Pharmacological evaluation of E2730, a novel selective uncompetitive GAT1 inhibitor, on epileptiform activities in resected brain tissues from human focal cortical dysplasia ex vivo.

Focal cortical dysplasia (FCD) is an important etiology of focal epilepsy in children and adults. However, only a few preclinical models sufficiently reproduce the characteristic histopathologic features of FCD. To improve the success rate of clinical trials for antiseizure medications (ASMs) in patients with FCD, more human-relevant preclinical models are needed, and epileptic foci resected from patients are a powerful tool for this purpose. Here, we conducted ex vivo studies using epileptic foci resected from patients with FCD type II to evaluate the pharmacologic effects of the ASM candidate E2730, a selective uncompetitive inhibitor of γ-aminobutyric acid transporter 1. We used the same ex vivo assay system to assess carbamazepine (CBZ), an ASM often prescribed for focal epilepsy, as a reference. At the higher dose tested (200 µM), both E2730 and CBZ suppressed spontaneous epileptiform activities almost completely. At the lower dose (100 µM), CBZ reduced the area of brain tissue showing epileptiform activity, whereas E2730 significantly decreased the number of epileptiforms. These findings suggest that E2730-both as a single agent and in combination with CBZ-merits evaluation in clinical trials involving patients with FCD.

USP10 Inhibits Aberrant Cytoplasmic Aggregation of TDP-43 by Promoting Stress Granule Clearance.

TAR DNA-binding protein 43 (TDP-43) is a causative factor of amyotrophic lateral sclerosis (ALS). Cytoplasmic TDP-43 aggregates in neurons are a hallmark pathology of ALS. Under various stress conditions, TDP-43 localizes sequentially to two cytoplasmic protein aggregates, namely, stress granules (SGs) first and then aggresomes. Accumulating evidence suggests that delayed clearance of TDP-43-positive SGs is associated with pathological TDP-43 aggregates in ALS. We found that ubiquitin-specific protease 10 (USP10) promotes the clearance of TDP-43-positive SGs in cells treated with proteasome inhibitor, thereby promoting the formation of TDP-43-positive aggresomes, and the depletion of USP10 increases the amount of insoluble TDP-35, a cleaved product of TDP-43, in the cytoplasm. TDP-35 interacted with USP10 in an RNA-binding-dependent manner; however, impaired RNA binding of TDP-35 reduced the localization in SGs and aggresomes and induced USP10-negative TDP-35 aggregates. Immunohistochemistry showed that most of the cytoplasmic TDP-43/TDP-35 aggregates in the neurons of ALS patients were USP10 negative. Our findings suggest that USP10 inhibits aberrant aggregation of TDP-43/TDP-35 in the cytoplasm of neuronal cells by promoting the clearance of TDP-43/TDP-35-positive SGs and facilitating the formation of TDP-43/TDP-35-positive aggresomes.

Deep learning-based diagnosis of temporal lobe epilepsy associated with hippocampal sclerosis: An MRI study.

PURPOSE: The currently available indicators-sensitivity and specificity of expert radiological evaluation of MRIs-to identify mesial temporal lobe epilepsy (MTLE) associated with hippocampal sclerosis (HS) are deficient, as they cannot be easily assessed. We developed and investigated the use of a novel convolutional neural network trained on preoperative MRIs to aid diagnosis of these conditions. SUBJECTS AND METHODS: We enrolled 141 individuals: 85 with clinically diagnosed mesial temporal lobe epilepsy (MTLE) and hippocampal sclerosis International League Against Epilepsy (HS ILAE) type 1 who had undergone anterior temporal lobe hippocampectomy were assigned to the MTLE-HS group, and 56 epilepsy clinic outpatients diagnosed as nonepileptic were assigned to the normal group. We fine-tuned a modified CNN (mCNN) to classify the fully connected layers of ImageNet-pretrained VGG16 network models into the MTLE-HS and control groups. MTLE-HS was diagnosed using MRI both by the fine-tuned mCNN and epilepsy specialists. Their performances were compared. RESULTS: The fine-tuned mCNN achieved excellent diagnostic performance, including 91.1% [85%, 96%] mean sensitivity and 83.5% [75%, 91%] mean specificity. The area under the resulting receiver operating characteristic curve was 0.94 [0.90, 0.98] (DeLong's method). Expert interpretation of the same image data achieved a mean sensitivity of 73.1% [65%, 82%] and specificity of 66.3% [50%, 82%]. These confidence intervals were located entirely under the receiver operating characteristic curve of the fine-tuned mCNN. CONCLUSIONS: Deep learning-based diagnosis of MTLE-HS from preoperative MR images using our fine-tuned mCNN achieved a performance superior to the visual interpretation by epilepsy specialists. Our model could serve as a useful preoperative diagnostic tool for ascertaining hippocampal atrophy in patients with MTLE.

