Pasko Rakic - US grants

The proposed Senator Jacob Javits Center will be dedicated to research on the basic neurobiology of the primate neocortex. Although the site of dysfunction in a large share of neurological diseases and human suffering, the neocortex has not been the focus of a specific Institute or of a Center devoted to multidisciplinary research. A team of investigators with wide experience (molecular biology, cell biology, biochemistry, immunocytochemistry, electron microscopy, endocrinology, neuroanatomy, neurophysiology, pharmacology, psychobiology, computer engineering, veterinary medicine, neurosurgery and neuropathology) have joined forces to explore three major themes: [1] Organization, [2] Development, and [3] Modifiability of the neocortex in the rhesus monkey. Each theme will be studied at the molecular, structural, physiological and behavioral levels, and the design of these studies generally will allow the use of the same set of animals for multiple experiments. The first theme includes basic analysis of input-output relationships and microcircuitry of selected cortical areas, their transmitters, receptors and information processing properties. The second theme is built upon the first and includes genetic and epigenetic determinants of (molecular, structural and functional) neuronal phenotypes and development of their laminar, modular and regional positions; studies in this theme will also examine the mode and mechanisms of establishment of axonal connections and the sequence, tempo and pattern of synaptogenesis in selected cortical regions. The third theme concerns the molecular, anatomical and physiological modifiability of neocortex with emphasis on interactions between ipsilateral and contralateral cortico-cortical connections, afferents, cortico-subcortical and intrinsic microcircuitry. The techniques of prenatal neurosurgery will enable experimental manipulation of cortical development, its connections and synaptic organization at critical fetal ages. Studies will be carried out on the rhesus monkey with the intent of enhancing understanding of cellular mechanisms in normal cortical development and pathogenesis of disorders affecting higher cortical functions in humans. The Center will promote research collaboration and enable comprehensive, multipronged and quantitative analyses that have not been possible before now, and that would not be practical for individual investigators supported by separate research grants.

The goal of this research is to elucidate fundamental principles governing the development of the visual system in primates including humans. Work on embryonic and fetal eye and visual brain centers directly in nonhuman primates is essential for the understanding of normal and pathological development of the visual system in man. The project consists of three integral parts conducted simultaneously: (1) normal prenatal development in rhesus monkey, (2) experimentally perturbed development in the monkey, and (3) correlation of development in monkey and human. In the first part, emphasis is on the mechanisms of neuronal genesis, migration, determination and differentiation including axonal growth and synaptogenesis in the retina, subcortical visual centers and visual cortex. In the second part, the consequences of selective destruction of visual centers and/or pathways sustained prenatally are evaluated in postnatal monkeys to determine the extent of neuronal plasticity and synaptic reorganization. Finally, in order to relate experimental data from nonhuman primates to man, we are performing correlative cytological analysis in both species using Golgi histochemistry and electron microscopy. The experimental analysis is conducted with a battery of modern neurological methods including 3H-thymidine autoradiography, Golgi impregnation, immunocytochemistry, electron microscopy, and retrograde, anterograde and transneuronal retrograde transport methods for tracing development of neuronal pathways. We have refined a prenatal neurosurgical procedure so that destruction and/or injection of radioactive tracers into selected brain regions during intrauterine life from the 50th day of pregnancy to birth is compatible with survival of fetus. Studies conducted this year include time of origin and cytology of interstitial neurons situated in the white matter subjacent to primary visual cortex, development of the superior colliculus, and genesis of afferent and efferent connections of the visual cortex in a series of fetal and postnatal monkeys.

