Abnormal activation of Yap/Taz contributes to the pathogenesis of tuberous sclerosis complex.

The multi-systemic genetic disorder tuberous sclerosis complex (TSC) impacts multiple neurodevelopmental processes including neuronal morphogenesis, neuronal migration, myelination and gliogenesis. These alterations contribute to the development of cerebral cortex abnormalities and malformations. Although TSC is caused by mTORC1 hyperactivation, cognitive and behavioral impairments are not improved through mTORC1 targeting, making the study of the downstream effectors of this complex important for understanding the mechanisms underlying TSC. As mTORC1 has been shown to promote the activity of the transcriptional co-activator Yap, we hypothesized that altered Yap/Taz signaling contributes to the pathogenesis of TSC. We first observed that the levels of Yap/Taz are increased in human cortical tuber samples and in embryonic cortices of Tsc2 conditional knockout (cKO) mice. Next, to determine how abnormal upregulation of Yap/Taz impacts the neuropathology of TSC, we deleted Yap/Taz in Tsc2 cKO mice. Importantly, Yap/Taz/Tsc2 triple conditional knockout (tcKO) animals show reduced cortical thickness and cortical neuron cell size, despite the persistence of high mTORC1 activity, suggesting that Yap/Taz play a downstream role in cytomegaly. Furthermore, Yap/Taz/Tsc2 tcKO significantly restored cortical and hippocampal lamination defects and reduced hippocampal heterotopia formation. Finally, the loss of Yap/Taz increased the distribution of myelin basic protein in Tsc2 cKO animals, consistent with an improvement in myelination. Overall, our results indicate that targeting Yap/Taz lessens the severity of neuropathology in a TSC animal model. This study is the first to implicate Yap/Taz as contributors to cortical pathogenesis in TSC and therefore as potential novel targets in the treatment of this disorder.

Models of KPTN-related disorder implicate mTOR signalling in cognitive and overgrowth phenotypes.

KPTN-related disorder is an autosomal recessive disorder associated with germline variants in KPTN (previously known as kaptin), a component of the mTOR regulatory complex KICSTOR. To gain further insights into the pathogenesis of KPTN-related disorder, we analysed mouse knockout and human stem cell KPTN loss-of-function models. Kptn -/- mice display many of the key KPTN-related disorder phenotypes, including brain overgrowth, behavioural abnormalities, and cognitive deficits. By assessment of affected individuals, we have identified widespread cognitive deficits (n = 6) and postnatal onset of brain overgrowth (n = 19). By analysing head size data from their parents (n = 24), we have identified a previously unrecognized KPTN dosage-sensitivity, resulting in increased head circumference in heterozygous carriers of pathogenic KPTN variants. Molecular and structural analysis of Kptn-/- mice revealed pathological changes, including differences in brain size, shape and cell numbers primarily due to abnormal postnatal brain development. Both the mouse and differentiated induced pluripotent stem cell models of the disorder display transcriptional and biochemical evidence for altered mTOR pathway signalling, supporting the role of KPTN in regulating mTORC1. By treatment in our KPTN mouse model, we found that the increased mTOR signalling downstream of KPTN is rapamycin sensitive, highlighting possible therapeutic avenues with currently available mTOR inhibitors. These findings place KPTN-related disorder in the broader group of mTORC1-related disorders affecting brain structure, cognitive function and network integrity.

SLC35A2 somatic variants in drug resistant epilepsy: FCD and MOGHE.