Reactive astrocytes contribute to epileptogenesis in patients with cavernous angioma.

OBJECTIVE: Patients with cavernous angioma (CA) often suffer from severe epilepsy, and surgical resection is often performed to attenuate these epileptic seizures. Several studies have suggested that surgical removal of the surrounding hemosiderin-pigmented tissues adjacent to CA achieves better seizure control than restricted lesionectomy. Pathological examination of the resected foci reveals not only hemosiderin pigmentation but also various degrees of inflammatory change, such as hemosiderin-laden macrophages, gliosis and fibrosis. However, there is some controversy regarding the epileptogenic potential of these regions due to the uncertain nature of the mechanisms contributing to these histopathological changes. METHODS: To investigate the correlations between neuron hyperexcitability and evident pathological changes, we performed ex vivo flavoprotein fluorescence imaging using surgically resected epileptogenic foci surrounding CA. The mirror surfaces of the tissues used for the physiological experiment were also subjected to morphological examination. RESULTS: Hemosiderin-laden macrophages and many gemistocytic astrocytes were observed in the area adjacent to CA, where horizontal spreading excitations were detected significantly more frequently. Outside these areas, we found fine granular iron deposits and only a few fibrillary astrocytes, and weakly propagating excitations were detected. Furthermore, areas of enhanced activation were more clearly correlated with the glial proliferation index than with iron deposition. CONCLUSION: These results suggest that the epileptogenesis in patients with CA may be based on a biological process, such as alteration of glial function, rather than direct chemical reactions involving iron deposition.

Longitudinal GluCEST MRI Changes and Cerebral Blood Flow in 5xFAD Mice.

Many of the focal neurological symptoms associated with Alzheimer's disease (AD) are due to synaptic loss. Glutamate chemical exchange saturation transfer (GluCEST) magnetic resonance imaging (MRI) is a candidate method to assess synaptic dysfunction. We assessed chronological changes in GluCEST in a 5xFAD mouse model of AD, comparing Glucest effects and regional cerebral blood flow (CBF). GluCEST effects and CBF in 5xFAD mice aged 1-15 months and their littermates (WT) were measured. Neurite orientation dispersion and density imaging (NODDI) MRI reflecting dendritic/axonal density was also measured and compared with GluCEST in 7-month-old mice. While regional CBF's decrease began at 7 months, GluCEST-reduction effects preceded hypoperfusion of the temporal cortex and hippocampus. While longitudinal 5xFAD mouse measurements revealed a correlation between the regional GluCEST effects and CBF, a generalized linear mixed model revealed statistically different correlations in cortical and basal brain regions. Further, NODDI-derived neurite density correlated with GluCEST effects in the parietal cortex, but not in the hippocampus, thereby revealing regional differences in pathophysiological mechanisms. Finally, GluCEST's effects correlated with regional synaptophysin. These results demonstrate that GluCEST can reflect subtle synaptic changes and may be a potential imaging method for AD diagnosis as well as serve as a biomarker of AD progression.

Proteomic profile differentiating between mesial temporal lobe epilepsy with and without hippocampal sclerosis.