This is a new program project with the aim of uncovering the basic principles of cellular and chemical organization, development and modifiability of primate neocortex. A team of investigators with experience in cell biology, biochemistry, immunocytochemistry electronmicroscopy, endocrinology, pharmacology, psychobiology, computer engineering, veterinary medicine, neurosurgery and neuropathology have joined forces to explore (1) determinants of laminar and areal positions and differentiation of cell phenotypes; (2) establishment of extrinsic and intrinsic axonal connections and synaptogenetic sequences; (3) regional development of putative transmitters and their receptors; (4) emergence and regional distribution of steriod hormone receptors; and (5) emergence of normal and abnormal functions. Each specific aim will consist of a series of interrelated experiments on developing rhesus monkeys using a battery of the most advanced neurobiological techniques each requiring participation of several investigators. The design of the projects allows use of each animal for multiple experiments. The techniques of prenatal neurosurgery on temporarily exteriorized fetuses that were refined in our department enable experimental manipulation of cortical development that will for the first time allow exploration of various epigenetic factors and mechanisms that govern organization and parcellation of primate neocortex. All studies will be done on the rhesus monkey with the conviction that the results may aid the understanding of mechanisms of normal cortical development as well as pathogenesis of developmental disorders affecting higher cortical functions in humans. It is expected that this program will: 1) promote interaction and collaboration by investigators with various scientific backgrounds; 2) support core facilities for sophisticated computer data and graphic analyses, and breeding of timed pregnancies in rhesus monkeys; and 3) stimulate sharing of ideas, equipment, know how and valuable primate tissue.

This request for a continuation of support is for longitudinal research designed to reveal basic principles and cellular mechanisms underlying development of the primate nervous system. In the next cycle of this grant, emphasis will be on (1) mechanisms of neuronal migration that include identification of binding molecule(s) and relative speed of cell translocation under normal and altered conditions; (2) the determinants of cell phenotypes and emergence of cytochemical individuality of neurons in developing primate brain under normal and experimentally altered conditions; (3) the capacity for postdevelopmental neurogenesis in primates; (4) development of synaptic connectivity and biochemical maturation of the "extrapyramidal" motor system in developing normal monkeys and monkeys lesioned at critical embryonic ages; and (5) correlation of critical developmental periods as defined in experimental studies on developing monkey with corresponding stages based on various morphological and cytochemical criteria applied in postmortem human fetal brain. Each Specific Aim consists of several interrelated experiments designed to use a battery of various light, electronmicroscopic and immunocytochemical methods including Golgi impregnation, Nissl-stain, histochemistry (e.g., AChE, monoamine fluorescence), immunocytochemistry (using a variety of antibodies), 3H-TdR autoradiography, anterograde and/or retrograde transport of radioactive tracers, HRP, or various fluorescent dyes. Our ability to perform intrauterine neurosurgery with short and long survival periods not only provides an opportunity to study the emergence of connections, but to change connectivity and explore experimentally the role of cell-to-cell interaction in morphological and biochemical maturation of the central nervous system. Until recently, experimental manipulation of early developmental processes has been the domain of research in simpler and peripheral nervous systems. In this project, as in the past, all studies will be performed on the developing rhesus monkey, with the conviction that the results will contribute to a greater understanding of mechanisms in normal brain development and help to uncover the etiology and pathogenesis of developmental brain disorders in man.

The research supported by this grant will continue our longterm commitment to understanding the prenatal development of the primate visual system and the genetic and epigenetic determinants of neuronal phenotype and connectivity. Our basic strategy remains to view the entire developing visual system in the rhesus monkey as an interacting unit. In this cycle we are emphasizing the specification of primate-specific color channels at the level of the retina (e.g., red-green, blue cones), the development of parallel color-opponent or parvocellular (P) and broad-band or magnocellular (m) thalamic pathways that are thought to underlay color and form/motion perception, and the incorporation of these channels into cortical maps. Experiments on the retina will focus on determinants of the photoreceptor mosaic and cell phenotypes as well as the establishment of separate channels of retinofugal connections. Studies on the lateral geniculate will examine the specification of the thalamic relay neurons that form the M and P system and may coordinate development of both retinal and cortical maps. In cerebral cortex we pursue the areal, modular, laminar and local synaptic specification of areas 17, 18 and MT and their color and noncolor processing channels. The issues addressed will require a battery of methods including: retroviral gene transfer technique to determine lineage relationship among cells in the retina and LGN, immunocyto- and histochemistry ( e.g. antibodies to red/green- and blue-sensitive cones to expose the emergence of photoreceptor phenotypes, or CAT-301 to display motion related components in the cortex), application of In vitro autoradiography and in situ hybridization to reveal the emergence and distribution of major neurotransmitter receptors, analysis of neuronal connectivity using axonal markers (e.g. HRP, fluorescent or carbocianin dyes) and confocal and electron microscopy to study the development of cell dendritic pattern and synaptic connectivity. The studies will be carried out over a five year period in a series of normal and experimentally perturbed developing macaque monkeys. The experiments are designed in a way that tissue from each animal is used for multiple purposes, providing correlative developmental data and maximizing the amount of information from scarce and valuable primates. The underlying strategy is to discern how developmental events are orchestrated between the periphery and brain centers and to elucidate their involvement in congenital and acquired defects of vision.

