Marianna Baybis

mTOR cascade activation distinguishes tubers from focal cortical dysplasia.

Balloon cells (BCs) in focal cortical dysplasia (FCD) and giant cells (GCs) in tubers of the tuberous sclerosis complex (TSC) share phenotypic similarities. TSC1 or TSC2 gene mutations in TSC lead to mTOR pathway activation and p70S6kinase (phospho‐S6K) and ribosomal S6 (phospho‐S6) protein phosphorylation. Phospho‐S6K, phospho‐S6, and phospho‐S6K–activated proteins phospho‐STAT3 and phospho‐4EBP1 were detected immunohistochemically in GCs, whereas only phospho‐S6 was observed in BCs. Expression of four candidate gene families (cell signaling, cell adhesion, growth factor/receptor, and transcription factor mRNAs) was assayed in single, microdissected phospho‐S6–immunolabeled BCs and GCs as a strategy to define whether BCs and GCs exhibit differential transcriptional profiles. Among 60 genes, differential expression of 24 mRNAs distinguished BCs from GCs and only 4 genes showed similar expression profiles between BCs and GCs. Tuberin mRNA levels were reduced in GCs from TSC patients with TSC2 gene mutations but were unchanged in BCs. Phospho‐S6K, ‐S6, ‐STAT3, and ‐4EBP1 expression in GCs reflects loss of hamartin‐tuberin–mediated mTOR pathway inhibition. Phospho‐S6 expression alone in BCs does not support mTOR cascade activation in FCD. Differential gene expression profiles in BCs and GCs supports the hypothesis that these cell types derive by distinct pathogenic mechanisms. Ann Neurol 2004

Expression of ICAM-1, TNF-α, NFκB, and MAP kinase in tubers of the tuberous sclerosis complex

Abstract Individuals affected with tuberous sclerosis complex (TSC) develop cortical tubers characterized by disorganized cytoarchitecture and morphologically abnormal cell types, such as dysplastic neurons (DNs) and giant cells (GCs). As part of ongoing cDNA array analysis to study the molecular pathogenesis of tuber formation, we detected increased expression of intercellular adhesion molecule-1 (ICAM-1) mRNA, a cell adhesion molecule (CAM) that functions in cytokine signaling, in tubers. Western and immunohistochemical analyses revealed that ICAM-1 protein was selectively expressed in tubers, but was only minimally expressed in control cortex, adjacent nontuberal cortex, or in non-TSC focal cortical dysplasia. Increased expression of ICAM-1 was found in mice in which the Tsc1 gene was conditionally inactivated in astrocytes. Expression of molecules involved in ICAM-1 activation and cytokine signaling were increased in tubers, including tumor necrosis factor alpha (TNF-α), mitogen activated protein kinase (MAPK), and nuclear factor kappa B (NF-κB). Numerous CD68-immunoreactive macrophages were observed clustered around GCs further supporting an inflammatory response in tubers. Expression of caspase 8 and Fas support cytokine activation and detection of TUNEL reactivity suggests ongoing cell death in tubers. Specific alterations in ICAM-1, TNF-α, NF-κB1, and MAPK expression coupled with the detection of numerous CD68-immunoreactive macrophages suggests activation of proinflammatory cytokine signaling pathways in tubers that may culminate in cell death.

Markers of cellular proliferation are expressed in cortical tubers.

p34cdc2, collapsin response mediator protein 4 (CRMP4), doublecortin (DCX), HuD, and NeuN expression was assessed in tuber (n = 16) and subependymal giant cell astrocytoma (SEGA; n = 6) specimens in tuberous sclerosis complex to define the developmental phenotype and lineage of giant cells (CGs) in these lesions. Many GCs exhibited HuD and NeuN immunolabeling suggesting a differentiated neural phenotype. Giant cells in tubers, SEGAs and subependymal nodules in the Eker rat model of TSC expressed CRMP4 and DCX. Tubers and SEGAs exhibit a heterogeneous profile of differentiation and may share a common cellular lineage. Tubers may contain a subpopulation of newly generated cells. Ann Neurol 2003;53:668–673

STRADα deficiency results in aberrant mTORC1 signaling during corticogenesis in humans and mice

