1998 — 2002 |
Anderson, Stewart A |
K08Activity Code Description: To provide the opportunity for promising medical scientists with demonstrated aptitude to develop into independent investigators, or for faculty members to pursue research aspects of categorical areas applicable to the awarding unit, and aid in filling the academic faculty gap in these shortage areas within health profession's institutions of the country. |
Neuronal Migration From Basal Forebrain to Neocortex @ University of California San Francisco
DESCRIPTION: (Adapted from applicant's abstract): Abnormalities of telecephalic development have been implicated in the etiology of psychiatric illnesses such as autism and schizophrenia. Previous studies have indicated that different telecephalic regions, including the basal ganglia and neocortex, are derived from spatially distinct proliferative zones. However, recent evidence suggests that a population of neocortical neurons originates within the ventral (subcortical) telencephalon in the primordia of the basal ganglia (Anderson et al., in press). The purpose of this proposal is to further investigate what types of neocortical cells originate in the subcortical telencephalon, where they originate within this region, and what are the mechanisms of this migration. Several approaches will be used, including the analysis of mutant mice lacking this migration and the injection of cell tracers into embryonic ferrets in vivo. In addition, mechanistic characteristics of subcortical to neocortical migration will be studied by combining slice culture experiments with electron microscopy and by testing the hypothesis that chemotaxant factors guide cellular migration from the subcortical telencephalon into the neocortex. The candidate is a psychiatrist with a long-standing interest in the biology of neuropsychiatric disorders. As a postdoctoral fellow, he has spent three years in the laboratory of John Rubenstein at UCSF examining the molecular genetic underpinnings of forebrain development. Short-term goals are to remain in this laboratory as an adjunct assistant professor to further develop basic research skills at the organ, cellular, and molecular levels. Through collaborations with three other laboratories, those of Peter Ralston and Marc Tessier-Lavigne at UCSF and Susan McConnell at Stanford University, this project will involve a significant broadening of research experience. In addition, the candidate is committed to enhancing clinical skills with a focus on psychotic disorders. These goals will be attained through a program which devotes 75-80% time to research and no more than 25% time to clinical work and teaching. The candidate's long-term goal is to establish an independent academic career studying neuronal migration and differentiation in the mammalian telencephalon. It is hoped this work will contribute to the investigation of abnormal brain development as it occurs in human neurodevelopmental disorders.
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0.961 |
2003 — 2006 |
Anderson, Stewart A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Specification of Interneurons of the Cerebral Cortex @ Weill Medical College of Cornell Univ
DESCRIPTION (provided by applicant): The cerebral cortex contains two types of neurons, excitatory projection neurons and inhibitory interneurons. Based on chemical, physiological and morphological criterion, the inhibitory interneurons occur in distinct subtypes that subserve distinct functions. Several common illnesses, including epilepsy and schizophrenia may involve the abnormal development and/or dysfunction of particular interneuron subtypes. The long-term objective of this research is to understand the molecular basis for interneuron subtype specification. Recent studies have determined that most cortical interneurons derive from the ventral forebrain, in the anlage of the basal ganglia. The Aims of this project are designed to investigate the following questions. Are the cortical interneurons fated to become specific subtypes within the ventral forebrain where they are born? Alternatively, (or in addition) do they migrate into the cortex as GABAergic "protointerneurons" that utilize local factors to determine their ultimate subtype? Aim 1: Regional and temporal influences on interneuron subtype specification. Two methods will be employed to examine whether distinct interneuron subtypes have distinct sources. First, cells from various cortical and subcortical regions of mouse telencephali, at various times over the age-range of cortical neurogenesis, will be transplanted into neonatal cortical environments in vivo and in vitro. The effect of the donor cell's place and time of birth on their differentiated fate will be assessed. Second, a transgenic mouse expressing the Cre-recombinase under control of the transcription factor Nkx2.1 under will be generated. This mouse will permit fate-mapping of cells that originated within a subregion of the subcortical telencephalon, the medial ganglionic eminence and preoptic area. Aim 2: Factors that specify cortical interneuron subtypes: A candidate molecule approach. Since preliminary evidence suggests that important aspects of interneuron subtype specification do occur in within the ventral telencephalon, a candidate molecule approach is being taken to study factors which may influence this process. The two main factors under investigation, by manipulations in slice cultures followed by transplantation, are the secreted signalling molecule Sonic Hedghog, and the transcription factor Lhx6.