The cerebral cortex, the organ of human intellect, is the site of many mental and neurological dysfunctions, the causes of which can be traced to genetic and acquired developmental abnormalities in early prenatal life. A fundamental question in understanding this phase of corticogenesis is how, after their last cell division in the proliferative ventricular zone, postmitotic neurons achieve their laminar and radial positions in the cortical plate, differentiate into specific phenotypes, generate a repertoire of neurotransmitters and receptors, and establish functional synaptic circuits. Previous findings from research in this program Project include i) development of novel surface neuron-glia junctional proteins that are involved in neuronal migration, ii) demonstration of the role of voltage and ligand-gated Ca2+ channels in neuronal migration, iii) evidence of the role of the cytoskeleton in cell motility, iv) identification of specific proteoglycans that are involved in activity-dependent cortical differentiation, v) discovery of adrenergic receptors in the transient embryonic zones of the primate embryos, vi) demonstration of a cortical role for various neurotransmitter systems ina the regulation and generation of synaptic and neuronal activity in developing forebrain circuits. These findings have generated novel concepts regarding developmental mechanisms or opened new areas of research. The present proposal is based on the premise that these are causally interrelated and overlapping events that can be understood best in relationship to each other . With the support of this Program Project, a team of investigators with expertise in molecular biology, immunocytochemistry, laser microscopy, electron microscopy, neuroanatomy, receptor pharmacology, computer imaging, and electrophysiology have joined forces to explore five major themes: (1) Surface mediated mechanisms of neuronal migration; (2) Regulation of cell phenotype in the developing neocortex; (3) Transmitter regulation of cortical phenotype; (4) Neurotransmitter receptors as sites of morphogenetic activity in the transient embryonic zones of the developing primate cerebral cortex; (5) Development of network activity in thalamocortical circuits. Thr proposed experiments utilize some of the most advanced neurobiological approaches that can, at this time, be applied to the complex developmental problems involved in cortical ontogeny. Our experience is that a program project of this scope promotes communication between investigators with different backgrounds, fosters research collaboration, and enables comprehensive, multifaceted analyses that are either not possible or practical for individual investigators working alone.

The Third World Congress of the International Brain Research Organization (IBRO) brings together outstanding neuroscientists from all over the world for a week of symposia, lectures, and workshops in all areas of neuroscience. It provides a valuable opportunity to promote or enhance international collaborations, to enrich the international dialog on current issues, and to exchange information on the latest techniques in this fast- moving, interdisciplinary field. The Society for Neuroscience will use this support to provide funding for travel to allow young investigators, particularly for minority, women, or other underrepresented groups, to take advantage of this major event to enlarge their horizons and career opportunities.

The Society for Neuroscience proposes to provide travel funds for outstanding U.S. Neuroscientists who are invited symposium speakers at the Third IBRO World Congress of Neuroscience, Montreal, Canada, August 4-9, 1991. The Scientific Program of the Congress has been developed under the leadership of Dr. Albert Aguayo, who has gathered suggestions from all over the world in assembling the Congress program of symposia and workshops. This will be the second large world-wide gathering of neuroscientists, and it is important to ensure that the most understanding U.S. scientists can attend. This Congress is designed to bring together both basic and clinical research, and it is hoped that progress will be made towards alleviating some of the many disorders of the brain and nervous system. Travel funds are requested for APEX travel only. The Society of Neuroscience will provide all administrative support for this program at no charge, and no indirect charges are requested.

