Tarik F. Haydar - US grants

This project is a live imaging and interventional approach to study the molecular mechanisms of cerebral cortical development. Mental retardation in several diseases and syndromes such as Downs syndrome and autism may be a result of environmental or genetic alterations in prenatal cortical proliferation. To better understand how proliferation is controlled during brain development, molecular biology and virology will be combined with a novel organotypic culture of the neocortex to establish that glutamate, GABA, and bFGF can affect neocortical growth by changing the proliferative properties of neuronal progenitor cells. In addition, it will be determined, through a mutagenic approach, whether these extracellular molecules act via the Notch pathway of signal transduction, an intracellular mechanism for the control of neuroprogenitor proliferation.

DESCRIPTION (provided by applicant): The cells of the cerebral cortex need to be generated in a timely fashion and then migrate to proper positions prior to the development of synaptic connections and functional circuits. Thus, disturbances in the proliferation, migration, or differentiation of cortical cells can lead to cognitive impairments such as those seen in Down syndrome, autism, and other developmental disabilities. The long-term objectives of this project are to identify the various stem and progenitor cells in the embryonic neocortical wall and then to characterize the molecules controlling neuronal and glial cell production from those progenitors during development of the cerebral cortex. In particular, we are studying how the movement of the mitotic spindle apparatus and expression of fate determining molecules combine to allow for the switch between symmetrical and asymmetrical divisions during neocortical neurogenesis. We will study this process using time-lapse multiphoton microscopy, which enables long term visualization of the stem cells in their intact environment. Combining this imaging technology with overexpression of wild-type and dominant negative gene constructs via stem cell transfection will allow us to determine the molecular requirements of stem cell proliferation in a rapid and straightforward manner.

[unreadable] DESCRIPTION (provided by applicant): The hippocampus, part of the limbic system, mediates learning and memory and is implicated in epilepsy. We are developing a multidisciplinary approach to study development and function of the excitatory and inhibitory neurons in the hippocampus. Using electroporation to transfect the hippocampal stem cells in utero, we will introduce fluorescent protein markers as well as mutated versions of the TrkB tyrosine kinase receptor into specific subregions of the hippocampal circuitry. The expression of these exogenous genes will be controlled temporally by placing them under the control of inducible gene promoters so that their expression coincides with particular phases of neuronal development. The functional effects of these molecular changes will be assessed by characterizing cell differentiation using immunochemical staining and by direct electrophysiogical measurements using patch clamp recording. These experiments will define a novel and rapid method for assessing the development, allocation and synaptic physiology of specific subpopulations of hippocampal neurons by introducing molecular changes in their germinal cells. This method will result in a technical advance for the study of hippocampal function and will be useful for understanding the underlying causes of abnormal synaptic transmission in diseases including epilepsy and mental retardation. PUBLIC HEALTH RELEVANCE:In this project we seek to develop methods for tracking the development and changing the gene expression of neurons in the hippocampus to enable rapid and cost-effective functional tests. In particular we will be assessing the role of the TrkB neurotrophin receptor in hippocampal neuronal migration and synaptic plasticity. To study both prenatal and postnatal effects of TrkB perturbation, we have engineered inducible plasmid constructs with which we can initiate expression of the transgene upon administration of tamoxifen. This work will enhance our ability to elucidate normal developmental mechanisms and allow rapid testing on causes of epilepsy and mental retardation. [unreadable] [unreadable]

Trisomy 21 results in the constellation of phenotypes collectively termed Down syndrome (DS) and is one of the most prevalent congenital birth defects. Motor and sensory deficits and often severe mental retardation are among the many debilitating sequelae of DS. Although the precise causes of cognitive impairment in DS are not known, these abnormalities are thought to be due to altered brain development as changes in cell number and volume are found in neocortex, hippocampus and cerebellum in the perinatal and juvenile postmortem brain. A comprehensive understanding of the . etiology of the DS cognitive phenotype therefore requires examination of early prenatal development, but this analysis has never been conducted as it is hampered by challenges obtaining staged human embryonic tissue and by breeding difficulties in mouse models of DS. We have addressed this problem by generating a breeding colony of the Ts65Dn mouse model of DS and have used this resource to determine the effect of trisomy on embryonic development of the cerebral cortex and hippocampus. We have uncovered substantial over-production of inhibitory neurons in the Ts65Dn cerebral cortex and hippocampus which may lead to increased inhibitory drive and abnormal morphogenesis of these forebrain regions. These two areas are known to be affected in the DS brain. Using cellular, molecular and electrophysiological techniques, we wi II determine how altered development in Ts65Dn leads to functional changes in the maturing neuronal circuits in the neocortex and hippocampus. These experiments will define the underlying developmental causative factors of cognitive impairment in DS at both the cellular and physiological levels and are therefore important for the future development of prevention or treatment strategies.

