Gail Mandel - US grants

The voltage-dependent sodium channel is the basis of electrical excitability in mammalian nerve and muscle cells. This proposal decribes experiments to elucidate the structure of the rat sodium channel, to measure expression of the sodium channel gene in response to neuronal growth factors, and to identify and characterize regulatory sequences in the gene. The structural gene (cDNA) for the rat muscle sodium channel will be isolated from a bacteriophage expression library by screening with polyclonal and monoclonal antibodies directed against purified sodium channel proteins. The nucleotide sequence of the muscle cDNA clones will be used to deduce the primary structure of the sodium channel proteins. Northern blot and S1 nuclease hybridization assays will be performed to determine whether muscle and nerve sodium channels are identical. A sensitive assay system for measuring changes in sodium channel mRNA production will be established using an NGF-inducible cell line (PC-12) and the characterized cDNA clones as probes. These probes will be used in Northern blot assays to detect changes in sodium channel mRNA levels after treatment of PC-12 cells with NGF. If an effect on transcription of the sodium channel gene is observed, experiments will be performed to identify the NGF-responsive sequences in the gene. These studies involve constructing expression vectors containing the sodium channel gene fused to bacterial gene sequences. The recombinants will be introduced into PC-12 cells and assayed for fusion gene products in the presence and absence of NGF. Because cAMP may be involved in the mechanism by which NGF exerts its effects on PC-12 cells, cAMP will also be tested for its ability to regulate expression of the sodium channel gene. These experiments will provide a framework for understanding the molecular mechanisms underlying the development of excitability in neuronal cells.

The long range goal of our research is to gain a better understanding of the regulation of membrane excitability. Our studies continue to focus on regulation of the voltage-dependent sodium channel, which is responsible for the generation of action potentials in most excitable tissues. Experiments in the previous funding period revealed that expression of a major sodium channel type in the central nervous system, type II, is likely due to a novel transcriptional repression mechanism that prevents expression of this channel in non-neural tissues. We showed that the molecular basis of this mechanism was the DNA-binding protein that we termed REST. Further, because a large number of other "neural-specific" genes contain REST-binding sites, we proposed that REST played an important role in determining the neural phenotype. The new specific aiins will test our hypothesis that REST is indeed a critical factor for acquisition of neural fate. We will perform gain-of-function and loss-of function studies of REST in cell lines and in transgenic mice to determine whether REST controls expression of target genes in the chromosome (aim l). We will also examine, as a measure of the extent of influence of REST, the ability of ectopic expression of REST to block the induction of sodium channel excitability by Nerve Growth Factor. Preliminary data suggests that REST expression is repressed at a developmental time when neural progenitor cells differentiate to acqulre neural fates. The elucidation of upstream factors that repress REST expression during neurogenesis is attacked in specific aim 2. The upstream regulatory region of REST will be isolated and tested in transient expression assays for the presence of DNA elements that regulate its cell type specific expression. The ability of the cloned REST sequences expressed in transgenic mice to recapitulate the temporal and spatial pattern of expression of the endogenous REST gene will be confirmed and subsequent experiments will identify the precise DNA elements required for the absence of REST expression in neuronal cells. The proposed studies will contribute importantly to the revelation of mechanisms controlling the appearance and maintenance of membrane excitability and of factors critical for regulation of mammalian neurogenesis.

With a Faculty Award for Women Scientists and Engineers from the National Science Foundation, Dr. Gail Mandel will continue to investigate the basis of regulated expression of sodium channel genes in the nervous system. Presently, Dr. Mandel is focusing on the molecular events leading to the first appearance of sodium channels in the developing embryonic nervous system. This examination will determine whether cell-cell interactions are critical for the initial activation of sodium channel gene expression and, if so, how signals are transmitted to the nucleus. The importance of molecular studies on channel genes may extend to other genes involved in commitment to the neuronal pathway, having a major impact on the field of molecular neurobiology in general and the understanding of ion channel regulation in particular.