This is a competing renewal for a longitudinal project dedicated to the study of the neurogenetic processes of the primate brain. The proposed research is focused on the macaque monkey brain and designed to provide information about timing, sequences and developmental mechanisms that ultimately may determine the wiring diagram of the human central nervous system. Although empirical data on cellular events in fetal, neonatal and adolescent primates are important in their own right, we are continuously exploring broad conceptual issues concerning determinants of cell phenotype, control of cell number, capacity for compensatory plasticity and recovery of function, the validity of the radial unit hypothesis and the postulate of a cortical protomap. In the next cycle of this grant, emphasis will be on (1) determining the duration of the cell mitotic cycle and the kinetics of proliferation during critical developmental stages in various structures of the primate brain using a combination of bromodeoxyuridine immunocytochemistry and 3H-thymidine autoradiography; (2) determinants of cell lineages and the emergence of phenotypic individuality of neuronal cell classes in developing primate brain using recombinant retroviral vectors inserted in the genome of dividing ventricular cells; (3) expression of developmentally regulated molecules (protooncogenes, homeoboxes, proteoglycans), biochemical differentiation of neurons and emergence of synaptic connectivity (neurotransmitters, neuromodulators and receptors) using immunocytochemistry, in vitro binding of various ligands and in situ hybridization; (4) crossregional transplantation of 3H-thymidine prelabeled tissue slabs in age-matched primate fetuses; and (5) correlation of critical developmental periods as defined in experiments on developing monkeys with the corresponding stages in human based on structural and molecular criteria applied to the postmortem fetal brain. Altogether, the proposed experiments should advance our understanding of the development of the human brain and the pathogenesis of abnormalities caused by genetic and environmental factors.

Neuronal migration is an extraordinary event which insures that postmitotic neurons generated in the embryonic ventricular zone acquire their proper positions in the mature brain and establish their adult phenotypic identities. In the mammalian embryo, most postmitotic neurons destined to form the cerebral cortex reach their permanent residences by following along the surface of elongated fibers that have been identified as transient radial glial cells. This project analyzed the molecular mechanisms of this dynamic process by focusing ont he molecular bonds that attract and adhere neurons to glia during migration. Using polyclonal antibodies (D4) to antigens on radial glial cells made in the previous cycle of this grant and two new mouse monoclonal antibodies (14D7 and 19G11), we have demonstrated the presence of two distinct "microdomain" antigens of 48 kD and 72 kD molecular weight on the free surface of radial glial cells in the embryonic cerebral wall in vitro. Up to now, these are the only identified recognition molecules isolated from the glial side of the neuron-glia junction. Here we propose to characterize further the emergence, distribution, and function of these polypeptides in the embryonic cerebral wall of living embryos. To attain this challenging goal, we propose the following logic for our studies: 1) examine expression and localization of the glial cell surface microdomain proteins at various stages during neuronal migration in vivo: 2) study the modulation of neuronal migration by antibodies to glial cell surface microdomains; 3) characterize glial cell surface microdomain proteins and identify the corresponding receptor molecules on the neuronal side of the microdomain junctions. We have all the necessary methods, personnel and equipment as well as a unique battery of antibodies to identified glial cell surface antigens to address these goals. These studies offer an opportunity to obtain a comprehensive view of the molecular mechanisms underlying the critical development event of neuronal migration and to gain insight into the pathogenesis of a variety of migratory disorders that underlie cortical (and subcortical) malformations.

tissues; imaging /visualization /scanning; eye; biomedical facility; vision; anatomy; computer data analysis; health science research; bioimaging /biomedical imaging; confocal scanning microscopy;

