Arturo Alvarez-Buylla - US grants

In primates including human, the generation of neurons occurs during development while the brain is growing. This imposes natural constraints on adult brain repair. Recently it was discovered that unlike primates, adult birds continue to generate new neurons throughout their forebrain. These cells originate from discrete regions in the ventricular zone (VZ). The young neurons then adopt an elongated shape as they separate from the VZ at 20-30 um/hr using radial glial fibers as guides. Differentiation into mature neurons occurs 20-40 days later up to 6 mm away from their site of birth. The present proposal takes advantage of this model system for neurogenesis to study the origins of the new neurons. The PI's objectives are: 1) Identify the stem cells in the VZ of adult birds that give rise to the young migrating neurons. 2) Develop monoclonal antibodies (Mab) as markers to these precursor cells and to the young migrating neurons.3) Test the significance of radial glial proliferation in the adult avian brain.4) Understand the local movement of precursor cells within the VZ in relation to their proliferation dynamics. 5) Develops a new experimental approach to study neuronal migration. Electron microscopy (EM) and immunocytochemistry combined with [3H]-thymidine autoradiography will be used to identify the precursor cells in the VZ that give rise to neurons.Disaggregated VZ cells and transplantation and culture techniques are combined to study the early events leading to neurogenesis. The cells that give rise to the new neurons, their site of origin and their proliferation time table will be determined. The migration of live young neurons as they separate from the VZ will be followed under the microscope in brain slices.Their behavior on different terrains will reveal orientation cues used during migration. Cells born in the adult avian VZ only give rise to neurons. Neurogenesis occurs against a distinct background of unchanging adult brain parenchyma. Establishing the cellular prerequisites for adult neurogenesis and the mechanism that brings neurons to sites where needed in an adult brain could have a significant impact on approaches to brain repair.

DESCRIPTION: The subventricular zone (SVZ) in juvenile and adult mammalian brains, including primates, contain a population of rapidly proliferative cells. New neurons and glia can be derived from these proliferating SVZ precursors in adult mice. Adult SVZ cells can also be grown in vitro with EGF or bFGF and these cells retain the potential to form neurons and glia in vitro. In rodents, SVZ cells migrate several mm to the olfactory bulb where they differentiate into neurons. These SVZ cells migrate associated with each other, forming chains of neuronal precursors (chain migration), faithfully reaching the olfactory bulb without raidal glia or axons for guidance. An extensive network for chain migration exits in the SVZ of adult mice suggesting that an unprecedented traffic of neuronal precursors exist in the adult mammalian brain. The goal is to determine the potential of the adult SVZ to generate new neurons for brain repair. To this end, the following question will be addressed. 1) Is the olfactory bulb required for the migration and proliferation of SVZ precursors? 2) Can SVZ precursors generate new neurons after transplantation into different regions of the adult brain? 3) Do chains of neuronal precursors exist in the brain of other adult vertebrates including primates and humans? 4) Which of the 5 cell types that the investigator has recently characterized in the SVZ of adult mice grow with EGF or bFGF in vitro? 5) Can SVZ precursors, either isolated directly from the brain or cultured in vitro with growth factors integrate and generate different neuronal types following transfer into the embryonic brain?