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0.961 |
2004 — 2008 |
Anderson, Stewart A |
K02Activity Code Description: Undocumented code - click on the grant title for more information. |
Specification of Cortical Interneurons @ Weill Medical College of Cornell Univ
DESCRIPTION (provided by applicant): Dr. Anderson is a physician-scientist who has a career focus on the molecular mechanisms of cerebral cortex development, as well as clinical and research expertise in schizophrenia. Continued salary support under the Research Career Award mechanism will be crucial to his career development. The cerebral cortex contains two types of neurons, excitatory projection neurons and inhibitory interneurons. Based on chemical, physiological and morphological criterion, the inhibitory interneurons occur in distinct subtypes that subserve distinct functions. Several common illnesses, including epilepsy and schizophrenia may involve the abnormal development and/or dysfunction of particular interneuron subtypes. Recent studies have determined that most cortical interneurons derive from the ventral forebrain, in the anlage of the basal ganglia. The long-term objective of this research is to understand the molecular basis for cortical interneuron subtype specification. Aim 1: Regional and temporal influences on interneuron subtype specification Two methods will be employed to examine whether distinct interneuron subtypes have distinct sources. First, cells from various cortical and subcortical regions of mouse telencephali, at various times over the age-range of cortical neurogenesis, will be transplanted into neonatal cortical environments in vivo and in vitro. The effect of the donor cell's place and time of birth on their differentiated fate will be assessed. Second, a transgenic mouse expressing the Cre-recombinase under control of the transcription factor Nkx2.1 under will be generated. This mouse will permit fate-mapping of cells that originated within a subregion of the subcortical telencephalon, the medial ganglionic eminence and preoptic area. Aims 2 and 3: The role of Sonic Hedgehog in interneuron subtype specification. Since evidence suggests that important aspects of interneuron subtype specification occur within the ventral forebrain, a candidate molecule approach is being taken to study factors, which may influence this process. The primary factor being examined is Sonic Hedgehog (Shh), a glycoprotein involved in cell fate determination in the ventral spinal cord, and which is also expressed in the ventral forebrain. Shh's role in interneuron specification will be studied by in vitro gain and loss of function manipulations followed by transplantation of neuronal progenitors into cortical environments in vivo and in vitro. In addition Shh function will be studied using conditional knockouts that target Shh expression within the ventral forebrain.
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0.961 |
2006 — 2010 |
Anderson, Stewart A |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Cortical Interneuron Fate Determination in the Medial Ganglionic Eminence @ Weill Medical College of Cornell Univ
GABAergic interneurons perform crucial roles in cerebral cortical development and function, but little is[unreadable] known about the molecular mechanisms that control interneuron fate determination. Most cortical[unreadable] interneurons originate in the medial ganglionic eminence (MGE) of the ventral forebrain, where recent[unreadable] evidence has begun to further define the origins of distinct subgroups of cortical interneurons[unreadable] (Xu...Anderson, 2004). The experiments described below are designed to examine how molecular signals[unreadable] specify interneurons within the MGE. To achieve this goal we will use a combination of in vivo and in vitro[unreadable] gain and loss of function studies focused on four proteins:[unreadable] 1) Sonic Hedgehog (Shh), a morphogen that promotes ventral neural tube development including the MGE,[unreadable] 2) NKX2.1, a transcription factor target of Shh signaling that is required for normal MGE development,[unreadable] 3) LHX6, a transcription factor that is, downstream of Nkx2.1 in the MGE and is expressed in interneurons[unreadable] migrating to the cerebral cortex,[unreadable] 4) ARX1, also expressed in migrating interneurons, and required for normal interneuron development.[unreadable] Since mutations inShh, Nkx2.1 and Arx1 have been linked to developmental forebrain abnormalities in[unreadable] humans, these studies lay the groundwork for identifying the "molecular code" for interneuron specification[unreadable] that will enhance our understanding of and treatment approaches for a variety of neuropathologic conditions.[unreadable] In addition, these studies are highly synergistic with other aims of the PPG including; the molecular control[unreadable] of cortical and subcortical proliferation by cyclin D2 and Shh (Projects 1 and 3), the role of migratory[unreadable] subcortical interneurons in cortical proliferation (Project 3) and the histological, physiological and behavioral[unreadable] effects of altered interneuron output by the MGE (Projects 1 and 4). In sum, the overarching goal of this[unreadable] project is to link clinically relevant alterations in embryonic forebrain development with postnatal histological[unreadable] and functional phenotypes.