During the past year we continued our research on developing primate brain that has been obtained from the New England Regional Primate Research Center. Among projects that were completed is a longitudinal study on synaptogenesis in the cerebral cortex of the macaque monkey. In addition, we also continued our investigation of the primate retinal photoreceptor mosaic in collaboration with Drs. K. Wikler and P. MacLeish, which is build on a novel cone-specific monoclonal antibody, 7G6 generated by our team. This antibody stains specifically sub-populations of cones in the fetal monkey retina (Wikler et al, 1997). We also made considerable progress in our research on one of our major goals, which is to determine when and how the circuitry of M and P streams in the primate visual system becomes coordinated from the periphery (retina) to the center (cortex). Now that we have establish that m and P ganglion cells in the embryonic macaque retina diverge into their respective subtypes independently and prior to the formation of synaptic contacts (Meissirel et al., 1997), we are investigating possible developmental mechanisms. For this purpose, we plan to manipulate the relative size of M and P streams by selectively reducing M and P neurons in the geniculate nucleus by timed X-irradiation in the embryo, as we did in the deletion of specific layers of the visual cortex (Algan and Rakic, 1997). Last year we published a longitudinal study of the cell-cycle parameters in the proliferative ventricular zone of fetal macaque monkeys. To our surprise, we found that cell-cycle duration in monkeys was as much as 5-times longer than those reported for the smaller rodent neocortex (Kornack and Rakic, 1988). However, we also found that substantially more successive cell divisions elapsed during neurogenesis in monkeys than in rodents. Moreover, cell division accelerated during neurogenesis for the enlarged cortical layers in monkeys, in contrast to the progressive slowing described in rodents. Thus, evolutionary modification of the duration and number of progenitor cell divisions underlies both the expansion and laminar elaboration of the primate neocortex. In the next year, we will continue our search for the regional patterns of expression of various transcription factors and signaling molecules in the developing primate cerebral cortex.

Glutamate excitotoxicity is the presumed mechanism underlying many neurological disorders including acute ischemia and chronic neurodegeneration. Recent work from our laboratories demonstrated that mice lacking the neural isoform of the stress activated protein kinsase SAPK/JNK (JNK3) has remarkable resistance to kainic acid-induced seizures, AP-1 transcriptional activity and apoptosis of hippocampal neurons. These results strongly suggest that the SAPK/JNK signaling pathway is a critical component in the pathogenesis of glutamate excitotoxicity. Based on this hypothesis, the present proposal comprises two sets of closely related in vitro and in vivo experiments. Specific Aim I: Mechanism of JNK -mediated glutamate neurotoxicity ( in vitro studies). Keys issues to be addressed include: (1) the JNK - mediated neurotoxicity through gene regulation and de novo biosynthesis; (2) the regulation of intracellular calcium oscillation through JNK signaling; (3) the interplay between JNK - signaling And oxidative stress, (4) the specificity of activation route of JNK signaling. These potential mechanisms will be tested in primary culture of dissociated neurons using a combination of morphological, physiological and biochemical approaches. Specific Aim II: Neuroprotection of the blockade JNK signaling pathway (in vivo studies) Potential clinical applications of inhibition of the JNK signaling may include: (1) the prevention of ischemic apoptosis; (2) attenuation of chronic glutamate excitotoxicity in amytrophic lateral sclerosis; (3) the prevention of neurodegneration (4) enhanced survival of grafted neurons in transplantation therapy. Preliminary evidence of feasibility has been obtained for each Specific Aim. The potential applications will be tested in experimental models of neuropathology using wildtype and mutant mice lacking the neural-specific JNK3 isoform. In summary, the overall goal of the present proposal is both to understand the mechanism underlying JNK- mediated glutamate neurotoxicity and to test the clinical potential of targeting the JNK signaling pathway for therapeutic intervention.