De novo somatic (post-zygotic) gene mutations affecting neuroglial progenitor cell types in embryonic cerebral cortex are increasingly identified in patients with drug resistant epilepsy (DRE) associated with malformations of cortical development, in particular, focal cortical dysplasias (FCD). Somatic variants in at least 16 genes have been linked to FCD type II, all encoding components of the mechanistic target of rapamycin (mTOR) pathway. FCD type II is characterized histopathologically by cytomegalic dysmorphic neurons and balloon cells. In contrast, the molecular pathogenesis of FCD I subtypes is less well understood, and histological features are characterized by alterations in columnar or laminar organization without cytomegalic dysmorphic neurons or balloon cells. In 2018, we reported somatic mutations in Solute Carrier Family 35 member A2 (SLC35A2) linked to DRE underlying FCD type I and subsequently to a new histopathological phenotype: excess oligodendrocytes and heterotopic neurons in subcortical white matter known as MOGHE (mild malformation of cortical development with oligodendroglial hyperplasia). These discoveries opened the door to studies linking somatic mutations to FCD. In this review, we discuss the biology of SLC35A2 somatic mutations in epilepsy in FCD and MOGHE, and insights into SLC35A2 epilepsy pathogenesis, describing progress to date and critical areas for investigation.

NPRL3 loss alters neuronal morphology, mTOR localization, cortical lamination and seizure threshold.

Mutations in nitrogen permease regulator-like 3 (NPRL3), a component of the GATOR1 complex within the mTOR pathway, are associated with epilepsy and malformations of cortical development. Little is known about the effects of NPRL3 loss on neuronal mTOR signalling and morphology, or cerebral cortical development and seizure susceptibility. We report the clinical phenotypic spectrum of a founder NPRL3 pedigree (c.349delG, p.Glu117LysFS; n = 133) among Old Order Mennonites dating to 1727. Next, as a strategy to define the role of NPRL3 in cortical development, CRISPR/Cas9 Nprl3 knockout in Neuro2a cells in vitro and in foetal mouse brain in vivo was used to assess the effects of Nprl3 knockout on mTOR activation, subcellular mTOR localization, nutrient signalling, cell morphology and aggregation, cerebral cortical cytoarchitecture and network integrity. The NPRL3 pedigree exhibited an epilepsy penetrance of 28% and heterogeneous clinical phenotypes with a range of epilepsy semiologies, i.e. focal or generalized onset, brain imaging abnormalities, i.e. polymicrogyria, focal cortical dysplasia or normal imaging, and EEG findings, e.g. focal, multi-focal or generalized spikes, focal or generalized slowing. Whole exome analysis comparing a seizure-free group (n = 37) to those with epilepsy (n = 24) to search for gene modifiers for epilepsy did not identify a unique genetic modifier that explained the variability in seizure penetrance in this cohort. Nprl3 knockout in vitro caused mTOR pathway hyperactivation, cell soma enlargement and the formation of cellular aggregates seen in time-lapse videos that were prevented with the mTOR inhibitors rapamycin or torin1. In Nprl3 knockout cells, mTOR remained localized on the lysosome in a constitutively active conformation, as evidenced by phosphorylation of ribosomal S6 and 4E-BP1 proteins, even under nutrient starvation (amino acid-free) conditions, demonstrating that Nprl3 loss decouples mTOR activation from neuronal metabolic state. To model human malformations of cortical development associated with NPRL3 variants, we created a focal Nprl3 knockout in foetal mouse cortex by in utero electroporation and found altered cortical lamination and white matter heterotopic neurons, effects which were prevented with rapamycin treatment. EEG recordings showed network hyperexcitability and reduced seizure threshold to pentylenetetrazol treatment. NPRL3 variants are linked to a highly variable clinical phenotype which we propose results from mTOR-dependent effects on cell structure, cortical development and network organization.

Somatic variants in diverse genes leads to a spectrum of focal cortical malformations.