Hippocampal sclerosis (HS) is the most common neuropathological condition in adults with drug-resistant epilepsy and represents a critical feature in mesial temporal lobe epilepsy (MTLE) syndrome. Although epileptogenic brain tissue is associated with glutamate excitotoxicity leading to oxidative stress, the proteins that are targets of oxidative damage remain to be determined. In the present study we designed comprehensive analyses of changes in protein expression level and protein oxidation status in the hippocampus or neocortex to highlight proteins associated with excitotoxicity by comparing MTLE patients with relatively mild excitotoxicity (MTLE patients without HS, MTLE-non-HS) and those with severe excitotoxicity (MTLE patients with HS, MTLE-HS). We performed 2-dimensional fluorescence difference gel electrophoresis, 2D-oxyblot analysis, and mass spectrometric amino acid sequencing. We identified 16 proteins at 18 spots in which the protein expression levels differed between sclerotic and non-sclerotic hippocampi. In the sclerotic hippocampus, the expression levels of several synaptic proteins were decreased, and those of some glia-associated proteins increased. We confirmed histologically that all MTLE-HS cases examined exhibited severe neuronal cell loss and remarkable astrocytic gliosis in the hippocampi. In all MTLE-non-HS cases examined, neurons were spared and gliosis was unremarkable. Therefore, we consider that decreased synaptic proteins are a manifestation of loss of neuronal cell bodies and dendrites, whereas increased glia-associated proteins are a manifestation of proliferation and hypertrophy of astrocytes. These are considered to be the result of hippocampal sclerosis. In contrast, the expression level of d-3-phosphoglycerate dehydrogenase (PHGDH), an l-serine synthetic enzyme expressed exclusively by astrocytes, was decreased, and that of stathmin 1, a neurite extension-related protein expressed by neurons, was increased in the sclerotic hippocampus. These findings cannot be explained solely as the result of hippocampal sclerosis. Rather, these changes can be involved in the continuation of seizure disorders in MTLE-HS. In addition, the protein carbonylation detection, an indicator of protein oxidation caused by excitotoxicity of multiple seizures and/or status epilepticus, revealed that the carbonyl level of collapsin response mediator protein 2 (CRMP2) increased significantly in the sclerotic hippocampus. In conclusion, protein identification following profiling of protein expression levels and detection of oxidative proteins indicated potential pathognomonic protein changes. The decreased expression of PHGDH, increased expression of stathmin 1, and carbonylation of CRMP2 differentiate between MTLE with and without HS. Therefore, further investigations of PHGDH, stathmin 1 and CRMP2 are promising to study more detailed effects of excitotoxicity on epileptogenic hippocampal tissue.

Versatile whole-organ/body staining and imaging based on electrolyte-gel properties of biological tissues.

Whole-organ/body three-dimensional (3D) staining and imaging have been enduring challenges in histology. By dissecting the complex physicochemical environment of the staining system, we developed a highly optimized 3D staining imaging pipeline based on CUBIC. Based on our precise characterization of biological tissues as an electrolyte gel, we experimentally evaluated broad 3D staining conditions by using an artificial tissue-mimicking material. The combination of optimized conditions allows a bottom-up design of a superior 3D staining protocol that can uniformly label whole adult mouse brains, an adult marmoset brain hemisphere, an ~1 cm3 tissue block of a postmortem adult human cerebellum, and an entire infant marmoset body with dozens of antibodies and cell-impermeant nuclear stains. The whole-organ 3D images collected by light-sheet microscopy are used for computational analyses and whole-organ comparison analysis between species. This pipeline, named CUBIC-HistoVIsion, thus offers advanced opportunities for organ- and organism-scale histological analysis of multicellular systems.

Skull diploë is rich in aquaporin-4.

Aquaporin-4 (AQP4) is a water conducting membrane integral protein channel which is widely expressed in the astrocyte system of the brain. During the development of the AQP4 positron emission tomography (PET) imaging agent [11C]TGN-020 (N-(1,3,4-thiadiazol-2-yl)pyridine-3-[11C]-carboxamide), significant radioligand uptake was observed in the skull, where there was no known distribution of any aquaporin family proteins. Herein we confirmed via a newly developed method for bone-tissue immunohistology, a hitherto unrecognized distribution of AQP4, and not AQP1, in the skull. Other bony structures, by contrast, showed virtually no uptake of [11C]TGN-020, and likewise, do not express either AQP4 or AQP1. Immunohistological analysis demonstrated that the AQP4 expression in the skull is restricted to the diploë. Consequently, we suspect AQP4 plays a pivotal role in the formation and maintenance of yellow marrow and the diploë. However, elucidating the exact nature of that role will require further studies.