One of the major problems with brain aging and neurodegenerative diseases is the cognitive impairment caused by the destabilization of neurites and synaptic connections followed by the degeneration and loss of neurons. Thus, elucidating the molecular mechanisms that regulate the growth and stabilization of neurites and synapses may not only help us better understand these events, but may also provide information for developing new preventive and therapeutic approaches. Recent evidence obtained in several laboratories, including ours, has implicated contact- dependent signaling via the Notch receptors, which has been traditionally associated only with embryonic development, in the growth and stabilization of neurites and synaptic connections in the cerebral cortex. We have also demonstrated that endogenous Notch activity in both developing and mature cortical neurons can be modulated by exogenously applying the Notch ligands Delta and Jagged, and the intracellular proteins Numb, Numb-like, and Deltex. In addition, numerous mutations causing early-onset Alzheimer's Disease (AD) have been identified in the presenilin (PS) genes, which have been shown to be necessary not only for gamma-secretase-mediated processing of amyloid precursor protein (APP), but also for the trafficking, endoproteolytic processing, and activity of the Notch receptors. Our working hypothesis is that in aging and many neurodegenerative disorders, the degradation of neuronal connections is associated with changes in the expression and activity of the Notch signaling pathway genes, which may directly-or indirectly contribute to the underlying pathogenesis. If so, then finding a way to control Notch signaling, either upstream, via ligands, or downstream, via intracellular proteins, could provide a means to slow down or change the outcome of aging and memory disorders in the cerebral cortex. We propose three logistically related Specific Aims: (I) The expression and subcellular localization of Notch signaling molecules in the normal and abnormal adult cerebral cortex; (II) The effect of manipulating Notch activity on the stability and modifiability of cortical neurites and synapses; and (III) The role of preseniiins in the expression, endoproteolytic processing, and activity of Notch receptors in the cortical neurons. We have promising preliminary data and our expectation is that research of this scope and methodological diversity will provide insight into the pathogenesis of neurodegeneration and possibly generate the means to alleviate or slow its progression.

This two-day workshop will define particular opportunities in the field of cognitive neuroscience. It is unique in its intent to explicitly identify particular linkages to promote among cognition, computation and biology. The three organizers are highly regarded leaders in their fields, and the speakers include a diverse group with many well-recognized names. Topics include how to define cognitive neuroscience and how to use biological, behavioral and computational approaches to study cognition, and the format provides adequate time to discuss the several topics proposed.
Results from the workshop will include a set of recommendations to NSF for programmatic and cross-directorate activity mechanisms to encourage productive new approaches to cognitive neuroscience.

The long range objective of this laboratory is to understand the cellular and circuit basis of cognitive operations of the prefrontal cortex, the brain area closely associated with the executive functions of the human brain. In the past cycle of this grant, we have defined separate spatial and nonspatial visual processing domains associated with the dorsolateral and inferior prefrontal convexities, respectively. The goal of the present application is to functionally and anatomically dissect the intrinsic circuit mechanisms by which the prefrontal cortex performs its basic cognitive operations and to test the hypothesis that the prefrontal cortex is composed of content- constrained modules not only at the level of cytoarchitectonic areas (macroarchitecture) but also at the level of columns and microcolumns (microarchitecture). In Specific Aim number 1, powerful new multielectrode technology will be applied in vivo to the study of the dorsolateral prefrontal cortex to examine the dynamic interactions of ensembles of neurons with cue, delay and action-related response properties within and across cortical columns during spatial working memory tasks. This method will also allow analysis of the contributions of fast-spiking (putative interneurons) and regular spiking (putative pyramidal) neurons to these dynamic interactions and spatial memory field formation in the dorsolateral cortex. In Specific Aim number 2, the same powerful method will be applied to the object processing system of the inferior prefrontal cortex (IFC) to assess ensemble encoding in relation to the object/face response properties of IFC neurons. Specific Aim number 3 will complement the in vivo approach by employing whole cell patch clamp recording combined with infrared microscopy to visualize and directly study local circuits among functionally connected neurons in cortical slices. Collectively, these aims will explore a new level of functional anatomy in prefrontal cortex with the goal of providing mechanistic explanations of cognitive phenomena and insight into pathological processes of mental illness.

