James H. Eberwine - US grants

1-(3,4-dihydroxyphenyl)ethane-1,2-diol; 2'-Nor-2'-deoxyguanosine; 2'NDG; 2-Amino-1,9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]-6H-purin-6-one; 3,4-dihydroxyphenylglycol; 6H-Purin-6-one, 2-amino-1,9-dihydro-9-((2-hydroxy-1-(hydroxymethyl)ethoxy)methyl)-; 9-[(1,3-Dihydroxy-2-propoxy)methyl]guanine; Aging; Antibodies; BDNF; Binding; Binding (Molecular Function); Binding Proteins; Blood Serum; Brain-Derived Neurotrophic Factor; CNS plasticity; Cell Body; Class; Complement; Complement Proteins; DHPG; DOPEG; Data; Dendrites; Drugs; Elements; Environment; Experimental Designs; Ganciclovir; Gancyclovir; Gel; Gene Expression; Gene Products, RNA; Genetics, in situ Hybridization; Genetics-Mutagenesis; Glia; Glial Cells; Grant; Imagery; In Situ Hybridization; In Vitro; Integral Membrane Protein; Intrinsic Membrane Protein; Kolliker's reticulum; Laboratories; Ligand Binding Protein; Localized; MGC34632; Medication; Membrane; Messenger RNA; Method LOINC Axis 6; Methodology; Microarray Analysis; Microarray-Based Analysis; Molecular; Molecular Biology, Mutagenesis; Molecular Interaction; Movement; Mutagenesis; Nerve Cells; Nerve Unit; Neural Cell; Neurocyte; Neuroglia; Neuroglial Cells; Neuronal Plasticity; Neurons; Non-neuronal cell; Nordeoxyguanosine; Peptide Biosynthesis, Ribosomal; Pharmaceutic Preparations; Pharmaceutical Preparations; Polyribosomes; Polysomes; Position; Positioning Attribute; Post-Translational Regulation; Posttranslational Regulation; Procedures; Protein Binding; Protein Biosynthesis; Protein Biosynthesis, Ribosomal; Protein Synthesis, Ribosomal; Proteins; RNA; RNA amplification; RNA, Messenger; RNA, Non-Polyadenylated; RNA-Binding Proteins; Rate; Ribonucleic Acid; Screening procedure; Senescence; Serum; Starvation; Stimulus; Structure; Synapses; Synaptic; Synaptic plasticity; Technology; Time; To specify; Translating; Translatings; Translations; Transmembrane Protein; Visualization; base; body movement; cell body (neuron); cross-link; crosslink; deprivation; dihydroxyphenylethylene glycol; dihydroxypropoxymethylguanine; drug/agent; experiment; experimental research; experimental study; gene product; in situ Hybridization Staining Method; in vivo; insight; language translation; mRNA; member; membrane structure; microarray technology; nerve cement; neural cell body; neural plasticity; neuronal; neuronal cell body; neuroplasticity; new technology; novel; presynaptic; protein synthesis; research study; response; screening; screenings; senescent; soma

The striatum is a complex neuroanatomical structure composed of at least two compartments called striasomal and matrix. There are currently no good molecular markers that distinguish between these subdivisions. It is clear that the striasomal neurons contain a higher abundance of u- opiate receptors than do the matrix compartment neurons. In this proposal the opiate responsiveness of the striatum will be characterized using a combination of electrophysiology and single cell molecular biology technique with particular attention paid to highlighting differences between the striasomal and matrix compartments. This is possible because of the anatomical precision of the techniques that will be employed. Initially dispersed rat striatal cells will be examined in a paradigm where opiates will be given acutely after which the opiate induced changes in electrophysiological responsiveness will be examined. In the same recording electrode we will have the reagents necessary to perform cDNA synthesis which will be followed by aRNA synthesis and expression profiling of several candidate genes. This analysis will yield a molecular fingerprint of the naive and opiate treated cells. The dispersed cell studies will be followed by examination of striatal cells in the live cortico-striatal slice preparation. In this paradigm the presence of synaptic connectivity will permit a determination of how chronic opiate treatment will alter the striatal responsiveness to afferent input from the cortex. Briefly, the cortex will be electrically stimulated while a recording electrode will be present in individual striatal cells which will be recorded from after which the mRNA contents analysed. Again the same experimental technology will be used. Finally, novel mRNAs whose abundance changes in response to opiates will be cloned and characterized from the dispersed cells in an effort to better understand the molecular differences in the physiological states of these cells due to opiate challenge. Additionally, this analysis may permit the characterization of striasomal and matrix specific markers. At the end of these studies the electrophysiological and molecular biological characterization of opiate responsive cells in the striatum will provide a molecular fingerprint of opiate responsiveness within striatal cells. This information will provide significant insight into how opiates modulate cellular physiology and may provide a framework in which it will be possible to modulate this responsiveness. Also, this detailed analysis of the striatum, with particular attention paid to the neuroanatomy of the structure, may provide insight into how the striatum is involved in different pathologies that affect the basal ganglia such as Parkinson's Disease and Huntington's Disease.

