1999 — 2011 |
Kriegstein, Arnold |
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. |
Intercellular Signaling in Neocortical Development @ University of California San Francisco
DESCRIPTION (provided by applicant): Regulation of cell cycle dynamics is a contributing mechanism for generating cortical diversity and regulating the balance between excitatory and inhibitory neurons. In early telencephalic development, symmetrical cell divisions expand the pool of neuronal progenitor cells. With the onset of neurogenesis in the cortex, the progenitors of the excitatory cells undergo asymmetric divisions within the ventricular zone (VZ) to generate neurons. The key feature of asymmetric divisions is the unequal inheritance of intrinsic determinants. However, both cell-intrinsic and cell-extrinsic factors influence neuronal production. In this proposal, we will use techniques of retroviral lineage analysis, optical imaging, time-lapse confocal microscopy, and electrophysiology to characterize progenitor cell divisions and to answer the following questions: How do patterns of asymmetric and symmetric progenitor cell divisions in the cortical proliferative zone in vivo generate neuronal diversity in the developing neocortex? Do spatially distinct neurogenic niches determine the mode of cell division and patterns of neurogenesis? Does cleavage plane determine or predict progenitor fate? Does segregation of the cell-intrinsic factor Numb determine symmetry/asymmetry of division and how does this differ at different stages of corticogenesis? Do cell-extrinsic fate-determining signals such as GABA activate cortical VZ cells and promote symmetric progenitor divisions to expand the precursor pool? Do GABAergic interneurons originating in the striatum promote the generation of excitatory pyramidal cells in the developing cortex? These related yet independent questions are the subject of the current proposal. The critical balance between excitation and inhibition underlies the regulation of excitability in the developing and mature cortex and an imbalance is known to have significant pathological effects ranging from subtle disorders associated with seizures, to devastating cortical malformations with intractable epilepsy. The data from these experiments will help us to understand how the critical balance of excitatory and inhibitory cells is regulated and how this affects brain development.
|
0.915 |
2003 — 2015 |
Kriegstein, Arnold |
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. R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
The Physiology of Radial Units in Corticogenesis @ University of California, San Francisco
DESCRIPTION (provided by applicant): The role of gap junction proteins in the regulation of neural stem and progenitor cell proliferation and neuronal migration are the subjects of the current proposal. It has long been known that neuroepithelial cells in the developing cortex express gap junction proteins and are coupled by gap junction channels to other cells, but the role of coupling is not well understood. We have developed a set of molecular tools to address this issue. The experiments in the current proposal build on our preliminary results to explore how gap junction coupling regulates proliferation, migration and fate determination in the developing embryonic neocortex. We have designed and tested a set of RNAi constructs that are able to knock-down expression of each of the connexin subunits known to be expressed by embryonic neural stem and progenitor cells. We also constructed and tested the necessary mutant RNAi controls and introduced these constructs, singly and in combination, into proliferate cells in the in vivo developing cerebral cortex using in utero, intraventricular injection and electroporation. Inspection of the developing brain following variable survival intervals has demonstrated remarkable cellular effects of the loss-of-function strategy, including alterations in glial-guided migration, progenitor cell proliferation, and neurogenesis. We propose to refine a set of molecular tools to disrupt gap junction function in order to test a series of hypotheses concerning cortical development, including that gap junction communication mediates radial glial cell proliferation, couples migrating neurons to radial glial guides, promotes the generation of neurons, and mediates small molecule exchange between progenitor cells and immature neurons. The specific experiments outlined in this proposal will provide new information concerning the way neurons and progenitor cells interact at embryonic stages of brain development. Many human neurodevelopment disorders are associated with defects in neurogenesis and migration, ranging from severe malformations with mental retardation and epilepsy, to more subtle disorders such as autism and dyslexia. The results of this study may help shed light on mechanisms relevant to the etiology of many of them.