This core facility will provide equipment, reagents, and technical support required for the successful completion of the molecular biology components of the individual projeCts. The rationale of the core is that all of the projects require either molecular cloning or expression studies using cloned cDNAs. The molecular biology core is a single laboratory space, to be shared by all of the groups, which will alleviate duplication costs for equipment, supplies, and salaries. By nature of its central location in the Life Sciences Building, the core facility will enhance scientific interactions among project members. The core is also designed to eliminate potential technical barriers which may exist between labs of different disciplines. The molecular biology core will provide the following specific services critical for each proposed project: a) Propagation and maintenance of plasmid vectors: plasmids will be purified in the core using either CsCl gradient ultra centrifugation or commercial kits which utilize chromatography. The core will be responsible for transformation of the DNAs into bacteria, for maintaining frozen stocks of the recombinant bacteria, and for mailing DNAs to other colleagues in the field, upon request. b) Sequencing: Sequencing reactions will be performed by the research assistant using oligonucleotide primers made with the core Millipore synthesizer. A new automated sequencer in the Life Science building will be used as soon as it is up-and-running. A computer work station in core, containing DNA software, is available for storage and analysis of data. c) Preparation of commonly used solutions (electrophoresis buffers, bacterial media) and preparation of bacterial plates containing different antibiotics. d) Preparation of synthetic RNAs for expression studies. e) Purification of GST-fusion proteins. f) Maintenance of all equipment, including periodic cleaning of centrifuges, rotors, gel drier, lyophilizers.

Voltage-dependent sodium channels are responsible for action potential generation in the axons and cell bodies of peripheral nervous system (PNS) neurons. Through collaborations described in this program project we have determined that the predominant sodium channel in PNS is one that we have named Peripheral Nerve type 1 (PN1). PN1 is the first example of a gene that is expressed exclusively in PNS neurons. PN1 gene expression is rapidly and selectively induced in PC12 cells in response to one-minute treatments of neuronal growth factors and the cytokine, interferon-gamma, leading to membrane excitability. The "triggered" induction of PN1 mRNA occurs through a novel signal transduction pathway that is being-defined in project 2. The overall goal of this project is to identify the DNA elements and cognate transcription factors that control expression of the PN1 sodium channel gene. Specific Aim 1 focuses on identifying, through genetic and biochemical approaches, the DNA elements and DNA-binding proteins that restrict the expression of PN1 reporter genes to PC12 cells. The yeast one-hybrid screen, developed originally in the PI's lab to isolate factors required for expression of CNS sodium channel genes, will be exploited for these studies. Specific Aim 2 focuses on elucidating the DNA elements and transcription factors that mediate the "triggered" induction of PN1 mRNA in PC12 cells. Reporter genes containing different PN1 gene sequences will be introduced into PC12 cells and then treated briefly with NGF or interferon-gamma. The DNA- binding proteins will be identified using the one-hybrid genetic screen. Specific Aim 3 focuses on determining whether the genetic elements that regulate expression of PN1 reporter genes in PC12 cells are sufficient to restrict expression of the same reporter genes to PNS neurons in vivo, in transgenic mice. Expression patterns of the reporter genes will be compared to the expression pattern of the endogenous gene and to expression of the type II silencer protein, REST. The results from these studies will provide insights into the basis for processing sensory, and particularly pain, information at the cellular and molecular levels. The knowledge of the DNA elements and proteins that restrict expression of the PN1 gene specifically to the PNS also provides a potential means to manipulate the phenotype of sensory or sympathetic neurons through overexpression of proteins in these neurons.

DESCRIPTION (provided by applicant): Rett Syndrome (RTT) is a severe neurological disorder in humans that results from mutations in the gene encoding methyl-CpG binding protein 2, MeCP2. One of the mysteries surrounding RTT is that although MeCP2 is a ubiquitous protein, the manifestations of the disease are restricted primarily to the central nervous system. Possible explanations have included increased sensitivity of neurons to loss of MeCP2 and lack of redundancy of MeCP2 function in neurons. Here we present an alternative idea, that the nervous system manifestation of RTT is due, at least in part, to loss of MeCP2 from glia, which in turn results in a negative influence on neurons. This idea is based on our preliminary findings that 1) MeCP2 is expressed in glia as well as in neurons, 2) that astroglia from RTT mice exhibit chromatin modifications similar to those in brains of mice lacking MeCP2, suggesting a less repressive chromatin status, and 3) that, in co- culture, RTT astroglia confer an aberrant morphology on wild type neurons. We will test this new idea in three specific aims. First, we will identify the defects induced in wild type (WT) neurons by astroglia from RTT mice and determine, conversely, whether defects in neurons from RTT mice are rescued by WT astroglia. Second, our conditioned medium experiments suggest that astroglia from RTT mice secrete a soluble protein inhibitory to neuronal morphology. We will analyze by microarray and biochemical approaches, the nature of the aberrant factor(s) secreted by astroglia from RTT mice. Finally, we will characterize RTT symptoms in a newly generated mouse model in which MeCP2 can be deleted either specifically from astroglia or from both neurons and astroglia. These mice will provide in vivo confirmation of a role for MeCP2-null astroglia in conferring aberrant neuronal morphology in RTT and permit functional studies of neurons in the context of abnormal astroglia. The proposed studies offer the potential for a new attack, through pharmacological inhibition of potential glial secreted inhibitory factors, on this debilitating neurological disorder that strikes one in 10,000-15,000 girls. PUBLIC HEALTH REVELANCE This grant identifies, for the first time, a potential role for a factor secreted from glia as an underlying cause of neuronal damage in Rett Syndrome. If, as our data suggest, the glial effects are due to secretion of an inhibitory growth factor, it raises the possibility of therapeutic intervention for treatment of RTT patients.

