Peter Crino - US grants

DESCRIPTION: (applicant's abstract) Human focal cortical dysplasia (FCD) is a developmental brain malformation characterized histologically by disorganized cerebral cortical cytoarchitecture and lamination. FCD is associated with several mental disorders including mental retardation (MR) and autism. FCD likely results from abnormal neuronal migration during corticoneogenesis although the molecular events that culminate in aberrant cortical lamination are unknown. Previous work has suggested that FCD neurons may have failed to terminally differentiate prior to migration since these cells express proteins, such as the intermediate filament nestin, that are typically identified in immature neuronal precursor cells. These, dysplastic neurons may not express other developmentally appropriate genes necessary to complete migration and lamination. This proposal will describe three approaches to study the developmental and molecular pathogenesis of FCD. First, expression of transcription factors, neurotrophic factors/receptors and cell adhesion molecules mRNAs will be assayed in single nestin- or MAP1B-immunolabeled cells in human FCD specimens and compared with normal cortical neurons, and neuronal precursors in the ventricular zone. Because expression of these candidate genes varies during early development and because they have been implicated in corticogenesis, coordinate change in their relative abundance will provide insights into molecular pathways altered in FCD. Second, developmentally inappropriate genes such as s nestin or MAP1B will be overexpressed in cortical slice cultures and effects on migration will be assayed. Finally, dynamic changes in gene expression will be assayed in actively migrating neurons in animal models of FCD including administration of exogenous neurotrophins or antisense oligonucleotides. It is anticipated that these studies will provide for the first time a view of altered gene expression in FCD, which will shed light on the pathogenesis of these lesions. Furthermore, in identifying alterations in specific genes, the relationship between FCD and mental disorders can be rendered. These analyses may point toward new avenues for therapy.

DESCRIPTION (Applicant's Abstract): Focal regions of disorganized cortical lamination and abnormal neuronal cytoarchitecture (focal cortical dysplasia, FCD) are in utero developmental brain malformations which have been identified in many neuropsychiatric disorders including mental retardation syndromes (MR), autism, and epilepsy. FCD likely results from abnormal neural migration during corticogenesis although the molecular events leading to aberrant cortical lamination in these divergent conditions are unknown. The major difficulty in addressing the molecular pathogenesis of FCD is that many epilepsy and mental retardation syndromes are of unclear inheritance pattern rendering a genetic or pedigree analysis complicated. Thus, novel strategies must be implemented to address two compelling questions regarding the pathogenesis of FCD: 1) what is the phenotype of dysplastic cells and 2) what mechanisms are responsible for disorganized cortical lamination during corticogenesis. Answers to these questions will shed light on the practical question of how altered laminar and cellular cytoarchitecture in FCD contributes to the neurological manifestations such as seizures, MR, and behavioral abnormalities in such a wide variety of neurological disorders. Three sets of experiments will investigate the phenotype of dysplastic cells in human FCD specimens and assess the expression of developmentally regulated genes necessary for cortical lamination in an experimental model of FCD. Because of the heterogeneous cell types within FCD, a central feature of the proposed experiments is the use of single cell mRNA amplification technology. This novel approach permits quantification of mRNA abundance in individual immunolabeled or live neurons that are of similar or distinct phenotype. Analysis of human FCD specimens will provide a direct avenue to investigate the developmental phenotype of FCD neurons. The model FCD system will permit assessment of gene regulation during the dynamic phases of cortical migration. The overall objective of the proposal is to determine the molecular pathways that lead to disorganized cortical cytoarchitecture in FCD. Furthermore, by identifying altered expression of select genes, the relationship between FCD and various mental disorders can be discerned. These analyses may point toward new avenues for therapy.