Post-zygotically acquired genetic variants, or somatic variants, that arise during cortical development have emerged as important causes of focal epilepsies, particularly those due to malformations of cortical development. Pathogenic somatic variants have been identified in many genes within the PI3K-AKT-mTOR-signalling pathway in individuals with hemimegalencephaly and focal cortical dysplasia (type II), and more recently in SLC35A2 in individuals with focal cortical dysplasia (type I) or non-dysplastic epileptic cortex. Given the expanding role of somatic variants across different brain malformations, we sought to delineate the landscape of somatic variants in a large cohort of patients who underwent epilepsy surgery with hemimegalencephaly or focal cortical dysplasia. We evaluated samples from 123 children with hemimegalencephaly (n = 16), focal cortical dysplasia type I and related phenotypes (n = 48), focal cortical dysplasia type II (n = 44), or focal cortical dysplasia type III (n = 15). We performed high-depth exome sequencing in brain tissue-derived DNA from each case and identified somatic single nucleotide, indel and large copy number variants. In 75% of individuals with hemimegalencephaly and 29% with focal cortical dysplasia type II, we identified pathogenic variants in PI3K-AKT-mTOR pathway genes. Four of 48 cases with focal cortical dysplasia type I (8%) had a likely pathogenic variant in SLC35A2. While no other gene had multiple disease-causing somatic variants across the focal cortical dysplasia type I cohort, four individuals in this group had a single pathogenic or likely pathogenic somatic variant in CASK, KRAS, NF1 and NIPBL, genes previously associated with neurodevelopmental disorders. No rare pathogenic or likely pathogenic somatic variants in any neurological disease genes like those identified in the focal cortical dysplasia type I cohort were found in 63 neurologically normal controls (P = 0.017), suggesting a role for these novel variants. We also identified a somatic loss-of-function variant in the known epilepsy gene, PCDH19, present in a small number of alleles in the dysplastic tissue from a female patient with focal cortical dysplasia IIIa with hippocampal sclerosis. In contrast to focal cortical dysplasia type II, neither focal cortical dysplasia type I nor III had somatic variants in genes that converge on a unifying biological pathway, suggesting greater genetic heterogeneity compared to type II. Importantly, we demonstrate that focal cortical dysplasia types I, II and III are associated with somatic gene variants across a broad range of genes, many associated with epilepsy in clinical syndromes caused by germline variants, as well as including some not previously associated with radiographically evident cortical brain malformations.

MicroRNA-34a activation in tuberous sclerosis complex during early brain development may lead to impaired corticogenesis.

AIMS: Tuberous sclerosis complex (TSC) is a genetic disorder associated with dysregulation of the mechanistic target of rapamycin complex 1 (mTORC1) signalling pathway. Neurodevelopmental disorders, frequently present in TSC, are linked to cortical tubers in the brain. We previously reported microRNA-34a (miR-34a) among the most upregulated miRs in tubers. Here, we characterised miR-34a expression in tubers with the focus on the early brain development and assessed the regulation of mTORC1 pathway and corticogenesis by miR-34a. METHODS: We analysed the expression of miR-34a in resected cortical tubers (n = 37) compared with autopsy-derived control tissue (n = 27). The effect of miR-34a overexpression on corticogenesis was assessed in mice at E18. The regulation of the mTORC1 pathway and the expression of the bioinformatically predicted target genes were assessed in primary astrocyte cultures from three patients with TSC and in SH-SY5Y cells following miR-34a transfection. RESULTS: The peak of miR-34a overexpression in tubers was observed during infancy, concomitant with the presence of pathological markers, particularly in giant cells and dysmorphic neurons. miR-34a was also strongly expressed in foetal TSC cortex. Overexpression of miR-34a in mouse embryos decreased the percentage of cells migrated to the cortical plate. The transfection of miR-34a mimic in TSC astrocytes negatively regulated mTORC1 and decreased the expression of the target genes RAS related (RRAS) and NOTCH1. CONCLUSIONS: MicroRNA-34a is most highly overexpressed in tubers during foetal and early postnatal brain development. miR-34a can negatively regulate mTORC1; however, it may also contribute to abnormal corticogenesis in TSC.

The role of somatic mutational events in the pathogenesis of epilepsy.

