lesions with molecular technologies. Our results showed that the mRNA and protein levels of Plexin-B2 expression were significantly increased both in TSC and FCD IIb lesions versus that in the control cortex. Notably, Plexin-B2 was also predominantly observed in GCs in TSC epileptic lesions and BCs in FCD IIb lesions. In contrast, the expression of Sema4C, the ligand of Plexin-B2, was significantly decreased in DNs, GCs, and BCs in TSC and FCD IIb epileptic lesions. Additionally, Plexin-B2 and Sema4C were expressed in astrocytes and microglia cells in TSC and FCD IIb lesions. Furthermore, the expression of Plexin-B2 was positively correlated with seizure frequency in TSC and FCD IIb patients. In conclusion, our results showed the Plexin-B2-Sema4C system was abnormally expressed in cortical lesions of TSC and FCD IIb patients, signifying that the Plexin-B2-Sema4C system may play a role in the pathogenic development of TSC and FCD IIb.

Expression and cellular distribution of multidrug resistance-related proteins in patients with focal cortical dysplasia

Focal cortical dysplasia (FCD) type IIb (FCD IIb) is an epileptogenic malformation of the neocortex that is characterized by cortical dyslamination, dysmorphic neurons (DNs) and balloon cells (BCs). Approximately 30–60% of lesions are associated with brain somatic mutations in the mTOR pathway. Herein, we investigated the transcriptional changes around the DNs and BCs regions in freshly frozen brain samples from three patients with FCD IIb by using spatial transcriptomics. We demonstrated that the DNs region in a gene enrichment network enriched for the mTOR signalling pathway, autophagy and the ubiquitin‒proteasome system, additionally which are involved in regulating membrane potential, may contribute to epileptic discharge. Moreover, differential expression analysis further demonstrated stronger expression of components of the inflammatory response and complement activation in the BCs region. And the DNs and BCs regions exhibited common functional modules, including regulation of cell morphogenesis and developmental growth. Furthermore, the expression of representative proteins in the functional enrichment module mentioned above was increased in the lesions of FCD IIb, such as p62 in DNs and BCs, UCHL1 in DNs, and C3 and CLU in BCs, which was confirmed via immunohistochemistry. Collectively, we constructed a spatial map showing the potential effects and functions of the DNs and BCs regions at the transcriptomic level and generated publicly available data on human FCD IIb to facilitate future research on human epileptogenesis.

Emergence of dysmorphic neurons is the primary pathology in focal cortical dysplasia (FCD) associated pediatric intractable epilepsy; however, the etiologies related to the development and function of dysmorphic neurons are not fully understood. Our previous studies revealed that the expression of vascular endothelial growth factor‐C (VEGF‐C) and corresponding receptors VEGFR‐2, VEGFR‐3 was increased in the epileptic lesions of patients with tuberous sclerosis complex or mesial temporal lobe epilepsy. Here, we showed that the expression of VEGF‐C, VEGFR‐2, and VEGFR‐3 was increased at both mRNA and protein levels in patients with cortical lesions of type I, IIa, and IIb FCD. The immunoreactivity of VEGF‐C, VEGFR‐2 and VEGFR‐3 was located in the micro‐columnar neurons in FCD type I lesions, dysplastic neurons (DNs) in FCD type IIa lesions, balloon cells (BCs) and astrocytes in FCD type IIb lesions. Additionally, the amplitude of evoked‐EPSCs (eEPSC) mediated by NMDA receptor, the ratio of NMDA receptor‐ and AMPA receptor‐mediated eEPSC were increased in the dysmorphic neurons of FCD rats established by prenatal X‐ray radiation. Furthermore, NMDA receptor mediated current in dysmorphic neurons was further potentiated by exogenous administration of VEGF‐C, however, could be antagonized by ki8751, the blocker of VEGFR‐2. These results suggest that VEGF‐C system participate in the pathogenesis of cortical lesions in patients with FCD in association with modulating NMDA receptor–mediated currents.