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0.961 |
2007 — 2011 |
Anderson, Stewart A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Specification of Gabergic Interneurons in the Developing Cerebral Cortex @ Weill Medical Coll of Cornell Univ
DESCRIPTION (provided by applicant): GABAergic interneurons perform crucial roles in cortical development and function. These roles are executed by diverse interneuron subtypes, and abnormal function of particular interneurons has been implicated in a variety of neuropsychiatric diseases. However, little is known about the molecular mechanisms that underlie interneuron fate determination. Many cortical interneurons originate in the ventral (subcortical) telencephalon, where recent studies have begun to further define the origins of distinct subgroups of cortical interneurons. The realization that distinct interneuron subgroups have distinct origins has begun to inform efforts to understand the molecular regulation of interneuron specification. For example, in the medial ganglionic eminence (MGE) of the subcortical telencephalon, the signaling molecule Sonic Hedgehog (Shh) maintains the expression of Nkx2.1, a transcription factor whose expression during neurogenesis is required for the specification of important subgroups of cortical interneurons (Xu &Anderson, 2004, 2005). The goal of the experiments described below is to determine the mechanisms behind the specification of distinct interneuron subgroups within the MGE. Our overriding hypothesis is that much of the fate diversity of MGE- derived interneurons is based on their distinct origins within the MGE, and by the molecular differences encountered by progenitors within these origins. Aim 1. Origins of Parvalbumin or Somatostatin-expressing interneuron subgroups in the MGE. Expression of the neuropeptide somatostatin (SST) or the calcium binding protein parvalbumin (PV) define two non-overlapping subgroups of cortical interneurons, both of which require Nkx2.1 for their specification. Preliminary data from transplantation studies suggest that there is a strong bias for the SST+ subgroup to originate from the dorsal MGE, and for the PV+ subgroup to originate from the ventral MGE. This aim will further examine the spatio-temporal origins of these interneuron subgroups in the MGE. Aim 2. Specification of Parvalbumin or Somatostatin-expressing interneuron subgroups in the MGE. Nkx6.2, like the Shh signaling effector Gli1, is selectively expressed in the dorsal-most region of the MGE. Both of these genes are downregulated when Shh signaling is inactivated in the neuroepithelium by NestinCre:SmoFl/Fl (Xu &Anderson, 2005). The role of Nkx6.2 in interneuron specification will be tested by loss of function studies-- analysis of Nkx6.2 nulls, and gain of function studies--analysis of the interneuron fate effect of transient misexpression of Nkx6.2 in the ventral MGE of wild type slices, and of the ability of Nkx6.2 to rescue interneuron fate in Nkx2.1-/- slices. Future experiments will test the functions of other genes with enriched expressed in the dorsal or ventral MGE that have been identified through an array based screen.
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0.961 |
2008 |
Anderson, Stewart A |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Treating Cortical Epilepsy With Interneuron Transplants @ Weill Medical College of Cornell Univ
[unreadable] DESCRIPTION (provided by applicant): Despite advances in the treatment of seizure disorders, medically intractable epilepsy requiring surgical treatment remains a serious problem. The concept of treating seizures by grafting inhibitory interneurons into seizure foci is more than a decade old, but has yet to lead to clinically useful approaches. However, recent studies on the origins and specification of cortical interneurons warrant a new examination of this issue. We propose to use intracerebral grafts of interneuron precursors to treat chronic seizures that are focally induced in the adult mouse cortex. This study combines the expertise of two laboratories at Weill-Cornell Medical College. The PI, an Assistant Professor of Neurological Surgery, studies the propagation of cortical seizures in rodent models of epilepsy using in vivo electrophysiology and intrinsic optical imaging. The co-investigator, an Assistant Professor of Psychiatry, studies the specification of cortical interneuron subtypes in mice using transplantation of interneuron progenitors from the subcortical embryonic forebrain into the postnatal cortex. [unreadable] [unreadable] In Aim 1 the investigators will optimize the protocol for transplantation of interneuron progenitors into Tetanus Toxin (TTx)-induced chronic epileptic foci. In addition, effects of chronic seizures on interneuron migration, survival, and differentiation will be examined. Preliminary data suggest that, like transplants interneuron progenitors into the cortex of normal adult mice, transplants into TTx-induced seizure foci also result in migration, survival, and interneuronal differentiation of substantial numbers cells. [unreadable] [unreadable] Aim 2 will test the system-level functionality of transplanted interneurons by determining their ability to suppress epileptiform activity in the TTx model. Epileptiform activity will be assessed by simultaneous telemetry and video monitoring for several weeks before and several months following transplantation. The capacity to reduce epileptiform discharges with transplants of interneuron progenitor pools that are enriched for distinct interneuron subgroups will be compared to vehicle and cell-based controls. The results will strengthen the rationale for future studies using interneuron-progenitor directed stem cells, and also to explore the use of interneuron progenitors as a cell-based delivery system of anti-epileptic agents. [unreadable] [unreadable] The advances resulting from this proposal should inform efforts to prevent or interrupt intractable epileptogenesis resulting from focal lesions. The application of this research to increasingly clinically-oriented studies will be achieved by consultation with Dr. Theodore Schwartz, a practicing neurosurgeon who is the Director of Research at the Center for Epilepsy Surgery at WMC. [unreadable] [unreadable] [unreadable]
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0.961 |
2009 — 2010 |
Anderson, Stewart A Studer, Lorenz P. |
RC1Activity Code Description: NIH Challenge Grants in Health and Science Research |
