[Show abstract][Hide abstract] ABSTRACT: The etiology of central nervous system (CNS) tumor heterogeneity is unclear. To clarify this issue, a novel animal model was developed of glioma and atypical teratoid/rhabdoid-like tumor (ATRT) produced in rats by non-viral cellular transgenesis initiated in utero. This model system affords the opportunity for directed oncogene expression, clonal labeling, and addition of tumor-modifying transgenes. By directing HRasV12 and AKT transgene expression in different cell populations with promoters that are active ubiquitously (CAG promoter), astrocyte-selective (GFAP promoter), or oligodendrocyte-selective (MBP promoter); thus, generating glioblastoma multiforme (GBM) and anaplastic oligoastrocytoma (AO), respectively. Importantly, the GBM and AO tumors were distinguishable at both the cellular and molecular level. Furthermore, proneural basic-helix-loop-helix (bHLH) transcription factors, Ngn2 (NEUROG2) or NeuroD1, were expressed along with HRasV12 and AKT in neocortical radial glia, leading to the formation of highly lethal atypical teratoid/rhabdoid-like tumors (ATRT). This study establishes a unique model in which determinants of CNS tumor diversity can be parsed out and reveals that both mutation and expression of neurogenic bHLH transcription factors contributes to CNS tumor diversity. Implications: A novel CNS tumor model reveals that oncogenic events occurring in disparate cell types and/or molecular contexts leads to different tumor types; these findings shed light on the sources of brain tumor heterogeneity.
Molecular Cancer Research 02/2014; · 4.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In utero RNAi of the dyslexia-associated gene Kiaa0319 in rats (KIA-) degrades cortical responses to speech sounds and increases trial-by-trial variability in onset latency. We tested the hypothesis that KIA- rats would be impaired at speech sound discrimination. KIA- rats needed twice as much training in quiet conditions to perform at control levels and remained impaired at several speech tasks. Focused training using truncated speech sounds was able to normalize speech discrimination in quiet and background noise conditions. Training also normalized trial-by-trial neural variability and temporal phase locking. Cortical activity from speech trained KIA- rats was sufficient to accurately discriminate between similar consonant sounds. These results provide the first direct evidence that assumed reduced expression of the dyslexia-associated gene KIAA0319 can cause phoneme processing impairments similar to those seen in dyslexia and that intensive behavioral therapy can eliminate these impairments.
PLoS ONE 01/2014; 9(5):e98439. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Variants in dyslexia-associated genes, including DCDC2, have been linked to altered neocortical activation, suggesting that dyslexia associated genes might play as yet unspecified roles in neuronal physiology.
Whole-cell patch clamp recordings were used to compare the electrophysiological properties of regular spiking pyramidal neurons of neocortex in Dcdc2 knockout (KO) and wild-type mice. Ribonucleic acid sequencing and reverse transcriptase polymerase chain reaction were performed to identify and characterize changes in gene expression in Dcdc2 KOs.
Neurons in KOs showed increased excitability and decreased temporal precision in action potential firing. The RNA sequencing screen revealed that the N-methyl-D-aspartate receptor (NMDAR) subunit Grin2B was elevated in Dcdc2 KOs, and an electrophysiological assessment confirmed a functional increase in spontaneous NMDAR-mediated activity. Remarkably, the decreased action potential temporal precision could be restored in mutants by treatment with either the NMDAR antagonist (2R)-amino-5-phosphonovaleric acid or the NMDAR 2B subunit-specific antagonist Ro 25-6981.
These results link the function of the dyslexia-associated gene Dcdc2 to spike timing through activity of NMDAR.
[Show abstract][Hide abstract] ABSTRACT: One in 15 school age children have dyslexia, which is characterized by phoneme-processing problems and difficulty learning to read. Dyslexia is associated with mutations in the gene KIAA0319. It is not known whether reduced expression of KIAA0319 can degrade the brain's ability to process phonemes. In the current study, we used RNA interference (RNAi) to reduce expression of Kiaa0319 (the rat homolog of the human gene KIAA0319) and evaluate the effect in a rat model of phoneme discrimination. Speech discrimination thresholds in normal rats are nearly identical to human thresholds. We recorded multiunit neural responses to isolated speech sounds in primary auditory cortex (A1) of rats that received in utero RNAi of Kiaa0319. Reduced expression of Kiaa0319 increased the trial-by-trial variability of speech responses and reduced the neural discrimination ability of speech sounds. Intracellular recordings from affected neurons revealed that reduced expression of Kiaa0319 increased neural excitability and input resistance. These results provide the first evidence that decreased expression of the dyslexia-associated gene Kiaa0319 can alter cortical responses and impair phoneme processing in auditory cortex.
[Show abstract][Hide abstract] ABSTRACT: Human embryonic stem cell-derived neuronal progenitors (hNPs) provide a potential source for cellular replacement following neurodegenerative diseases. One of the greatest challenges for future neuron replacement therapies will be to control extensive cell proliferation and stimulate cell migration of transplanted cells. The doublecortin (DCX) gene encodes the protein DCX, a microtubule-associated protein essential for the migration of neurons in the human brain. In this study, we tested whether increasing the expression of DCX in hNPs would favorably alter their proliferation and migration. Migration and proliferation of hNPs was compared between hNPs expressing a bicistronic DCX/IRES-GFP transgene and those expressing a green fluorescent protein (GFP) transgene introduced by piggyBac-mediated transposition. The DCX-transfected hNPs showed a significant decrease in their proliferation and migrated significantly further on two different substrates, Matrigel and brain slices. Additionally, a dense network of nestin-positive (+) and vimentin+ fibers were found to extend from neurospheres transplanted onto brain slices, and this fiber growth was increased from neurospheres containing DCX-transfected hNPs. In summary, our results show that increased DCX expression inhibits proliferation and promotes migration of hNPs.