In the present CCNMD we propose to conduct a new program of translational research concerned with the endogenous processes responsible for cognitive dysfunctions of the dorsolateral prefrontal cortex (DLPFC) in schizophrenia and related disorders. The central hypothesis of the proposal is based on a new set of findings pointing to deficiencies in Ca2-associated intracellular cascades in schizophrenia, among them the recent discovery of a new class of dopamine (DA) receptor interacting proteins (DRIPs) which affect calcium signaling in model systems. We propose that such deficiencies, which may arise from many causes, constitute the underlying causal mechanism in the disorder and can account for both diverse and widespread neuropathologies as well as prominent vulnerability of higher-order functions. Extensive research inspired by the DA hypothesis of schizophrenia has yet to isolate a clear disease-associated abnormality in any particular component of the DA system, but the role of dopamine in the core cognitive dysfunctions of mental diseases cannot be denied. The present CCNMD is comprised of projects at 4 different universities designed to integrate dopamine pharmacology, cell biology, circuit mechanisms and neuronal processes with behavioral endpoints in animals and patients. State-of-the-art methodologies range from the isolation of novel DRIPs and characterization of their cellular functions in cell cultures through a clinical trial in schizophrenia patients based on a broad array of preclinical research. One subproject interrogates the status of DRIPs in postmortem brains from patients and controls with molecular, neurochemical and anatomical tools. Another subproject examines DA modulation and DA mediated calcium signaling of neural interactions in identified neurons and circuits in PFC cortical slices. Studies of PKA and PKC signaling in rodents, including mice genetically engineered to express DRIPs, are carried out. Other projects will interrogate similar pharmacological as well as physiological mechanisms in nonhuman primates performing cognitive tasks under normal and chronic drug regimens designed to elucidate basic properties of cognitive function and simultaneously parallel the clinical study in Project 9. These multidimensional projects based on novel concepts and discoveries are challenging and risk-taking, but all Pl's have a long record of effective interactions and productive collaboration to justify investment in the potential of this proposal.

This proposal is a competitive renewal application for our research program on the role of the Caspase family of proteases, the c-Jun NH2-terminal kinase (JNK), and the phosphatidylserine receptor (PSR) in neuronal apoptosis in brain development and neurological diseases. In the present project, we will extend our research of these signaling pathways with a new emphasis on the mechanism of apoptosis and renewal of neural progenitor cells. Aim 1: We will investigate the mechanism downstream of phosphatidylserine and phosphatidylserine receptor (PS-PSR) recognition leading to the apoptosis of neural progenitor cells. We will express PS on the outer cell surface of neural progenitor cells and examine the signaling events following PS-PSR recognition. In addition, we will test whether the disruption of PS-PSR recognition in the embryonic brain will alter the apoptosis of neural progenitor cells. Aim 2: We will examine the interaction between the JNK and canonical Wnt/beta-catenin signaling in the decision of self-renewal or cell-cycle exit by neural progenitor cells. We will determine whether the canonical Wnt/beta-catenin pathway mediates self-renewal, and whether JNK activation attenuates the canonical Wnt/ beta-catenin signaling to promote differentiation of the daughter cells. Aim 3: We will use a transgenic system to calculate the net increase of adult-generated neurons in the mouse brain. In addition, we will test whether EOF and FGF2 infusion can accelerate adult neurogenesis and attract newly born neurons into stroke damaged brain areas. In summary, the present project is built on our research of JNK, phosphatidylserine receptor (PSR), and canonical Wnt/beta-catenin signaling with an emphasis on the mechanism governing the adjustment of the number of neural progenitor cells (Aim 1), the decision of cell-cycle exit by neural progenitor cells (Aim 2), and the accelerated neurogenesis after brain injury (Aim 3). These studies will enhance our understanding of neural plasticity and functional recovery after brain injury.