DESCRIPTION (provided by applicant): Neurogenesis continues in the adult brain within an extensive germinal zone on the walls of the lateral ventricles called the Subventricular Zone (SVZ). SVZ astrocytes (B cells) function as adult neural stem cells (NSCs), generating transit amplifying C cells which give rise to neurons (Type A cells). New SVZ neurons migrate through the rostral migratory stream (RMS) to the olfactory bulb (OB) where they differentiate into multiple types of local interneurons. It remains unknown if B cells self-renew in vivo, how many times C cells divide before they generate new neurons, and whether generation of different neuronal lineages involves different patterns of proliferation for B and C cells. Aim 1 will address these important gaps in our understanding of the SVZ. A recent study has redefined the architecture for the SVZ, revealing a pinwheel arrangement of B and ependymal cells and the apical-basal organization of B cells. The core of these pinwheels contains the apical compartments of B cells with primary cilia contacting the ventricle. Primary cilia concentrate essential components for Shh signal transduction and other pathways known to regulate B cell proliferation. Aim 2 will explore the role of primary cilia in adult SVZ NSCs. SVZ astrocytes with NSC properties in vitro have also been identified in adult human brain, suggesting that SVZ adult NSCs could be used for therapeutic neuronal and glial replacement. However, the structure of the human SVZ is different from that in rodents. In addition, the extent of SVZ NSC proliferation in vivo and the ability of these cells to generate neurons that migrate long distances into the OB in adult humans remains highly controversial. This controversy is addressed by work proposed in Aim 3. Experiments in Aim 1 will investigate the cell cycle times of different SVZ progenitors, determine whether B cells self-renew in vivo and the number of times C cells divide before generation of different types of neurons in the rodent SVZ. Aim 2 uses genetic methods to remove the primary cilia in adult rodent NSCs to investigate the role of this apical organelle in the proliferation of adult NSCs. Aim 3 will determine the developmental origins of the unique organization of the adult human SVZ and will investigate age dependent changes in the levels of SVZ proliferation and possible fates of young neurons derived from the SVZ. We also intend to clarify whether an RMS exists in adult human brain and how this structure changes through development. Preliminary results suggest that: 1) B cells are consumed with age and their proliferation may be regionally regulated;2) primary cilia play a key role in the proliferation of adult NSCs;3) young neurons are present in the developing human SVZ, but interestingly very scarce along the olfactory track;4) a possible medial extension of the human RMS may exist. The above experiments will provide basic information on the most extensive germinal niche in the postnatal brain. PUBLIC HEALTH RELEVANCE: Neural Stem Cells (NSCs) that continually generate new neurons exist in the walls of the liquid filled cavities (ventricles) in the adult brain. This germinal region, called the subventricular zone (SVZ), is the largest single source of NSCs in the adult brain. In rodents, new neurons born in the SVZ migrate to the olfactory bulb, where they continually replace older neurons. The progenitor cells involved in adult neurogenesis in rodents have been identified, but we do not know how frequently they divide or the precise pattern of cell divisions and cell cycle length that leads to the generation of different types of neurons. Experiments proposed here will specifically determine the cell cycle time for adult SVZ progenitors and determine if cell cycle dynamics vary for different regions of the SVZ. The regulation of SVZ NSCs proliferation remains poorly understood. Recent work indicates that the NSCs contact the ventricle directly with a specialized extension called the primary cilium. Primary cilia are like cellular antennas that are receptive to external signals and multiple important growth factor receptors and signaling molecules are thought to be concentrated in primary cilia. We propose experiments to remove the primary cilium in adult NSCs and study the effects on the proliferation of adult NSCs. It remains controversial whether neurogenesis and long-range migration to the olfactory bulb, like those observed in adult rodents, occurs in adult humans. We propose to study how the SVZ develops in humans and to identify migratory routes and levels of proliferation at different ages. This information is essential to translate some of our observations in animal models to the clinic. This work will help in the development of novel strategies for brain repair and suggest how derailed proliferation of endogenous precursors might lead to tumor formation.