Derivation of Cerebral Cortical Gabaergic Interneurons From Human Ips Cells @ Weill Medical Coll of Cornell Univ
DESCRIPTION (provided by applicant): Broad challenge area (14), Stem Cells. Specific challenge topic 14-MH-101: Developing iPS cells for mental disorders. Dysfunction of GABAergic interneurons of the cerebral cortex has been implicated in a variety of major neuropsychiatric illnesses, including schizophrenia, autism, anxiety, and epilepsy (where depression is a major source of morbidity). However, our lack of knowledge of how human interneurons develop and function, and how disease-related genes influence this process, greatly hinder our ability to understand, prevent or to treat interneuron-related mental illness. In addition, since neuropsychiatric disorders such as schizophrenia and autism appear to largely result from the combinatorial effects of polygenic risk factors with a significant component of environmental influence (perhaps mainly in utero), techniques for addressing this complicated intermix of effects would be tremendously useful. Induced pluripotent stem cells (iPSCs) are an important potential source of human interneurons that could be used to address both genetic influences on interneuron development and function, and to study genetic-environmental interactions in this process. Two specific examples of ways in which an iPSC based method could be used to study schizophrenia are provided in the Future Directions section below, but the ideal future objective would be to;1) derive iPSCs from patients known to harbor particular risk alleles for the given neuropsychiatric illness together with unaffected and non-risk allele carrying controls, 2) insert a fluorescent reporter construct known to express at relevant stages of interneuron development (i.e. genesis, migration, elaboration of processes and connectivity, activity dependent refinement, mature function, senescence) that are implicated in how that risk allele affects the development of disease, 3) direct the differentiation of the iPSCs to the particular stage of interneuron development, and 4) collect cells at that stage by FACS for the study of disease-related gene and protein expression, neuronal development or function, susceptibility to models of environmental insults, and testing of preventative or corrective agents. As a first step towards this idealized objective, the goal of this proposal is to develop and validate methods of generating, isolating, and studying putative cortical interneurons from human fibroblast derived iPSCs. The proposal builds on the progress in the Anderson and Studer labs at i) using modified bacterial artificial chromosomes (BACs) to obtain tissue-specific expression of reporter genes in transgenic mice as well as in mouse and human embryonic stem cells ii) developing highly efficient protocols for the conversion of human ES cells and human iPSCs into neural cells with forebrain identities, We propose the following Aims: Specific Aim 1. Use of an Lhx6-GFP reporter to isolate iPS cells directed to interneuron lineages Specific Aim 2. Use of an Nkx2.1-GFP reporter to isolate iPS cells directed to interneuron lineages Nkx2.1 is expressed in mitotic interneuron progenitors, whereas Lhx6 is expressed by the some of the same progenitors at the time of cell cycle exit and maintained during subsequent development. By directing iPSCs to putative interneuron progenitor fates (Nkx2.1+), we can prospectively isolate cells at this critical stage for genetic and transplantation studies aimed at defining their fate potential. These studies will be complemented by the isolating cells at Lhx6-expressing stages, when most of them have down-regulated Nkx2.1. We can again perform genetic and fate potential studies particular to these later stages of interneuron development. Despite the tremendous potential for using iPS-based approaches to study mental illness, several key challenges must be overcome to realize this promise. Among the key questions are: First, what is the optimal method for deriving interneuron-progenitor like cells from iPSCs? Second, how do we differentiate them into distinct interneuron types, and what system can we use to define those types outside of the human brain? Third, how much variability is present in this method when multiple interneuron-differentiated samples derived from the same human source are compared? These questions must be addressed to achieve significant utility in the use of iPSCs to study interneuron-related disease. High variability within multiple samples from the same source will render comparisons between patient and control-derived samples meaningless, or even misleading. To address these challenges we have assembled a team with considerable experience at each level of the project;1) embryonic and iPS-derived stem cell biology (Co-PI Dr. Studer), 2) cortical interneuron development (PI-Dr. Anderson), 3) electrophysiology in forebrain slices (Co-Investigator Dr. Goldstein), and 4) human cerebral cortex/interneuron and pathobiology of schizophrenia (Consultant David Lewis). Success in achieving the goals of the project will enable a wealth of future studies by our and many other groups, directed at the interaction of disease-related genes and environment on interneuron genesis, maturation, and function. Such iPSC-based approaches could have a major impact on understanding the tremendously complex, and difficult to study, role of interneurons in neuropsychiatric diseases. PUBLIC HEALTH RELEVANCE: Dysfunction of GABAergic interneurons of the cerebral cortex has been implicated in a variety of major neuropsychiatric illnesses, including schizophrenia, autism, anxiety, and epilepsy (where depression is a major source of suffering). However, both our general lack of knowledge regarding how human interneurons develop and function, and our specific lack of knowledge of how disease-related genes may influence this process, greatly hinder our ability to understand, prevent or to treat interneuron-related mental illness. Induced pluripotent stem cells (iPSCs) are an important potential source of human interneurons that could be used to address both genetic influences on interneuron development and function, and to study genetic-environmental interactions in this process. The goal of this proposal is to develop methods and protocols for the consistent derivation of function inhibitory interneurons from human iPSCs, to lay critical ground word for future studies on the role of inhibitory interneurons in neuropsychiatric disease.