DESCRIPTION (provided by applicant): Two major goals of developmental neurobiology are to identify the germinal cells which form the central nervous system and to characterize the cellular and molecular mechanisms by which these cells generate the proper numbers and types of neurons and glial cells. Recently, retroviral and genetic studies in mice have demonstrated that radial glial cells (RGCs) contribute significantly to neocortical development by generating postmitotic neurons and then serving as the substrate for the migration of those daughter cells into the neocortical wall. Furthermore, RGCs are thought now to be the prenatal stem cell of the neocortex, and several groups have suggested that the neocortical ventricular zone (VZ) is composed solely of RGCs during embryonic neurogenesis. However, it remains unclear how a single VZ precursor cell type can generate the vast diversity of neocortical neurons. In addition, since multiple types of precursors have been found in the human and monkey VZ, whether this heterogeneity in the primate VZ represents evolutionary divergence of rodents and primates also remains a question. We have uncovered significant precursor diversity in the in vivo murine VZ. Through the use of multiple histological, genetic and ultrastructural techniques - many of which we innovated for this project - we now show that RGCs are joined by another type of resident VZ cell which we have named the short neural precursor cell (SNP). We demonstrate that RGCs and SNPs can be distinguished morphologically, molecularly, and with respect to proliferation kinetics and lineage potential. In addition, we have uncovered in vivo differences between classes of RGCs which suggest that not all RGCs are multipotent stem cells. Taken together, our published and Preliminary Data fundamentally alter our understanding of the composition of the mammalian VZ and clearly indicate that diversity in this germinal compartment is required for proper neocortical growth and function. This project will comprehensively test the overall hypothesis that the VZ becomes heterogeneous during embryonic development through diversification of a common VZ ancestor and that this complexity is necessary for proper neocortical growth. PUBLIC HEALTH RELEVANCE: How the rich diversity in types of forebrain neurons is achieved during development is not well understood. In this project, we build upon our novel findings that different types of neural stem and progenitor cells exist in the embryonic brain and generate neurons via distinct mechanisms. Using in vivo molecular approaches to measure proliferation, neurogenesis and allocation of progeny from individual precursor groups, this study will uncover the spatiotemporal inter-relationships between these precursor cells and determine how interaction with the extracellular matrix in the germinal zone controls their neuronal output.

DESCRIPTION (provided by applicant): Down syndrome (DS), or Trisomy 21, is the most common genetic cause of cognitive disability, afflicting 1 in every 800 live births. Importantly, the cause(s) of the cognitive disability in DS have not yet been uncovered. Our group has been studying the Ts65Dn mouse model of DS which has triplication of 184 of the 364 genes triplicated in DS. These mice have a large number of DS- relevant phenotypes, including cardiac and craniofacial defects as well as spatial learning/memory and motor abnormalities, the latter two of which have been principally measured in adult animals. We now report two major developmental forebrain phenotypes in Ts65Dn which precede these neurological deficits: 1) a dorsal forebrain abnormality which results in under-production of excitatory neurons and 2) a ventral forebrain defect which results in over-production of inhibitory interneurons. Together, these defects substantially shift the excitatory:inhibitory neuron ratio in Ts65Dn; we hypothesize that this is the primary cause for cognitive dysfunction in Ts65Dn/DS. Through a comprehensive set of experiments on cell proliferation, neural differentiation, gene expression and neuronal electrophysiology, we uncovered that two specific triplicated genes (the structurally linked transcription factors Olig1 and Olig2) cause the ventral Ts65Dn forebrain phenotype; we rescued the inhibitory defect in the embryonic and adult brain at the cellular and electrophysiological levels by generating Ts65DnOlig1/2+/+/- animals (reducing Olig1 and Olig2 genes from 3 to 2 alleles within the Ts65Dn background). In this period of study, we will determine whether Olig1/2 triplication influences the learning/memory and synaptic plasticity defects in Ts65Dn and investigate the role of the Dyrk1a minibrain gene in the (dorsal) excitatory neuron deficit in Ts65Dn.