|
0.915 |
2009 — 2010 |
Kriegstein, Arnold Noble, Linda J. |
RC1Activity Code Description: NIH Challenge Grants in Health and Science Research |
Spinal Cord Injury: Targeting Local Inhibition to Improve Outcome @ University of California, San Francisco
DESCRIPTION (provided by applicant): Project Summary/Abstract This proposal addresses Broad Challenge Area: 15: Translational Science and Specific Challenge Topic: Demonstration of "proof-of-concept" for a new therapeutic approach in a neurologic disease: 15-NS-104 It is estimated that 11,000 Americans suffer spinal cord injury (SCI) every year. According to the Centers for Diseases Control and Prevention, SCI costs the nation an estimated $9.7 billion each year. Most patients survive their injury, leaving as many as 250,000 people in the United States who suffer from chronic spinal cord injury. The majority of people who survive a spinal cord injury will develop symptoms of spasticity, a pathologic exaggeration of normal spinal reflexes that develops in nerve cells below the level of injury. Spasticity is a major health problem for SCI patients. It limits their mobility and independence and can cause pain, muscle contractures, disabling muscle spasms, and bowel and bladder spasticity. Bladder spasticity is a particular problem, since it can cause urine reflux and kidney damage. The mechanisms of muscle spasticity after spinal cord injury are not well understood, but recent studies indicate that the loss of particular descending axonal pathways most likely results in the decreased activity of inhibitory interneurons, which causes overreaction of motor neurons. The primary medications used to treat spasticity either enhance neural inhibition or inhibit muscle contraction. All have significant systemic side effects and do not completely eliminate spasticity. Surgical treatments used in medically intractable cases include intrathecal baclofen, which can cause loss of residual walking ability, tolerance, withdrawal syndrome, and infectious and other complications related to indwelling catheters;and posterior rhizotomy which can cause weakness, sensory loss, urinary dysfunction, sexual dysfunction, and spinal instability. An effective local therapy that avoids systemic effects and spares residual function is an unmet need for SCI patients. A novel approach to spinal injury treatment would be to transplant inhibitory interneuron progenitor cells in segments below the injury site where they could migrate, integrate, and establish new inhibitory circuits capable of reducing spastic activity. Work in our laboratories has demonstrated that precursors of inhibitory GABAergic interneurons derived from the rodent medial ganglionic eminence (MGE), a brain region specialized to produce large numbers of inhibitory interneurons, can migrate, integrate and modulate neural circuits when grafted into the adult brain. This is a unique and robust property of MGE progenitor cells that has not been demonstrated in any other type of neural precursor cell, and our preliminary data show that MGE cells can also integrate in adult spinal cord grey matter. Our proposal will determine how these cells migrate, integrate, and function when grafted to the spinal cord grey matter (Aim 1). We will determine whether MGE progenitor cells, grafted below the level of a spinal cord injury, can provide inhibitory modulation of spinal circuits (Aim 2). Finally, we will determine if MGE cell grafts can reduce neurogenic bladder dysfunction, a measurable index of spasticity following spinal cord injury (Aim 3). If successful, our studies will lay the groundwork for a potential novel therapy for chronic spinal cord injury. PUBLIC HEALTH RELEVANCE: There is an estimated 250,000 individuals who currently live with disability associated with chronic spinal cord injury. This proposal addresses a novel stem cell transplantation strategy to ameliorate bladder dysfunction and involuntary muscle spasms that accompany chronic spinal cord injury. A long-term goal of these studies is to translate this effort to the spinal cord injured patient, with a specific focus on improving bladder function and reducing muscle spasms, both of which can profoundly affect quality of life.