DESCRIPTION (provided by applicant): The human cortex is distinguished from other species by its size, addition of specialized cytoarchitectonic areas, new subtypes of neurons and increased number of connections that subserve unique functions such as language and higher order cognition. The mechanism by which human cortical distinction emerges during development and how it has evolved during evolution is not known. The evolutionary novelties in the cortex are likely to be an outcome of relatively small genetic differences effecting the timing, sequence and level of gene expression at early embryonic stages that can be uncovered only by studying unique features of cortical formation in human and non-human primates in comparison to rodent and other species. Genes, transcription factors and cellular mechanisms that generate species-specific differences and human distinction in cortical development have not been previously analyzed and we are in the unique position to address this biomedically significant, but neglected issue in modern neuroscience. The human telencephalic primordium can be distinguished from that of the mouse and monkey by its size, morphology, neural stem cell types, and hitherto unrecognized abventricular stem cells two weeks before the onset of neurogenesis from the local proliferative ventricular zone. Thus, we will focus on the initial stages of cortical development in the rodent (mouse), non-human primate (macaque) and human telencephalon by using the most advanced genetic molecular and in vitro and in vivo cell biological methods available. More specifically, we will perform: 1) Isolation, in vivo lineage analysis and histochemical characterization, fate determination and testing function and potential of the early ventricular, subventricular and abventricular neuronal stem cells in these three species;2) Identification of genes differentially regulated among human, macaque and mouse early stem cell types using high throughput microarray gene expression analysis and in situ hybridization analysis of putatively differentially regulated genes;and, 3) Functional studies of genes screened in Specific Aim 1 &2. Abnormal genesis and initial patterning of the human cerebral cortex can give rise to uniquely human neuropsychiatric disorders such as schizophrenia, autism, developmental dyslexia, mental retardation and response to drugs for therapy and abuse. PUBLIC HEALTH RELEVANCE: The proposed research focuses on the initial steps of cellular and molecular events that establish anatomical and functional differences between the embryonic human, non-human primates and rodent cerebral cortex. The expected set of unique data on the origin and nature of species-specific differences are not only of theoretical, but also of considerable clinical significance, as many of the newly evolved traits may be more vulnerable to genetic and environmental factors that can initiate certain human-specific neuropsychiatric disorders such as schizophrenia, autism, developmental dyslexia, mental retardation and response to drugs used in therapy and abuse.

DESCRIPTION (provided by applicant): The primary goal of this competitive grant renewal remains to identify genes and regulatory non-coding sequences that generate species-specific differences in development and organization of the cerebral cortex, particularly association areas, such as the prefrontal cortex. The prefrontal cortex is considered the crucible of human cognitive capacities with well-documented neuroanatomical connectivity and unique cellular features that are thought to be undermined in neuropsychiatric disorders and hijacked by drugs of abuse. We start with the assumption that basic principles of cortical development in all mammals are remarkably similar. It i, however, reasonable to expect important quantitative (e.g. the number of neurons, tempo and sequence of cellular events) and qualitative changes (e.g. the introduction of new neuronal subtypes, elaboration of synaptic connections and addition of functionally specialized cortical areas) since primates split from the rodent lineage about 100 million years ago. Thus, our strategy has been to study in parallel developmental events in the rodent (mouse), non-human primate (macaque) and human embryonic telencephalon by using the most advanced molecular and cell biological methods available, including comparative high-resolution mRNA-sequencing, in utero and ex utero gene manipulation (Loss or Gain of function) and heterologous transplantation of neural stem cells. We will complete and further augment our ongoing high-resolution mRNA-sequencing, confirm results by qRT-PCR, and gain further insight through bioinformatics network analysis in three species, which was initiated in the first cycle of this grant (Aim #1). Then, based on our discovery of several primate-specific cortical neuronal subtypes, we now plan to identify their genetic determinants by performing lineage analysis and use of ex-utero electroporation to overexpress and/or knockdown selected genes to examine their interactions and identify downstream transcription factors (Aim #2). Finally, we will proceed to the next stage f this research by identifying regulatory changes driving human and nonhuman primate-specific gene expression and binding sites on downstream targets followed by comparison of ChIP-seq data to differential gene expression (Aim #3). Although the proposed research is extremely time-consuming, logistically difficult and costly, it is realistic based on our published record and our progress, which shows that we succeeded to establish the unique facilities, master and modify essential methodology and already have obtained a substantial amount of feasibility data. We argue that abnormal genesis and initial formation of the evolutionarily novel, human- specific traits of the cerebral cortex ay be particularly vulnerable to genetic mutations and environmental influences each of which alone, or in combination, can give rise to elusive neuropsychiatric disorders and neuronal response to prenatal exposure to drugs of therapy and abuse.