DESCRIPTION (provided by applicant): Neuronal birth, migration, and differentiation continue in restricted regions of the postnatal mammalian brain. An understanding of neural stem cells and the mechanisms of migration and maturation of their progeny will lead to new neuroregenerative therapies. The subventricular zone (SVZ) contains the largest pool of neural stem cells in the adult brain. These cells are astrocytes, which constantly generate transient-amplifying cells (C cells) for the formation of new neurons that migrate to the olfactory bulb (OB). The proposed experiments aim to answer the following questions (1) How do astrocytes from germinal and non-germinal brain regions differ in structure and in their ability to function as neural stem cells? Grafting, cell culture, and molecular markers will be used to characterize different populations of postnatal astrocytes. Preliminary studies show that cultured SVZ astrocytes maintain the capacity to form neurons after transplantation. (2) Are SVZ neural stem cells multipotent in vivo? Can they generate oligodendrocytes and neurons? Preliminary evidence suggests that SVZ astrocytes also produce oligodendrocytes. In vivo and in vitro clonal analyses will determine if individual SVZ astrocytes generate both cell classes. The migratory pathways of young oligodendrocytes will also be determined. (3) How is the survival of neuroblasts and new neurons in the OB regulated in adult mice? The role of the neurotrophin, BDNF, in the survival of migrating neuroblasts and young OB neurons will be investigated. The cells in the SVZ and OB that express BDNF and its receptor, TrkB, will be identified. A novel method to selectively mutate TrkB in a subpopulation of newly formed neurons will be used to study the function of TrkB signaling in vivo. (4) What is the cellular composition, organization, and identity of the neural stem cells in the SVZ of the juvenile and adult human brain? Preliminary data indicate that human SVZ astrocytes also function as neural stem cells. Their location and properties will be extensively studied. Together these studies will help explain how new neurons and glia are produced, migrate, and survive in the postnatal brain. This work will also help elucidate the function of the SVZ, a germinal region that produces many brain cells during juvenile and adult life.

DESCRIPTION (provided by applicant): The subventricular zone (SVZ) is the most extensive germinal region in the adult mammalian brain and contains the largest postnatal repository of neural progenitor cells (NPCs). These primary progenitors continue to generate new neurons and glial cells throughout life. Adult SVZ NPCs, frequently called adult neural stem cells, have been considered homogenous and multipotent. However, preliminary results in the laboratory indicate that adult NPCs are regionally specified generating specific types of neurons in different locations of the SVZ. The proposed work focuses on understanding this heterogeneity: 1) Generating a comprehensive fate map of NPCs in different regions of the postnatal SVZ. Preliminary observations hint to novel neurogenic domains and the postnatal production of novel neuronal types. The site of birth of different types of neurons formed in the postnatal SVZ and the developmental origin of SVZ regional specification will be investigated. 2) Studying how the anatomy of the SVZ varies regionally and how NPCs differ in gene and marker expression in different domains of the SVZ preliminary results suggest regionally regulated expression of transcription and growth factors. Heterotopic transplantation of regionally labeled NPCs will be used to determine if NPC potential is cell-autonomous or is determined by the niche environments preliminary evidence suggests that specification is cell autonomous. 3) Investigating how the postnatal ventricular wall is patterned. Whole mounts of the ventricular wall will be used to visualize ependymal cells and SVZ NPCs differentiation. Gradients of maturation of this germinal wall in relation to the regional specification of the SVZ and the molecular events that regulate the patterning and differentiation of this epithelium will be elucidated. This work is a continuation of research on the mechanisms of adult neurogenesis in the SVZ. Young neurons born in this germinal layer migrate rostrally into the olfactory bulb (OB) where they differentiate into several types of local interneurons. These new neurons originate from NPCs, identified as SVZ astrocytes (type B cells), that are distributed throughout the SVZ. In order to reach the OB, young neurons form an extensive network of chain migration pathways. The new preliminary data uncovers an unexpected level of spatial specification, and explains the need for such a complex network of migratory pathways: to collect neuronal progeny from regionally specified SVZ progenitors. Investigating how NPCs become spatially specified and how the SVZ is patterned during development is an important next step to explain adult neurogenesis. SVZ NPCs have been implicated in brain repair and tumor initiation. Therefore, a thorough understanding of SVZ patterning will help understand the development and therapeutic potential of this postnatal neural germinal niche. Public Health Relevance: Neural stem cells persist in the adult brain and offer new perspectives for the treatment of neurodegenerative diseases like multiple sclerosis, Parkinson's, epilepsy and stroke. These progenitor cells can generate neurons and glial cells and it has been assumed that they are homogeneous and multipotent. However, recent preliminary observations in our laboratory indicate that neural stem cells in the adult brain are heterogeneous and produce different types of neurons depending on their location within the adult germinal niche. Proposed work will reveal the different types produced, their location, gene profile and origin of these different primary progenitor cells. Understanding this heterogeneity is fundamental to any future attempt to use adult neural stem cells for brain repair.