Epilepsy is a neurologic disorder that effects between 1-4% of the population and is associated with significant morbidity and even mortality. The birth of new neurons and astrocytes (cellular proliferation) is a unique and central pathologic process that occurs in the hippocampus of patients with epilepsy that likely contributes to epileptogenesis and may be accentuated by recurrent seizures. The genes that regulate cell proliferation in epilepsy have not been identified and understanding why cell proliferation occurs in epilepsy will yield insight into seizure initiation in select patients. Indeed, the molecular pharmacologic phenotype of new cells may be distinct from existing hippocampal astrocytes and neurons ad thus, may provide critical changes in receptor or ion channel expression that fosters seizure initiation. Thus, one term goal of the proposed studies is to identify select cell populations in epilepsy that can be targeted for cell specific therapeutic modulation. The objective of this proposal is to study the molecular basis of neuronal and glial proliferation in human epilepsy and to define the molecular pharmacologic phenotype of newly born neurons and astrocytes. The goals of our proposal match well with the mission statement of the RFA and provide an intensive plan to investigate the mechanisms that modulate cellular proliferation in epilepsy. To accomplish these goals, we will assay cellular proliferation in a well-characterized rodent seizure model system and then in human epilepsy samples. The experiments in the proposal will: 1) identify select populations of newly divided neurons and astrocytes in the dentate gyrus by BrdU and immunohistochemical approaches, 2) determine the electrophysiologic properties of these proliferating cells so they can be readily identified in human epilepsy samples, 3) use a targeted strategy to define altered expression of several candidate gene families in experimental and human epilepsy, and 4) corroborate the changes in gene expression with protein assays including Western analysis and immunohistochemistry.

[unreadable] DESCRIPTION (provided by applicant): The tuberous sclerosis complex (TSC) is an autosomal dominant, multisystem disorder that affects the brain and results from mutations in one of two genes, TSC1, encoding hamartin, and TSC2, encoding tuberin. Neurological complications of TSC are the most disabling and include epilepsy in over 70-80% of TSC patients, as well as autism and mental retardation in half of TSC patients. These neuropsychiatric abnormalities in TSC result from the effects of cortical tubers, the characteristic brain lesions of TSC, on brain function. Tubers are developmental abnormalities of cerebral cortical cytoarchitecture (a form of cortical dysplasia) characterized histologically by disorganized cortical lamination and cells with aberrant morphologies. The prominent abnormal cell types in tubers are dysplastic neurons (DN), giant cells (GC), and abnormal astrocytes. Tubers are epileptogenic and seizures in TSC patients are often refractory to medical management despite anticonvulsant polytherapy. Surgical resection of tubers may be necessary to achieve adequate seizure control. The number of tubers present in TSC patients seems to correlate with the onset and severity of mental retardation and autism in TSC patients. The broad goal of this grant proposal is to investigate how hamartin and tuberin mutations contribute to tuber formation using 3 experimental paradigms. We will determine whether tubers form as a result of a "second hit" somatic mutation or haploinsufficency using a high resolution analysis of TSC1 and TSC2 genes in single microdissected GCs, DNs, and astrocytes. Second, we define the expression of five candidate gene and protein families that are pivotal in normal corticogenesis including cell adhesion molecules, transcription factors, growth factors, and cytoskeletal elements in single DNs, GCs, and astrocytes and then relate these changes in expression to the mutational state of these cell types. Third, the expression of the five candidate gene and protein families will be determined during distinct epochs of cortical developmental in 3 transgenic mouse strains in which tuberin or hamartin have been completely or conditionally knocked out. These experiments provide a strategy to define the molecular mechanism of tuber formation as a direct consequence of TSC gene mutations and the downstream effects on gene expression within distinct populations of cells at defined developmental timepoints.