We have previously characterized coordinate changes in the expression of multiple genes that occur during the ageing process in single live neurons from the aged rat hippocampi. In an effort to correlate the glucocorticoid model of ageing with actual aged neurons we have shown that coordinate changes in mRNA abundance occur in the sub-regions of the hippocampus in response to glucocorticoid challenge in a time-dependent manner. Of the changes that have been observed there are several which are consistent between the two experimental systems - including changes in the excitatory to inhibitory response potential of the hippocampus. We have further examined the effects of glucocorticoids on the expression profiles of individual hippocampal pyramidal cells in primary culture. Based upon these preliminary expression profiles we propose to expand upon these studies by performing add-back experiments in which we attempt to alter the expression of particular mRNAs by using antisense oligonucleotides to manipulate the production of mRNA and functional protein. This will be accomplished in three ways 1) expression profiling of individual dispersed neurons (from hippocampi isolated from glucocorticoid treated rats) which have been inhected with the particular antisense oligonucleotides combined with electrophysiological recordings of these same cells 2) isolation of novel mRNAs whose abundances are altered by antisense manipulation using cDNA enrichment techniques and 3) protein profiling of individual cells using immuno-aRNA to determine whether particular antisense oligonucleotides alter the amount of detectable protein for the targeted mRNA. These particular experiments are biologically extremely selective and sensitive because of the specificity of the starting cDNA and protein, i.e. that from a single cell. These experiments will investigate the mechanisms of antisense function by determine whether administration of oligonucleotides into the nucleus or cytoplasm of cells differentially affects the expression profile. The hypothesis to be examined is that coordinate changes in mRNA levels which provide a fingerprint of glucocorticoid challenged neurons can be manipulated in a predictable manner by addition of antisense oligonucleotides resulting in the alteration of levels of specific mRNAs and proteins within these cells. These data will likely have therapeutic implications for long term steroid treatments.

DESCRIPTION (provided by applicant): Neuronal dendrites are the initial post-synaptic interpreters and integrators of presynapfic information. Over the last several decades, data has been generated from both in vitro and in vivo experiments that show that dendrites increase in number and size in response to various behavioral and pharmacological manipulations. More recently mRNAs have been localized in dendrites. Further data shows that mRNAs can be translated locally, in the dendrite, in response to various types of modulators. We have recently shown that dendritic translation occurs in immobile hotspots along the length of the dendrite through the monitoring of fluorescence from GFP that was synthesized from GFP that had been transfected into isolated dendrites. Further these hotspots were heterogeneous in their translational rate in response to DHPG, a mGluR1 agonist. Most of the hotspots exhibited exponential translation rates (EXP) while the remaining hotspots were linear (LIN) in their response. The existence of these translational hotspots suggests a subdendritic specificity to post-translational responsiveness. We propose to further characterize these translational hotspots to determine 1) do the EXP hotspots always respond in an EXP manner or can they also exhibit LIN translation rates and vice-versa, 2) whether known modulators of dendritic translation alter the ratio of linear exponential translation hotspots 3) what translational machinery component co-localize with each type of hotspot, 4) do dendritic spine associated proteins colocalize with either type of hotspot preferentially and finally, 5) do changes in mRNA structure alter the distribution or number of EXP and LIN hotspots. In addition, we will go one step further and determine the protein profile of dendrites using a novel proteomics method recently developed in our lab called Immuno-Detectlon Amplified by T7 RNA Polymerase (IDAT). We propose to utilize two different phage display libraries that are enriched (panned) for dendritic protein detection in conjunction with IDAT to determine the identity of proteins in the dendritic compartment and, using a differential screening procedure, changes in the abundance of these proteins after treatment with pharmacological modulators of dendritic function. These data will be compared with the mRNA expression profiles that have been generated to distinguish between the potential for somatic or dendritically synthesized proteins to contribute to the predominant protein profile of dendritic responsiveness to modulation. Included in our analysis is the possibility of looking at post-translational modification of dendritically localized proteins. These data will likely have significant impact upon how we think about post-synaptic involvement in regulation of the Hebbian synapse

