Erik M. Ullian - US grants

Synapses throughout the human brain are closely associated with glia. Yet, the role of glia in the development and maintenance of synapses has not been thoroughly investigated. Preliminary data from Dr. Barres' laboratory indicate that synapses formed in the absence of glia in vitro have low efficacy. Glial cells can strongly enhance the efficacy of these synapses. We propose to further characterize how glial cells enhance synaptic efficacy. In particular, we will test the hypothesis that glia enhance synaptic efficacy by increasing the probability of presynaptic transmitter release. We will investigate how glial cells regulate synaptic efficacy. a) Do glial cells regulate transmitter synthesis? b) Do glial cells regulate transmitter release? c) Is electrical activity required for the glial enhancement of synaptic efficacy? d) Do glial cells enhance presynaptic calcium currents? e) Do glial cells upregulate expression of one or more presynaptic proteins.

DESCRIPTION (provided by applicant): Nearly 90% of the human genome is composed of non-coding regions that are not translated into proteins. Previously referred to as "junk" DNA it has recently become apparent that some of this DNA is used to make small app. 22-nucleotide single stranded RNAs known as micro-RNAs (miRNAs). Within the last several years researchers have discovered that these miRNAs are not only important for a variety of processes, but essential. Indeed, removing miRNAs early in development by knocking out the key RNAseIII enzyme required for their synthesis, Dicer, is lethal at the very early stages of gastrulation. Importantly, these miRNAs are continuously expressed throughout the life of mammals and it is clear that they continue to play important regulatory roles in cellular function. The greatest variety of miRNAs are expressed in the nervous system, including the cortex, making it paramount to understand what role the miRNAs play in normal cortical function and mental and behavioral disorders. To date only a few miRNAs has been investigated in hippocampal neurons in culture and these miRNA was found to be required for proper neurite outgrowth and synapse morphology. Importantly computational analyses of likely targets indicate numerous signaling pathways that impact neuronal survival and function are regulated by miRNAs. We propose to use the power of mouse genetics to specifically remove all miRNAs from pyramidal cortical neurons after they have differentiated using a conditional knock out of Dicer and another key gene for the miRNA pathway DGCR8 and then ask what role miRNAs play in dendrite morphology, synaptic spines, and function. Finally, we will identify the complement of miRNAs that are expressed in purified pyramidal neurons that may underlie the observed phenotypes. These data will provide the first inroad to understanding the role of small RNA biology in normal brain function and disease. These initial studies will allow us to develop and perfect the tools to extend these studies to other, layer specific, pyramidal neurons in the cortex and to specific miRNAs that we have identified as candidates for impacting synaptic transmission or affecting schizophrenia-linked genes. PUBLIC HEALTH RELEVANCE. The small RNA pathway is a poorly understood, but essential translational repression pathway heavily expressed in mammalian neurons. This pathway is likely to be important for normal neuronal function and is almost certainly affected in a variety of behavioral and mental disorders such as autism and schizophrenia. These studies will be among the first to provide a detailed cellular and molecular description of the small RNA pathways in normal neuronal development and function in vivo.

DESCRIPTION (Provided by the applicant) Abstract: Astrocytes are the most abundant cell type in the human brain, yet we still do not fully understand the impact of astrocytes on human disease. In this proposal we will begin to uncover the role of astrocytes in regulating cortical plasticity using several new technologies including quantitative mass spectroscopy and microfluidic chambers developed to rapidly identify astrocyte factors that are released in response to the paracrine signals acetylcholine (ACh) or norepinephrine (NE) and that are likely to impact plasticity in both the developing and adult brain. Additionally, we will take advantage of the outstanding stem cell and proteomic centers here at UCSF to ask whether astrocytes derived from somatic cells from individuals on the autism spectrum secrete altered levels of synaptogenic factors. Using a combined microfluidic chamber and imaging system we will screen for effects on synapse formation and function using astrocytes derived from autism patients and familial controls. These studies have the potential to uncover the role of glial cells both in regulating normal plasticity and in disease states. Public Health Relevance: These studies will investigate the role of astrocytes in autism spectrum disorders and in cortical plasticity. Astrocytes can profoundly regulate synapse formation and function and both of these are disrupted in autism spectrum disorders.

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. ABSTRACT Recent studies indicate that astrocytes are spatially diversified, which may have consequence for psychiatric diseases with regional brain dysfunction. However, establishing diversified functions of astrocytes in brain development and disease has been limited by a lack of tools for lineage-specific astrocyte manipulation. Recently, we developed novel transgenic methods to label, purify and characterize regional astrocyte populations in mouse brain. The midbrain and brainstem contain dopaminergic nuclei of the ventral tegmental area (VTA) and substantia nigra (SN) that are implicated in schizophrenia, ADHD and Parkinson's disease. We will test the hypothesis that regionally-specified astrocytes from the ventral midbrain are uniquely suited to support survival and connectivity of dopaminergic projections from the VTA and SN. We will determine molecular diversity of region-restricted midbrain astrocytes as a first step towards uncovering the role of regionally diverse astrocytes in neurological disorders. Deliverables include validated transgenic mouse tools for conditional temporal- spatial labeling, purification of astrocytes throughout the CNS as well as markers of regionally heterogeneous astrocytes subtypes of mid/hindbrain compiled into a public database. The project is intended to reveal signatures of astrocyte subtypes and provide insight into the role of astrocytes in mental health.

