Ronald P. Hart - US grants

The research project will focus on the structure and regulation of the gene encoding tryptophan hydroxylase (TPH), the rate-limiting enzyme in the synthesis of serotonin. While TPH is known to be regulated through protein kinase-mediated phosphorylation, this key enzyme may also be regulated at the level of gene expression. Using oligonucleotide probes based on cloned, pineal cDNAs encoding TPH, we can detect mRNA from the CNS. We have shown that the CNS mRNA is altered at the 5' end when compared with the cloned, pineal cDNAs. Furthermore, we have shown that pharmacological agents which alter serontonin levels alter steady-state TPH mRNA levels. We now propose to obtain genomic- and CNS-specific cDNA- clones specific for TPH and to study the mechanism of gene regulation. Lastly, we will study the potential involvement of receptor events in the regulation of TPH mRNA levels.

This application is a request for support for the fifth Federation of American Societies for Experimental Biology Summer Research Conference on Neuroimmunology to be held at Wilsonville, Oregon, in July 1998. Scientists from a variety of disciplines have become increasingly interested in the dialogue between the immune system and the nervous system. In the past, the major effort in this field came from immunologists, who attempted to explain how the immune system affects nervous system behavior under pathological conditions such as autoimmune diseases of the nervous system. Little is known about the reciprocal effect of the nervous system on the immune system and even less is known about the bi-directional communication. This subject has recently attracted the attention of neurobiologists in general, and those working on CNS trauma and degenerative diseases in particular. As a result, it has now become clear that: (a) some components once thought to be exclusive to the immune system are also expressed by the nervous system; (b) there is a cross-talk between the immune system and nervous system, both in the intact and the injured CNS; and (c) the nature of the dialogue is unique in certain aspects to CNS while other aspects might be common to all tissues. In addition, the rapid advances in the field make it necessary to reassess a number of basic aspects of CNS-immune interactions, including anatomical, physiological and functional aspects of the CNS in relation to immune activity and interaction. In the past, interest in immune involvement in brain diseases was restricted to autoimmune diseases. Recently it became evident that almost any nerve-related disease involves an immune-associated component as part of its pathogenesis. This alters our view of therapeutic approaches currently accepted in the pharmaceutical industry for treatment of degenerative diseases of the CNS as well as treatment of CNS trauma. The basic aspects which should be reassessed include the role of microglia in degenerative and autoimmune diseases, CNS immune privilege, microglia in differentiation, their accessibility to the CNS and their role in trauma and degenerative diseases. It is important to assemble neuroimmunologists in their own forum, particularly since these processes involve input from several disciplines.

DESCRIPTION (provided by applicant): Epigenetic regulation of microRNAs in neurogenesis Histone deacetylase (HDAC) inhibitors such as valproate (VPA) are commonly used to treat epilepsy. One me- chanism of their efficacy may result from epigenetic effects on differentiation of neural precursors. While HDAC inhibitors are likely to affect mRNA-encoding genes, we believe that the rapid regulation of microRNAs by VPA treatment suggests a novel complementary mechanism. Hypothesis: Acetylated histones allow expression of microRNAs that enhance or support neurogenesis from neural precursors. Therefore, adding an HDAC inhibitor allows the accumulation of acetyl marks on microRNA-encoding sites on the chromatin, in turn enhancing expression of the marked microRNAs, which contribute to neural differentiation. We will test the hypothesis by using "deep sequencing" to identify the full complement of VPA-regulated microRNAs, including hundreds of novel microRNAs that we recently identified in neural differentiation. Finally, we will build on our preliminary studies with microRNA inhibitors and screening techniques to evaluate candidate microRNAs for their requirement for VPA-induced neuronal differentiation. Results from these three aims will identify both known and putatively novel microRNAs that are regulated after HDAC inhibition, the acetylation status of histones located near microRNA promoter regions, and whether the regulated microRNAs contribute to neuronal differentiation. Understanding the regulatory networks required for neurogenesis is important for determining how differentiation of neural precursors could be impaired in conditions such as dementia, Parkinson's, or stroke. Alternatively, the use of HDAC inhibitors in conditions such as epilepsy, bipolar disorder, or migraines may have unexpected effects on neurogenesis from adult precursors, and this potential benefit should be understood. PUBLIC HEALTH RELEVANCE: Epigenetic regulation of microRNAs in neurogenesis. Epilepsy drugs such as valproate (VPA) are known to inhibit histone deacetylases, which control gene expression epigenetically. Since VPA also increases the production of neurons from adult stem cells or other precursors, we believe that VPA may regulate microRNA genes, in turn affecting decisions controlling cell differentiation. We will use "deep sequencing" of microRNAs and gene regulatory regions to identify novel neurogenesis mechanisms.