Objective To analyze the MR imaging features of epileptogenic focal cortical dysplasia (FCD)and to optimize the scanning protocols by correlating MRI appearance with pathological findings.Methods MRI findings and the relative scanning protocols in 36 patients with surgically and pathologically proved 40 lesions of FCD were retrospectively analyzed. According to Palmini classification system,all 40 lesions were pathologically categorized as FCD type Ⅰ (including FCD Ⅰ A and FCD Ⅰ B) and FCD type Ⅱ (including FCD Ⅱ A and FCD ⅡB ). The distribution of cerebral or dual lesions accompanied hippocampal sclerosis were observed.Differences of the distribution of cerebral in FCD type Ⅰ and FCD type Ⅱ were compared by using Fisher exact probabilities. MR scans in all patients consisted of routine and optimized protocols.Axial FSE T2WI,axial SET1WI and axial FLAIR were recognized as routine scanning protocols,while adding oblique coronal FSE T2WI and FLAIR were recognized as optimization scanning protocols.Both routine and optimization scanning protocols were performed in all patients.The conspicuity of main findings of FCD on different imaging plane and sequences of each protocol were assessed.The detection of cerebral or dual lesion and the accompanied hippocampal sclerosis were compared between the routine protocol and the optimized protocol by using McNemar test.Results Forty lesions were found in 36 cases with FCD,29 had temporal lobe lesion (72.5％ ),9 had frontal lobe lesion(22.5％ ) and 2 had parietal lobe lesion (5.0％).According to Palmini classification system,29 lesions in 27 patients (72.5％ ) were FCD type Ⅰ,11 lesions in 10 patients (27.5％) were FCD type Ⅱ lesions.There were 25 temporal lobe lesions in FCD type Ⅰ,while 4 in FCD type Ⅱ.There were statistically significant differences between FCD type Ⅰ group and FCD type Ⅱ group in the distribution of cerebral (P =0.002 ).Fourteen cases were found to have hippocampal sclerosis simultaneously,with 13 cases found in FCD type Ⅰ patients and 1 case in type Ⅱ patients.The detection rate of temporal lobe lesion was 65.5 ％ (19/29) and 44.8 ％ ( 13/29 ) respectively on optimized protocol and routine protocol.There was statistically significant difference ( x2 =4.167,P =0.031 ).The detection rate of hippocampal sclerosis was 85.7％ (12/14) and 42.9％ (6/14) respectively on optimized protocol and routine protocol respectively.There was statistically significant difference ( x2 =4.167,P =0.031 ).The detection rate of frontal lobe lesion showed no statistically significant difference between optimized protocols and routine protocols (x2 =0.304,P =1.000 ).Conclusions FCD was frequently involved the temporal lobe,followed by the frontal lobe.FCD type Ⅰ lesion was frequently found in the temporal lobe,with a higher incidence of concomitant hippocampal sclerosis. The optimized whole temporal lobe scanning with imaging plane perpendicular to the hippocampus long axis was a highly desired scanning protocol specifically for FCD,which is helpful for the detection of the FCD lesions. Key words: Cerebral cortex; Nervous system abnormalities; Magnetic resonance imaging