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0.961 |
2009 — 2013 |
Anderson, Stewart A |
K02Activity Code Description: Undocumented code - click on the grant title for more information. |
Fate Determination of Interneruons in the Mammalian Telecephalon @ Weill Medical Coll of Cornell Univ
DESCRIPTION (provided by applicant): Dr. Anderson is a physician-scientist who has a career focus on the molecular mechanisms of cerebral cortex development, as well as clinical and research expertise in schizophrenia. Continued salary support under the Research Career Award mechanism will be crucial to his career development. The cerebral cortex contains two types of neurons, excitatory projection neurons and inhibitory interneurons. Based on chemical, physiological and morphological criteria, the inhibitory interneurons occur in distinct subtypes that subserve distinct functions. Several common illnesses, including epilepsy and schizophrenia may involve the abnormal development and/or dysfunction of particular interneuron subtypes. Recent studies have determined that most cortical interneurons derive from the ventral forebrain, in the anlage of the basal ganglia. The long-term objective of this research is to understand the molecular basis for cortical interneuron subtype specification. Aim 1: Regional and temporal influences on interneuron subtype specification. Two methods will be employed to examine whether distinct interneuron subtypes have distinct sources. First, cells from various cortical and subcortical regions of mouse telencephali, at various times over the age-range of cortical neurogenesis, will be transplanted into neonatal cortical environments in vivo and in vitro. The effect of the donor cell's place and time of birth on their differentiated fate will be assessed. Second, a transgenic mouse expressing Cre-recombinase under control of the transcription factor Nkx2.1 will be generated. This mouse will permit fate-mapping of cells that originated within a subregion of the subcortical telencephalon, the medial ganglionic eminence and preoptic area. Aims 2 and 3: The role of Sonic Hedgehog in interneuron subtype specification. Since evidence suggests that important aspects of interneuron subtype specification occur within the ventral forebrain, a candidate molecule approach is being taken to study factors which may influence this process. The primary factor being examined is Sonic Hedgehog (Shh), a glycoprotein involved in cell fate determination in the ventral spinal cord which is also expressed in the ventral forebrain. Shh's role in interneuron specification will be studied by in vitro gain and loss of function manipulations followed by transplantation of neuronal progenitors into cortical environments in vivo and in vitro. In addition Shh function will be studied using conditional knockouts that target Shh expression within the ventral forebrain.
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0.961 |
2011 — 2015 |
Anderson, Stewart A |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
The Regulation of Mge Proliferation and Cortical Interneuron Fate Determination @ Weill Medical Coll of Cornell Univ
This Program examines the interaction of proliferation and cortical interneuron fate determination and probes the functional consequences of altering interneuron subpopulations. Generating the correct number and subtypes of these neurons is crucial for the development of a normally functioning brain, and Project 2 focuses on the interacting roles of several signaling systems, Notch, Wnt, and Sonic hedgehog (Shh), that critically influence this process. Aim 1. Notch signaling regulates proliferation and cell fate in many organs, but a role for Notch in interneuron generation by the medial ganglionic eminence (MGE), the source of critical cortical interneuron subpopulations, is not known. We identified the Notch ligand Jagged-1 in a microarray screen for genes differentially expressed in the dorsal versus the ventral MGE, raising the possibility that Notch signaling regulates interneuron fate determination. In Aim 1, we examine conditional loss of Jagged-1 function in the dorsal MGE. Via interactions with Project 1, we explore abnormalities of cyclin D2 expression that we have identified in preliminary studies with these mutants. Via interactions with Project 3, we will further explore Notch-related alterations in proliferative behavior using live imaging in organotypic slice cultures. Aim 2. During the first four years of this Program we have shown that the expression of the interneuron fate-determining transcription factor, Nkx2.1, requires Shh signaling during interneuron genesis. We also found that proliferation of Nkx2.1-expressing, MGE progenitors requires canonical Wnt signaling. In the other systems, Shh signaling can be necessary for the expression of Tcf4, an effector of canonical Wnt signaling, that we have shown to be expressed in the subcortical telencephalon. Tcf4, in turn, has been shown to activate the expression of Jagged 1 in non-neural tissue. In Aim 2 we examine potential interactions of Shh, Wnt, and Notch signaling effectors as they relate to MGE proliferation and interneuron fate. Again, interaction with Projects 1 & 3 will be critical for teasing out the mechanisms underlying defects in cell cycle and modes of progenitor division that are generated through our various signaling manipulations. As effectors of all three of these signaling systems, like cortical interneurons themselves, are associated with neurological and neuropsychiatric disease, Project 2 will generate several novel mouse models of selective cortical interneuron losses, one of which is expected to produce an inducible, titratable, and time-limited reduction of interneuron genesis, for detailed investigation by the Neurobehavioral Analysis Core. The overarching goal of this project is to link critical mechanisms in neurogenesis and neural subtype fate with clinically germane aspects of brain function.