DESCRIPTION (provided by applicant): Excitatory neurons of the cerebral cortex are generated prenatally from a diverse group of neural precursors, some of which have only been identified recently. Radial glia stem cells (RGCs) generate neurons directly and at least three separate lineages of Intermediate Neural Precursor Cells (IPCs), which are themselves produced from RGC, also produce neurons. Intriguingly, neurons within each cortical lamina are derived from these different parent cells. The reason why the neocortex requires so many individual precursor cell types, and whether the diverse ancestry of neurons within each layer plays a functional role in neocortical circuitry, has not been established. The numbers of IPCs are thought to be abnormal in several developmental disabilities, including Fragile X and Down's syndromes. In this project, we identify specific lineages of neocortical pyramidal neurons with novel genetic fate mapping tools and determine their structural, functional and connectional characteristics with patch clamp electrophysiology and high resolution 3D imaging. Our preliminary data indicate that neurons from individual precursor lineages, even within the same lamina, are imparted with specific functional properties and that the multiple IPC groups therefore directly underlie neuron and circuit complexity in the neocortex. Morphological and electrophysiological parameters will be quantitatively examined using a multidisciplinary approach and a two-laboratory collaboration.

? DESCRIPTION (provided by applicant): Newborns with Down syndrome (DS) are hypotonic and exhibit disturbances in movement production and postural control. Their acquisition of motor skills in infancy is significantly delayed, negatively impacting their gait, reflexes, and fie motor control throughout life. Work investigating the etiology of DS-related motor deficits has been largely focused on the cerebellum, yet preliminary work in our lab has for the first time elucidated substantial cytoarchitectural as well as gene expression differences between spinal cords (SCs) in trisomic mice and their euploid controls. Furthermore, two genes critically involved in SC development, known as Oligodendrocyte transcription factor 1 and 2 (Olig1/2) are triplicated in both people with DS and trisomic mouse models. Both Olig genes are expressed in a progenitor domain by a bipotential population of cells that can differentiate into either motor neurons (MNs) or oligodendrocytes (OLs). Additionally, Olig2 plays a role in interneuron (IN) specification through cross-repression. The fate-designation and maturation of precursor cells of these three cell types are essential for the proper development and function of the SC. Thus, the triplication of Olig1/2, coupled with their role in SC development, suggests that the Olig genes play a major role in SC abnormalities in DS. In this project, we propose to identify biomarkers of trisomic SC development and function in the adult, perinatal, and embryonic Ts65Dn mouse model of DS. We also plan to elucidate the specific role of Olig1/2 in trisomic SC development by utilizing a `gene dosage reduced' Ts65Dn model disomic for Olig1/2. Using a combination of behavioral testing, gene expression analyses, and immunohistochemical staining we will comprehensively assess how changes in multiple neuronal and glial populations influence SC development and motor behavior in trisomic animals while examining the degree to which triplication of Olig1/2 affects cell population dynamics in the cord. By targeting the Olig genes, this work is slated to reveal the specific molecular underpinnings of SC-related motor deficits in DS with potential for therapeutic exploitation to correct such debilitating and impactful life-long deficits.

Zika Virus (ZIKV) is a mosquito-borne Flavivirus. Outbreaks across Southeast Asia and the Western Pacific have been reported over the past 10 years and case reports of associated Guillain-Barré syndrome have suggested that these virus strains may have pathological effects on neural tissue. The most recent migration of Zika into South and Central America has rapidly expanded the area of virus transmission. In addition, more than 400 cases have been reported in the US since late 2015, a number that continues to increase. This rise in transmission is of critical concern since infection with ZIKV during pregnancy is thought to lead to microcephaly and ocular abnormalities. It is still unknown how prenatal ZIKV brain infection leads to morphological abnormalities in the developing brain. Here we propose a multidisciplinary study using imaging, genetic fate mapping and in vivo neurodevelopmental approaches to study development of the murine neocortex in a precise and quantitative fashion. Our recent studies demonstrate that these techniques robustly measure critical cellular and molecular processes of fetal brain growth and function. We will leverage this expertise in collaboration with virus experts to determine the consequences of ZIKV infection on the developing brain in a model of known ZIKV susceptibility. We will determine which cells are most vulnerable to ZIKV and how infection of these cells translates to abnormal brain growth and intellectual disability.

