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Multi-level functional studies of candidate dyslexia susceptibility genes in the rat

Project I - Dyx1c1 and Mechanisms of Neuronal Migration in Neocortex

Project II - Postnatal Anatomic and Connectional Effects of Disruption of Candidate Dyslexia Susceptibility Genes

Project II - Behavioral Consequences of Embryonic Disruption of Candidate Dyslexia Susceptibility Genes -

Transfected NeuronsThe proposed Program Project is to study a unique rat model of developmental learning disability that uses methods of developmental neurobiology, structural anatomy, and behavior to analyze the functions of three candidate dyslexia susceptibility genes (CDSGs). Neuropathologic studies in human dyslexic brains and previous animal models have underscored the importance of focal neuronal migration defects and developmental plasticity for some of the dyslexic deficits. The discovery of CDSGs challenges us to analyze the effects of this genetic variation on brain development, structure, and behavior with respect to learning disability. Using an in utero electroporation method developed in our laboratories, we will transfect into young neurons in the ventricular zone short hairpin RNAs or over-expression constructs targeted against homologs in the rat of CDSG Dyx1c1, Kiaa0319, or Dcdc2. We have already seen that this procedure leads to abnormal neuronal migration, alters neuronal morphology, and causes secondary effects in untouched neighboring neurons, thus producing a picture reminiscent of dyslexic brains. Interesting behavioral alterations are also seen. Project I (J.J. LoTurco, PI) will analyze Dyx1c1's interaction with genes with known molecular pathways involved in process extension, nuclear movement, and cell adhesion, the domains on the Dyx1c1 critical to function. Project II (A.M. Galaburda, PI) will characterize anatomic changes (cortical architecture, cell identity, morphology, and connectivity) associated with knockdown or overexpression of CDSGs. Project III (H. Fitch, PI) will uncover behavioral consequences of CDSG disruption (auditory processing and learning), and will attempt to ameliorate the effects of these genetic manipulations by behavioral interventions. The three interactive projects will be supported by an Administrative Core, an In Utero Electroporation Core, and a Neurohistology, Morphometry, and Data Processing Core. A better understanding of the functions of CDSGs will shed a broader light on mechanisms of normal brain development and on the abnormalities seen in developmental dyslexia, but also offering the possibility of earlier detection, biologically-based subtyping, and improved treatment.s.

Project I - Candidate Dyslexia Susceptibility Genes and Mechanisms of Neuronal Migration in Neocortex

Dyx1c1The specific causes of dyslexia are not yet known. Recent genetic and neurobiological studies strengthen a working hypothesis that dyslexia is caused by early developmental disruptions that subsequently cause functional impairments in neocortical circuits. Within the past four years, 4 candidate dyslexia susceptibility genes with roles in neuronal development have been proposed (DYX1C1, KIAA0319, DCDC2 and ROBO1). Rodent homologs of three of these, Dyx1c1, Kiaa0319 and Dcdc2 have been shown by our group to play a role in neuronal migration in developing neocortex, and Robo1 was previously shown to be important for axon growth and guidance. The three aims of this project will further define the cellular and developmental roles of Dyx1c1. The proposed experiments will define the compo-nents of neuronal migration and differentiation regulated by Dyx1c1, and identify functional links between Dyx1c1 and other proteins essential to migration. The aims will be executed by a combination of in utero RNAi, imaging, protein-protein interaction assays, and cell culture approaches. Novel in vivo conditional RNAi and overexpression methods will be used to explore the temporal dependence of Dyx1c1 function, and potential developmental reversibility of Dyx1c1 dysfunction. Together, these experiments will lead to a comprehensive molecular and cellular understanding of the function of a gene linked to reading and learning disability.

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Project II - Postnatal Anatomic and Connectional Effects of Disruption of Candidate Dyslexia Susceptibility Genes

