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 TS

Co-expression of cyclin D1 and phosphorylated ribosomal S6 proteins in hemimegalencephaly

Hemimegalencephaly (HMEG) is a developmental brain malformation highly associated with epilepsy. Balloon cells (BCs) and cytomegalic neurons (CNs) are frequently observed in HMEG specimens. Cytomegaly in developmental brain malformations may reflect in aberrant activation of the mTOR and beta-catenin signaling cascades, known regulators of cell size. We hypothesized that there is aberrant co-expression of phospho-ribosomal S6 (P-S6) protein, a downstream effector of the mTOR cascade, as well as cyclin D1, a downstream effector of the beta-catenin pathway, in BCs and cytomegalic neurons in HMEG. We hypothesized that mutations in PTEN (a cause of HMEG associated with Proteus syndrome), TSC1 or TSC2 (tuberous sclerosis complex) genes, which are known to modulate beta-catenin and mTOR signaling could cause sporadic HMEG. Expression of cyclin D1, phospho-p70 S6 kinase (P-p70S6K, another mTOR cascade kinase), P-S6, MAP2, NeuN, or GFAP was determined by immunohistochemistry in HMEG brain tissue (n = 7 specimens). Cyclin D1, P-p70S6K, and P-S6 proteins were co-localized in BCs and CNs in the enlarged hemisphere but not in the unaffected hemisphere or in morphologically normal tissue. Cyclin D1 and P-S6 proteins were not detected in GFAP-labeled astrocytes. Sequencing of PTEN, TSC1, and TSC2 genes in cytomegalic cells co-expressing cyclin D1 and P-S6 proteins did not reveal mutations. Selective expression of cyclin D1 and P-S6 in cytomegalic cells in HMEG suggests co-activation of the beta-catenin and mTOR cascades. PTEN, TSC1, or TSC2 gene mutations were not detected suggesting that sporadic HMEG is distinct from HMEG associated with Proteus syndrome or tuberous sclerosis comple

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. ANN NEUROL 2012;72:881-89

Developmental lineage of cell types in cortical dysplasia with balloon cells

Focal cortical dysplasia type IIB with Ballon cells is a developmental malformation of the cerebral cortex highly associated with epilepsy. As a strategy to define the embryonic origin and neurochemical phenotype of cells in this disease, we probed specimens (n = 10) resected during epilepsy surgery with a panel of 13 antibodies recognizing proteins associated with (i) specific progenitor cell types including brain lipid binding protein (BLBP), collapsin response mediator protein 4 (CRMP4), Dlx1, Dlx2, GFAPdelta, MASH1, Otx1, Pax6, vimentin and phosphorylated vimentin and (ii) excitatory or inhibitory neurochemical phenotypes such as the vesicular glutamate transporters-1 and 2 (VGLUT-1, VGLUT-2), or the vesicular GABA transporter (VGAT). Balloon cells and large dysplastic neurons in all specimens expressed Otx1, phospho-vimentin, Pax6 and BLBP, proteins normally expressed by cells in the embryonic ventricular zone. A subpopulation of balloon cells expressed MASH-1 also expressed in the ventricular zone. Most balloon cells and dysplastic neurons were VGLUT2 immunoreactive, whereas none expressed Dlx1 or Dlx2, markers for inhibitory cells derived from the medial ganglionic eminence and few expressed VGAT, found in GABAergic interneurons. Otx1 mRNA expression and Dlx1 mRNA absence was confirmed by single cell RT-PCR. A subpopulation of balloon cells was labelled with CRMP4 and GFAPdelta, markers specific for newly generated cells derived from the adult subventricular zone. Detection of Otx1, phospho-vimentin, Pax6 and BLBP expression but absence of Dlx1/Dlx2 expression suggests that balloon cells and dysplastic neurons derive from radial glial cells in the telencephalic ventricular zone and not the medial ganglionic eminence. VGLUT expression argues that dysplastic neurons may be glutamatergic. CRMP-4 and GFAPdelta expression suggests that new cells may arrive in focal cortical dysplasia, perhaps deriving in part from the subventricular zone. These findings provide a developmental lineage model in which balloon cells and dysplastic neurons are derived from radial glial progenitor cell

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 mechanism

Targeted gene expression analysis in hemimegalencephaly: activation of beta-catenin signaling

Hemimegalencephaly (HMEG) is a developmental brain malformation characterized by unilateral hemispheric enlargement, cytoarchitectural abnormalities, and an association with epilepsy. To define the developmental pathogenesis of HMEG, the expression of 200 cell signaling, growth, angiogenic, and transcription factor genes was assayed in HMEG samples (n=8) with targeted cDNA arrays. Differential expression of 31 mRNAs across the 4 gene families was identified in HMEG compared with control cortex. Increases in growth and transcription factor genes included JNK-1, cyclic AMP response element binding protein (CREB), and tuberin mRNAs and decreases included insulin-like growth factor-1 (IGF-1), transforming growth factor beta-3 (TGF-beta3), and NFkB mRNAs. Increased expression of cyclin D1, c-myc, and WISP-1 mRNAs in HMEG suggested activation of the Wnt-1/beta-catenin cascade. Western analysis demonstrated increased levels of non-phosphorylated beta-catenin, which transcriptionally activates cyclin D7 and c-myc genes, but reduced levels of Ser33/Ser37/Thr41 phospho-beta-catenin, which is essential for beta-catenin-inactivation, in HMEG. Altered expression of 31 mRNAs from 4 gene families in human HMEG may lead to aberrant cell growth and hemispheric enlargement during brain development. Enhanced cyclin D1 and c-myc transcription likely reflects increased transcriptionally active beta-catenin due to decreased Ser33/Ser37/Thr41 phospho-beta-catenin and suggests activation of the Wnt-1/beta-catenin cascade in HME

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 of 2 mTORC1 substrates, S6 and S6 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 specimen