The University of Pennsylvania Cancer Center has planned a Microarray Core based on extensive analysis and planning. This effort has been strongly supported by the School of Medicine, Cancer Center and Department of Pathology and Laboratory Medicine, all of which have committed resources to ensure the successful implementation and the maintenance of a state-of-the-art facility in which technological advances occur rapidly. Penn has contributed substantially to the field of microarray technology. One of the leading investigators in this field, James Eberwine, PhD, will serve as the Facility Director for the new core. In addition, Penn already has the sophistical informatics capability required to support such a core without the need for additional external funding. Several important aspects have been incorporated into this core to make its services have even greater value to a wide range of users while being cost-effective. This Microarray ore will be readily differentiated from other microarray cores at other institutions through the following offerings: 1) RNA amplification technology developed here at Penn, which will allow analyses of a few or even single cells; 2) pathology-directed microdissection of experimental and clinical malignant tissues; 3) microarrays of conventional and ultimately cancer relevant design; and 4) access to the Penn Center for Bioinformatics for detailed data analysis that goes beyond that provided by conventional microarray analysis software packages. Based on surveys conducted during the past year, this new Shared Resource will have significance usage and will contribute greatly to the innovative basic and translational research conducted at the Cancer Center. Usage by Cancer Center members with peer reviewed funding is expected to be 70% of total facility usage.

DESCRIPTION (Applicant's abstract): The amygdaloid complex is involved in the "processing" of multiple behaviors including fear, anxiety and learning and memory. There is also evidence that biochemical changes occur in the amygdala as a result of disease including schizophrenia, bipolar disease and cocaine and opiate abuse. The neuronal circuitry connecting various nuclei of the amygdala allowing internuclei communication has also been elucidated for aspects of fear conditioning. Given the central role of the amygdala in processing various types of CNS information an elucidation of the relative levels of mRNA abundance for the mRNAs that are present in the amygdala might provide insight into what neurochemical signals the amygdala responds to. Further since neuronal pathways have been dissected characterization of the expression profile in individual interconnected nuclei may provide information about how one region of the amygdala transfers information to another. We propose to generate molecular fingerprints of neurons within different regions of the amygdala using single cell and single nuclei aRNA amplification and microarray analysis in normal mice. Additionally we will determine how the profiles of these nuclei change in a mouse pharmacological model of bipolar disease involving amygdala response to withdrawal from lithium after chronic lithium treatment. As part of this proposal we propose to robotize the aRNA amplification procedure so that we can generate l000 individual sample templates each partitioned into an individual well in 96 well plates. While we are not proposing to perform 1000 microarray experiments (samples will be pooled as described in the text) having individual cells will permit each to be analyzed separately as required. In an effort to create a National resource of expression profiling data and amplified cells from the amygdala for use by other investigators the normal sample plates will be made available to anyone who requests them on a cost basis. The normal sample expression profiling data will be made available on a public website as soon as the data is confirmed by two array analyses. These data should be useful to investigators studying the amygdala as well as to "bioinformaticists" who are in need of large data sets from multiple samples. Such large data sets should prove useful in the development of pattern search algorithms that are sorely needed to property analyze such complex gene expression data sets.