Background: Pharmacoresistant MRI-negative focal epilepsies present significant challenges for neurologists. While surgical intervention can potentially render patients’ seizure-free, outcomes often vary significantly. The underlying pathology, aside from the localization of the epileptogenic zone, is a crucial factor influencing these outcomes. Certain etiologies are known to correlate with lower rates of post-surgery seizure freedom. Objective: This study evaluated the anatomopathological findings in a cohort of MRI-negative patients who underwent epilepsy surgery at the Porto Alegre Epilepsy Surgery Program (PAESP) from 2012 to 2019. Methods: The study included all MRI-negative patients who underwent surgery and had anatomopathological exams reviewed. Reports were available for 57 patients. Results showed Focal Cortical Dysplasia (FCD) I was the most common etiology, found in 18 (31.6%) patients. Mild Malformation of Cortical Development type II (MOGHE) was seen in 14 (21.1%), normal pathology in 10 (17.5%), FCD IIA in 9 (15.8%), mMCD I in 4 (6.5%), FCD IIB in 2 (3.2%), and FCD IIIA in 2 (3.5%). Rates of seizure-freedom were: 38.9% for FCD I, 22% for FCD IIA, 100% for FCD IIB, 33.3% for MOGHE, 20% for normal pathology, 25% for mMCD I, and 50% for FCD IIIA, with significant differences between etiologies (p=0.464). Median follow-up was 44 months (12-146). Results: In this cohort of MRI-negative patients, the pathology was consistent with previous studies, with FCD type I being the most common pathological substrate. Our 12-month seizure-free rate was 47.4%, with late recurrence in 30% of initially seizure-free patients. This could be due to more widespread underlying diseases, such as FCD type I, which is prevalent in nonlesional epilepsies. In a large cohort of MRI-negative patients, Wang et al. found FCD I in 40% of cases, corroborating our findings. Additionally, mild malformations of cortical development (mMCD) were a common finding in 27.6% of nonlesional cases. mMCD, as described in Palminis classification, includes heterotopic neurons in subcortical white matter (mMCD 1) or oligodendrocyte hyperplasia with blurring of the gray matter-white matter junction (mMCD II or MOGHE). Our study corroborates that mMCD II may be more common in nonlesional epilepsies than previously shown. Conclusion: In this southern Brazil cohort, FCD type I and MOGHE were the most common pathological substrates. These conditions may present a less distinct transition to normal cortex compared to other etiologies (e.g., FCD IIB), potentially leading to incomplete surgical resection of the epileptogenic zone and explaining late surgical recurrences in these patients.

Localizing and Lateralizing Value of Seizure Onset Pattern on Surface EEG in FCD Type II

1 Veronique M. Andre, 2 My Huynh, 1 Michael S. Levine, 2 Gary W. Mathern, and 1 Carlos Cepeda ( 1 Mental Retardation Research Center, David Geffen School of Medicine at UCLA ; and 2 Department of Neurosurgery, David Geffen School of Medicine, Los Angeles, CA ) Rationale: In pediatric cortical dysplasia (CD), we previously found more GABA relative to glutamate spontaneous synaptic activity (Cepeda et al., 2005; Dev. Neurosci., In Press). In this study, we determined the proportion of GABAergic neurons and their morphology in non-CD and CD patients. In addition, we examined functional properties of GABAA receptor signaling in normal-appearing and cytomegalic neurons. Methods: Double immunofluorescence stainings and cell counts for NeuN and for GAD were performed on non-CD and CD patients. Additional brain sections were processed for parvalbumin, calretinin and GAT-1. We separated the CD tissue into CD normal (containing only normal-appearing neurons) and CD cytomegalic (containing some cytomegalic neurons). Electrophysiological recordings were performed on acutely dissociated pyramidal neurons from non-CD and CD patients. Patch electrodes were filled with N-methyl-D-glucamine for whole-cell voltage clamp recordings. Different concentrations of GABA were applied to the cells. Results: The percent of GAD-labeled cells was similar in non-CD and CD normal tissue. In contrast, there were fewer GAD-labeled cells in the CD tissue containing cytomegalic neurons. However, a dense plexus of GABAergic fibers was observed in CD tissue, mostly around cytomegalic neurons, but not in non-CD cases. We also noticed the presence of abnormally large neurons positive for GAD that could be cytomegalic interneurons. GABA-induced currents were recorded in pyramidal neurons from non-CD and CD cases and in cytomegalic neurons. GABA peak current amplitudes were similar in normal-appearing pyramidal neurons from the non-CD and CD groups while they were larger in cytomegalic neurons. However, when the currents were normalized by cell capacitance, GABA peak current densities were smaller in CD cytomegalic neurons. Normal-appearing pyramidal neurons from non-CD and CD tissue showed the same desensitization rate while it was significantly slower in the cytomegalic neurons. Conclusions: We found that the number of GABAergic cells was similar in non-CD and CD tissue containing only normal-appearing neurons, whereas it was reduced in regions containing cytomegalic neurons. However, we also observed dense networks of GABAergic fibers around cytomegalic pyramidal-shaped neurons and the presence of putative cytomegalic interneurons. These findings suggest that some GABAergic inputs originate outside the most affected areas and/or that important synaptic reorganization occurs within these regions, including dense innervation from cytomegalic GABAergic interneurons. These findings could account for the higher frequency of GABA relative to glutamate spontaneous synaptic activity shown by previous electrophysiological studies in slices. Finally, data from dissociated cells indicate that GABAA receptor subunit composition is also altered in cytomegalic neurons. (Supported by NIH NS38992.)