Neural stem cells (NSCs) are retained in the walls of the lateral ventricle in the largest postnatal germinal niche: the ventricular-subventricular zone (V-SVZ). Past work - supported by this proposal - identified a population of astroglia (Bl cells) as the NSCs in the adult mouse brain. During the past few years, the epithelial organization of the V- SVZ was demonstrated; in particular, the apical, intermediate, and basal domains of NSCs were identified. 81 cells' apical compartment shares the ventricular surface with ependymal cells, which unlike Bl cells do not divide, but are essential for cerebrospinal fluid (CSF) circulation and homeostasis. The CSF, in turn, contains factors that regulate adult neurogenesis and neuronal migration within this niche. By adult stages ependymal cells form conspicuous rosettes that surrounds Bl cells' apical compartment in structures referred to as pinwheels. The lineage leading to the formation of these two important cell populations, and the cellular and molecular mechanisms that pattern the ependymal layer, remain unknown. Preliminary data suggests that NSCs and ependymal cells share common progenitors. More importantly, these data suggest that postnatal Bl cells are not a simple continuation of embryonic lineages of NSCs. Instead, the preliminary results suggest that postnatal B1 cells are produced during a restricted period of embryonic development and set apart from embryonic lineages to specifically function as postnatal NSCs. Curiously, the time of birth of ependymal cells coincides with the time of production of postnatal Bl cells. The goal of Aim 1 is to confirm the time during development when postnatal NSCs are produced and determine their lineage relationships to other embryonic progenitors. In Aim 2, we will investigate the lineage relationship between ependymal cells and Bl cells. Aim 3 will extend findings from the past few years indicating that primary cilia in embryonic progenitors are required for the patterning of the V-SVZ. Specifically, we present preliminary data that shows how mechano-sensory function of this organelle, through Polycystin 1 & 2, is essential for the patterning of the ependymal layer. The work will provide fundamental insights on the lineage relationship between embryonic NSCs, postnatal Bl cells and ependynrial cells. In addition, we will investigate how physical forces acting upon the primary cilia on fetal progenitors cells, help pattern this important epithelium that controls the circulation of CSF. Aberrant pinwheel development could result in dysfunctionalpostnatal NSCs and aberrant CSF flow, both conditions that can have devastating effects in early brain development. The understanding of how this epithelium, with its combine germinal and circulatory functions, develops will provide new insights on early postnatal human brain development and brain repair. RELEVANCE (See instructions): Two pooriy understood cell types, with apparently very dissimilar functions, share the epithelium that lines the adult' forebrain ventricles: ependymal cells and neural stem cells (NSCs). These two cell types, that during development become organized into polarized rosettes called pinwheels, combine two functions of great clinical interest: cerebrospinal fluid circulation and the postnatal generation of new neurons. In this proposal, the origin of adult neural stem cells in the fetal brain, their lineage relationship to ependymal cells and the mechanism by which they become organized into polarized pinwheels will be investigated. These findings could help understand the mechanism of hydrocephalus, a condition that afflicts 1 in 300 infants, and help develop new strategies for the use of postnatal neural stem cells for brain repair.