Polyhydramnios, megalencephaly, and symptomatic epilepsy syndrome (PMSE) is a rare human autosomal-recessive disorder characterized by abnormal brain development, cognitive disability, and intractable epilepsy. It is caused by homozygous deletions of STE20-related kinase adaptor alpha (STRADA). The underlying pathogenic mechanisms of PMSE and the role of STRADA in cortical development remain unknown. Here, we found that a human PMSE brain exhibits cytomegaly, neuronal heterotopia, and aberrant activation of mammalian target of rapamycin complex 1 (mTORC1) signaling. STRADalpha normally binds and exports the protein kinase LKB1 out of the nucleus, leading to suppression of the mTORC1 pathway. We found that neurons in human PMSE cortex exhibited abnormal nuclear localization of LKB1. To investigate this further, we modeled PMSE in mouse neural progenitor cells (mNPCs) in vitro and in developing mouse cortex in vivo by knocking down STRADalpha expression. STRADalpha-deficient mNPCs were cytomegalic and showed aberrant rapamycin-dependent activation of mTORC1 in association with abnormal nuclear localization of LKB1. Consistent with the observations in human PMSE brain, knockdown of STRADalpha in vivo resulted in cortical malformation, enhanced mTORC1 activation, and abnormal nuclear localization of LKB1. Thus, we suggest that the aberrant nuclear accumulation of LKB1 caused by STRADalpha deficiency contributes to hyperactivation of mTORC1 signaling and disruption of neuronal lamination during corticogenesis, and thereby the neurological features associated with PMSE.

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.

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

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.

Early Progenitor Cell Marker Expression Distinguishes Type II from Type I Focal Cortical Dysplasias

Type I and type II focal cortical dysplasias (FCDs) exhibit distinct histopathologic features that suggest different pathogenic mechanisms. Type I FCDs are characterized by mild laminar disorganization and hypertrophic neurons, whereas type II FCDs exhibit dramatic laminar disorganization and cytomegalic cells (balloon cells). Both FCD types are associated with intractable epilepsy; therefore, identifying cellular or molecular differences between these lesion types that explains the histologic differences could provide new diagnostic and therapeutic insights. Type II FCDs express nestin, a neuroglial progenitor protein that is modulated in vitro by the stem cell proteins c-Myc, sex-determining region Y-box 2 (SOX2), and Octamer-4 (Oct-4) after activation of mammalian target of rapamycin complex 1 (mTORC1). Because mTORC1 activation has been demonstrated in type II FCDs, we hypothesized that c-Myc, SOX2, and Oct-4 expression would distinguish type II from type I FCDs. In addition, we assayed the expression of progenitor cell proteins forkhead box G1 (FOXG1), Kruppel-like factor 4 (KLF4), Nanog, and SOX3. Differential expression of 7 stem cellproteins and aberrant phosphorylation of2mTORC1 substrates, S6 andS6 kinase 1 proteins, clearly distinguished type II from type I FCDs(n = 10 each). Our results demonstrate new potential pathogenic pathways in type II FCDs and suggest biomarkers for diagnostic pathology in resected epilepsy specimens.

Effects of rapamycin on gene expression, morphology, and electrophysiological properties of rat hippocampal neurons.

PURPOSE We assayed the effects of rapamycin, an immunomodulatory agent known to inhibit the activity of the mammalian target of rapamycin (mTOR) cascade, on candidate gene expression and single unit firing properties in cultured rat hippocampal neurons as a strategy to define the effects of rapamycin on neuronal gene transcription and excitability. METHODS Rapamycin was added (100nM) to cultured hippocampal neurons on days 3 and 14. Neuronal somatic size and dendritic length were assayed by immunohistochemistry and digital imaging. Radiolabeled mRNA was amplified from single hippocampal pyramidal neurons and used to probe cDNA arrays containing over 100 distinct candidate genes including cytoskeletal element, growth factor, transcription factor, neurotransmitter, and ion channel genes. In addition, the effects of rapamycin (200nM) on spontaneous neuronal activity and voltage-dependent currents were assessed. RESULTS There were no effects of rapamycin on cell size or dendrite length. Rapamycin altered expression of distinct mRNAs in each gene family on days 3 and 14 in culture. Single unit recordings from neurons exposed to rapamycin exhibited no change from baseline. When spontaneous activity was increased by blocking GABA-mediated inhibition with bicuculline, a fraction of the neurons exhibited a decreased duration of spontaneous bursts and a decrease in synaptic inputs. Rapamycin did not appear to alter voltage-dependent Na(+) or K(+) currents underlying action potentials. CONCLUSIONS These data demonstrate that rapamycin does not produce neurotoxicity nor alter dendritic growth and complexity in vitro and does not significantly alter voltage-gated sodium and potassium currents. Rapamycin does affect neuronal gene transcription in vitro. Use of rapamycin in clinical trials for patients with tuberous sclerosis complex warrants vigilance for possible effects on seizure frequency and neurocognitive function.