Glial pathology in a novel spontaneous mutant mouse of the Eif2b5 gene: a vanishing white matter disease model.

Vanishing white matter disease (VWM) is an autosomal recessive neurological disorder caused by mutation(s) in any subunit of eukaryotic translation initiation factor 2B (eIF2B), an activator of translation initiation factor eIF2. VWM occurs with mutation of the genes encoding eIF2B subunits (EIF2B1, EIF2B2, EIF2B3, EIF2B4, and EIF2B5). However, little is known regarding the underlying pathogenetic mechanisms or how to treat patients with VWM. Here we describe the identification and detailed analysis of a new spontaneous mutant mouse harboring a point mutation in the Eif2b5 gene (p.Ile98Met). Homozygous Eif2b5I98M mutant mice exhibited a small body, abnormal gait, male and female infertility, epileptic seizures, and a shortened lifespan. Biochemical analyses indicated that the mutant eIF2B protein with the Eif2b5I98M mutation decreased guanine nucleotide exchange activity on eIF2, and the level of the endoplasmic reticulum stress marker activating transcription factor 4 was elevated in the 1-month-old Eif2b5I98M brain. Histological analyses indicated up-regulated glial fibrillary acidic protein immunoreactivity in the astrocytes of the Eif2b5I98M forebrain and translocation of Bergmann glia in the Eif2b5I98M cerebellum, as well as increased mRNA expression of an endoplasmic reticulum stress marker, C/EBP homologous protein. Disruption of myelin and clustering of oligodendrocyte progenitor cells were also indicated in the white matter of the Eif2b5I98M spinal cord at 8 months old. Our data show that Eif2b5I98M mutants are a good model for understanding VWM pathogenesis and therapy development. Cover Image for this issue: doi: 10.1111/jnc.14751.

Reciprocal connectivity between secondary auditory cortical field and amygdala in mice.

Recent studies have examined the feedback pathway from the amygdala to the auditory cortex in conjunction with the feedforward pathway from the auditory cortex to the amygdala. However, these connections have not been fully characterized. Here, to visualize the comprehensive connectivity between the auditory cortex and amygdala, we injected cholera toxin subunit b (CTB), a bidirectional tracer, into multiple subfields in the mouse auditory cortex after identifying the location of these subfields using flavoprotein fluorescence imaging. After injecting CTB into the secondary auditory field (A2), we found densely innervated CTB-positive axon terminals that were mainly located in the lateral amygdala (La), and slight innervations in other divisions such as the basal amygdala. Moreover, we found a large number of retrogradely-stained CTB-positive neurons in La after injecting CTB into A2. When injecting CTB into the primary auditory cortex (A1), a small number of CTB-positive neurons and axons were visualized in the amygdala. Finally, we found a near complete absence of connections between the other auditory cortical fields and the amygdala. These data suggest that reciprocal connections between A2 and La are main conduits for communication between the auditory cortex and amygdala in mice.

G3BP1 inhibits ubiquitinated protein aggregations induced by p62 and USP10.

The aberrant accumulation of ubiquitinated protein aggregates in cells plays a critical role in the pathogenesis of several degenerative diseases, including Parkinson disease (PD) and cystic fibrosis (CF). In this study, we found that Ras GTPase-activating protein-binding protein 1 (G3BP1) inhibits ubiquitinated protein aggregations induced by p62 and USP10 in cultured cells. p62 is a ubiquitin receptor, and p62 and its binding partner USP10 have been shown to augment ubiquitinated protein aggregation. G3BP1 interacted with p62 and USP10 and inhibited p62/USP10-induced protein aggregation. The G3BP1 inhibition of protein aggregations targeted two aggregation-prone proteins, α-synuclein and CFTR-ΔF508, which are causative factors of PD and CF, respectively. G3BP1 depletion increased the amounts of ubiquitinated α-synuclein and CFTR-ΔF508 protein. A proteasome reporter indicated that G3BP1 depletion inhibits the proteasome activity. We herein present evidence that G3BP1, p62 and USP10 together control ubiquitinated protein toxicity by controlling both ubiquitination and aggregation. Taken together, these results suggest that G3BP1, p62 and USP10 could be therapeutic targets for ubiquitinated protein aggregation disorders, including PD and CF.