The molecular biology of the glial component of the tripartite synapse has been woefully neglected. It's clear that the interactions of the post-synaptic and pre-synaptic sites of neurons balance with those of the astrocyte to produce a dynamic functional synapse. We propose to extend our previous work on neuronal dendrites to determine the capacity of astrocytic processes to locally control mRNA movement and protein translation. These studies will be divided into two specific aims with the first Specific Aim devoted to characterization of the mRNA expression profile in astrocytic processes and the associated cell soma. This will be accomplished using the single cell aRNA nucleic acid amplification procedure coupled with micro- and macro-array analysis. The second Specific Aim will assess the ability of neuronal stimulation to modulate translation of mRNAs in astrocytic processes. These experiments will utilize primary cultures of mouse astrocytes as vehicles for the transfection of mRNAs that upon translation give rise to detectable proteins. Astrocytic processes will be mechanically microdissected and various mRNAs transfected followed by fluorescent and immunodetection of protein locally translated from transfected mRNAs. Subsequent experiments will use intact astrocytes that have been transfected with caged rnRNA that inhibits the mRNA translation. Upon in vivo laser uncaging the mRNA can be translated at the site of uncaging. Such experiments in combination with siRNA knock-down of selective gene expression will also permit an analysis of the functional significance of astrocytic process synthesized proteins. At the end of this granting period we will have determined the complement of mRNAs that are localized to astrocytes and astrocytic processes. Further we will have determined whether astrocytic processes can translate these localized mRNAs as well as the spatiotemporal aspects of translation in this subcellular region. Finally using RNA transfection methodologies we will have determined whether neuronal activity modulates astrocytic process mRNA translation and using novel caged mRNAs, in concert with siRNA knock-down experiments, determined whether astrocytic translation of mRNAs can modulate synaptic activity.

The overall goal of this proposal is to advance a new and powerful paradigm in pulmonary physiology; one we call Image-Functional Modeling (IFM). Conceptually, IFM synthesizes imaging data and mechanical and ventilation function data taken in the same subject with anatomically specific three-dimensional models of the lung. Boston University, as the lead institution will partner with the Massachusetts General Hospital, Brigham and Women's Hospital, Tufts University School of Veterinary Medicine, and the University of Aukland to exploit IFM via the following imaging modalities: Positive Emission Tomography (PET), High Resolution Computer Tomography (HRCT), and Hyperpolarized Helium Magnetic Resonance Imaging (Hyp 3/He MRI). The IFM will be applied to asthma and to respiratory distress syndrome (RDS). Our specific aims are to: . Advance a 3D anatomically specific computational model of the lung that can predict overall and dynamic lung mechanical and ventilation function while permitting the imposition of a heterogeneous insult to explicit anatomic locations. This model will consist of a scaffold of modules across multiple biological scales resulting in an open source simulation resource for the general respiratory structure-function community. . Synthesize our computational models with imaging and mechanical functional data taken simultaneously in the same subjects. The imaging data will quantify ventilation distribution and/or airway geometry while the mechanical function data will include standard clinical lung function indices (eg., spirometry) and dynamic lung function. Applications of IFM to asthma and RDS will quantify the likelihood of two hypotheses: a) The primary cause of functional degradation in both ventilation and mechanics during asthma lies within the smafl airways (d<2mm), namely, their constriction pattern; and b) During mechanical ventilation, setting the level of positive end-expiratory pressure to reduce dynamic heterogeneity will minimize risk of lung injury' while optimizing ventilation distribution. . Perform a rigorous sensitivity analysis to a) examine the impact of local and distributed disease induced changes in geometry and the biomaterial properties on function; and b) predict the likely impact and outcome of specific clinical interventions or therapies on total and local lung function. The IFM paradigm represents a breakthrough in quantitative image interpretation and in model based understanding of lung function. Comprehensive and integrative new hypotheses and insights into lung pathophysiology and in medical practice will evolve in ways unachievable by considering any of these domains alone. The long term goal is to provide guidance on interventions such as pharmaceutics and mechanical ventilation, and to do so on a personalized basis.

