Joseph J. LoTurco - US grants

DESCRIPTION (Adapted from applicant's abstract): Several disorders in mental function have been correlated with cellular aberrations in the cerebral cortex. The nature of these aberrations indicates that they arise early in development during the genesis and migration of neurons. All neurons within the cerebral cortex are generated within a proscribed region, the ventricular zone (VZ), throughout a discrete and defined period. It is becoming increasingly clear that signals acting within the VZ regulate both the proliferation and ultimate identity of cortical progenitors. The experiments proposed here will elucidate cellular signaling mechanisms within the VZ that are mediated by activation of GABA and glutamate receptors. GABA and glutamate depolarize, increase intracellular calcium concentration, and decrease the synthesis of DNA in the dividing population of cells within the VZ. In the experiments described here, a combination of patch-clamp techniques, calcium imaging, and assays of cell cycle kinetics will be applied to an in vitro explant preparation of developing cerebral cortex in order to further understand the mechanisms by which GABA and glutamate regulate cortical neurogenesis. The specific objectives of the proposed experiments are to (1) identify the circuits in the developing cortex that release the endogenous GABA and glutamate agonists which regulate the cell cycle of VZ cells, (2) specify effects of GABA and glutamate on cell cycle kinetics, and (3) determine whether the rise in intracellular calcium initiated by GABA and glutamate subserves the alterations in cell cycle. The results from these experiments will show how the genesis of neurons in the neocortex is regulated by diffusible signals released from circuits within the developing brain.

9729129 NISENBAUM A fundamental question in developmental neurobiology is how axons from specific neurons in one region of the brain form precise connections with target neurons in another region of the brain. Experiments examining the development of target specificity have revealed two types of mechanisms that contribute to the wiring of neural connections. The first type of mechanism, which occurs during the embryonic and early postnatal period, relies on guidance molecules to direct axonal projections along a path to their correct target region. The second type of mechanism, which takes place later in development, involves activity-dependent events to refine the precise pattern of neuronal connections. Although a great deal is known regarding the development of neuronal circuits within the brain, the development of the connections between the cerebral cortex and the basal ganglia have been largely ignored. The basal ganglia play a critical role in the control of movement and cognitive behaviors. Thus, the formation of precisely patterned connections from the cortex to the primary input structure of the basal ganglia, the striatum, is necessary for the proper functioning of the motor system. The goal of Dr. Nisenbaum's research is to determine when crucial target selection and pattern formation decisions are made during the course of corticostriatal development. The results from these studies will provide a better understanding of the types of mechanisms that are responsible for the precision of corticostriatal connectivity, and also provide insight into how motor systems become organized during development.

IBN 98-07941 KORN Heart, brain, endocrine and muscle cells are called excitable cells, and rely on the precisely coordinated action of many ion channels for their function. These channels in the plasma membrane of the cell open and close in response to physiological stimulation, and allow the flux of ions into or out of the cell. Flux of calcium and sodium ions through their respective channels tends to subserve excitatory functions, and flux of potassium through potassium channels underlies many inhibitory functions. Although control of excitable behavior is extremely complex, the magnitude and time-course of potassium flux through potassium channels is often a critical factor in determining the duration of excitatory events. There are many different potassium channels, with subtly different structures. This subtle structural variation among channels results in a large degree of functional diversity. Both the magnitude and time-course of potassium channel activity is sensitive to the concentration of potassium and sodium ion in the extracellular space (the region just outside of a cell), and this sensitivity also varies among channels. Two potassium binding sites have been functionally isolated, a low affinity site that underlies some channel functions and a high affinity site that underlies others. In particular, the high affinity site, located at the "selectivity filter," underlies the ability of potassium to prevent sodium from passing through the cell. The low affinity site is not involved in selectivity but is involved in several other channel functions. Recent structural studies have identified the molecular location of the selectivity filter, and the associated high affinity site(s). These studies were unable to identify a cation binding site external the site of selectivity. Consequently, either one of these putative high affinity sites contributes to the low affinity site functions or there is an as yet unknown cation binding site, external to the st ructurally described sites, which cannot be observed in structural studies. The primary focus of these studies will be to determine whether the structurally described selectivity filter site(s) interconvert between low affinity and high affinity sites, or whether a low affinity site exists external to and independent from the high affinity site. The studies will combine molecular biological techniques with electrophysiological recordings and biophysical analysis to determine whether the different functionally- identified binding sites are structurally distinct. Results from this project will enhance our understanding of the nature of cation binding sites in the channel pore, and will thus enhance our knowledge of the molecular mechanisms that underlie phenotypic diversity in potassium channels.