[Show abstract][Hide abstract] ABSTRACT: Methods that combine lineage tracing with cellular transgenesis are needed in order to determine mechanisms that specify neural cell types. Currently available methods include viral infection and Cre-mediated recombination. In utero electroporation (IUE) has been used in multiple species to deliver multiple transgenes simultaneously into neural progenitors. In standard IUE, most plasmids remain episomal, are lost during cell division, and so transgenes are not expressed in the complete neural lineage. Here we combine IUE with a binary piggyBac transposon system (PB-IUE), and show that unlike conventional IUE, a single embryonic transfection of neocortical radial glia with a piggyBac transposon system results in stable transgene expression in the neural lineage of radial glia: cortical neurons, astrocytes, oligodendrocytes, and olfactory bulb interneurons. We also developed a modular toolkit of donor and helper plasmids with different promoters that allows for shRNA, bicistronic expression, and trangenesis in subsets of progenitors. As a demonstration of the utility of the toolkit we show that transgenesis of epidermal growth factor receptor (EGFR) expands the number of astrocytes and oligodendrocyrtes generated from progenitors. The relative ease of implementation and experimental flexibility should make the piggyBac IUE method a valuable new tool for tracking and manipulating neural lineages.
Journal of neuroscience methods 04/2012; 207(2):172-80. · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The ability to introduce gain- or loss-of-function constructs into developing organisms is responsible for the majority of advances made in the field of developmental biology. Here we provide step-by-step methods on how to achieve cellular transgenesis in the developing mammalian neocortex. In utero electroporation (IUE) is a surgical preparation that allows for rapid manipulation of gene expression and is becoming an experimental standard in the field of neocortical development. We provide detailed description of the equipment required and the procedures necessary for successful embryonic brain transfection. In addition, we will discuss several different experimental approaches that demonstrate how IUE can be adapted to spatially and temporally control gene expression.
[Show abstract][Hide abstract] ABSTRACT: Myoclonin1/EFHC1 mutations cause 9% of juvenile myoclonic epilepsy (JME) by impairing apoptosis in neurons/synapses where R type voltage dependent calcium channel function is altered, imbuing susceptibility to myoclonic and grand mal seizures. By disrupting mitotic spindle assembly and radial migration of neuroblasts, mutations could produce abnormal intracortical architecture upon which epileptogenesis is established. For an expanded treatment of this topic see Jasper’s Basic Mechanisms of the Epilepsies, Fourth Edition (Noebels JL, Avoli M, Rogawski MA, Olsen RW, Delgado-Escueta AV, eds) published by Oxford University Press (available on the National Library of Medicine Bookshelf [NCBI] at http://www.ncbi.nlm.nih.gov/books).
[Show abstract][Hide abstract] ABSTRACT: Epilepsy-associated glioneuronal malformations (malformations of cortical development [MCD]) include focal cortical dysplasias (FCD) and highly differentiated glioneuronal tumors, most frequently gangliogliomas. The neuropathological findings are variable but suggest aberrant proliferation, migration, and differentiation of neural precursor cells as essential pathogenetic elements. Recent advances in animal models for MCDs allow new insights in the molecular pathogenesis of these epilepsy-associated lesions. Novel approaches, presented here, comprise RNA interference strategies to generate and study experimental models of subcortical band heterotopia and study functional aspects of aberrantly shaped and positioned neurons. Exciting analyses address impaired NMDA receptor expression in FCD animal models compared to human FCDs and excitatory imbalances in MCD animal models such as lissencephaly gene ablated mice as well as in utero irradiated rats. An improved understanding of relevant pathomechanisms will advance the development of targeted treatment strategies for epilepsy-associated malformations.
Epileptic disorders: international epilepsy journal with videotape 10/2009; 11(3):206-14. · 1.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This chapter presents recent works on Myocloni1/ EFHC1 a protein encoded by an epilepsy causing gene of juvenile myoclonic epilepsy (JME), one of the most frequent forms of idiopathic or genetic generalized epilepsies. Myoclonin 1/EFHC1 is a microtubule-associated protein involved in the regulation of cell division. In vitro, EFHC1 loss of function disrupted mitotic spindle organization, impaired M phase progression, induced microtubule bundling and increased apoptosis. EFHC1 impairment in the rat developing neocortex by ex vivo and in utero electroporation caused a marked disruption of radial migration. This effect was a result of cortical progenitors failing to exit the cell cycle. On the other hand, defects in the radial glia scaffold organization and in the locomotion of postmitotic neurons. Mutant analysis of Defhc1 loss- and gain-of-function alleles in vivo in Drosophila revealed a number of neuronal defects, including abnormal synaptic development characterized by extensive satellite bouton formation, increased frequency of spontaneous neurotransmitter release, and aberrations in dendritic arbour morphogenesis. Thus, Myoclonin 1/ EFHC1 is a regulator of cell division and neuronal migration during cortical development synaptic bouton and dendritic morphogenesis. Disruption of these properties lead to JME, being now therefore considered as a developmental disease.