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0.961 |
2012 — 2016 |
Anderson, Stewart A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Generating and Studying Gabaergic Forebrain Interneurons With Human Stem Cells @ Children's Hosp of Philadelphia
DESCRIPTION (provided by applicant): Multiple lines of evidence implicate dysfunction of forebrain interneurons in the symptomatology of major neuropsychiatric illnesses, including schizophrenia, autism, and epilepsy. This dysfunction involves subtypes of interneurons that differ in their neurochemistry, connectivity, and physiology. Unfortunately, gaining a detailed molecular and cellular grasp of how alterations of interneuron-related disease genes actually affect interneuron development or function has been exceedingly difficult. This is due, in part, to our general inability to obtain biopsy-like brain specimens from diseased individuals for in vitro studies. Recently, advances in the derivation of neurons from mouse and human stem cells suggest that pluripotent cells can be used to study developmental neurogenetics and function. My lab and others have begun to make progress in deriving cortical interneurons from mouse and human stem cells. In particular, we can generate cells that express molecular markers of interneuron progenitors of the basal forebrain of both mouse and human, and we can show that these cells can migrate and survive extensively after transplantation into mouse cortex, express GABA and other interneuron markers and, in the case of mouse ES derived interneurons, have interneuron subtype-like physiological characteristics. However, major hurdles must be solved before the stem cell system is ready for broad usage in the search for causes and treatments of interneuron-related disorders. For example, since distinct interneuron subtypes are differentially affected in various disorders, how will we generate these subtypes from human stem cells? Since key aspects of interneuron subtype function depend on specific patterns of inputs, intrinsic activity, and axonal targeting onto other neurons, what assays will allow us to study these features? Here we will use the experimentally facile mouse ES system to learn how to enrich stem cell differentiations for distinct subgroups or subtypes of cortical interneurons, and will apply that system to identify additional markers of fate-committed but highly immature interneuron precursors (Aim 1). We will then apply this information, together with results of ongoing studies, to generate interneuron subclasses from human stem cells (Aim 2). Aim 2 will involve both embryonic stem cells, that have the advantage of being better characterized and are not affected by the genetic disruptions associated with induced pluripotent stem cell (IPSC) reprogramming; and IPSCs, that are derivable from diseased individuals. In Aim 3 we will study loss of function effects of two disease-related genes on human interneuron migration and synaptogenesis. Success in this endeavor would enable a host of future studies on developmental and functional aspects of human interneurons, resulting in major advances in the etiology, prevention, and treatment of interneuron-related neuropsychiatric disease.
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0.901 |
2016 — 2018 |
Anderson, Stewart A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Ipsc Phenotype, Mitochondrial Haplotype and Psychosis in 22q11 Deletion Syndrome @ Children's Hosp of Philadelphia
Metabolic compromise, including mitochondrial dysfunction, may contribute to the development of symptoms in schizophrenia. However, approaches to explore the relationship between mitochondrial dysfunction, neural dysfunction, and schizophrenia risk are needed. The most common genetic risk factor for schizophrenia is the 22q11.2 deletion syndrome (22q11DS), associated with a 25% risk of developing this disorder. Interestingly, in 22q11DS six of the roughly 40 deleted genes encode for mitochondrial-localizing proteins. We have assembled a multidisciplinary team to combine the use of stem cell derived neurons from 22q11DS patients with or without schizophrenia, and controls, with genetic analyses of 22q11DS patients with and without schizophrenia. We will test the hypothesis that a ?second hit? within mitochondrial-related genes in the 22q11DS context increases metabolic dysfunction in neurons and is associated with an increased risk for schizophrenia in patients. Aim 1. Investigation of mitochondrial function in 22q11DS derived forebrain neurons. Existing IPSC lines from 3 groups will be compared: 1) 22q11DS with schizophrenia, 2) 22qDS without schizophrenia or history of psychosis (and over the age of 25), and healthy controls. Forebrain-like neurons from these lines will be compared for evidence of mitochondrial dysfunction that is most pronounced in the 22q11DS+SZ group. Consistent with the fact that one of the 22q11-deleted genes is MRPL40, a subunit of the mitochondrial ribosome, our preliminary evidence suggests that IPSC-derived neurons from the 22q11DS+SZ group have reduced translation of COX1, a key mitochondrial DNA-encoded protein. They also have significantly reduced cytochrome C oxidase activity, and strong evidence of oxidative damage. Aim 2. Are genetic ?second hits? to mitochondrial function associated with psychosis in 22q11DS? We will use whole genome sequence data from about 600 22q11DS patients, evenly split between those with and without chronic psychosis, from the International 22q11.2DS Brain and Behavior Consortium (22QIBBC). Bioinformatic processing will determine whether the risk of developing SZ in 22q11DS is potentially influenced by the mitochondrial haplogroup, or is associated with an increased presence of mutations in mtDNA, or is associated with an increased presence of mutations in nuclear-encoded genes that generate mitochondrial- functioning proteins. Future studies would employ mitochondrial cybrids (`swapping?) technology, genome editing, ?putback? experiments, and other approaches to test the functional relevance of findings from Aim 2 on mitochondrial function in 22q11DS-derived IPSCs, and could be extended to IPSCs from other high-risk SZ groups. These results could also be used to examine the prospective risk of 22q11DS children to develop chronic psychosis, using the large cohort of subjects currently being enrolled and studied by the 22QIBBC. Significance The results of this study could lead to improved prediction, treatment, and prevention of psychosis in 22q11DS, and could generalize to the treatment or prevention of psychosis beyond 22q11DS.