The molecular biology of the glial component of the tripartite synapse has been woefully neglected. It's clear that the interactions of the post-synaptic and pre-synaptic sites of neurons balance with those of the astrocyte to produce a dynamic functional synapse. We propose to extend our previous work on neuronal dendrites to determine the capacity of astrocytic processes to locally control mRNA movement and protein translation. These studies will be divided into two specific aims with the first Specific Aim devoted to characterization of the mRNA expression profile in astrocytic processes and the associated cell soma. This will be accomplished using the single cell aRNA nucleic acid amplification procedure coupled with micro- and macro-array analysis. The second Specific Aim will assess the ability of neuronal stimulation to modulate translation of mRNAs in astrocytic processes. These experiments will utilize primary cultures of mouse astrocytes as vehicles for the transfection of mRNAs that upon translation give rise to detectable proteins. Astrocytic processes will be mechanically microdissected and various mRNAs transfected followed by fluorescent and immunodetection of protein locally translated from transfected mRNAs. Subsequent experiments will use intact astrocytes that have been transfected with caged rnRNA that inhibits the mRNA translation. Upon in vivo laser uncaging the mRNA can be translated at the site of uncaging. Such experiments in combination with siRNA knock-down of selective gene expression will also permit an analysis of the functional significance of astrocytic process synthesized proteins. At the end of this granting period we will have determined the complement of mRNAs that are localized to astrocytes and astrocytic processes. Further we will have determined whether astrocytic processes can translate these localized mRNAs as well as the spatiotemporal aspects of translation in this subcellular region. Finally using RNA transfection methodologies we will have determined whether neuronal activity modulates astrocytic process mRNA translation and using novel caged mRNAs, in concert with siRNA knock-down experiments, determined whether astrocytic translation of mRNAs can modulate synaptic activity.

[unreadable] DESCRIPTION (provided by applicant): The discovery of transcription factor protein and mRNA in neuronal dendrites led to an elaboration of the "dendritic imprinting" hypothesis which suggests that transcription factors that are synthesized in the dendrite move to the nucleus to elicit a transcriptional response. The biological rationale for dendritic synthesis of a transcription factor that functions in the nucleus may be that the dendritic transcription factor would produce a transcriptional response distinct from that that would be generated through the normal convergence of stimulus-induced second messenger system signaling that modulates nuclearly localized transcription factors. Such distinct functioning may be associated with a unique dendritically-induced post-translational modification of the transcription factor. To examine the viability of this hypothesis, dendrites were screened for the presence of mRNAs that encode transcription factors. Among the dendritically localized transcription factors that we found, Elk-1 mRNA was particularly intriguing given that preliminary data shows selective dendritic translation of Elk-1 protein causes neuronal cell death. This discovery was enabled by our development of a novel photoporation technique that permits the introduction of small amounts of RNA into any subregion of a live neuron. The ability of a dendritically translated protein to elicit cell death was unexpected. The mechanism(s) by which dendritically synthesized Elk-1 protein causes cell death will be assessed through three Specific Aims. These Aims are to 1) Define the neuronal morphological parameters associated with the ability of dendritically synthesized Elk-1 to elicit cell death, 2) Characterize the post-translational modifications of dendritically synthesized Elk-1 that permit it to associate with mitochondria and to move into the nucleus to elicit cell death and 3) Determine the genes that are modulated by dendritically synthesized Elk-1 protein versus that made in the cell soma. The ability of the genes whose abundances are altered selectively by dendritic Elk-1 to recapitulate cell death will be assessed using a novel "global mixed RNA pool" functional screen. Upon completion of these studies we anticipate that the data will inform our understanding of the role of dendrites in modulating cell viability and may provide insight into the neurodegenerative processes associated with selected human diseases. The biological consequences of the localization of transcription factor mRNA and local synthesis in neuronal dendrites will be assessed. A combination of standard and novel cell biological, anatomical and molecular approaches will be used in this determination. [unreadable] [unreadable]

The Cellular Logic of Phenotype The goal of this application is to develop a strategy for predictably and reproducibly altering the phenotype of primary cells in culture. Differentiated cell types differ from each other in their RNA profiles (relative as well as absolute abundances of the RNAs they express). I hypothesize that, by the transferring entire RNA profiles from donor to recipient cells in a way that makes the recipient cells'survival dependent on donor RNA, the donor RNA will change the recipient into a destination phenotype that mimics the donor cell phenotype. This procedure is called Transcriptome Induced Phenotype Remodeling (TIPeR). Having the ability to transfer cell phenotypes between cells would provide important new insights into mechanisms controlling cell differentiation. The theory and technical strategies to accomplish this are being developed in my laboratory. Specifically, using laser light induced phototransfection (developed in my lab), we transiently produce pores in the host primary cell, through which RNA populations (in which RNA species and abundances are carefully controlled), can diffuse. Preliminary data shows that donor cell RNA populations carry "memory functions" in that, donor RNA can induce long-term changes in genomic transcription of the host cells thereby changing the functional phenotype of the host cells to that of the destination phenotype. This is due in part to the activity and abundances of the specific proteins made from the host cell RNA mixture. Through developing various high- throughput quantitative "Omics" level phenotyping technologies coupled with the TIPeR procedure it is anticipated that the "genomics logic" of phenotype will be discerned. An understanding of this logic will permit the creation of specific cell types at will. The ability to selectively and rationally create cellular phenotypes promises to provide important insights into the fundamental mechanisms underlying cellular polarity, functioning and phenotype stability and may yield novel "individualized medicinal therapeutics".