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (08) Genomics, and specific Challenge Topic 08-CA-104: Regulatory functions of small RNAs. Our goal is to identify small RNAs (smRNAs) as regulators of histone modifying enzymes, mediating interactions with promoters and/or non-coding RNAs present in promoter proximal regions and mediating long-term effects on gene expression. This is a novel model that would tie together genome-encoded small RNAs, non-coding RNAs, and the regulation of histone modifications, leading to long-term modulation of gene expression. Epigenetic modifications are inheritable alterations to the genome in the form of enzymatic manipulations of key histone residues, or the methylation of specific cytosines in the DNA sequence. Carcinogenesis is likely to include aberrant use of this pathway to cause long-term changes in gene expression in transformed cells. In fact, epigenetic dysregulation is a hallmark of numerous cancers, and compounds which broadly affect histone modifications are under intense scrutiny as cancer therapies. While this strategy may be effective, we believe that it will be possible to therapeutically target epigenetic modulation of selected genes by exploiting the existing RNAi machinery, as demonstrated for several smRNAs and genes. This project aims to understand the relationship between small, non-coding RNA sequences and epigenetic histone modifications, and explore the mechanisms by which epigenome-modifying complexes may be recruited by small RNAs to their target DNA sequences. Comparison of smRNAs identified in human embryonic stem cells by deep sequencing with genomic alignment sites and previously published epigenomic data of key histone modifications has identified striking association between these two previously independent pathways. In this proposal we will test the hypothesis that smRNAs mediate the placement of epigenetic marks. Specifically, we will (1) identify and characterize classes of smRNA sequences associated with specific chromatin modifications in human ES Cells during neural differentiation;(2) bioinformatically predict as yet unobserved smRNA-mediated epigenetic effects;and (3) experimentally confirm the functional interaction between small non-coding RNAs and epigenetic modifications in hESC. Epigenetic marking is one of the next great questions in biology and medicine. We propose that endogenous smRNAs are the key link between histone modification mechanisms and individual genomic loci. If smRNAs do in fact direct epigenetic modifications, this may represent a novel mechanism by which cells are capable of transcriptional regulation. This will provide both additional therapeutic targets and enhanced understanding of disease etiologies for regions of altered histone modification during cancers. PUBLIC HEALTH RELEVANCE: Epigenetic dysregulation is a hallmark of several human disorders and numerous cancers. Histone modifications represent one mechanism of epigenetic regulation that directly affects the transcriptional availability of adjacent genomic elements. This project aims to identify the relationship between small, non-coding RNA sequences and epigenetic histone modifications, and explore the mechanisms by which epigenome modifying complexes may be recruited by small RNAs to their target genomic regions. The association of small RNAs with specific histone modifications provides understanding of disease etiologies and suggests mechanisms to transcriptionally regulate target genes in patients.

DESCRIPTION (provided by applicant): Identification of genetic variations associated with addictive behaviors provides a novel opportunity for using cell cultures to model molecular and cellular mechanisms that underlie addiction. The observed genetic variations have been mapped to amino acid changes in cell surface receptors, presumably affecting neuronal circuits involved in addiction behaviors. However, cellular context is likely to be important in determining the function of these genes. Construction of induced pluripotent stem cells (iPSC) from adult cells derived from drug abusing individuals carrying known genetic variants provides a means for developing physiologically relevant culture systems for understanding addiction. For the culture cell model to be useful it must not only express the affected gene but it must also provide an appropriate cellular context for studying pharmacology or cell signaling. There is no clear expectation about what type of iPSC-derived cultures would be useful in studying the cellular physiology altered by gene variants. Our hypothesis is that the altered physiology of genetic variants associated with addiction liability can be modeled in cultured neurons derived from iPSCs constructed from donor lymphocyte samples. We propose to construct several iPSC lines from donor lymphocyte samples, differentiate these iPSC into functional neurons, and then to develop methods to assay possible phenotypic differences between variant-derived cells and wild-type. These cells will be valuable for identifying cell and molecular responses to substances of abuse, to examine the effects of a known genotype on the cellular phenotype, as well as to develop novel approaches for pharmacologic intervention.

DESCRIPTION (provided by applicant): This is an exploratory/developmental (R21) application under the Cutting-Edge Basic Research Awards (CEBRA) program. The pathogenesis of drugs associated with abuse behavior, including nicotine and alcohol addictions, remains elusive in humans because studies of the human brain are limited to functional brain imaging and post-mortem analysis. These types of analyses make it difficult or impossible to prove hypotheses directly since the system usually cannot be manipulated or sufficiently controlled. A large number of genetic variants have been identified to be risk factors for addictive behavior in human, however, little is known about how these genetic variations impact the development of addictive behavior in humans. Recent advances in stem cell biology allow construction of induced pluripotent stem cells (iPSC) from adult cells derived from addicted individuals carrying identified genetic variants and provide possibilities for developing cell-based models of addiction. Addictive behavior in human is not only related to cellular level modifications in a specific cell type in the brain but it also affects neuronal function such as synaptic plasticity at the neurocircuitry level. However, there are currently no such in vitro neurocircuitry models that have been established using human neurons. We hypothesize that neurons derived from subjects with risk-associated genetic variants will desensitize reward circuit modulation in an in vitro mini-neurocircuitry model. By using a compartmentalized culturing system, we propose to construct a mini-neurocircuitry model mimicking mesolimbic nucleus accumbens (NAc) neurons and their synaptic inputs. Cellular and synaptic phenotypes of neurons derived from addictive patients will be investigated under the context of neurocircuitry and compared with wild-type controls. This mini-neurocircuitry model will be essential to identify mechanisms underlying risk-associated gene variants and addictive behavior. It will also serve to develop and screen novel interventions for drug abuse therapies.