USP10 is a critical factor for Tau-positive stress granule formation in neuronal cells.

Tau aggregates in neurons of brain lesions is a hallmark pathology of tauopathies, including Alzheimer's disease (AD). Recent studies suggest that the RNA-binding protein TIA1 initiates Tau aggregation by inducing the formation of stress granules (SGs) containing Tau. SGs are stress-inducible cytoplasmic protein aggregates containing many RNA-binding proteins that has been implicated as an initial site of multiple pathogenic protein aggregates in several neurodegenerative diseases. In this study, we found that ubiquitin-specific protease 10 (USP10) is a critical factor for the formation of Tau/TIA1/USP10-positive SGs. Proteasome inhibition or TIA1-overexpression in HT22 neuronal cells induced the formation of TIA1/Tau-positive SGs, and the formations were severely attenuated by depletion of USP10. In addition, the overexpression of USP10 without stress stimuli in HT22 cells induced TIA1/Tau/USP10-positive SGs in a deubiquitinase-independent manner. In AD brain lesions, USP10 was colocalized with Tau aggregates in the cell body of neurons. The present findings suggest that USP10 plays a key role in the initiation of pathogenic Tau aggregation in AD through SG formation.

USP10 Is a Driver of Ubiquitinated Protein Aggregation and Aggresome Formation to Inhibit Apoptosis.

Accumulation of ubiquitinated proteins is cytotoxic, but cells inactivate these cytotoxicities by inducing aggresome formation. We found that ubiquitin-specific protease 10 (USP10) inhibits ubiquitinated protein-induced apoptosis by inducing aggresome formation. USP10 interacted with the ubiquitin receptor p62 and the interaction augmented p62-dependent ubiquitinated protein aggregation and aggresome formation, thereby cooperatively inhibiting apoptosis. We provide evidence that USP10/p62-induced protein aggregates inhibit proteasome activity, which increases the amount of ubiquitinated proteins and promotes aggresome formation. USP10 induced aggresomes containing α-synuclein, a pathogenic protein in Parkinson disease, in cultured cells. In Parkinson disease brains, USP10 was colocalized with α-synuclein in the disease-linked aggresome-like inclusion Lewy bodies, suggesting that USP10 inhibits α-synuclein-induced neurotoxicity by promoting Lewy body formation. Collectively, these findings suggest that USP10 is a critical factor to control protein aggregation, aggresome formation, and cytotoxicity in protein-aggregation-related diseases.

Pathophysiological Characteristics Associated With Epileptogenesis in Human Hippocampal Sclerosis.

Mesial temporal lobe epilepsy (MTLE) is the most frequent focal epileptic syndrome in adults, and the majority of seizures originate primarily from the hippocampus. The resected hippocampal tissue often shows severe neuronal loss, a condition referred to as hippocampal sclerosis (HS). In order to understand hippocampal epileptogenesis in MTLE, it seems important to clarify any discrepancies between the clinical and pathological features of affected patients. Here we investigated epileptiform activities ex vivo using living hippocampal tissue taken from patients with MTLE. Flavoprotein fluorescence imaging and local field potential recordings revealed that epileptiform activities developed from the subiculum. Moreover, physiological and morphological experiments revealed possible impairment of K+ clearance in the subiculum affected by HS. Stimulation of mossy fibers induced recurrent trans-synaptic activity in the granule cell layer of the dentate gyrus, suggesting that mossy fiber sprouting in HS also contributes to the epileptogenic mechanism. These results indicate that pathophysiological alterations involving the subiculum and dentate gyrus could be responsible for epileptogenesis in patients with MTLE.

Biallelic Variants in CNPY3, Encoding an Endoplasmic Reticulum Chaperone, Cause Early-Onset Epileptic Encephalopathy.