DESCRIPTION (provided by applicant): There is currently no way to correct disease-causing mutations in the nervous system without altering the physiological level of the endogenous mRNA. This is a serious challenge because haplo-insufficiency or two-fold over-expression is often sufficient to cause neurological disorders. An example is Rett Syndrome, caused by mutations in the Mecp2 gene. Mecp2 gene duplication, as well as loss-of-function, results in severe disease. We propose to meet the challenge by harnessing the natural ability of RNA editing enzymes to site-specifically fix mutations in endogenous mRNAs. As a target for gene therapy, mRNA offers advantages over DNA. Messenger RNA is cytoplasmic, a readily available substrate, and unlike DNA in which 'mistakes' will be maintained, mRNAs turnover, replenishing the therapeutic target. Our new approach, Site Directed RNA Editing (SDRE), offers enormous untapped potential for correcting mutations, particularly those affecting the nervous system, and for exploring fundamental biological questions. RNA editing, which occurs through adenosine or cytidine deamination, is a natural process. When it occurs within the coding sequence of an mRNA specific codons can be re-coded to produce an altered amino acid sequence. For example, excitatory neurotransmission absolutely depends on the editing of a single adenosine within AMPA-type glutamate receptor mRNAs. Recognizing the power of this activity, we engineered a hybrid modular adenosine deaminase. When used in combination with a small antisense guide RNA we can site-specifically target any chosen adenosine. A similar strategy will be employed to create a site-directed cytidine deaminase. Unlike established therapies that focus strictly on regulating gene expression, SDRE can also fine-tune protein function. Inherited mutations that underlie diseases due to amino acid substitutions or premature stop codons can be corrected, and second-site suppressor mutations that restore function can be selectively introduced. We will demonstrate the power of SDRE within the context of neurobiology, but importantly, it applies to any biological system.

? DESCRIPTION (provided by applicant): Despite advances in identifying defective genes underlying neuropathologies, how these defects underlie symptoms is not known, and this gap is a formidable stumbling block for therapeutics. A case in point is Rett Syndrome (RTT), a severe neurological disease in girls. The disease is due to sporadic mutations in the transcription factor, MeCP2, but why loss of MeCP2 causes neuropathology is enigmatic. Further, RTT holds a unique place in neurological disease because key symptoms are reversible in mice by expressing MeCP2 throughout the brain or just in astrocytes, the prominent glial cell type in brain. The rescue opens the door to therapeutic approaches, but requires a better understanding of what is deficient in RTT and precisely what is rescued upon MeCP2 restoration. Traditional approaches, such as microarray analysis, have focused almost exclusively on individual gene transcript changes, primarily in neurons. This approach has not led to clear answers about the functions of MeCP2 or the cellular basis of the disease, in part due to cellular heterogeneity. It also ignores work indicating a role for astrocytes in contributin to symptoms. In no case is there a molecular benchmark for extent of rescue. Our goal is to attack these issues head on by focusing specifically on rescue of RTT symptoms by astrocytes. Here, we perform a co-expression network analysis, using RNA seq combined with membrane proteomics, on brain and on pure populations of cells sorted from murine brain (aim 1). With an eye towards human-specific therapies, we identify the molecular and cellular consequences of loss and gain of MeCP2 in neural cells from RTT patient IPSCs, and test predictions from these studies in human/mouse xenografts (aim 2). Finally, we test a new hypothesis (aim 3), based on recent preliminary results, that reduced excitatory signaling between astrocytes and neurons may be a functional outcome of the alterations in molecular and membrane properties of these cells (aims 1 and 2).