DESCRIPTION (provided by applicant): Neural stem cells (NSCs) in the ventricular-subventricular zone (V-SVZ), an extensive germinal zone lining the walls of the lateral ventricles, continue to produce neurons and oligodendrocytes throughout juvenile and adult life (Ming and Song, 2011; Ihrie and Alvarez-Buylla, 2011). Identification of the NSCs, their progeny, and the mechanisms of adult neurogenesis, provide basic insight into brain repair and cancer (Sohur et al., 2006, Ma et al., 2009; Jacques et al., 2010). NSCs correspond to a subpopulation of V-SVZ astrocytes called B1 cells which generate specific types of neurons depending on their localization in different V-SVZ domains along the rostro-caudal and dorso-ventral axes. NSCs correspond to a subpopulation of V-SVZ astrocytes (B1 cells). B1 cells in different V-SVZ domains along the rostro-caudal and dorso-ventral axes generate specific types of neurons. Lineage expansion occurs through intermediate progenitors (C cells) and neuroblasts ( A cells) (Lois et al., 1996; Luskin et al., 1997; Doetsch et al., 1999b; Ponti et al. 2013a) and in rodents results in the generation of large numbers of new neurons that migrate to the olfactory bulb (OB) (Lois et al., 1994). In the infant human brain, V-SVZ-derived neurons not only migrate to the OB, but also into the cortex (Sanai et al.). The mode of division of B1 cells, the cell-cell interactions that occur during their division, and whether B1 cells self-renew - or ae consumed with age - is not known. The lineages of individual adult NSCs, whether they generate both neurons and glia, and which domains of the V-SVZ generate oligodendrocytes is also unknown. To address these questions, we have developed and validated a live-imaging method to directly visualize the behaviors of B1 and C cells within their niche and we have adapted and tested a barcode- retroviral lineage tracing method to investigate lineage relationships of cells derived from B1 cells in vivo. Aim 1 will determine whether B1 cells self-renew and whether they divide symmetrically or asymmetrically, and will study their interaction with other cells in the V-SVZ. In Aim 2 we present preliminary evidence of a transient postnatal (P0-P7) dorsal domain of the V-SVZ that is regulated by Sonic Hedgehog (Shh); initial observations indicate that this V-SVZ domain is an important source of oligodendrocytes in addition to neurons. We will examine whether single B1 cells in this domain are multipotent, and will analyze the function of Shh in the regulation of these progenitors. Unexpectedly, we encountered synaptic-like contacts between an extensive network of intraventricular axons containing serotonin (5HT) and B1 cells. In Aim 3, we will investigate the organization of these supraependymal axons, characterize the specific sets of 5HT receptors expressed by B1 cells, and determine the contribution of 5HT to postnatal V-SVZ neurogenesis. Self-renewal and multipotency are fundamental questions in the field. How new neurons and oligodendrocytes continue to be produced during postnatal life is essential to our understanding of postnatal brain development and suggests new approaches for brain repair. 2011)

Project Summary (Project 1) Project 1 will determine the contribution of neonatal and early childhood neuronal migration to human brain development and will develop an animal model to study the effects of hypoxia on these processes. We previously identified and characterized a novel ventral migratory stream of neurons in the early childhood brain. We now present preliminary data for a prominent dorsal migratory stream. This project will answer the following questions: (1) What is the precise location of young migrating interneurons in the developing human dorsal forebrain, and in which regions do these cells arrive? (2) Using the ferret as an experimental model for this uncharacterized postnatal neuronal migration in the human, what are the origins, identities, and destinations of these cells? (3) Do oxygen-regulated pathways impact postnatal interneuron generation, migration and/or differentiation? The Specific Aims and hypotheses to be tested are: Aim 1: Generate precise three dimensional maps of the location and extent of dorsal migratory streams in the infant human brain. Hypothesis: extensive postnatal neuronal migration exists in humans and these cells are late-arriving interneurons that complete circuitry in specific brain regions. Aim 2: Develop an animal model to study postnatal migratory streams. Hypothesis: postnatally migrating neurons in the ferret brain are also late-arriving interneurons that migrate to specific brain regions during discrete periods of postnatal development. Aim 3: Determine the effect of hypoxia on migrating neurons in the ferret. Hypothesis: neonatal hypoxic injury affects postnatal production and/or migration of interneurons and this changes circuit composition. We will characterize the regions of postnatal neuronal migration in the neonatal and infant human brain, determine possible contributions of these migrations to specific neuronal circuits, and develop an animal model to trace their lineage and determine their susceptibility to hypoxia during early postnatal life. Our preliminary findings reveal that there is a significant contribution of late migrating neurons and that this population is susceptible to hypoxic injury.