Early-onset epileptic encephalopathies, including West syndrome (WS), are a group of neurological disorders characterized by developmental impairments and intractable seizures from early infancy. We have now identified biallelic CNPY3 variants in three individuals with WS; these include compound-heterozygous missense and frameshift variants in a family with two affected siblings (individuals 1 and 2) and a homozygous splicing variant in a consanguineous family (individual 3). All three individuals showed hippocampal malrotation. In individuals 1 and 2, electroencephalography (EEG) revealed characteristic fast waves and diffuse sharp- and slow-wave complexes. The fast waves were clinically associated with seizures. CNPY3 encodes a co-chaperone in the endoplasmic reticulum and regulates the subcellular distribution and responses of multiple Toll-like receptors. The amount of CNPY3 in lymphoblastoid cells derived from individuals 1 and 2 was severely lower than that in control cells. Cnpy3-knockout mice exhibited spastic or dystonic features under resting conditions and hyperactivity and anxiolytic behavior during the open field test. Also, their resting EEG showed enhanced activity in the fast beta frequency band (20-35 Hz), which could mimic the fast waves in individuals 1 and 2. These data suggest that CNPY3 and Cnpy3 perform essential roles in brain function in addition to known Toll-like receptor-dependent immune responses.

Aquaporin Positron Emission Tomography Differentiates Between Grade III and IV Human Astrocytoma.

BACKGROUND: Aquaporin (AQP) water channels play a significant role in mesenchymal microvascular proliferation and infiltrative growth. AQPs are highly expressed in malignant astrocytomas, and a positive correlation is observed between their expression levels and histological tumor grade. OBJECTIVE: To examine the utility of aquaporin positron emission tomography (PET) for differentiating between astrocytoma grade III and grade IV using the AQP radioligand [11C]TGN-020. METHODS: Fifteen astrocytoma patients, grade III (n = 7) and grade IV (n = 8), and 10 healthy volunteers underwent [11C]TGN-020 aquaporin PET imaging. Surgical tissues of astrocytoma patients were examined for histopathological grading using the WHO classification standard and expression of AQP1 and AQP4 immunohistochemically. RESULTS: Mean standardized uptake values of astrocytoma grade III and IV (0.51 ± 0.11 vs 1.50 ± 0.44, respectively) were higher than normal white matter (0.17 ± 0.02, P < .001) for both tumor grades. Importantly, mean standardized uptake values of astrocytoma grade IV were significantly higher than grade III (P < .01). CONCLUSION: Our study demonstrated that [11C]TGN-020 aquaporin PET imaging differentiated between astrocytoma grades III and IV. We suggest its clinical application as a noninvasive diagnostic tool would lead to advancements in the management of these malignant brain tumors.

Features of amygdala in patients with mesial temporal lobe epilepsy and hippocampal sclerosis: An MRI volumetric and histopathological study.

OBJECTIVE: It is well-known that there is a correlation between the neuropathological grade of hippocampal sclerosis (HS) and neuroradiological atrophy of the hippocampus in mesial temporal lobe epilepsy (mTLE) patients. However, there is no strict definition or criterion regarding neuron loss and atrophy of the amygdala neighboring the hippocampus. We examined the relationship between HS and neuronal loss in the amygdala. MATERIALS AND METHODS: Nineteen mTLE patients with neuropathological proof of HS were assigned to Group A, while seven mTLE patients without HS were assigned to Group B. We used FreeSurfer software to measure amygdala volume automatically based on pre-operation magnetic resonance images. Neurons observed using Klüver-Barrera (KB) staining in resected amygdala tissue were counted. and the extent of immunostaining with stress marker antibodies was semiquantitatively evaluated. RESULTS: There was no significant difference in amygdala volume between the two groups (Group A: 1.41±0.24; Group B: 1.41±0.29cm3; p=0.98), nor in the neuron cellularity of resected amygdala specimens (Group A: 3.98±0.97; Group B: 3.67±0.67 10×-4 number of neurons/µm2; p=0.40). However, the HSP70 level, representing acute stress against epilepsy, in Group A patients was significantly larger than that in Group B. There was no significant difference in the level of Bcl-2, which is known as a protein that inhibits cell death, between the two groups. CONCLUSIONS: Neuronal loss and volume loss in the amygdala may not necessarily follow hippocampal sclerosis. From the analysis of stress proteins, epileptic attacks are as likely to damage the amygdala as the hippocampus but do not lead to neuronal death in the amygdala.