? DESCRIPTION (provided by applicant): The purpose of this project is to develop multicompartment neuronal cell microculture chambers in a 96 well format to produce high throughput (HTP) in vitro neurocircuitry models using human neuronal cells. This platform allows HTP phenotyping of neurons derived from induced pluripotent stem (iPS) cell lines carrying risk gene alleles associated with susceptibility towards substance abuse. In particular, this work will aid the understanding of synaptic plasticity changes associated with the pathogenic behaviors of drugs of abuse, including addiction to cocaine, nicotine and alcohol. Synaptic plasticity changes, especially in the mesolimbic system, are associated with addictive behavior and are not recapitulated with existing cell culture systems using human neurons. We have constructed a multicompartment, microfluidic system that allows us to model the neuronal circuitry of the mesolimbic reward system. However, given the increasing number of iPS cell lines available, the need to include genome edited versions of each genotype with isogenic genetic background, the multiple subtypes of neurons required, as well as the need for HTP screening for small molecule compounds that can reverse the alterations associated with the gene variants, the current analysis is difficult or impossible to scale up to meet with the need fo HTP analysis of defined neurocircuitry. Thus, in this CEBRA application, we propose to build upon our previous success in creating neurocircuitry models by constructing a microfabricated HTP platform, compatible with the GE IN CELL high-content imaging system, for both morphological and functional (Ca2+-imaging) analyses. Following construction of the HTP system, we will validate it by focusing on a well-studied genetic variant associated with increased alcohol consumption, nicotine and cocaine abuse, containing an altered ¿-opioid receptor (MOR) sequence that alters intracellular signaling. The Single Nucleotide Polymorphism (SNP) rs1799971 produces a non-synonymous amino acid substitution in MOR (OPRM1 A118G), replacing Asparagine 40 (MOR N40) with a variant with Aspartate (MOR D40). We have prepared multiple iPS cell lines carrying either homozygous MOR N40 or MOR D40 from subjects with well-characterized alcohol abuse, nicotine dependence-related behaviors, and known ethnicity. Our goal in this exploratory project is to develop a novel HTP experimental paradigm to support in vitro mini- neurocircuits mimicking the midbrain DA neurocircuitry that is formed by neurons generated from patient specific iPS cells carrying defined risk gene variants, to allow the screening of small molecule compounds to reduce phenotypes consistent with addictive behaviors.

Project Summary/Abstract Even with the advent of successful combination antiretroviral therapy (c-ART), infection by Human Immunodeficiency Virus (HIV-1) remains a global public health crisis. HIV infection of the brain results in important clinical symptoms including HIV-associated neurocognitive disorder or HAND, a constellation of symptoms affecting cognitive, behavioral, and motor functions, as well as pediatric HIV encephalopathy (PHE) in children, leading to neurodevelopmental deficits. These disorders may still occur even in patients receiving c- ART, although with less severity. The molecular and cellular mechanisms underlying the pathophysiology of HIV- HAND and PHE remain poorly understood, in large part because of limitations of the existing model systems to study them. Animal models are generally not infected by HIV, expensive and impractical or poorly recapitulate human brain infection. Cultured cell models provide some insights, but do not recapitulate many aspects of cell- cell and cell-matrix interactions found in human brain. Advances in stem cell technologies, including the use of human induced pluripotent stem cells (iPSC or iPS cells), now enable generation of patient-specific neural lineage cells and microglia (brain immune cells), as well as the assembly of these cells into 3-dimensional (3D) brain organoids. These cerebral organoids mimic important features of immune, glial and neuronal cell interactions, uniquely enabling us to examine how these interactions are affected by HIV infection and to model key aspects of HAND and PHE pathogenesis. The overarching goal of this project is to leverage our strengths in neurosciences, virology, stem cell biology and gene expression to understand the pathophysiology of HAND and PHE using a human neural-microglial system. We have established human 3D brain organoids prepared with iPS cell-derived microglial cells to study neurodevelopment and neuroimmune interactions and have successfully infected these mixed organoids with HIV. We hypothesize that microglial infection by HIV causes altered neuronal function which can be modeled in these microglial-containing brain organoids, providing a new system for mechanistic understanding of the pathogenesis of HAND and PHE. In this proposal, we will characterize the effects of HIV infection on microglia activation and cytokine production, neuronal cell populations, organization and gene expression, and the specific effects of candidate cytokine mediators. We will apply cutting- edge approaches drawn from stem cell technologies, brain organoid models, and single cell RNA sequencing to develop this model system, which will allow long-term, detailed analyses of the molecular and neurophysiologic mechanisms responsible for the pathogenesis of HIV-associated neurological disorders.