DESCRIPTION (provided by applicant): Most forebrain interneurons originate in the developing medial ganglionic eminence (MGE), from where they migrate into cortex, hippocampus, striatum, and amygdala to form local inhibitory circuits. When transplanted into the juvenile or adult mouse cortex, MGE cells retain the ability for migration, functional integration, and differentiation primarily into parvalbumin (PV) and somatostatin (SOM) expressing GABAergic cells. Previous work has shown that GABAergic inhibition is required for the induction of cortical plasticity and brain repair. Recent work from our laboratories has shown that transplantation of MGE cells into the neonatal or juvenile mouse visual cortex can induce a new period of ocular dominance plasticity (ODP). The transplantation of MGE cells can also enhance the brain's capacity for functional recovery and is being developed as a possible cell therapy for several brain disorders. The ability of these cells to induce plasticity de novo also offers a powerful tool to study the mechanisms and limits of cortical plasticity. The present proposal has three Aims. Aim (1) will determine which type of cortical interneuron is responsible for the induction of cortical plasticity. We have developed and validated genetic tools to ablate PV, SOM, or both cell types from the MGE grafts. Previous research suggests that PV cells may be responsible for the induction of ODP, but this hypothesis has not been formally tested. Our preliminary studies suggest that ODP can still be rekindled, even when most PV cells are eliminated from MGE grafts. We will determine if SOM interneuron depletion is sufficient for the elimination of ODP, or whether both these populations have the capacity to induce ODP. This ODP is induced in young animals weeks after the end of the critical period, but it remains unknown if the adult brain is similarly receptive to interneuron-induced plasticity. Aim (2) will determine if the transplantation of cortical interneurons in the adult brain can induce ODP and contribute to recovery of function. We have developed and validated optical recording techniques to study ODP induction in adult mice. We also have preliminary evidence that MGE cells grafted into the adult mouse cortex migrate and integrate, suggesting that they could also modify cortical circuits and possibly induce ODP. Aim (3) will determine the normal pattern of interneuron maturation in the human visual cortex. If cortical interneurons are key to the induction of critical period plasticity in humans, what is normal pattern of interneuron maturation in infants? Studies from our labs suggest that interneurons continue to be recruited into some regions in the infant brain and this could underlie extended periods of plasticity. Using a collection of brain samples from our developmental tissue bank, we have validated staining and stereological methods to quantify and map PV, SOM, and other markers of immature and mature interneurons. Identification of cortical interneurons responsible for the induction of plasticity, the age range when plasticity can be induced, and the normal patterns of human interneuron maturation will provide new information for the use of MGE cells in brain repair.

Project Summary (Core A) CORE A: This core component functions as an administrative and coordinating center for all the components of the program project. Functions include program oversight, including budget management and planning, data processing and manuscript preparation. It will serve to facilitate interactions among the investigators in the center as well as coordinate activities with the internal and external advisory committees. The core will function as a centralized facility where resources can be utilized to provide support and integration of services for all investigators. The specific aims of the administrative core are to coordinate the following activities in support the Projects: Aim 1: Provide grant administration and project coordination Aim 2: Promote access to project core services, other institutional resources Aim 3: Database management and sharing within the Project Aim 4: Promote research training opportunities and diversity Aim 5: Oversight and course correction to achieve overall goals The administrative committee will make decisions regarding the use of core services. This committee is chaired by the program director and is composed of the principal investigators of each project and the core directors. For any adjudication, the program director will decide when there is incomplete agreement on matters that relate to the overall program. Cost-effectiveness will be achieved through budgetary oversight by a single administrator who communicates with program director and the project leaders. Quality control is provided as a service of the UCSF Core Financial Unit through the internal auditing of budgets and processes. Additional quality control is realized through the use of our internal and external advisory committees. .