DESCRIPTION (provided by applicant): Mammalian behavior spans a fantastic range of function and ability, from complex linguistic processing, to social and sexual behavior, to simple stimulus-response. Traditional explanations of the mechanisms for this diversity include brain size, neuro- anatomy, and functional neuro-anatomy including connectivity patterns. Establishing these neuro-anatomical differences requires evolutionary differences in the genes guiding developmental processes. However, there has been little comparative studies focused on individual neuronal function in a non-developmental context. Previously, we initiated a project to understand what sequence motifs govern sub-cellular localization of mRNA to dendrites in rat neurons. Surprisingly, we found evidence that an evolutionarily novel element may partly govern dendritic localization. Furthermore, this element is abundant in the rat genome but an order of magnitude less abundant in the mouse genome. A micro-dissection and expression array survey of the mouse neurons seem to suggest that there is only 36% overlap between the homologous mRNA found in the mouse dendrites and the rat dendrites. Thus, we hypothesize that the genome-scale molecular physiology of neurons from different tissues and closely related species have broad differences and functional non-coding RNA derived from evolutionarily novel elements plays a role in establishing these differences. If true, this would have important consequences for translating animal neurobiological studies to humans and also suggest that evolutionarily novel elements such as retroviral-derived elements may be important in brain function and dysfunction. We propose to test our hypothesis using comparative single-cell localization assays, single-cell transcriptome assays, whole-transcriptome sequencing, and functional analysis. PUBLIC HEALTH RELEVANCE: In this project, we hypothesize that the genome-scale molecular physiology of neurons from different tissues and closely related species have broad differences and functional non-coding RNA derived from evolutionarily novel elements plays a role in establishing these differences. We propose to test our hypothesis using comparative single-cell localization assays, single-cell transcriptome assays, whole-transcriptome sequencing, and functional analysis. The results of our investigation will have important consequences for translating animal model neurobiological studies to humans diseases and also suggest that viral-derived elements may be important in brain function and neurodegenerative diseases.

DESCRIPTION (provided by applicant): The goal of this U01 is to characterize and understand the variability in the expressed transcriptome of human excitable cells. There are two predominant types of excitable cell in the human body, neurons and muscle cells, including cardiac cells. Many human CNS diseases result from modulation of the electrical responsiveness of neurons while cardiac arrhythmias account for most of heart associated deaths. However, at the level of individual cells there is considerable heterogeneity in function, response, and dysfunction. Here, we present preliminary data showing large-scale single cell variability that is difficult to explain as simple molecular noise. We hypothesize that there is a many-to-one relationship between transcriptome states and a cell's phenotype. In this relationship the functional molecular ratios of the RNA are determined by the cell systems' stoichiometric constraints, which underdetermine the transcriptome state. Because a broad set of multi-genic combinations support a particular phenotype, changes in the transcriptome state do not necessarily lead to changes in the phenotype potentially explaining cellular heterogeneity in phenotype response to variant conditions such as the application of therapeutic molecules. To test this hypothesis we propose to investigate the extent of single cell variation for the whole transcriptome for excitable cells that are in their natural environment using a novel mRNA capture methodology (TIVA-tag), and on a subset of the transcriptome, the mRNAs encoding the therapeutically important and manipulable G protein-coupled receptor (GPRC) pathways. The use of functional genomics techniques developed in the Eberwine and Kim labs (TIPeR) will permit an assessment of the biological role of multigenic transcriptome variation. These studies are truly interdisciplinary involving the collaboration of two clinicians (Drs. Grady, Neurosurgeon and Kuhn, Cardiologist), two genomicists (Drs. Eberwine and Kim) one of whom is a computational scientist (Dr. Kim), a neuro/cardio- pharmacologist (Dr. Bartfai) and a biophotonics expert (Dr. Sul).!