PURPOSE OF REVIEW: There has been rapid progress in defining novel causative gene variants responsible for a large spectrum of human epilepsy syndromes and subtypes. Of particular interest is the discovery that somatic mutations, for example, noninherited mutations occurring in neuroglial progenitor cells during embryonic brain development, are highly linked to malformations of cortical development (MCD) such as focal cortical dysplasia (FCD) type II and hemimegalencephaly. RECENT FINDINGS: Somatic gene variants have been identified in genes encoding regulatory proteins within the mechanistic target of rapamycin (mTOR) signaling cascade and have thus comprised the group classified as mTORopathies. FCD II and hemimegalencephaly often result from mutations in identical genes suggesting that these are spectrum disorders. An exciting recent development has been the identification of somatic mutations causing both FCD Ia and nonlesional neocortical epilepsy. SUMMARY: Defining somatic gene mutations in brain tissue specimens has shed new light on how MCD form and the mechanisms of epileptogenesis associated with MCD. Trials of mTOR inhibitors in tuberous sclerosis complex have demonstrated that inhibition of mTOR activation in mTORopathies can reduce seizure frequency. New somatic mutations found for a variety of epilepsy syndromes may provide new targets for clinical therapeutics.

Homozygous boricua TBCK mutation causes neurodegeneration and aberrant autophagy.

OBJECTIVE: Autosomal-recessive mutations in TBCK cause intellectual disability of variable severity. Although the physiological function of TBCK remains unclear, loss-of-function mutations are associated with inhibition of mechanistic target of rapamycin complex 1 (mTORC1) signaling. Given that mTORC1 signaling is known to regulate autophagy, we hypothesized that TBCK-encephalopathy patients with a neurodegenerative course have defects in autophagic-lysosomal dysfunction. METHODS: Children (n = 8) of Puerto Rican (Boricua) descent affected with homozygous TBCK p.R126X mutations underwent extensive neurological phenotyping and neurophysiological studies. We quantified autophagosome content in TBCK-/- patient-derived fibroblasts by immunostaining and assayed autophagic markers by western assay. Free sialylated oligosaccharide profiles were assayed in patient's urine and fibroblasts. RESULTS: The neurological phenotype of children with TBCK p.R126X mutations, which we call TBCK-encephaloneuronopathy (TBCKE), include congenital hypotonia, progressive motor neuronopathy, leukoencephalopathy, and epilepsy. Systemic features include coarse facies, dyslipidemia, and osteoporosis. TBCK-/- fibroblasts in vitro exhibit increased numbers of LC3+ autophagosomes and increased autophagic flux by immunoblots. Free oligosaccharide profiles in fibroblasts and urine of TBCKE patients differ from control fibroblasts and are ameliorated by treatment with the mTORC1 activator leucine. INTERPRETATION: TBCKE is a clinically distinguishable syndrome with progressive central and peripheral nervous system dysfunction, consistently observed in patients with the p.R126X mutation. We provide evidence that inappropriate autophagy in the absence of cellular stressors may play a role in this disorder, and that mTORC1 activation may ameliorate the autophagic-lysosomal system dysfunction. Free oligosaccharide profiles could serve as a novel biomarker for this disorder as well as a tool to evaluate potential therapeutic interventions. Ann Neurol 2018;83:153-165.

Somatic SLC35A2 variants in the brain are associated with intractable neocortical epilepsy.

OBJECTIVE: Somatic variants are a recognized cause of epilepsy-associated focal malformations of cortical development (MCD). We hypothesized that somatic variants may underlie a wider range of focal epilepsy, including nonlesional focal epilepsy (NLFE). Through genetic analysis of brain tissue, we evaluated the role of somatic variation in focal epilepsy with and without MCD. METHODS: We identified somatic variants through high-depth exome and ultra-high-depth candidate gene sequencing of DNA from epilepsy surgery specimens and leukocytes from 18 individuals with NLFE and 38 with focal MCD. RESULTS: We observed somatic variants in 5 cases in SLC35A2, a gene associated with glycosylation defects and rare X-linked epileptic encephalopathies. Nonsynonymous variants in SLC35A2 were detected in resected brain, and absent from leukocytes, in 3 of 18 individuals (17%) with NLFE, 1 female and 2 males, with variant allele frequencies (VAFs) in brain-derived DNA of 2 to 14%. Pathologic evaluation revealed focal cortical dysplasia type Ia (FCD1a) in 2 of the 3 NLFE cases. In the MCD cohort, nonsynonymous variants in SCL35A2 were detected in the brains of 2 males with intractable epilepsy, developmental delay, and magnetic resonance imaging suggesting FCD, with VAFs of 19 to 53%; Evidence for FCD was not observed in either brain tissue specimen. INTERPRETATION: We report somatic variants in SLC35A2 as an explanation for a substantial fraction of NLFE, a largely unexplained condition, as well as focal MCD, previously shown to result from somatic mutation but until now only in PI3K-AKT-mTOR pathway genes. Collectively, our findings suggest a larger role than previously recognized for glycosylation defects in the intractable epilepsies. Ann Neurol 2018.