|
0.915 |
2010 — 2014 |
Kriegstein, Arnold |
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. |
Molecular and Cellular Mechanism of Microcephaly @ University of California, San Francisco
DESCRIPTION (provided by applicant): Autosomal recessive primary microcephaly (MCPH) is a genetically and clinically heterogeneous disease defined by a decrease in head circumference at birth. Patients often have a broad spectrum of neurological problems, including mental retardation, focal or generalized seizures, hyperactivity, and attention deficit disorder. The decrease in brain volume without major architectonic abnormalities most likely stems from a primary defect in neurogenesis and or neuronal migration. Five of eight MCPH genes localize to the centrosome during all or part of the cell cycle. In vitro studies provide evidence that these genes play roles in essential centrosomal functions such as cell cycle regulation. Nonetheless, the mechanism of MCPH in brain development is still poorly understood. The long-term goal of this project is to profile the role of MCPH genes in neocortical development and disease pathogenesis. The objectives are to uncover the molecular and cellular controls of MCPH genes on neurogenesis and to define how centrosomal proteins regulate the mode of division (symmetric or asymmetric), neuronal migration and differentiation. Recent studies from our lab and others have demonstrated that radial glial cells are a major population of neuronal progenitor cells. They divide asymmetrically to self-renew and give rise to cortical neurons. Asymmetric centrosome inheritance is believed to regulate the differential behavior of self-renewing progenitors versus differentiating progeny in the embryonic mouse neocortex. Centrosome defects in Drosophila do not dramatically perturb mitosis in most somatic cells, but the asymmetric division of larval neuroblasts is noticeably disrupted, underscoring the particular significance of centrosome behavior for asymmetric cell division of progenitor cells and determination of daughter cell fate. Furthermore, the centrosome is the primary anchor for microtubules, enabling the differentiating neuron to initiate and extend an axon, a key process of neuron differentiation. Based on these observations, the central hypothesis of this application is that the MCPH genes control neurogenesis, neuronal migration, and differentiation in the developing cortex. Guided by strong preliminary data this hypothesis will be tested by pursuing four specific aims: 1) To determine the molecular and cellular mechanism by which MCPH genes regulate radial glial cell division; 2) To explore the function of MCPH genes in regulating asymmetric inheritance of mother versus daughter centrosomes and daughter cell fate; 3) To define the function of MCPH genes in regulating neuronal migration and differentiation in the developing cortex; and 4) To validate the relevance of findings in the mouse to the pathogenesis of human MCPH using patient induced pluripotent stem (iPS) cells. With innovative approaches including high-temporal time-lapse imaging and molecular genetic techniques, the proposed research will provide new insights into the pathogenesis of MCPH and expand our knowledge of brain development. Moreover, the results of this study may shed light on mechanisms relevant to the etiology of many neurological and psychiatric disorders related to cortical function.
|
0.915 |
2011 — 2016 |
Kriegstein, Arnold |
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. |
Stem Cells of the Developing Human Neocortex @ University of California, San Francisco
DESCRIPTION (provided by applicant): The most highly evolved structure in the human brain is the neocortex, which is responsible for the higher cognitive functions unique to humans. Despite its importance, little research has been done to understand how the human neocortex develops, especially during the period when most neurogenesis takes place, the second trimester of gestation. We have recently characterized a unique population of neural stem cells (oRG cells) in the developing human neocortex responsible for the majority of cortical neuron production. These cells reside in a germinal region known as the outer subventricular zone (OSVZ). Notably, the OSVZ is not found in the rodent, implying that using the rodent as a model system to understand human neocortical development has significant limitations. The aim of the proposed research is to further understand the mechanisms by which OSVZ progenitor cells contribute to human neocortical development, focusing on four areas: 1) the origin of oRG cells and their neural stem cell properties; 2) the cellular and molecular mechanisms that maintain oRG cell identity and self-renewal during neurogenesis; 3) the amplifying divisions that oRG cell daughters undergo to increase neuronal production; and 4) how oRG cells are required for the production and correct layering of neuronal subtypes in the neocortex. With innovative approaches such as cell labeling and clonal analysis, real-time imaging of cellular behaviors, and molecular genetic techniques in organotypic slice-cultures, the proposed research aims to provide a solid new foundation for understanding human neocortical development. This framework will be essential to correctly understand human neurological diseases that have genetic or developmental origins, ranging from cortical malformations such as lissencephaly and microcephaly to more subtle defects like epilepsy, autism, and schizophrenia. Knowing the proper developmental sequences by which cortical neurons are generated will also be important for cellular regeneration or transplantation therapies for neurological diseases.