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0.901 |
2017 |
Anderson, Stewart A Spinner, Nancy Bettina |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Human Chromosome 14 Analysis in Neuronal Cells @ Children's Hosp of Philadelphia
Project Summary/Abstract Human ring chromosomes are abnormal structures formed by intrachromosomal fusions, creating a circular chromosome. Ring chromosome syndrome is a debilitating disorder resulting in severe drug-resistant epilepsy, intellectually disabilities (IQ <70), and developmental delay. Ring chromosome patients and families deal with life-long challenges of prolonged seizures, learning and memory issues, severe psychological disruption, and a feeling of powerlessness, resulting in a great deal of stress and strain. Clinically this disorder affects multiple organ systems including the brain (microcephaly, early onset epilepsy, and intellectual disabilities), the eye, the immune system, and growth. Our laboratory and others, have focused on mapping and characterizing the molecular and cytogenetics of ring chromosome structures; however, to date no published studies have focused on investigating the cellular neuropathology due to lack of appropriate models. To investigate this disorder, we have created a large biobank of cell lines from ring chromosome 14 (r(14)) patients and family members. From these cell lines, we have produced the only model available by generating patient-specific induced pluripotent stem cells, which provide the foundation for studying this devastating disorder that lacks alternative models. In our preliminary work, we find that by differentiating ring chromosome iPSCs toward a forebrain fate we can generate populations of neuronal precursor cells (NPCs) that can be further differentiated into post-mitotic neurons. Neuronal precursor cells harboring the ring chromosome 14 have reduced cellular growth and decreased expression of key telencephalon neuronal markers. Interestingly, a number of genes located on chromosome 14 are involved in neuronal differentiation or maintenance, leading us to postulate that the ring structure modifies expression of this key gene responsible for in vitro cortical cell generation. We have also begun to generate an in vitro cerebral cortex organoid model, which will be important for studying the early embryonic events perturbed in a R(14) developmental model. Mammalian genomes are organized in distinct architectures which allow for the orchestration of proper gene and epigenetic regulation. Our proposed studies will identify ring chromosome 14 orientation in the nucleus, epigenetic marks on chromosome 14, and establish a 3-dimensional (3D) cerebral cortex developmental model for studying phenotypes of Ring chromosome 14 syndrome. We hypothesize that the altered higher order chromosome structure resulting from ring formation disrupts normal chromatin compaction, looping, localization within the nucleus and ultimately gene regulation. The proposed work will provide important tools for studying R(14) syndrome, and yield a comprehensive view of the role a ring chromosome has on DNA positioning, chromatin modifications, and in vitro neuronal development. In addition, this innovative model should provide a strong foundation for future work fully characterizing 3D organoid development in R(14) and provide essential reagents (neuronal tissues) to study the neural circuits and electrophysiological patterns in R(14) patient samples.
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0.901 |
2018 — 2019 |
Anderson, Stewart A Jordan-Sciutto, Kelly L (co-PI) [⬀] |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
A Novel System For the Study of Neuro-Hiv; Human Stem Cell Derived Microglia @ Children's Hosp of Philadelphia
Major neuropsychiatric disorders, including Aids Dementia, Alzheimer's Disease, depression and schizophrenia, may involve aberrant phagocytosis of cerebral cortical synapses by monocytes and/or microglia. However, efforts to target this process for therapeutic intervention have been limited. One reason for the limited success in the development of therapeutic interventions for CNS microglia/macrophage activation is that species differences between the immune systems of humans and model organisms render the co-culture of human immune cells with non-human neurons less informative. Fortunately, recent progress in generating cortical cells, including neurons, astrocytes and microglia, from human stem cells provides a new opportunity to address this challenge. This proposal is a collaborative effort between the labs of Kelly Jordan-Sciutto, who studies neuro-AIDS and monocyte activity in vitro, and Stewart Anderson, who studies forebrain development in relation to neuropsychiatric disorders using mouse and human stem cells. Differentiation of iPSCs into forebrain-like excitatory neurons (iNrns), and their co-culture with astrocyte-like cells (iAstrs), results in a synaptically dense network. By plating human monocytes over those neurons we observe IBA1+ cells that appear to have ingested synaptic markers. Moreover, we are able to generate human microglia like cells (iMgl), infect them with HIV, and find that they react to this infection by cytokine release and the formation of multinucleated cells. Importantly, exposure to an antiretroviral agent reduces their reverse transcriptase activity. We also find that we can co-culture iMgl together with i-Nrns and i-Astrs. In this exploratory R21 we will fine-tune this human iMgl, iAstr, iNrn culture system to test the hypothesis that HIV infected microglia decrease excitatory synapse density in vitro. If so, we will determine whether the effect is mimicked by conditioned media, is associated with increased compliment deposition, and is affected by ART. We will also test whether viral suppression occurs in the iMgl within the triculture context. Success would lead to new approaches for mechanistic studies and therapeutics development for the multiple neuropsychiatric disorders, such as HIV Associated Dementia, in which monocyte/microglial activation may play a crucial role in their pathogenesis.