GATORopathies: The role of amino acid regulatory gene mutations in epilepsy and cortical malformations.

The mechanistic target of rapamycin (mTOR) pathway has been implicated in a growing number of malformations of cortical development (MCD) associated with intractable epilepsy. Mutations in single genes encoding mTOR pathway regulatory proteins have been linked to MCD such as focal cortical dysplasia (FCD) types IIa and IIb, hemimegalencephaly (HME), and megalencephaly. Recent studies have demonstrated that the GATOR1 protein complex, comprised of DEPDC5, NPRL3, and NPRL2, plays a pivotal role in regulating mTOR signaling in response to cellular amino acid levels and that mutations in DEPDC5, NPRL3, or NPRL2 are linked to FCD, HME, and seizures. Histopathological analysis of FCD and HME tissue specimens resected from individuals harboring DEPDC5, NPRL3, or NPRL2 gene mutations reveals hyperactivation of mTOR pathway signaling. Family pedigrees carrying mutations in either DEPDC5 or NPRL3 share clinical phenotypes of epilepsy and MCD, as well as intellectual and neuropsychiatric disabilities. Interestingly, some individuals with seizures associated with DEPDC5, NPRL3, or NPRL2 variants exhibit normal brain imaging suggesting either occult MCD or a role for these genes in non-lesional neocortical epilepsy. Mouse models resulting from knockdown or knockout of either Depdc5 or Nprl3 exhibit altered cortical lamination, neuronal dysmorphogenesis, and enhanced neuronal excitability as reported in models resulting from direct mTOR activation through expression of its canonical activator RHEB. The role of the GATOR1 proteins in regulating mTOR signaling suggest plausible options for mTOR inhibition in the treatment of epilepsy associated with mutations in DEPDC5, NPRL3, or NPRL2.

Mechanistic target of rapamycin (mTOR) signaling in status epilepticus.

The mechanistic target of rapamycin (mTOR) pathway plays a critical role in brain development, neuronal shape and size, and synaptic plasticity, as well as learning and memory. Mutations in mTOR pathway genes (MPG) cause malformations of cortical development (MCDs) that are highly associated with often intractable epilepsy, thus highlighting an association between the mTOR pathway and establishment of the epileptic network. A growing body of preclinical evidence in in vitro and rodent model systems suggests that mTOR signaling may be altered in status epilepticus (SE) and that modulation of mTOR activation with mTOR inhibitors such as rapamycin (sirolimus) could provide new therapeutic avenues for treatment of both refractory epilepsy and SE. Rapamycin may have ubiquitous effects on all neuronal subtypes as well as astrocytes and seems to prevent the development of seizures following experimentally induced SE. To date, there have been no human studies focused on mTOR signaling in SE, but clearly, preclinical data support investigation into this pivotal cell signaling pathway. Thus, modulation of the mTOR pathway may provide a new strategy for treatment of SE and could have implications for the prevention of epilepsy in patients with SE. This article is part of the Special Issue "Proceedings of the 7th London-Innsbruck Colloquium on Status Epilepticus and Acute Seizures".

DEPDC5 and NPRL3 modulate cell size, filopodial outgrowth, and localization of mTOR in neural progenitor cells and neurons.