|
0.915 |
2014 — 2016 |
Kriegstein, Arnold |
U01Activity 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. |
Mapping the Developing Human Neocortex by Massively Parallel Single Cell Analysis @ University of California, San Francisco
? DESCRIPTION (provided by applicant): This proposal seeks to create a single cell resolution map of the developing human neocortex. We propose to determine the number of different subtypes of neural stem and progenitor cells that generate the cerebral cortex, and then follow the developmental trajectories of the newborn neurons they produce to obtain an understanding of the diversity of cortical neurons that will ultimately form the adult cortex. We plan a novel approach to this problem by integrating surveys of single cell gene expression and physiology in human cortical cells from multiple brain regions at a series of developmental stages. In collaboration with Fluidigm Corporation, we have developed innovative strategies for massively parallel profiling of molecular and physiological properties of primary human cortical cells using microfluidic technologies, cellular barcoding, and timelapse microscopy. We now propose to conduct an integrated survey of human fetal cortical cells in prefrontal, motor, and visual cortex to classify cell types and lineages. Our work will shed light on the developmental origins of cell diversity in the human cortex by addressing three specific aims. First, we will use unbiased cell type classification to provide a realistic estimate of the number of defined progenitor and immature cell types in specific brain regions. We hypothesize that progenitor diversity influences the development of structural and connectivity differences in cortical areas, but the relationship between the diversity of progenitor cells and adult neurons has been difficult to study in the human brain. We propose to sequence >100,000 single cells collected from specific lamina of the developing cortex and determine single cell gene expression using cellular barcodes for efficient low-coverage mRNA sequencing. By analyzing genes, microRNAs, and chromatin states, we anticipate to be able to distinguish discrete populations of progenitor and postmitotic cells. Second, we hypothesize that a combined understanding of physiological and molecular properties will improve cell type classification and reveal molecular factors most predictive of functional maturation. To this end we will use a novel high-throughput screen to measure physiological responses to a range of ligands and neurotransmitters in single cells captured directly on microfluidic chips. The cells will then be lysed, and mRNA reverse-transcribed, amplified, and sequenced. This approach to profiling and classification of single cells will integrate information on molecular properties with physiological responses across anatomical locations. Finally, we will classify cells as belonging to specific lineage trajectories, in additin to discrete categories, using cellular resolution maps of development. We will validate predicted functional and molecular lineages using timelapse microscopy and electrophysiology in cultured primary human cells and slices. This approach will further integrate the molecular and physiological identity of cells from distinct cortical areas with their lineage identity and will provide clues to the determinants that specify neuronal subtypes and connectivity patterns in the maturing human cortex.
|
0.915 |
2014 — 2015 |
Kriegstein, Arnold |
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. |
Project 2: Contribution of the 3rd Trimester Fetal Subventricular Zone to Human @ University of California, San Francisco
Project Summary (Project 2) The overall goals of Project 2 are to study the origins and later development of human oligodendrocyte precursor cells (OPCs) in fetal human cortex during 20-40 weeks gestation age (GA). We will study processes of OPC proliferation, the nature of their migratory behavior and differentiation in situ the relationship to the outer subventricular zone (oSVZ); a germinal zone present in 3rd trimester human brain that may account for significant gliogenesis. Specific aims include a detailed exploration of OPC lineage ontogeny in human cortex, based on anatomical and immunolabeling marker analysis of both acutely harvested cortical tissue and specimens from the Core B tissue bank. We will perform dynamic imaging of proliferation in fetal slice cultures to test our hypothesis that a transit amplifying OPC undergoes multiple rounds of symmetric divisions to provide the very large number of OL cells needed to myelinate axons in human cortex. We also plan to develop the ferret as an animal model of OL development with the expectation that oligodendrogenesis will mimic that of the developing human brain but on a compressed timescale. We will use the fetal gyrencephalic ferret cortex that can be manipulated experimentally; so that the combined study of human and ferret will provide a more complete understanding of OL development and maturation. Finally, We will use both human and ferret cortex in complementary fashion to determine if the developing ferret cortex can be used to model the effects of hypoxia in the preterm human. We will test the hypothesis that low O2 tension promotes fetal oligodendrocyte proliferation and inhibits differentiation, while high O2 inhibits oligodendrocyte proliferation and promotes differentiation. White matter injury is associated with cerebral palsy in premature infants, but the specific effects of O2 tension on the cell lineage leading to the production of a myelinating oligodendrocyte has not been well defined in animals or humans. The results may eventually help us better understand the causes of and guide the clinical management for premature newborn infants at risk for these disorders.