PROJECT SUMMARY Our recent transcriptome analysis of the brains of people with Down syndrome (DS), conducted from fetal stages to 40 years old, identified approximately 800 dysregulated genes across all chromosomes, each with specific temporal and regional profiles. These altered genes form co-expression networks, the most prominent of which indicates defective oligodendrocyte (OL) development and myelination. This finding is consistent with imaging studies demonstrating reduced white matter integrity in individuals with DS. In this collaborative study between the Haydar and Gallo labs, we will answer key questions regarding the timing and source of OL dysmaturation, and particularly whether cell-autonomous or non-cell autonomous mechanisms lead to altered cellular differentiation and myelination. The Aims of the project progress from defining the developmental time course of OL dysmaturation to comprehensive and integrated transplantation, behavioral and functional tests to evaluate the mechanism(s) of the defect. Whether these changes can be rescued by gene dosage normalization or by using newly identified pharmacological tools to prompt OL maturation will be studied in the last Aim.

The list of developmental events that may lead to intellectual disability in Down syndrome (DS) ranges from the over expression of single genes to altered synaptic and firing properties of neurons throughout the nervous system. Recently, our laboratory has discovered intrinsic defects in oligodendrocyte precursor cell (OPC) maturation that lead to changes in myelination and white matter tract formation in a mouse model of DS. This dysmyelination, confirmed using human brain gene expression profiling and myelin imaging, has been shown to lengthen the time it takes for action potentials to reach their postsynaptic targets. Since this novel cellular defect presumably underlies at least part of the intellectual disability in DS, a straightforward therapeutic approach would be to treat with compounds that promote OPC maturation. Initial results in mouse models look promising, but whether OPCs from humans with DS exhibit the same defects found in mouse model cells is critical for this effort. In this project, we will take advantage of pre-existing human stem cell lines and rapid OPC programming protocols to evaluate the maturation of human DS-derived OPCs for the first time. Next, in a series of in vitro experiments, we will test two FDA-approved drugs for their ability to prompt OPC differentiation and then test their myelination potential by transplanting these cells into the myelin-deficient Shiverer mouse brain. The results of these studies will generate a novel cellular resource for future study in the DS field, test the hypothesis that human DS-derived OPCs exhibit the same defects found in mouse model cells, and evaluate the efficacy of two potential drugs that could be used to improve myelination in the brains of people with DS.

Project Summary/Abstract The main objective of our T32 Training Program is to train MD and PhD post-doctoral fellows in research focused on Neurodevelopmental Disabilities (NDDs). This program, the District of Columbia NDD T32, is associated with and fully integrated into our District of Columbia Intellectual and Developmental Disabilities Research Center (DC-IDDRC). The rationale for this program is as follows: 1) Neurodevelopmental disabilities affect a significant percentage of the US population, and 10% of households live with an individual with an NDD, therefore these conditions represent a substantial financial and emotional burden in our society; 2) The biological causes of NDDs range from genetic to acquired insults, requiring an interdisciplinary neuroscience approach to define underlying causes and mechanisms of disease; and 3) There is significant overlap of symptoms amongst the various neurodevelopmental disorders, suggesting overlapping mechanisms. Our trainees and mentors use cutting-edge techniques, including genetic, cellular/molecular, physiological, functional/behavioral, and structural/dynamic imaging to engage in basic and translational NDD research projects. Multidisciplinary training is emphasized, based on the variety of training opportunities and core facilities available at the participating institutions. Children?s National Hospital (CNH) is particularly well positioned to lead this program, based on: 1) Strengths in basic, translational and clinical research, and in mentorship in all the proposed areas of NDD research; 2) Substantial institutional resources for research and training in NDDs; 3) Established, strong collaborations with all participating institutions; and 4) Its leading role in the DC-IDDRC and in many NIH awards focusing on conditions causing NDDs. Thirty mentors from CNH, George Washington, Howard and Georgetown Universities will be involved in the program to help develop and support careers of the trainees, including submission of a K Award application. We request funding for 3 postdoctoral fellows/year who participate in a 3- year program. The trainees will obtain: 1) specific and integrated training in NDDs and 2) formal and practical training in basic and essential skills required for independence, with a focus on research methodology, statistics, and rigor and reproducibility. The training components include a core curriculum consisting of: 1) Subject matter expertise; 2) Research and quantitative skills, and 3) Communication and writing skills. In addition, trainees will have customized and experiential components to deepen their training. These include a cross-training rotation for trainees (e.g., preclinical researchers rotate in the clinic and vice versa); a regular discussion group with a world-renowned leader and author in the field of NDDs (Mark Batshaw, MD); and we also offer trainees opportunities to participate in activities that are critical for broadening their perspective on how to conduct research by interacting with the community they are aiming to help in different venues beyond the lab or clinic, which will enhance relationships and promote advocacy for the NDD community.