PROJECT SUMMARY Cortical interneurons are a diverse set of local inhibitory cells essential for proper balance of excitation and inhibition and key to brain function. Abnormal development of interneurons can lead to severe neuropsychiatric disorders (interneuropathies). Identification of the molecular pathways and cellular populations implicated in these disorders is necessary to understand their etiology and to improve our ability to predict which individuals are at risk. Work from our original grant demonstrated that the infant frontal lobe maintains a large population of migratory interneurons for several months after birth. This collection of migrating young neurons (Arc) targets many areas of the infant frontal lobe. These cells contribute importantly to the final interneuron composition of the human brain (e.g. cingulate gyrus). The human cortex, therefore, continues to receive interneurons for several months after birth, especially in areas of higher cognition implicated in neurodevelopmental disorders. Similarly, our recent evidence shows that the human amygdala continues to receive many young neurons postnatally. These observations significantly change how we view the development of the infant brain and raise the need to better understand the origins of human cortical interneurons, including those that migrate postnatally in the Arc. Young neurons in the human Arc and amygdala are postmitotic (Ki67-), supporting the hypothesis that they are not generated within these regions. The expression pattern of regional transcription factors in the Arc, suggests that they come from the developing human ventral forebrain; the Medial and Caudal Ganglionic Eminences (hMGE and hCGE). The project's overall goal is to understand how the human GE generates large numbers and diverse types of cortical interneurons by studying its development during the mid- late gestation and early postnatal life. We will determine the genetic and molecular properties of proliferative populations in the hMGE, hLGE, and hCGE using human postmortem brain samples from neonatal and infant cases (up to 6 months after birth), define how interneuron subtypes arise in the perinatal human brain. The proposed studies will identify the unique and sustained properties of human inhibitory neuron development. By using a multi-disciplinary approach (including transcriptomic, histological, and acute slice cultures), we aim to establish how interneurons are made in the human brain and provide the fundamental knowledge needed to understand their role in disease.

The mechanisms that regulate the production and migration of human interneurons (IN) and glial sub-types and the functional integration into neural circuits during perinatal stages remains poorly understood. These processes are likely disrupted in neonatal neurological injuries, which can result in severe long-term cognitive disabilities and high social and financial burden. The proposed program will investigate the developmental origins, diversity and cellular interactions of IN, OPCs and microglia using post-mortem human brain tissue and rodent experimental systems. Key past findings include: (i) Discovery of extensive postnatal migration of INs to specialized cortical regions, suggesting that formation of neural circuits takes place over a protracted period (Paredes, Science; Sorrels, Nature); (ii) that the HIF pathway is a critical regulator of oligodendrocyte precursor cell (OPC) maturation and that OPCs under hypoxic conditions become angiogenic in cortical white matter (Yuen, Cell). Our results indicated that OPCs use vasculature as a scaffold to traffic through the developing brain (Tsai, Science), and (iii) that ins migrate in clusters along large vessels in human neonatal brain (Paredes, Science). For the competing renewal we have expanded the investigator team under direction of Arturo Alvarez- Buylla to recruit promising junior investigators (Mercedes Paredes, Steve Fancy, Tom Nowakowski) and new Project Leader Xian Piao. Project 1 investigates origins and diversity of migrating young INs to human newborn cortex and amygdala. Project 2 investigates the mechanisms underlying angiogenesis and IN migration along the blood vessels in human developing cortex and HIE. Project 3 will provide evidence for microglial-encoded GPR56 pathway function in regulation of IN maturation. The administrative core (A) provides budgetary oversight, coordination and access to resources. All projects will use postmortem neonatal human neuropathological specimens supported by a neuropathology core (B) and transcriptomic core (C) to support studies in cellular diversity (Velmeshev, Science; Schirmer, Nature). The studies are intended to reveal cellular and genetic mechanisms impacted by neonatal brain injury and congenital neurogenetic disease.