DESCRIPTION (provided by applicant): The broad objective of this NEI Center Core Grant for Vision Research application is to facilitate study of the structure, development and function of the visual system in health and in blinding diseases, with the aim of preventing, mitigating or curing such diseases, or the restoration of lost vision, through the application of the most sophisticated available techniques. Four resource and service Cores will help achieve the broad objective, as follows: I. Imaging Resource Core (morphometric analysis; computer-aided image analysis; production of graphics for data analysis, presentation and publication); II. Morphology Resource Core (ocular imaging, including slit lamp photography, fundus examination and fluorescein angiography with Micron III, and optical coherence tomography; paraffin and plastic embedding, sectioning and staining; cryosectioning for immunohistochemistry; light microscopy and photomicrography, including brightfield, darkfield, phase contrast, DIC and florescence; electron microscopy; and confocal microscopy); III. Computer/IT Resource Core (programming for custom research needs; assistance in computer and information technology hardware and software selection, installation, instruction in use, maintenance and minor repairs); IV. Machine Shop Service Core (design, manufacture, maintenance and repair of specialized research instruments and devices using state of the art computer numerically controlled machines). This is a resubmission application of a NEI Center Core Grant for Vision Research competing renewal submitted by the Principal Investigator and 13 other vision scientists who hold 17 active NEI ROI research grants. In addition, the UCSF vision research community supported by the NEI Vision Core Grant comprises 6 NEI-supported scientists with grant mechanisms other than ROI, 2 with other NIH funding, 1 with FDA R01 funding and 7 with private funding. There are 30 Core Investigators with 33 active research programs, overall, each using at least one Core at a moderate or extensive level. Using traditional and innovative approaches, this Core Vision Research Grant has been highly successful and instrumental in enhancing the productivity and impact of vision research, attracting scientists to vision research and facilitating collaborative studies on the visual syste at UCSF.

PROJECT SUMMARY The P30 Vision Core was established in 1985, and continues to be an invaluable resource to all UCSF vision scientist investigators. Over the years it has provided critical resources and services that could not have been supported by individual laboratories. The Cores have evolved over the years based on the changing needs of the community and continues to offer cutting edge technologies, services and equipment to all UCSF vision investigators.

Project Summary Astrocytes are the most abundant non-neuronal cell types in the brain. Astrocytes promote neuronal survival, provide neurons with metabolic substrates, help recycle synaptic neurotransmitters, regulate blood flow, buffer extracellular ions, induce or eliminate synapses and regulate synaptic transmission and synaptic plasticity. Most recently, it has been shown that when specific signaling pathways are compromised, astrocytes can become reactive and can start eliminating synapses ultimately; leading to neuronal cell death during both natural aging and age-related neurodegenerative diseases. Our research aims to understand how astroglial activation is initiated in Alzheimer?s Disease (AD). We will focus on this question by studying a gene called granulin (GRN). This gene has been linked to aging in the human cortex and loss of GRN inevitably leads to astroglial activation and a devastating neurodegenerative disease termed Frontal Temporal Lobar Degeneration (FTLD). To begin to tackle this question, we propose investigating the molecular mechanisms of GRN signaling on astroglial activation. The goal of our work is to understand how normal GRN function prevents protein mislocalization in healthy aging human neurons, and how loss of GRN causes these same proteins to mislocalize in neurons: leading to neuronal death in AD and FTLD. Based on recent findings that rodent astrocytes and human astrocytes are genetically and morphologically very different, we will use human cells to study this question and set up a novel 3D human induced pluripotent stem cell (iPSC) organoid system to incorporate human neurons, astrocytes and microglia. We will then analyze the mislocalization of specific protein in GRN positive and GRN knockout organoids. We will also study the signaling differences between GRN positive and GRN knockout astrocytes, and focus on the innate immune complement pathway and how GRN affects this pathway. Collectively, these studies will allow us to identify the factors in astrocytes that promote brain homeostasis, aging and disease.

PROJECT SUMMARY The broad objective of this NEI Center Core Grant for Vision Research application is to facilitate study of the structure, development and function of the visual system in health and in blinding diseases, with the aim of preventing, mitigating or curing such diseases, or the restoration of lost vision, through the application of the most sophisticated available techniques. Four resource and service Cores will help achieve the broad objective, as follows: I. Image Analysis and Graphics Core: Morphometric analysis; computer-aided image analysis; production of graphics for data analysis, presentation and publication II. Morphology Core: Histology (paraffin and plastic embedding, sectioning and staining, frozen sectioning for immunohistochemistry), ocular imaging (slit lamp examination and photography, Micron III fundus photography (with fluorescein angiography) and optical coherence tomography (OCT), biometric Bioptigen OCT) and visual functional testing (ERG, OptoMotry and IOP) and in vivo rodent eye injection with electroporator set up. Microscopy (light microscopy - brightfield, darkfield, phase contrast, DIC and fluorescence) and photomicrography, and advanced microscopy using a combination of spinning disk and confocal microscopes). III. Computer/IT Core: Programming for custom research needs; assistance in computer and information technology hardware and software selection, installation, instruction in use, maintenance and minor repairs. IV. Rapid-Prototyping and Design Core: Design of new unique research equipment plus education and training of users, empowering them to participate in the manufacturing, construction, maintenance, and upgrading of their unique research equipment designs. This is an application of a NEI Center Core Grant for Vision Research competing renewal submitted by the Principal Investigator and 19 other vision scientists who hold 23 active NEI R01 research grants. In addition, the UCSF vision research community supported by the NEI Vision Core Grant comprises 14 NEI- supported scientists with grant mechanisms other than active R01, 4 with other NIH funding, 1 with FDA R01 funding and 5 with private funding. There are 41 Core Investigators with active research programs, overall, each using at least one Core at a moderate or extensive level. Using traditional and innovative approaches, this Core Vision Research Grant has been highly successful and instrumental in enhancing the productivity and impact of vision research, attracting scientists to vision research and facilitating collaborative studies on the visual system at UCSF.