|
0.915 |
2017 — 2021 |
Huang, Eric J (co-PI) [⬀] Kriegstein, Arnold |
U01Activity 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. |
A Cellular Resolution Census of the Developing Human Brain @ University of California, San Francisco
Project Summary/Abstract We aim to create a spatiotemporal single cell resolution map of the developing human neocortex in order to establish how many distinct cell types are present and to unravel their complex developmental history. We will build our analysis on a multimodal classification of cells types based on transcriptomic signatures but complemented where possible by physiological and epigenetic features. We will also examine transient cell populations present only during developmental stages, and we will retain positional information for all our cell data to create a developmental cell atlas that plots the diversity of cell types according to their locations in the growing human brain. We have developed innovative strategies for massively parallel profiling of molecular and physiological properties of primary human cortical cells using droplet based capture technologies, high content microscopy, and paired physiological responses to transcriptional state. We propose to conduct our integrated cellular survey of developing human brain in specific regions of the cortex, as well as in the striatum, thalamus, hypothalamus and cerebellum, and we will use single nuclei sequencing to unlock developmental time points that have been traditionally difficult to study. Our project will shed light on the origins of cellular diversity in the human cortex by addressing three specific aims: 1) We will use single cell RNA-sequencing to interrogate how neurogenesis and gliogenesis proceed and give rise to key cell types in the developed brain. We hypothesize that key events promoting regionalization and connectivity can be transcriptionally distinguished from the first trimester to postnatal stages, providing insights into how cell identity is determined. 2) Our developmental approach to the human brain cell atlas provides an opportunity to characterize transient cell populations that appear early in development in the marginal zone and subplate regions, and disappear at neonatal stages. These cell types are presumed to play important roles in establishing brain architecture and function, but they remain poorly characterized in developing human brain. We hypothesize the heterogeneity of these populations can be identified transcriptionally and can explain a diverse set of roles for these transient populations. 3) Transcriptional states are a powerful tool for cell type identification, but they do not capture the entire complexity of molecular features. We will profile cell-specific agonist responses and chromatin state that reflect heterogeneity within defined transcriptional classes. We hypothesize that the intersection of physiological state and epigenetic state to transcription will provide additional nuance to cell type classification. Our results will provide a framework of cellular taxonomy in the developing human brain and create a comprehensive cellular resolution map of molecularly defined cell types throughout functional regions of the human brain during development. This unique resource will serve as a blueprint for studies of human brain function, selective vulnerability of cell types in disease, and the features of brain evolution that make us unique.
|
0.915 |
2017 — 2021 |
Kriegstein, Arnold |
R35Activity Code Description: To provide long term support to an experienced investigator with an outstanding record of research productivity. This support is intended to encourage investigators to embark on long-term projects of unusual potential. |
Development and Expansion of the Human Cerebral Cortex @ University of California, San Francisco
A major long-term goal of this proposal is to understand human brain development and the origins of neurodevelopmental diseases. The cerebral cortex is a structure where model systems, such as mouse or rat, may not capture the complexity of architecture and function relevant for understanding human development and disease. This proposal aims to address the gap in our understanding of human cortical development through the study of primary tissue complemented by human stem cell-derived in vitro model systems, using ?cerebral organoids?. Understanding human-specific aspects of brain development is not only critically important for understanding the etiology of neurodevelopmental disorders, including autism and schizophrenia and ultimately developing therapies, but will also benefit our understanding of human cortical evolution, the diversity and lineage of neural cell types, and the mechanisms of cortical expansion - it will help define what makes us unique. The developing human brain contains an enlarged proliferative region, the outer subventricular zone (OSVZ) that is not present in rodents. This study will target two recently discovered neural progenitor cell types found in the OSVZ, outer radial glia (oRG) and intermediate progenitor (IP) cells. These cell types are particularly important as they underlie the huge developmental and evolutionary expansion of the human brain. This proposal seeks to illuminate the complexity of human cortical development in terms of the genomic, cellular, and behavioral features of its constituent oRG and IP neural progenitor cells and their progeny through the key stages of neurogenesis. We plan to discover lineage trajectories that define progenitor-progeny relationships and determine the cellular fates of clonal descendants. We will use novel oRG and IPC markers to enrich progenitor cell populations for analysis, explore the intracellular signaling networks that regulate IP cell expansion, investigate the role of distinct neurogenic niches in creating neuronal diversity, and examine neuron to progenitor signaling pathways that may regulate IPC neurogenesis. Additionally, we will explore the role of oRGs and IPCs in lissencephaly and related neurodevelopmental diseases, and pursue an intriguing relationship between oRG cells and invasive glioblastoma. These ambitious goals are attainable due to recent technological advances, including improvements in single cell genomics, bioinformatics, real time imaging of primary tissue samples, and in vitro models of human cortical development. The outcome holds promise to transform our understanding of human brain development in health and disease.