PROJECT SUMMARY The laboratory has identified a novel type of ependymal cell (E2) that has two long cilia anchored by two basal bodies that are 30-100 fold larger than those in other cells (Mirzadeh et al. 2008, 2017). E2 cells are found in strategic locations of the ventricular system, next to Neural Stem Cells (NSCs) in the walls of the lateral ventricle and in regions of the third and fourth ventricle critical to feeding and glucose regulation, circadian rhythms, consciousness, alertness and sleep (Mirzadeh et al. 2017). Interestingly, E2-like cells have been also observed in ependymomas, suggesting a link to proliferating progenitors and cancer (Alfaro-Cervelló et al. 2015; Ho, Caccamo, and Garcia 1994). E2 cells' genetic profile, the composition and organization of their unique cilia and basal bodies, their developmental origin, their regenerative capacity, and their function are not known. Ependymal (E) cells remain one of the least understood glial cell types in the brain, yet these cells are involved in functions that are essential for proper brain function. Multiciliated ependymal (E1) cells, through the coordinated beating of their ~50 motile cilia, contribute to cerebrospinal fluid (CSF) flow, and are required to prevent hydrocephalus (Jiménez et al. 2014; Ohata and Alvarez-Buylla 2016; Banizs et al. 2005). In the lateral ventricles, E cells contribute to the regulation of adult neural stem cells (NSCs) and neuronal migration in the largest germinal zone of the adult brain: the ventricular-subventricular zone (V-SVZ). How E cells sense and transmit CSF signals to this germinal niche remains unknown. It is unlikely that E2 cells through their two cilia contribute significantly to CSF flow. Instead, we propose that E2 cilia and basal body could play a key role in the detection of CSF signals. Their location at the interface between the CSF and important brain regions strongly suggests they have pivotal, as-yet unidentified, roles in brain function. Surprisingly, preliminary data indicate that the lateral ventricle E2 cells are relatively short-lived, decrease in number with age, and are constantly regenerated in adult mice. We propose to: 1) characterize E2 cells and their cilia and basal bodies using single cell gene expression analysis, electron and ultra-high resolution microscopy; 2) determine the development and adult population dynamics of E2 cells, and identify the progenitor cells giving rise to new E2 cells in the adult (preliminary evidence suggests that E2 cells are derived from adult NSCs); and 3) investigate whether E2 cell cilia signaling modulates adult stem cell niche function, using conditional deletion of a key cilia signaling molecule enriched in E2 cells. This new knowledge will be essential to decipher the function of E2 cells in the adult V-SVZ. In addition, molecular markers and signaling pathways identified in E2 cells could help understand the cell of origin and growth control of some ependymomas. Given the presence of E2 cells in the third and fourth ventricles, and central canal, next to regions of great functional importance, this new understanding will also help studies of E cell function throughout the brain.

Adult; Affect; Apoptosis; Brain; Bypass; Calcium; Cell Adhesion Molecules; Cell Death; Cells; Cerebral cortex; Code; Cognition; combinatorial; conditional knockout; critical period; Data; Development; Electrophysiology (science); Elements; Esthesia; Excision; Family; Functional disorder; Ganglia; Gene Cluster; Goals; GTP-Binding Protein alpha Subunits, Gs; hippocampal pyramidal neuron; Homologous Gene; Image; Impairment; In Vitro; in vivo; Interneurons; Life; Link; Medial; Mediating; Mental disorders; Microelectrodes; migration; Morphology; Mus; mutant; Mutant Strains Mice; Mutation; Neocortex; Nervous system structure; neural circuit; neuronal survival; Neurons; Pattern; postnatal; postnatal development; Process; Prosencephalon; Protein Isoforms; Pyramidal Cells; Regulation; Resolution; Retina; Role; Signal Transduction; Slice; Spinal Cord; Structure; System; Testing; time use; Transfection; Transplantation; two-photon; Visual Cortex; visual information;