DESCRIPTION (provided by applicant): Analysis of spontaneous mutations in rodents and humans has proven invaluable in identifying molecular mechanisms essential to brain development. Most notably, proteins essential to patterned migration of cortical neurons have been identified through genetic analyses and positional cloning. In contrast, there have been few spontaneous mutations that have led to the identification of pathways involved in the control of cytokinesis and associated cellular dynamics that occur throughout neurogenesis at the surface of the ventricles. The regulation of cytokinesis plays a central role in neocortical neurogenesis, and may determine the fates of newly generated daughter cells. In the first period of this grant we discovered a novel mutant, flathead, which has now revealed an essential molecular mechanism that operates to regulate cytokinesis and neurogenesis in neuronal progenitors. The CNS-specific phenotype of the flathead mutant is characterized by severe micrencephaly with alterations in the relative numbers of different neuronal cell types, and in abnormal neuronal hypertrophy. A positional cloning strategy was used to identify the flathead mutation as a mutation in the Citron-kinase gene (CitronK or CitK) located on the long arm of rat and human chromosome 12. The flathead mutation is a single base deletion in exon 1 that results in a nonsense codon and absence of CitronK protein expression in proliferative zones. CitronK protein is concentrated at the VZ surface at adherens unctions and cytokinesis furrows. We hypothesize that CitK, which contains multiple protein interaction domains, links fate determining signals with cytoskeletal restructuring at the VZ surface. We propose to use a combination of imaging experiments, in vivo transfection, and biochemical experiments to further elucidate a cellular and molecular pathway necessary for neurogenesis.

DYSLEXIA SUSCEPTIBILITY GENES AND MECHANISMS OF NEURONAL DEVELOPMENT Reading disability (RD) or dyslexia is the most common learning disorder in children. While the 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 three years four candidate dyslexia susceptibility genes have been proposed (DYX1C1, KIAA0319, DCDC2 and ROBO1), and all four play roles in neuronal development. Rodent homologs of three of these, Dyx1c1, Kiaa0319 and Dcdc2 have been shown to play a role in neuronal migration in developing neocortex, and Robo1 was previously shown to be important for axon growth and guidance. The first three aims of the project will further define the cellular and developmental roles of Kiaa0319 and Dcdc2, the two genes currently with strongest genetic support as dyslexia susceptibility genes. These three aims are to determine the components of neuronal migration regulated by dcdc2 and kiaa0319, to determine functional links between kiaa0319 and dcdc2 in neuronal migration, and to determine the functionally necessary protein domains of dcdc2 and kiaa0319. The aims will be carried out by a combination of in utero RNAi, imaging, and cell culture approaches. Novel combinatorial methods of RNAi and electroporation are proposed to investigate interaction between Kiaa0319 and Dcdc2. Finally, in collaboration with groups currently working on identifying additional dyslexia susceptibility genes in human popualtions, we propose to test the developmental roles of new candidate dyslexia susceptibility genes in neuronal migration and development. Together, results form these experiments will reveal the cellular functions of candidate dyslexia susceptibility genes in neuronal development, and this should contribute to an eventual understanding of the underlying causes of this learning disorder. Reading disability (RD) or dyslexia is the most common learning disorder in children. While the 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. Results form these proposed experiments will reveal the cellular functions of candidate dyslexia susceptibility genes in neuronal development, and this should contribute to an eventual understanding of the underlying causes of this learning disorder.

DESCRIPTION (provided by applicant): Mutations in several genes have now been identified to cause a massive and specific reduction in the total number of neurons in the human brain. The specific role of these primary microcephaly (MCPH) genes in neural progenitors has yet to be fully defined. We have found that that the products of these genes, MCPH proteins, are localized to the point of abscission in dividing cells, and we hypothesize that asymmetric cell abscission in neocortical progenitors is regulated by interactions between MCPH proteins. We will pursue this hypothesis by addressing 4 specific aims: 1) Define the complex of microcephaly proteins at the midbody ring during neurogenesis;2) Examine the role of microcephaly proteins in the timing and location of cell abscissions in neural progenitors;3) Determine the effects of midbody ring inheritance on subsequent neural progenitor outcomes;4) Identify mechanisms that link cell abscissions to apical junctions in neocortical neuroepithelium. PUBLIC HEALTH RELEVANCE: Mutations in several genes have now been identified to cause a massive and specific reduction in the total number of neurons in the human brain. The specific role of these primary microcephaly (MCPH) genes in neural progenitors has yet to be fully defined. We have found that that the products of these genes, MCPH proteins, are localized to the point of abscission in dividing cells, and we hypothesize that asymmetric cell abscission in neocortical progenitors is regulated by interactions between MCPH proteins.