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0.901 |
2018 — 2020 |
Anderson, Stewart A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Generating and Accelerating the Maturation of Pvalb-Fast Spiking Interneurons From Human Stem Cells @ Children's Hosp of Philadelphia
Multiple lines of evidence implicate dysfunction of cerebral cortical inhibitory interneurons in the symptomatology of major neuropsychiatric illnesses, including schizophrenia, autism, Tourette disorder, bipolar disorder, and epilepsy. This dysfunction involves subtypes of interneurons that differ in their neurochemistry, connectivity, and physiological characteristics. Recent advances in the derivation of interneurons from human pluripotent stem cells (PSCs) demonstrate their utility in studies of neuronal developmental genetics, function, and disease. Unfortunately, there has been limited success at generating the parvalbumin-expressing, fast- spiking (PV-FS) subgroup, even though this is the most plentiful subgroup of cortical interneuron and their dysfunction is strongly implicated neuropsychiatric disease. Despite tremendous progress in enriching for mouse embryonic stem cell (mESC)-derived PV-FS interneurons, no approach for enriching for their human counterparts is available. This shortfall may be secondary to the length and complexity of human forebrain neural subtype-specific directed differentiation protocols, but also to the protracted maturation of PV-FS cells that normally require strong excitatory inputs to mature. The goal of this highly focused, 3-year, modular budget, renewal application is to build directly upon our substantial progress in deriving PV-FS from mESCs the previous grant cycle, together with our progress at accelerating the maturation of human stem cell derived interneuron-like cells by conditional activation of mTOR signaling, to enrich for the derivation of PV-FS interneurons from human PSCs. Through this scalable process we will also establish experimental systems, including co-culture with prenatal rat cortical neurons and astrocytes, transplantation into neonatal mouse neocortex, and engraftment into human forebrain organoids, that will enable complimentary scientific questions to be addressed. Success in this endeavor will make possible a host of future studies on the etiology, prevention, and treatment of interneuron-related neuropsychiatric disease.
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0.901 |
2021 |
Anderson, Stewart A |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Predicting Psychosis in 22q11.2 by Failed Mitochondrial Compensation @ Children's Hosp of Philadelphia
The 22q11.2 deletion syndrome (22qDS), one of the most common copy number variations at 1:4000 births, is associated with a roughly 25% risk of developing schizophrenia-related symptoms. Since the features of schizophrenia (SZ) in the context of 22qDS are largely shared with non-syndromic SZ in terms of typical onset in later adolescence or early adulthood, symptoms, and brain changes, the high rate of SZ in 22qDS provides an opportunity for longitudinal studies that identify cognitive and physiological changes that predict SZ risk. Such identification can lead to mechanistic studies behind the variable penetrance for SZ in 22qDS, and lead to preventative measures that may extrapolate to some instances of non-syndromic SZ. Multiple lines of evidence, from studies of human blood, human genetics, IPSC-derived neurons, and mouse models, suggest that aspects of the 22qDS neural phenotype involves mitochondrial dysfunction. Indeed, 6 of the 46 genes in the deleted region encode for mitochondrial localizing proteins. We find in an ongoing study of IPSC-derived neurons that while mitochondrial OXPHOS is reduced in the 22+SZ group relative to control (Li et al., 2019), the 22q without SZ (22q(-)SZ) group has control-levels of OXPHOS. Remarkably, relative to both controls and to the 22q+SZ groups, the 22q(-)SZ group has upregulated expression of multiple genes involved in OXPHOS, and upregulation of PGC1a, a master regulator of mitochondrial biogenesis. These results suggest that variable penetrance for SZ in 22qDS may be influenced by an individual's capacity for mitochondrial compensation, a feature of some mitochondrial genetic diseases. To test this idea with a far higher throughput approach than possible with IPSC-derived neurons, we examined 20 lymphoblastoid cell lines (LCLs) from 22qDS adults, equally split between those with 22q+SZ and those with 22q(-)SZ. By a targeted analysis of a few measures of OXPHOS activity and related gene expression, we found that mitochondrial complex I activity is higher in the 22q(-)SZ group, as are the levels of the complex 1 gene NDUFV1, PGC1a, and its co-factor PPARa. Statistical analyses revealed that a composite Mito-score based on these 4 measures has over 90% predictability for the presence or absence of SZ-related symptoms in 22qDS. These results raise the compelling question, can failed mitochondrial compensation identified in lymphoblastic cell lines from teenagers with 22q11.2 deletion syndrome predict their likelihood of developing schizophrenia? In this R21 proposal, using existing LCL lines and existing longitudinal follow up data, we seek to determine whether mitochondrial function/gene expression in LCLs from mid to younger teenagers predicts their risk of developing SZ-related symptoms as later teenagers or young adults. Positive results would lead directly to a clinical trial designed to prevent or ameliorate the development of SZ in 22qDS, and would likely have implications for the treatment or prevention of some instances of non-syndromic schizophrenia.
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0.901 |