Malformations of cortical development (MOD) are the most common cause of intractable epilepsy in children. Focal cortical dysplasia (FCD) and hemimegalencephaly (HMEG) affect restricted brain regions. FCD and HMEG are sporadic disorders and have no identified cause. FCD and HMEG share similar histological features including cells exhibiting cytomegaly and abnormal morphology called balloon cells (BCs). We have shown that there is cell selective activation of the PI3K-AKT-TOR-p70S6kinase-ribosomal S6 pathway in FCD and HMEG as evidenced by expression of phosphorylated ribosomal S6 protein (phospho-S6) only in BCs. We have also identified increased activity of the Wnt/beta-catenin pathway in FCD and HMEG. We propose that FCD and HMEG result from related pathogenic mechanisms and that BCs are specifically generated by somatic gene mutations leading to activation of PI3K-AKT-TOR- p70S6kinase-ribosomal S6 or Wnt/beta-catenin cascades. To test this hypothesis, we will first demonstrate immunohistochemically that the PI3K-AKT-TOR-p70S6kinase-ribosomal S6 and Wnt/beta-catenin cascades are activated in BCs compared with cytomegalic neurons and astrocytes in FCD and HMEG. Second, we will use single cell gene expression analysis to identify altered expression of candidate genes that modulate the TOR and Wnt pathways as well as to investigate other candidate gene pathways that may contribute to increased cell size in FCD and HMEG. Third, we will use 4 mutation analysis strategies to define whole gene, multi-exon, single exon, or point mutations that may be causative in FCD and HMEG. Single nucleotide polymorphism (SNP) array analysis will permit a whole genome approach to define candidate gene loci. Loss of heterozygosity and multiplex-ligation dependent probe analysis (MLPA) assays will detect whole gene and exonic deletions. We have developed atechnique to sequence genes from cDNA isolated from single microdissected cells or from genomic DMAisolated from pooled cells so that we can directly sequence at least 20 candidate genes that modulate the PI3K-AKT-TOR-p70S6kinase-ribosomal S6 and Wnt/beta-catenin pathways. These studies provide a targeted, pathway directed strategy to identify altered protein expression, gene transcription, and gene sequence that lead to the formation of FCD and HMEG during brain development.

DESCRIPTION (provided by applicant): Tuberous sclerosis complex (TSC) is an autosomal disorder resulting from mutations in the TSC1 or TSC2 genes that is associated with epilepsy, cognitive disability, and autism. TSC1/TSC2 gene mutations lead to developmental alterations in brain structure known as tubers in over 80% of TSC patients. Loss of TSC1 or TSC2 function in tubers results from biallelic TSC gene inactivation and leads to activation of the mTOR cascade as evidenced by phosphorylation of ribosomal S6 protein (P-S6). Several new findings warrant further investigation of the mechanisms through which TSC gene mutations lead to developmental alterations in brain structure. Recent MRI studies suggest that there are subtle widespread abnormalites in TSC brains that contribute to neurocognitive deficits and in vitro evidence suggests that reduction of Tsc1 in rat neurons leads to altered dendritic structure. First, we will define subtle structural alterations distinct from tubers in post-mortem TSC brain specimens in the cortex, thalamus, basal ganglia, and cerebellum which may contribute to neurocognitive abnormalites in TSC using neuronal and astrocytic protein markers. Then, in these non-tuber brain lesions we will define P- S6 expression as a strategy to determine whether cells in non-tuber brain lesions exhibit mTOR cascade activation similar to tubers. Next, we will identify somatic second hit mutations in single microdissected P-S6 labeled cells in non-tuber brain lesions as a strategy to define whether all structural abnormalites in TSC require biallelic TSC gene inactivation. Then, we will determine whether P-S6 labeled giant cells in tubers and non- tuber brain lesions express a single or multiple somatic second hit mutations to test the hypothesis that structural lesions form by a clonal cellular expansion. In the second experiments, we will transfect cultured rat neocortical neurons at embryonic day 16 with Tsc1 or Tsc2 shRNA to define the effects of reduced Tsc1 or Tsc2 on dendrite outgrowth and expression of dendritic mRNAs. In the third experiments, we will define the differential expression of microRNAs (miRNAs) in P-S6 labeled giant cells and in P-S6 labeled astrocytes from the Tsc1GFAP cre mouse strain. These short non-coding RNAs play a pivotal role in translational regulation and interact with several proteins including EIFs and STAT3 that mediate the effects of TSC1/TSC2 in neurons. These experiments provide new mechanistic strategies to define how loss of TSC1/TSC2 leads to altered brain structure is TSC. PUBLIC HEALTH RELEVANCE Tuberous sclerosis complex (TSC) is an autosomal dominant, multisystem disorder that affects the brain, skin, kidney, heart, and lungs. The neurological manifestations include epilepsy, autism, hydrocephalus, and cognitive impairments. We propose to more fully define the extent of brain involvement in TSC by analyzing the brain structure of 10 post- mortem TSC brains with protein specific antibodies. In a second set of experiments we will define novel mechanisms that regulate dendrite outgrowth in neurons regulated by the TSC encoded proteins. These studies will provide new insights into the mechanisms leading to epilepsy, autism, and cognitive impairment in TSC.