Oxidative stress (OS) occurs in brains of patients with epilepsy and coincides with brain inflammation, and both phenomena contribute to seizure generation in animal models. We investigated whether expression of OS and brain inflammation markers co-occurred also in resected brain tissue of patients with epileptogenic cortical malformations: hemimegalencephaly (HME), focal cortical dysplasia (FCD) and cortical tubers in tuberous sclerosis complex (TSC). Moreover, we studied molecular mechanisms linking OS and inflammation in an in vitro model of neuronal function. Untangling interdependency and underlying molecular mechanisms might pose new therapeutic strategies for treating patients with drug-resistant epilepsy of different etiologies. Immunohistochemistry was performed for specific OS markers xCT and iNOS and brain inflammation markers TLR4, COX-2 and NF-κB in cortical tissue derived from patients with HME, FCD IIa, IIb and TSC. Additionally, we studied gene expression of these markers using the human neuronal cell line SH-SY5Y in which OS was induced using H2 O2 . OS markers were higher in dysmorphic neurons and balloon/giant cells in cortex of patients with FCD IIb or TSC. Expression of OS markers was positively correlated to expression of brain inflammation markers. In vitro, 100µM, but not 50µM, of H2 O2 increased expression of TLR4, IL-1β and COX-2. We found that NF-κB signaling was activated only upon stimulation with 100µM H2 O2 leading to upregulation of TLR4 signaling and IL-1β. The NF-κB inhibitor TPCA-1 completely reversed this effect. Our results show that OS positively correlates with neuroinflammation and is particularly evident in brain tissue of patients with FCD IIb and TSC. In vitro, NF-κB is involved in the switch to an inflammatory state after OS. We propose that the extent of OS can predict the neuroinflammatory state of the brain. Additionally, antioxidant treatments may prevent the switch to inflammation in neurons thus targeting multiple epileptogenic processes at once.

Objective To investigate the MRI characteristics of different pathologic types of patients with focal cortical dysplasia (FCD). Methods The postoperative clinical data of 23 patients with FCD were retrospectively analyzed and they were divided into patient groups of 4 types according to Palmini' s classification. The MRI characteristics of different pathologic types of patients with FCD were concluded. Results Among these 23 patients, 2 were diagnosed as having FCD ⅠA, 6 as having FCD IB, 8 as having FCD ⅡA and 7 as having FCD IE. As compared with that in patients with FCD Ⅰ, obviously increased signal of Flair images in patients with FCD Ⅱ was more prevalent (2:11,P=0.039). Increased signal in T2 images and tapering of abnormal white matter signals to ventricles were more prevalent in patients with FCD Ⅱ as compared with those in patients with FCD Ⅰ, but no significant differences were noted (P= 0.074, 0.058). As compared with patients with other 3 types of FCD, blurring of the gray matter-white matter junction, increased signal in T2 images, deep sulcus, thickening of the cortex and tapering of abnormal white matter signals to ventricles in patients with FCD ⅡB were more prevalent (P<0.05). Conclusion Different MRI characteristics in each type of FCD are noted, especially in patients with FCDⅡB. These different MRI characteristics are helpful to make preoperative diagnosis and planning of FCD. Key words: Epilepsy; Cortical dysplasia; MRI