Periventricular Nodular HeterotopiasRecent evidence indicates that candidate dyslexia susceptibility genes (CDSGs) have roles in the development of the cerebral cortex, especially in neuronal migration and maturation. In Project II, we will investigate postnatal anatomic consequences of neuronal migration disorders induced by embryonic transfection with small hairpin RNAs (shRNA) targeted against CDSG homologs Dyx1c1, Kiaa0319, or Dcdc2 in the rat cerebral cortex. Based on preliminary results, and because it is not yet known in humans whether all of these gene variants result in loss of function, we will also investigate the effects of CDSG overexpression. Since all CDSGs share among them an association with dyslexia, in Aim 1 we will address anatomical RNAi and overexpression phenotypes that appear to be shared among the genes—namely a bimodal distribution of transfected cells that either undermigrate or migrate past their expected laminar locations. We will use molecular and birthdate markers to assess the phenotypes of these mismigrated neurons, whether or not layer appropriate. In addition, we will co transfect gain and loss of function neurons with a wheat germ agglutinin transgene that will allow determination of the connectivity of transfected neurons in both control and experimental cases. We will compare the intra- and inter-hemispheric, cortico cortical, cortico thalamic, and thalamo-cortical connections in rats transfected with different CDSG shRNAs, as well as between experimentals and controls. In the expectation that this work can guide research on dyslexia subtyping, Aim 2 will focus on systematic differences that are seen in the brains of rats embryonically transfected with shRNA or overexpression plasmids for each of the CDSG homologs. Following completed work in embryos, we will use in situ hybridization and immunohistochemistry to compare the genes’ temporal and spatial expression patterns in the postnatal rat. We will also assess the neuronal morphology of transfected neurons and their processes. Aim 3 examines widespread changes in anatomic organization, which are hypothesized to arise directly from local transfections of shRNA or overexpression constructs and as a result of secondary plasticity-related effects. We will use efficient and accurate stereologic probes to estimate neuron number, neuron size, and regional volume throughout the neocortex and thalamus. An accurate description of the forebrain anatomy that results from either knockdown or overexpression of rat homologs of CDSGs, both cell autonomous and secondary effects, and the course of their development, serve as a good bridge between genetics and behavior and will help to shed a broader light on the neurobiological substrates underlying developmental dyslexia in humans. We will link results from this project down to developmental and molecular mechanisms studied in Project I and up to behavioral changes to be characterized in Project III.

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Project III- Behavioral Consequences of Embryonic Disruption of Candidate Dyslexia Susceptibility Genes

NeuronsAccumulating evidence that core phonological processing problems in language-disabled subjects may relate to more basic deficits in rapid auditory processing has introduced new possibilities for the use of animal models in the study of developmental language disorders (e.g., dyslexia). Studies performed in our lab over the past decade reveal that cortical neuronal migration anomalies (similar to those seen in post mortem brains of dyslexics) are associated with behavioral deficits in rapid auditory processing (RAP), as well as in short-term memory (STM), in rodents. Moreover, RAP deficits are larger in juvenile as compared to adult male rats, are seen following cortical neuronal migration disruption in various species, and are larger in male as compared to female rats and mice. RAP deficits are also consistently seen in the absence of overall auditory processing impairments (e.g., performance on longer-stimulus acoustic discrimination tasks is normal). Thus, convergent findings from rodent models parallels behavioral and anatomic findings from human language disabled populations in a variety of ways. These data led us to perform behavioral assessments in rats following embryonic interference with the functions of gene homologs associated (in humans) with dyslexia. We found that E14/15 transfection with RNAi for the candidate dyslexia susceptibility rat gene homolog, Dyx1c1, led to subsequent impairments of rapid/complex acoustic discrimination in male rats (though no deficits for discriminating longer gap stimuli were seen). Persistent deficits were also found for Dyx1c1 subjects on a complex short-term memory (STM) task. Such findings have enormous translational potential for dyslexia research, by linking data across levels of genetic disruption, neurodevelopmental disruption, and disruption of cognition/behavior.

The proposed studies will continue to address the neuropathological/behavioral consequences of embryonic manipulation of rat homologs for three candidate dyslexia susceptibility genes (Dyx1c1, Kiaa0319, and Dcdc2). Rats undergoing embryonic transfection with RNAi (or induced gene over- expression) will be evaluated on auditory, visual, and learning/memory tasks, as well as for post mortem neuropathology. Results will be assessed for evidence of genetic and neuropathological factors associated with specific behavioral deficits in RAP and STM that parallel deficits in language-disabled humans (in contrast to more general cognitive, motor, and/ or sensory deficits). Such studies may bridge the gap between disrupted brain function/behavior in dyslexics, epidemiological evidence of genetic associations with dyslexia, and the critical intervening neurodevelopmental processes that are so difficult to study in humans, but so amenable to study in rodent models

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© 2008. Page created by Alena Rosen and maintained by Glenn Rosen. Last updated Febrruary, 2009.