Mutations in DEPDC5 and NPRL3 subunits of GATOR1, a modulator of mechanistic target of rapamycin (mTOR), are linked to malformations of cortical development (MCD). Brain specimens from these individuals reveal abnormal cortical lamination, altered cell morphology, and hyperphosphorylation of ribosomal S6 protein (PS6), a marker for mTOR activation. While numerous studies have examined GATOR1 subunit function in non-neuronal cell lines, few have directly assessed loss of GATOR1 subunit function in neuronal cell types. We hypothesized that DEPDC5 or NPRL3 shRNA-mediated knockdown (DEPDC5/NPRL3 KD) leads to inappropriate functional activation of mTOR and mTOR-dependent alterations in neuronal morphology. Neuronal size was determined in human specimens harboring DEPDC5 or NPRL3 mutations resected for epilepsy treatment. DEPDC5/NPRL3 KD effects on cell size, filopodial extension, subcellular mTOR complex 1 (mTORC1) localization, and mTORC1 activation during nutrient deprivation were assayed in mouse neuroblastoma cells (N2aC) and mouse subventricular zone derived neural progenitor cells (mNPCs). mTORC1-dependent effects of DEPDC5/NPRL3 KD were determined using the mTOR inhibitor rapamycin. Changes in mTOR subcellular localization and mTORC1 pathway activation following DEPDC5/NPRL3 KD were determined by examining the proximity of mTOR to the lysosomal surface during amino acid starvation. Neurons exhibiting PS6 immunoreactivity (Ser 235/236) in human specimens were 1.5× larger than neurons in post-mortem control samples. DEPDC5/NPRL3 KD caused mTORC1, but not mTORC2, hyperactivation, soma enlargement, and increased filopodia in N2aC and mNPCs compared with wildtype cells. DEPDC5/NPRL3 KD led to inappropriate mTOR localization at the lysosome along with constitutive mTOR activation following amino acid deprivation. DEPDC5/NPRL3 KD effects on morphology and functional mTOR activation were reversed by rapamycin. mTOR-dependent effects of DEPDC5/NPRL3 KD on morphology and subcellular localization of mTOR in neurons suggests that loss-of-function in GATOR1 subunits may play a role in MCD formation during fetal brain development.

Familial cortical dysplasia caused by mutation in the mammalian target of rapamycin regulator NPRL3.

We describe first cousin sibling pairs with focal epilepsy, one of each pair having focal cortical dysplasia (FCD) IIa. Linkage analysis and whole-exome sequencing identified a heterozygous germline frameshift mutation in the gene encoding nitrogen permease regulator-like 3 (NPRL3). NPRL3 is a component of GAP Activity Towards Rags 1, a negative regulator of the mammalian target of rapamycin complex 1 signaling pathway. Immunostaining of resected brain tissue demonstrated mammalian target of rapamycin activation. Screening of 52 unrelated individuals with FCD identified 2 additional patients with FCDIIa and germline NPRL3 mutations. Similar to DEPDC5, NPRL3 mutations may be considered as causal variants in patients with FCD or magnetic resonance imaging-negative focal epilepsy.

mTOR pathway inhibition prevents neuroinflammation and neuronal death in a mouse model of cerebral palsy.

BACKGROUND AND PURPOSE: Mammalian target of rapamycin (mTOR) pathway signaling governs cellular responses to hypoxia and inflammation including induction of autophagy and cell survival. Cerebral palsy (CP) is a neurodevelopmental disorder linked to hypoxic and inflammatory brain injury however, a role for mTOR modulation in CP has not been investigated. We hypothesized that mTOR pathway inhibition would diminish inflammation and prevent neuronal death in a mouse model of CP. METHODS: Mouse pups (P6) were subjected to hypoxia-ischemia and lipopolysaccharide-induced inflammation (HIL), a model of CP causing neuronal injury within the hippocampus, periventricular white matter, and neocortex. mTOR pathway inhibition was achieved with rapamycin (an mTOR inhibitor; 5mg/kg) or PF-4708671 (an inhibitor of the downstream p70S6kinase, S6K, 75 mg/kg) immediately following HIL, and then for 3 subsequent days. Phospho-activation of the mTOR effectors p70S6kinase and ribosomal S6 protein and expression of hypoxia inducible factor 1 (HIF-1α) were assayed. Neuronal cell death was defined with Fluoro-Jade C (FJC) and autophagy was measured using Beclin-1 and LC3II expression. Iba-1 labeled, activated microglia were quantified. RESULTS: Neuronal death, enhanced HIF-1α expression, and numerous Iba-1 labeled, activated microglia were evident at 24 and 48 h following HIL. Basal mTOR signaling, as evidenced by phosphorylated-S6 and -S6K levels, was unchanged by HIL. Rapamycin or PF-4,708,671 treatment significantly reduced mTOR signaling, neuronal death, HIF-1α expression, and microglial activation, coincident with enhanced expression of Beclin-1 and LC3II, markers of autophagy induction. CONCLUSIONS: mTOR pathway inhibition prevented neuronal death and diminished neuroinflammation in this model of CP. Persistent mTOR signaling following HIL suggests a failure of autophagy induction, which may contribute to neuronal death in CP. These results suggest that mTOR signaling may be a novel therapeutic target to reduce neuronal cell death in CP.