R25 Advanced Techniques in Single Cell Transcriptomics (J. Eberwine/J. Kim) Project Summary The Penn Genome Frontiers Institute (PGFI) will offer an Advanced Techniques in Single Cell Transcriptomics five-day course in 2014, 2015 and 2016 that would be open to researchers (PIs, Postdocs, and Graduate Students) nationally. Course participants will be trained to successfully perform the entire process of quantifying RNA from individual cells from multi- cellular organisms. The base set of techniques that will be taught include single cell isolation, single cell RNA isolation, RNA amplification by aRNA and PCR, NextGen seq library construction, sequencing and data analysis. Lectures and discussions on the broader context of single cell transcriptomics within single cell analysis and on the most recent technological developments will enhance the hands-on bench training. The final session of the course will cover RNAseq data analysis and interpretation. Course materials, including basic curriculum, protocols and datasets, will be available on PGFI-hosted, publically accessible web pages. This Single Cell Transcriptomics course will provide a research training opportunity for which we expect there to be broad interest and applicability among researchers working in gene expression and functional genomics in a diverse range of fundamental and biomedical disciplines. Researchers constrained by access to very small cell populations or directly interested in cell heterogeneity (e.g., characterization of transcriptomes, development and normal states, cancer and other disease-state genomics) and its consequences would gain valuable tools from this workshop. Dissemination of tools for single cell analysis is critical for enabling this promising area to expand and deliver on its potential.

DESCRIPTION (provided by applicant): Neurons have been known to have distinct anatomical specifications for over a century. As neurons can have many dendrites and each dendrite can have many synapses it is clear that dendrites are an important modulator of cellular communication and function. How these morphological features modulate cellular function has been a mystery since the time of Cajal's initial observations. Progress has been made in showing that dendrites exhibit chemical compartmentalization. This compartmentalization is exemplified by stimulated changes in Ca++ levels in specific dendritic areas. These features show that dendrites are not homogeneous and indeed not only exhibit morphological heterogeneity but also functional heterogeneity. One of the dominant questions in dendrite biology is how does stimulation of selected regions of dendrites in the intact tissue result in a cellular response? This has been termed dendritic integration with much of its characterization using electrophysiological and Ca++ outputs as indicators of dendritic function. There are however other physiological processes that occur in dendrites with mRNA targeting and local translation that are also important modulators of dendrite-mediated physiologies including synaptic plasticity. Dendritic translation occurs at sites along the length of the dendrites called hotspots first demonstrated simultaneously in the Schuman and Eberwine labs. Recent, in vitro studies from the Eberwine and Kim labs have demonstrated a highly complex dendritic translational process. These data, and those of others, highlight the fundamental need to analyze the temporal and spatial dynamics of translation in dendrites to understand the mechanism of post-synaptic responsiveness and dendritic integration. Much of the translation work to date has utilized dispersed neurons in culture and while appropriate for many experimental questions, it is increasingly clear that cells in their normal microenvironment can be functionally distinct from their in vitro counterparts in their cell biology including RNA expression. Experiments in this application will define the fundamental aspects of multi-mRNA translation in intact dendrites of neurons that are in their natural microenvironment. These data will be the first to quantify the role of the microenvironment in modulating neuronal physiology through modulation of the dynamics of localized dendritic protein synthesis.