Ca2+ -permeable AMPA receptors associated with epileptogenesis of hypothalamic hamartoma.

Hypothalamic hamartoma (HH), composed of neurons and glia without apparent cytologic abnormalities, is a rare developmental malformation in humans. Patients with HH often have characteristic medically refractory gelastic seizures, and intrinsic epileptogenesis within the lesions has been speculated. Herein we provide evidence to suggest that in HH neurons, Ca2+ permeability through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors is aberrantly elevated. In needle biopsy specimens of HH tissue, field potential recordings demonstrated spontaneous epileptiform activities similar to those observed in other etiologically distinct epileptogenic tissues. In HH, however, these activities were clearly abolished by application of Joro Spider Toxin (JSTX), a specific inhibitor of the Ca2+ -permeable AMPA receptor. Consistent with these physiologic findings, the neuronal nuclei showed disappearance of adenosine deaminase acting on RNA 2 (ADAR2) immunoreactivity. Furthermore, examination of glutamate receptor 2 (GluA2) messenger RNA (mRNA) revealed that editing efficiency at the glutamine/arginine site was significantly low. These results suggest that neurons in HH may bear Ca2+ -permeable AMPA receptors due to dislocation of ADAR2.

Characteristic expression of p57/Kip2 in balloon cells in focal cortical dysplasia.

Balloon cells are a pathognomonic cellular feature of various cortical malformations, including focal cortical dysplasia type IIb (FCD IIb), cortical tubers of tuberous sclerosis (TSC) and hemimegalencephaly (HME). In the present study, we investigated the immunohistochemical expression of p57/Kip2, a member of the Cip/Kip family of cyclin-dependent kinase inhibitory proteins, in balloon cells in surgical specimens taken from 26, 17 and six patients with FCD IIb, TSC and HME, respectively. Characteristic dot-like reactivity with a faint, intense, reticular and process-like pattern was confined to the proximal portion of the cytoplasmic processes of the cells. Immunoelectron microscopy revealed the p57/Kip2 reactivity on intermediate filaments in the proximal portion of the processes. The immunohistochemical profile appeared similar to that of CD34; however, a double immunofluorescence study demonstrated that no cells showed reactivity for both p57/Kip2 and CD34. The frequencies of the p57/Kip2-positive cells in FCD IIb and HME were significantly higher than those in TSC, suggesting that the balloon cells may be heterogeneous. These findings suggest some functional significance of the protein on the cytoplasmic processes of balloon cells and appear consistent with the notion that the cells are abnormally differentiated progenitor cells.

Somatic Mutations in the MTOR gene cause focal cortical dysplasia type IIb.

OBJECTIVE: Focal cortical dysplasia (FCD) type IIb is a cortical malformation characterized by cortical architectural abnormalities, dysmorphic neurons, and balloon cells. It has been suggested that FCDs are caused by somatic mutations in cells in the developing brain. Here, we explore the possible involvement of somatic mutations in FCD type IIb. METHODS: We collected a total of 24 blood-brain paired samples with FCD, including 13 individuals with FCD type IIb, 5 with type IIa, and 6 with type I. We performed whole-exome sequencing using paired samples from 9 of the FCD type IIb subjects. Somatic MTOR mutations were identified and further investigated using all 24 paired samples by deep sequencing of the entire gene's coding region. Somatic MTOR mutations were confirmed by droplet digital polymerase chain reaction. The effect of MTOR mutations on mammalian target of rapamycin (mTOR) kinase signaling was evaluated by immunohistochemistry and Western blotting analyses of brain samples and by in vitro transfection experiments. RESULTS: We identified four lesion-specific somatic MTOR mutations in 6 of 13 (46%) individuals with FCD type IIb showing mutant allele rates of 1.11% to 9.31%. Functional analyses showed that phosphorylation of ribosomal protein S6 in FCD type IIb brain tissues with MTOR mutations was clearly elevated, compared to control samples. Transfection of any of the four MTOR mutants into HEK293T cells led to elevated phosphorylation of 4EBP, the direct target of mTOR kinase. INTERPRETATION: We found low-prevalence somatic mutations in MTOR in FCD type IIb, indicating that activating somatic mutations in MTOR cause FCD type IIb.