|
0.915 |
2018 — 2021 |
Huang, Eric J [⬀] Kriegstein, Arnold |
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. |
Single Cell Analyses of Neuroimmune Dysfunctions in the Thalamocortical Circuit in Ftld @ University of California, San Francisco
PROJECT SUMMARY Aberrant glial activation is a prominent feature in neurodegenerative diseases. But, what triggers glial activation in the aging brain and how it contributes to neuronal degeneration remains unclear. The scientific premise of this proposal is based on previous studies that dominant mutations in human Progranulin gene (GRN [gene], PGRN [protein]) cause a drastic reduction in PGRN levels in CSF and brain tissues in patients with frontotemporal lobar degeneration (FTLD), leading to profound gliosis, aggregation of RNA binding protein TDP-43, and neurodegeneration. In support of this idea, our recent studies show that Grn knockout (Grn-/-) mice is a valid model that captures several key disease features in FTLD caused by GRN mutations (FTLD- GRN), including microglial activation, microglia-mediated synaptic pruning and dysfunction in the thalamocortical circuit. Our ongoing work further revealed that Grn-/- mice and FTLD-GRN patients also shows a robust astroglial activation that positively correlates with microglial activation. Similar to Grn-/- microglia, Grn-/- astrocytes exhibit an age-dependent up-regulation of innate immunity genes, including complements C3 and C4b, which together with C1qa from Grn-/- microglia, activate both classical and alternative complement pathways to promote neurodegeneration. Taken together, these results support the hypothesis that PGRN deficiency is a feasible disease model to uncover the intricate neuroimmune interactions and how perturbation to these interactions leads to neuronal degeneration. To test this hypothesis, we propose a comprehensive single cell transcriptomic approach to survey the dynamic changes of glial and neuronal cell types in the thalamocortical circuit that is most severely impacted by PGRN deficiency. This approach will provide critical insights into the intrinsic mechanism of glial activation, neuronal degeneration and neural circuit dysfunction in Grn-/- mice and in FTLD-GRN patients. This innovative strategy involves high throughput profiling of transcriptomic and physiological properties of glia and neurons using droplet-based capture technology, microscopy and dynamic imaging of cell intrinsic physiological responses. These results will provide an unprecedented resolution to directly test the hypothesis that disruptions to the dynamic neuroimmune interactions between microglia, astrocytes and neurons in the thalamocortical circuit lead to neurodegeneration in FTLD caused by PGRN deficiency.