The cellular requirement for mRNA diversity is apparent, as the evolutionarily conserved process of mRNA splicing generates mRNA and protein diversity through alternative mRNA splicing. Indeed it has been established that >90% of mammalian genes are alternatively spliced. The abundance of the alternatively spliced forms varies extensively, but a large fraction (~85%) of these alternatively spliced RNAs exist in the range of 5-15% of that particular gene's mRNA transcript population. The biological roles of alternatively spliced mRNAs are varied for example different spliced forms of channels and receptors give rise to differentially responsive proteins, spliced cadherin RNAs facilitate specific cell-cell interactions and distinct splice forms of individual transcription factors modulate distinct gene sets. With such examples of molecular diversity, there has been increased effort to characterize additional splicing events resulting in the recent discovery of three different types of alternatively spliced RNAs including 1) circular RNAs, 2) exitrons and 3) a complex population of alternatively spliced RNAs containing retained introns (ciRNAs) that was identified in the cytoplasm of cells through the use of highly sensitive NextGen sequencing on isolated neuronal dendrite RNA populations. This last class of RNAs is the topic of this proposal. The discovery of a large population of ciRNAs was unexpected, yet led to the hypothesis that they may exert a here to for unknown biological function. An example of a ciRNA that provides insight into functionality of this class of RNAs is one that comprises part of BKCa mRNA population. Preliminary evidence suggests a physiological role for the ciRNA in BK channel functioning but little is known about the intrinsic mechanisms involved and whether multiple ciRNAs that possess different retained introns for a particular RNA exert similar or distinct functions. The robust biological impact of this ciRNA isolated from dispersed cultured neurons highlights the need to identify and characterize the ciRNAs from cells in their native tissue microenvironment to explore how they may regulate the cells' natural physiological responsiveness. We propose to investigate these events in situ using our newly developed Transcriptome In Vivo Analysis (TIVA) to isolate RNA from individual dendrites resident in the live mouse brain slice. The identity of dendritically localized ciRNAs (including depolarization induced ciRNAs) will be determined by single cell RNAseq. A second goal is to start to dissect the mechanism(s) of action of ciRNAs by manipulating their expression and measuring function. While we expect to discover new ciRNAs in the course of this project, the ciRNAs encoding channels are among the most easily examined for a functional role and provide a starting point for functional assessments of this novel class of RNAs.  

A cell is a highly complex system with distributed molecular physiologies in structured sub- cellular compartments whose interplay with the nuclear genome determine the functional characteristics of the cell. A classic example of distributed genomic processes is found in neurons. Learning and memory requires modulation of individual synapses through RNA localization, localized translation, and localized metabolites such as those from dendritic mitochondria. Dendrites of neurons integrate distributed synaptic signals into both electrical and nuclear transcriptional response. Dysfunction of these distributed genomic functions in neurons can result in a broad spectrum of neuropsychiatric diseases such as bipolar and depressive disorders, autism, among others. Understanding complex genomic interactions within a single cell requires new technologies: we need nano-scale ability to make genome-wide measurements at highly localized compartments and to effect highly localized functional genomic manipulations, especially in live tissues. To address this need, we propose to establish a Center for Sub-Cellular Genomics using neurons as model systems. The center will develop new optical and nanotechnology approaches to isolate sub-cellular scale components for genomic, metabolomics, and lipidomic analyses. The center will also develop new mass spectrometry methods, molecular biology methods, and informatics models to create a platform technology for sub-cellular genomics.

RNA metabolism, from its synthesis in the nucleus, through its role in cellular homeostasis, to degradation in the lysosome, is a regulated process that is inherently controlled by RNA structure. We know this, in part, from the plethora of papers detailing disease-causing deficiencies in RNA metabolism. Modeling of the ?structural landscape of RNA metabolism? to enable experimental regulation of the process, requires knowledge of what RNAs are expressed, their 3D-structures, their subcellular location and how they interact with their local interacting partners. To date, most efforts that generate information about these RNA regulatory processes, such as RNA interactions with RNA binding proteins, use purified fractions of cellular homogenates from groups of cells. Such cell-ensemble information is useful, however, the cell-selectivity of these processes and the dynamics of RNA structural changes across this structural landscape is unknown. The uniqueness of individual cells and subcellular environments requires that such studies be performed at the level of single cells. There is currently no experimental approach that allows for structural analysis of RNA molecules across the RNA metabolic landscape within the natural microenvironment of individual cells. We propose to map the structural landscape of RNA metabolism in single cells, in vivo, by developing a suite of sensitive, high- resolution molecular approaches that yields a quantitative 3-D map of all RNA-associated structures within single cells and subregions of these cells. This approach, called In Vivo Structural Analysis mapping, or VISTA mapping, uses a combination of protein, RNA and organelle markers to direct subcellular function of light- activated in situ RNA amplifiers, the product of which is RNA-structure sensitive and informative. The structural analysis of all RNAs and RNA-associated organelles in a single cell will permit a determination the overall logic of the RNA metabolic landscape within a cell. The goal of the proposed studies is to create and understand the ?Topological Map of Single Cell RNA Metabolism?. This will be accomplished using newly developed VISTA mapping. Cell-type specific VISTA maps, generated from neurons in their natural microenvironment, will provide novel insights into and opportunities for manipulating normal cell biology as well as disease-associated RNAopathies.! !