Rapamycin Prevents Seizures After Depletion of STRADA in a Rare Neurodevelopmental Disorder

Blocking mTORC1 rescues the neural progenitor cell migratory defect caused by depletion of the STRADA pseudokinase and reduces seizures in patients with a rare neurodevelopmental disorder. Preventing Seizures with Rapamycin The discovery of new treatments for rare neurodevelopmental disorders associated with epilepsy and intellectual disability is often limited by small patient sample sizes that delay initiation of clinical trials. Mutations in the gene STRADA cause brain malformations, seizures, and failure to develop social language in children, with no known successful treatment. In a new study, Parker and colleagues now show that the protein STRADA modulates the mammalian target of rapamycin (mTOR) signaling pathway and that loss of STRADA results in unchecked mTOR activity. Depletion of STRADA in mouse neural progenitor cells resulted in loss of polarity, impaired migration, and inability to form layers in the cerebral cortex. Impaired migration was also identified in fibroblasts from patients lacking STRADA, and all of these effects were prevented with the mTOR inhibitor rapamycin, an immunosuppressant drug in clinical use. The authors then treated five children with rapamycin (sirolimus) beginning at 3 to 8 months of age, and abatement of seizures was observed in all of the children. Early treatment with mTOR pathway inhibitors may be beneficial for children with this neurodevelopmental disorder or with other conditions associated with enhanced mTOR signaling such as tuberous sclerosis complex and fragile X syndrome. A rare neurodevelopmental disorder in the Old Order Mennonite population called PMSE (polyhydramnios, megalencephaly, and symptomatic epilepsy syndrome; also called Pretzel syndrome) is characterized by infantile-onset epilepsy, neurocognitive delay, craniofacial dysmorphism, and histopathological evidence of heterotopic neurons in subcortical white matter and subependymal regions. PMSE is caused by a homozygous deletion of exons 9 to 13 of the LYK5/STRADA gene, which encodes the pseudokinase STRADA, an upstream inhibitor of mammalian target of rapamycin complex 1 (mTORC1). We show that disrupted pathfinding in migrating mouse neural progenitor cells in vitro caused by STRADA depletion is prevented by mTORC1 inhibition with rapamycin or inhibition of its downstream effector p70 S6 kinase (p70S6K) with the drug PF-4708671 (p70S6Ki). We demonstrate that rapamycin can rescue aberrant cortical lamination and heterotopia associated with STRADA depletion in the mouse cerebral cortex. Constitutive mTORC1 signaling and a migration defect observed in fibroblasts from patients with PMSE were also prevented by mTORC1 inhibition. On the basis of these preclinical findings, we treated five PMSE patients with sirolimus (rapamycin) without complication and observed a reduction in seizure frequency and an improvement in receptive language. Our findings demonstrate a mechanistic link between STRADA loss and mTORC1 hyperactivity in PMSE, and suggest that mTORC1 inhibition may be a potential treatment for PMSE as well as other mTOR-associated neurodevelopmental disorders.

Cytoarchitectural alterations are widespread in cerebral cortex in tuberous sclerosis complex

Tubers are cerebral cortical developmental malformations associated with epilepsy and autism in tuberous sclerosis complex (TSC). The disparity between tuber number and severity of neurological impairment often observed in TSC led us to hypothesize that microscopic structural abnormalities distinct from tubers may occur in TSC. Serial frontal to occipital lobe sections were prepared from five postmortem TSC brain specimens. Sections were probed with cresyl violet stain or NeuN antibodies to define cytoarchitectural abnormalities and phospho-S6 (Ser235/236) antibodies to define mammalian target of rapamycin complex 1 (mTORC1) pathway activation. Tubers identified in all specimens (mean, 5 tubers per brain specimen) were defined by abnormal cortical lamination, dysmorphic neurons, and giant cells (GCs) and exhibited robust phospho-S6 immunolabeling. Histopathological analysis of non-tuber cortices demonstrated that 32% of the sections exhibited microscopic cytoarchitectural alterations, whereas 68% of the sections did not. Four types of morphological abnormalities were defined including: (1) focal dyslamination, (2) heterotopic neurons, (3) small collections of giant cells (GCs) and neurons we termed “microtubers”, (4) isolated GCs we termed “sentinel” cells. When compared with control cortex, phospho-S6 labeling was enhanced in microtubers and sentinel cells and in some but not all areas of dyslamination. There are microscopic cytoarchitectural abnormalities identified in postmortem TSC brain specimens that are distinct from tubers. mTORC1 cascade activation in these areas supports a widespread effect of TSC1 or TSC2 mutations on brain development. Tubers may represent the most dramatic developmental abnormality in TSC; however, more regionally pervasive yet subtle abnormalities may contribute to neurological disability in TSC.