|
0.915 |
2020 — 2021 |
Kriegstein, Arnold |
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. |
Core C Transcriptomics @ University of California, San Francisco
Project Summary/Abstract Transcriptomic Core (C) provides critical state-of-the-art services to support all of the Projects in this proposal. Core C will apply whole cell and nuclear single cell transcriptomics to evaluate molecular identity and activated gene networks in specific neural and non-neural human brain cells, and will use cutting-edge `Large area spatial transcriptomics (LaST)' to map gene expression across cell types and brain regions at key stages of human 3rd trimester-term neonatal brain development. The use of these technologies will advance our understanding of the mechanisms of cell lineage development in preterm and term neonatal human brain and serve as a high-resolution molecular map against which to measure how perinatal injuries, such as hypoxic ischemic encephalopathy (HIE) impact the development of the brain. Additionally, Core C will work with project leaders to develop a battery of novel cell-specific markers for already defined cell types, such as neural progenitor cells, newly born neurons, microglia, and glial cell lineages, as well as novel markers that will serve as tools to identify and characterize the newly-described cell types of the neonatal human brain such as the migrating neuronal streams that have already been discovered during the initial tenure of this proposal. Core C will be directed jointly by Drs. Kriegstein and Rowitch who have expertise in human brain development and in single cell transcriptomics and spatial transcriptomics respectively. Dr. Kriegstein will provide oversight and guidance of day-to-day operations, scheduling and experimental design. Dr. Kriegstein's laboratory has developed and validated both single cell and single nuclei mRNA sequencing technology recently documented in two joint publications with the Rowitch laboratory (Velmeshev et al, Science, 2019; Schirmer et al, Nature 2019); the Rowitch laboratory has designed a unique pathway for Large-area spatial transcriptomic (LaST) mapping of mouse and human brain (Bayraktar et al., 2019, Nat Neurosci; Schirmer et al, Nature 2019);. The combined use of both of these technologies will enable Projects 1-3 to molecularly profile diverse cell types including interneurons, oligodendrocytes, and microglia, discover new cell type specific markers, and map the in situ expression patterns of genes identified through single cell mRNA sequencing. The Core Directors, assisted by Dr. Dmitry Velmeshev, a talented bio-informatician who will serve as Core Manager, have developed an organizational scheme for Core C that is designed to meet the complementary needs of all the projects. They will also assist project leaders and trainees in the PPG in order to optimize design and execution of their single cell sequencing and mapping projects. In addition, Core C will work with the Scientific Advisory Committee to maintain the highest standard of quality control and with the Administrative Core (Core A) to manage operational and budgetary issues. Core C will provide critical support to help achieve the goals of Projects 1-3 that will, together, provide new insights into human brain development at clinically important neonatal stages of development.
|
0.915 |
2021 |
Huang, Eric J [⬀] Kriegstein, Arnold |
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. |
Diversity Supplement: Single Cell Analyses of Neuroimmune Dysfunctions in the Thalamocortical Circuit in Ftld @ University of California, San Francisco
Project Summary Frontotemporal dementia (FTD) is an early onset neurodegenerative disease, and the second most common cause of dementia in patients 60 years or younger. The majority of familial FTD are caused by intronic hexanucleotide (CCCCGG) repeat expansion in chromosome 9 open reading frame 72 (C9orf72) gene and by dominant mutations in the Progranulin (GRN) gene, which account for 25% and 15% of familial FTD cases, respectively. These mutations cause haploinsufficiency in both genes and lead to abnormal protein aggregation involving RNA binding protein TDP-43 in neuronal nuclei and cytoplasm. The goal of the parent grant (R01 AA027074-03) is to test the hypothesis that loss of PGRN disrupts neuroimmune interaction in the thalamo- cortical circuit in Grn-/- mice. The purpose of this Diversity Supplement is to expand the scope of the parent grant and characterize the potential interaction of C9orf72 and progranulin in neurodegeneration. To this end, the proposed trainee, Naznin Jahan ? a 4th year graduate student in the BMS Graduate Program at UCSF, has established an aging cohort of C9orf72-/-, Grn-/-, and C9orf72-/-;Grn-/- double KO (DKO) mice. The trainee?s results showed that C9orf72-/-;Grn-/- DKO mice exhibit age-dependent neuroinflammation and neuronal loss that are more pronounced than those seen in C9orf72-/- and Grn-/- mice. These findings support the intriguing hypothesis that simultaneous loss-of-function (LOF) in C9orf72 and GRN genes synergistically disrupts glial homeostasis and promote neuronal degeneration in an age dependent manner. The scope of this Diversity Supplement includes (1) to determine the transcriptomic changes regulated by C9orf72 and Grn in glia-neuron homeostasis, and (2) to expand the transcriptomic data using in situ hybridization (ISH), immunohistochemistry and Western blots. In addition, this Diversity Supplement includes a well-defined 2-year timeline, a detailed Mentoring Plan, and Individual Career Development Plan (ICDP) that will significantly enhance the candidate?s research capabilities and complete her dissertation work on the genetic interactions that cause the age dependent neurodegeneration in mice. Working within the proposed timeline, this supplement will prepare the candidate for her long-term career goal as an academic scientist in the field of neuroimmune interaction and neurodegeneration.