Focal Cortical Dysplasia.

Focal cortical dysplasias are common malformations of cerebral cortical development and are highly associated with medically intractable epilepsy. They have been classified into neuropathological subtypes (type Ia, Ib, IIa, IIb, and III) based on the severity of cytoarchitectural disruption--tangential or radial dispersion, or loss of laminar structure--and the presence of unique cells types such as cytomegalic neurons or balloon cells. Most focal cortical dysplasias can be identified on neuroimaging and many require resective epilepsy surgery to cure refractory seizures. The pathogenesis of focal cortical dysplasias remains to be defined, although there is recent evidence to suggest that focal cortical dysplasias arise from de novo somatic mutations occurring during brain development. Some focal cortical dysplasia subtypes show a link to the mammalian target of rapamycin signaling cascade; this has now extended to other cortical malformations, including hemimegalencephaly.

Fetal brain mTOR signaling activation in tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is characterized by developmental malformations of the cerebral cortex known as tubers, comprised of cells that exhibit enhanced mammalian target of rapamycin (mTOR) signaling. To date, there are no reports of mTORC1 and mTORC2 activation in fetal tubers or in neural progenitor cells lacking Tsc2. We demonstrate mTORC1 activation by immunohistochemical detection of substrates phospho-p70S6K1 (T389) and phospho-S6 (S235/236), and mTORC2 activation by substrates phospho-PKCα (S657), phospho-Akt (Ser473), and phospho-SGK1 (S422) in fetal tubers. Then, we show that Tsc2 shRNA knockdown (KD) in mouse neural progenitor cells (mNPCs) in vitro results in enhanced mTORC1 (phospho-S6, phospho-4E-BP1) and mTORC2 (phospho-Akt and phospho-NDRG1) signaling, as well as a doubling of cell size that is rescued by rapamycin, an mTORC1 inhibitor. Tsc2 KD in vivo in the fetal mouse brain by in utero electroporation causes disorganized cortical lamination and increased cell volume that is prevented with rapamycin. We demonstrate for the first time that mTORC1 and mTORC2 signaling is activated in fetal tubers and in mNPCs following Tsc2 KD. These results suggest that inhibition of mTOR pathway signaling during embryogenesis could prevent abnormal brain development in TSC.

Is focal cortical dysplasia sporadic? Family evidence for genetic susceptibility.

Focal cortical dysplasia is a common cortical malformation and an important cause of epilepsy. There is evidence for shared molecular mechanisms underlying cortical dysplasia, ganglioglioma, hemimegalencephaly, and dysembryoplastic neuroepithelial tumor. However, there are no familial reports of typical cortical dysplasia or co-occurrence of cortical dysplasia and related lesions within the same pedigree. We report the clinical, imaging, and histologic features of six pedigrees with familial cortical dysplasia and related lesions. Twelve patients from six pedigrees were ascertained from pediatric and adult epilepsy centers, eleven of whom underwent epilepsy surgery. Pedigree data, clinical information, neuroimaging findings, and histopathologic features are presented. The families comprise brothers with focal cortical dysplasia, a male and his sister with focal cortical dysplasia, a female with focal cortical dysplasia and her brother with hemimegalencephaly, a female with focal cortical dysplasia and her female first cousin with ganglioglioma, a female with focal cortical dysplasia and her male cousin with dysembryoplastic neuroepithelial tumor, and a female and her nephew with focal cortical dysplasia. This series shows that focal cortical dysplasia can be familial and provides clinical evidence suggesting that cortical dysplasia, hemimegalencephaly, ganglioglioma, and dysembryoplastic neuroepithelial tumors may share common genetic determinants.