Despite advances in model systems for identifying genes involved in aging, many unanswered questions still remain about the biology and underlying mechanisms in humans, particularly related to age-related cognitive decline. This is, in part, because even between mouse and human genomes, genetic regulatory elements have diverged, in sequence, numbers, and their presence in different genes. Additionally, the extreme heterogeneity of cell types in brain is a factor. In this grant, we take on the challenging problem of genetic mechanisms underlying human brain aging. First, we exploit the accumulated wealth of knowledge of the transcriptional repressor, REST, to fully characterize its function in neurons in adult brain. We, and others, have been studying REST for decades as a model for understanding fundamentals of gene regulation because of some prominent features. REST has an easily recognizable unusually large binding site that occurs in thousands of neuronal genes and it robustly signals its function when bound to genes by recruiting chromatin modifiers. While others have reported an activator function for REST in neurons, we have only found repressor function, so we will also settle this discussion in Aim 1. Aim 1 also includes studies to identify ?orphan? REST binding sites of unknown function. The results from this aim will expand the neuronal functions under REST control, as well as provide a potentially new model of repressor function, generally. Second, our preliminary results indicate REST is expressed to different levels in different neuronal types, human and mouse, and that its loss in mice results in differential transcriptional effects in these neurons. This parsing of REST function in human neurons is studied in depth in Aims 2 and 3. We recently found that REST protein levels in human hippocampus increase with age sometime in the second decade after birth, remaining elevated even in centenarians, whereas REST levels in mouse brain are greatly reduced by 4-6m after birth, and stay extremely low for life, suggesting human-specific REST functions and/or target genes with age. We will identify REST functions with age in Aim 2. Third, polymorphisms in REST have been associated with the age-related disease, Alzheimer's Disease (AD). A recent study suggests that REST is involved directly in cognitive decline, although some of the published findings are at odds with our results. We will test hypotheses regarding REST gene regulation in AD in Aim 3. In total, our proposed experiments will provide new information on REST function and its potential impact on neuronal gene expression and chromatin changes during brain aging and related pathologies. The experiments will also provide new information on fundamental questions regarding repressor function.  

Project Summary In this revised proposal, we continue to develop an approach to repair base mutations at the level of RNA, for attenuating symptoms in mouse models of human neurological disease. The experiments are an outgrowth of a pilot NIH Director?s Transformative Research Award that supported both recently published and preliminary results attesting to feasibility of this approach. Our work is currently focused on Rett syndrome, a devastating neurological disease due to mutations in the gene encoding the transcription factor, MECP2. We focused initially on a human patient guanosine (G) to adenosine (A) mutation in MECP2, MECP2317G>A, which interferes severely with its ability to bind to chromatin and results in Rett syndrome. We showed, for the first time, that endogenous Mecp2317G>A RNA can be recoded to the wild type amino acid efficiently in non-dividing neurons cultured from a Mecp2317G>A mouse line that exhibits severe Rett-like symptoms. The recoding occurred through site- directed deamination by a hijacked catalytic domain of Adenosine Acting on RNA 2 (ADAR2) (Editase), fused to a bacteriophage RNA binding peptide, which we targeted to the Mecp2 mutation by an RNA guide. In this revised application, we present new data indicating that recoding also occurs in vivo, in 3 different hippocampal neuronal populations, after direct hippocampal injection of AAV encoding the hybrid ADAR2 protein and Mecp2 RNA guides. Moreover, recoding resulted in amount of MeCP2 localization to chromatin consistent with amount of editing at the RNA level. We have developed the tools and reagents that now place us in an ideal position to address critical unanswered questions for reversing neurological phenotypes of Rett syndrome, and for testing hypotheses related to site-directed repair. In Aim 1, we test the hypotheses that brain-wide repair of Mecp2317G>A RNA, by site-directed editing, can be tuned to high efficiency and specificity in mice, and restores proper chromatin interaction. For this purpose, we perform whole transcriptomic RNA seq analysis across the brain after peripheral injections of an efficient brain AAV serotype virus encoding optimized editing or control components. In Aim 2, we test new guides for the ability to recruit endogenous ADAR2 to mutant Mecp2 RNA in vivo, circumventing potential immune responses to the bacteriophage moiety in the hybrid ADAR2 protein and potentially minimizing off-target editing. Our initial model is Mecp2311G>A that has the ideal nonsense codon for this approach. In Aim 3, we inject peripherally the virus encoding our current and optimized editing components, or controls, to test our hypothesis that site-directed RNA editing can stabilize/reverse Rett-like symptoms in both Mecp2G>A mouse lines. In addition to Rett syndrome, our approach has the potential to cure thousands of additional pathogenic G>A mutations.