|
0.915 |
2021 |
Kriegstein, Arnold Shen, Yin [⬀] |
U01Activity 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. |
Charting the 3d Epigenome in Human Brain Development and Diseases @ University of California, San Francisco
Project Summary Cis-regulatory elements control Cell-type-specific gene regulation via looping with their targeting genes. Therefore, mapping the 3D chromatin interactions between promoters and cis-regulatory elements will be pivotal to understand the functions of regulatory regions. Besides, many genetic variants associated with neuropsychiatric diseases reside in the putative cis-regulatory elements. They may contribute to disease by affecting regulatory sequences function, but the exact mechanisms of how they contribute to diseases via gene regulation remain unknown. Here, we aim to make substantial advances in understanding how the 3D epigenome contributes to brain development and diseases by mapping and analyzing the dynamic changes during the human prefrontal cortex development. We will first map transcriptome, chromatin accessibility, and 3D chromatin loops in six distinct cell types from the developing prefrontal cortex and perform an integrative analysis to interrogate how chromatin interaction control gene expression and development. Second, we will integrate the 3D epigenomic datasets with medical genetics resources to gain insights into cell types, genomic loci, and biological pathways that are causal to diseases, link GWAS SNPs with their target genes. Third, to establish cell-type-specific functional links between chromatin loops and target gene expression, we will test the biological consequences of distal regulatory regions interacting with promoters in iPSC models and primary cells. Our project will reveal new insight into the biological functions of the 3D epigenome in brain development and diseases.
|
0.915 |
2021 |
Kriegstein, Arnold Nowakowski, Tomasz (co-PI) [⬀] Sanders, Stephan [⬀] |
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. |
Assessing Genomic, Regulatory and Transcriptional Variation At Single Nuclei Resolution in the Brains of Individuals With Autism Spectrum Disorder @ University of California, San Francisco
ABSTRACT Autism spectrum disorder (ASD) is a highly heritable neurodevelopmental disorder of unknown etiology and with limited effective therapeutic options that affects millions of individuals. Our research team has a longstanding commitment to understanding the cause of ASD and the molecular processes underlying brain development, function, and pathology. We will use this experience to apply the latest molecular techniques to samples from a new repository of brain tissue from individuals with ASD to create the largest and most detailed analysis of the molecular consequences of ASD. Genetic analyses of gene disrupting de novo mutations have identified over one hundred genes associated with ASD with three main functional groups: regulation of gene expression, neuronal communication, and cytoskeleton. Prior analyses of brain tissue from individuals with ASD have identified a group of downregulated neuronal communication genes, that overlap with ASD-associated genes, and a group of upregulated glial genes that do not overlap with ASD-associated genes or variants. It is unclear if these changes reflect altered cell composition or cell function and how they relate to genetic factors. We propose to analyze post-mortem brain samples from 40 individuals with ASD and 40 unaffected controls, sourced from the Autism BrainNet BioBank, to assess the molecular changes that occur. We will use whole-genome sequencing to identify gene disruptive variants in genes previously associated with ASD and to identify rare and common variants that may alter gene expression or splicing. In tissue samples the prefrontal cortex and striatum in from 40 cases and 40 controls, we will use recently developed single-nuclei methods to perform RNA-seq and ATAC-seq at single-cell resolution to identify ASD-related changes in gene regulation and expression in specific cell types and brain regions. For tissue samples from the prefrontal cortex of 20 cases and 20 controls we will also use cutting-edge single nuclei long-read RNA-seq (Iso-seq), along with bulk tissue RNA-seq, for an in-depth analysis of how gene isoforms differ between ASD cases and controls. Finally, we will assess how single-nuclei gene expression varies in brain organoids grown from pluripotent stem cells edited to contain mutations in three ASD-associated genes. Integrating these data, we will profile the molecular changes associated with ASD and assess how these changes vary by cell type, brain region, age, sex, seizure status, and genotype. We will use RNAscope in situ hybridization to validate the molecular and cell composition changes we observe and a lentivirus-based massively parallel reporter assay to test the function of regulatory regions or variants in proximity to genes with ASD-related differences in expression to validate these effects and assess causality. We hope that these insights will provide a basis for understanding the heterogeneity of ASD and the neurobiological features of this disorder and provide molecular signatures that could be developed into future biomarkers for ASD model systems.
|
0.915 |