DESCRIPTION (provided by applicant): A causal link between malformations of cerebral cortex and seizures is well established. Surgical removal of operable malformations can significantly reduce seizures in human patients, however for inoperable or diffuse cortical malformations, often refractory to pharmacological therapy, alternative therapies must be developed. New treatments may evolve from either the discovery of pharmacological agents that target dysplastic or displaced neurons, or from approaches to prevent or eliminate formed or forming malformations. We hypothesize that during development there is a sustained period of structural plasticity during which time neuronal migration can be restarted in displaced neurons. We further hypothesize that during this period early neocortical malformations can be regressed by the appropriate activation or expression of proteins that promote migration, or by elimination of cells that interfere with the migration of other cells. We propose to test this hypothesis by focusing our studies on a novel model of sub cortical band heterotopia (sbh) or double cortex syndrome. In preliminary studies we have found that malformations in this model can be prevented from forming by re-expressing doublecortin (DCX) in migrationally impaired neurons. Most importantly, we observe that malformation regression can occur after a malformation begins to form in early development. Understanding the developmental limits and mechanisms of heterotopia regression in this animal model is an important step to determining whether epileptogenic neuronal malformations in humans may be reversed by reactivating normal neuronal migration. PUBLIC HEALTH RELEVANCE: Seizure disorders caused by developmental malformation of the cerebral cortex are one of the most challenging forms of epilepsy to manage. Such malformations are often formed by early deficits in migration of neurons during early brain maturation. If neurons stalled in there migration during early development can be re-started, then this opens up the possibility of regressing or reversing malformations even after they have begun to form. Experiments proposed here will use a novel pre-clinical model to establish the developmental limits of restoring normal migration and of reversing formation of potentially epileptogenic malformations.

Every cell has a unique combination of mutations that have accumulated by imperfect DNA repair. If these somatic mutations occur in a critical early time in cerebral cortical development they can affect enough cells in the brain to impair function and result in pathophysiology. Perhaps the best demonstrated examples of somatic mutations causing pathophysiology in the human brain are seizures caused by glial and glial-neuronal tumors and by focal cortical dysplasias. The most common somatic mutations identified to date in these lesions include activating BRAF kinase mutations in approximately 30-50% of resected gangliogliomas, and activating mutations in MTOR, AKT, and PIK3CA kinases in focal cortical dysplasias. Current evidence suggests that these mutations are drivers of the underlying pathologies responsible for focal epilepsies. Consistent with the idea that focal somatic mutations in a subset of neurons and/or glia are sufficient to cause seizures, recent studies have shown that expression of mutations identified in resected human tissue in relatively small numbers of cortical neurons in mice is sufficient to cause seizures. What remains largely unknown is precisely how and whether different somatic mutations lead to neuronal hyperexcitability in cortical neurons and hypersynchrony in cortical circuits. Using novel animal models of focal somatic mutation in neural progenitors we propose to test three hypotheses focused on defining the underlying developmental, cellular and molecular causes of seizures resulting from somatic mutations in cortex: 1) cellular phenotypes and seizure severity are a function of the neocortical progenitors in which epileptogenic mutations arise, 2) elevated cortical excitability is a direct consequence of overactive MAPK/ERK and MTOR pathways in either or both neurons and astrocytes, and 3) epileptiform activity spreads from perilesional zones by altered connections to inhibitory interneuron networks that result in hypersynchronous interneuron activity.

Abstract/Project Summary This is a new proposal to start the first T34 MARC U-STAR program at the University of Connecticut (UCONN). The UCONN program will be led by the Department of Physiology and Neurobiology (PNB) and will bring together some of our best mentors from four well-integrated academic units: the departments of PNB, Molecular and Cellular Biology, Biomedical Engineering, Psychology and Psychological Sciences, and Pharmaceutical Sciences. The UCONN MARC U-STAR program features a comprehensive recruitment plan, a well-supported mentoring structure, a group of dedicated mentors with strong funding histories, individual and programmatic experience in mentoring under-represented minority students, an immersive educational program, and collaborations with existing programs at the University focused on maximizing underrepresented student recruitment and retention in STEM fields. The MARC U-STAR program will benefit from our previous success in implementing a Beckman Scholars Program and a Research Experience for Undergraduates (REU) program within the department, and our departmental involvement in university wide diversity programs including LSAMP and the McNair Scholars Program. At its core, the MARC U-STAR program at UCONN will provide mentoring and hands-on research experience to 7 under-graduate students per year with each trainee receiving 24 months of direct support from this mechanism. The program will boast a wide scope of proposed research and didactic activities, a recruitment and retention plan that leverages local minority-serving programs currently present at UCONN including the Louis Stokes Alliance for Minority Participation (LSAMP), the McNair Scholars Program, and the BRIDGE program for incoming freshman. Each student's training and mentoring plan will be informed by an Individualized Development Program (IDP). We expect that each trainee will complete an honors thesis in the biomedical sciences, will present and participate in scientific conferences, will contribute to original published works, and will gain the confidence, skills-training and experience needed to go on to gain advanced degrees in biomedical research. We also expect 70% of our trainees will pursuit a Ph.D. degree in Biomedical Sciences. Our specific goals are to increase the recruitment, retention, and graduation rates of underrepresented honors students, provide trainees with an extended opportunity to be engaged in scientific inquiry, and to develop a community of underrepresented undergraduate scholars with a shared commitment to contribute to society by pursuing advanced training in biomedical research.