Loss of Slc35a2 alters development of the mouse cerebral cortex.

Brain somatic variants in SLC35A2, an intracellular UDP-galactose transporter, are commonly identified mutations associated with drug-resistant neocortical epilepsy and developmental brain malformations, including focal cortical dysplasia type I and mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (MOGHE). However, the causal effects of altered SLC35A2 function on cortical development remain untested. We hypothesized that focal Slc35a2 knockout (KO) or knockdown (KD) in the developing mouse cortex would disrupt cortical development and change network excitability. Through two independent studies, we used in utero electroporation (IUE) to introduce CRISPR/Cas9/targeted guide RNAs or short-hairpin RNAs into the embryonic mouse brain at day 14.5-15.5 to achieve Slc35a2 KO or KD, respectively, from neural precursor cells. Slc35a2 KO or KD caused disrupted radial migration of electroporated neurons evidenced by heterotopic cells located in lower cortical layers and in the sub-cortical white matter. Slc35a2 KO in neurons did not induce changes in oligodendrocyte number, importantly suggesting that the oligodendroglial hyperplasia observed in MOGHE originates from distinct cell autonomous effects of Slc35a2 mutations. Adult KO mice were implanted with EEG electrodes for 72-hour continuous recording. Spontaneous seizures were not observed in focal Slc35a2 KO mice, but there was reduced seizure threshold following pentylenetetrazol injection. Here we demonstrate that focal Slc35a2 KO or KD in vivo disrupts corticogenesis through altered neuronal migration and that KO leads to reduced seizure threshold. Together these results demonstrate a direct causal role for SLC35A2 in cortical development.

Detection of human papillomavirus in human focal cortical dysplasia type IIB.

OBJECTIVE: Focal cortical dysplasia type IIB (FCDIIB) is a sporadic developmental malformation of the cerebral cortex highly associated with pediatric epilepsy. Balloon cells (BCs) in FCDIIB exhibit constitutive activation of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. Recently, the high-risk human papillomavirus type 16 oncoprotein E6 was identified as a potent activator of mTORC1 signaling. Here, we test the hypothesis that HPV16 E6 is present in human FCDIIB specimens. METHODS: HPV16 E6 protein expression was assayed by immunohistochemistry in FCDIIB specimens (n = 50) and control brain specimens (n = 36). HPV16 E6 DNA was assayed by polymerase chain reaction (PCR) and in situ hybridization; HPV16 E6 mRNA was assayed by reverse transcriptase PCR. HPV16 E6 was transfected into fetal mouse brains by in utero electroporation to test the effects of E6 on cortical development. RESULTS: HPV16 E6 protein was robustly expressed in all FCDIIB specimens in BCs, but not in regions without BCs or in control tissue specimens including normal brain, lymphoblasts, and fibroblasts, cortical tubers, and U87 glioma cells. E6 expression in FCDIIB colocalized with phosphoactivated S6 protein, a known mTORC1 substrate. HPV16 E6 DNA and mRNA were detected in representative specimens of FCDIIB but not control cortex, and were confirmed by sequencing. Transfection of E6 into fetal mouse brains caused a focal cortical malformation in association with enhanced mTORC1 signaling. INTERPRETATION: Our results indicate a new association between HPV16 E6 and FCDIIB and demonstrate for the first time HPV16 E6 in the human brain. We propose a novel etiology for FCDIIB based on HPV16 E6 expression during fetal brain development.