Michael S. Levine - US grants

The aim of this research proposal is to assess some of the morphological and neurophysiological correlates of the aging process in the central nervous system. To study these the basal ganglia will be used as a model system. In one set of experiments we will determine the effects of the aging process on the cytoarchitectural and ultrastructural characteristics of basal ganglia neurons in cats. In a second set of experiments the effects of the aging process on the neurophysiological characteristics of basal ganglia neurons in cats will be determined. Wherever possible the morphological and functional alterations that occur in aged animals will be correlated to provide a picture of the neuronal dysfunctions that occur in senescence. In a third set of experiments th anatomical and physiological effects of aging will be related to some of the neurological disturbances that occur.

The Drosophila larva is composed of a series of discrete head, thoracic and abdominal segments. The Antannapedia locus (Antp) of the Antannapedia Complex (ANT-C) is required for proper differentiation of the larval thoracic segments. Genes of the bithorax complex (BX-C) are required for the morphogenesis of the metathorax and abdominal segments. The embryonic segments and segment anlage that accumulate transcripts specified by ANT-C and BX-C correspond to those that are most affected by mutations of these genes. Restricted spatial expression of ANT-C and BX-C appears to involve hierarchial interactions of the genes contained within these complexes. BX-C- embryos display an epidermal and neural transformation of the metathorax and first seven abdominal segments into the homologous tissues of the mesothorax. It appears that this transformation results from a posterior extension of the normally thoracic realm of Antp+ expression since transcripts specified by Antp accumulate in the metathoracic and first seven abdominal ganglia of BX-C- embryos. The experiments described in this proposal will permit a detailed identification of the BX-C genes involved in the inhibition of Antp expression in the posterior ganglia of wild-type embryos. In addition, we will use P-mediated transformation to define the sequences within the Antp transcriptional unit that are required for interaction with BX-C. Additional homeotic loci appear to be involved in segmental determination of the anterior embryonic regions that will form the prothorax and head. By localizing transcripts specified by the Scr locus and putative 'head forming' loci within mutant embryos that are deficient for various ANT-C and BX-C genes we will determine whether the anterior realms of expression that are thought to be acquired by these genes result from inhibitory interactions with Antp and bithorax products. Garcia-Bellido has proposed that homeotic genes control developmental pathways by the activation of 'realisator loci' which in turn specify morphogenetic cell properties such as surface recognition characteristics. A prediction of this model is that particular genes become selectively expressed in the mesothoracic ganglion of developing embryos in response to Antp+ products. The experiments proposed in this grant should allow us to test this model by isolating and characterizing neural-specific genes that are present in BX-C embryos and absent in Antp- embryos.

These studies are directed toward the regulatory protein (Gs) of adenylate cyclase. Adenylate cyclase is composed of at least three protein components: 1) hormone receptors, 2) a catalytic moiety, and 3) a regulatory protein (Gs) that binds guanine nucleotides. Gs functions to modulate agonist-occupied receptor affinity, and to couple hormone-receptor complexes to the catalytic unit. Gs thus regulates the hormonal activation of adenylate cyclase. Strong evidence supports a defect in adenylate cyclase as the cause of hormone resistance in pseudohypoparathyroidism (PHP), a metabolic disorder characterized by resistance of the target organs bone and kidney to the actions of parathyroid hormone. Recently, a deficiency in Gs activity has been described in cell membranes from many subjects with PHP. Unlike tissue-specific hormone receptors, Gs is similar in all cells, and deficient Gs activity is implicated as the cause of decreased synthesis of cAMP and hormone resistance in diverse tissues in many patients with PHP. For these reasons it is both interesting and important to study further the structure and mechanism(s) of action of Gs. These studies propose 1) to purify Gs from human erythrocyte membranes by a novel approach: cholera toxin catalyzes the covalent ADP-ribosylation of a 42,000-dalton subunit of Gs; T4 RNA ligase can attach polyadenylate specifically to the ADP-ribosylated Gs peptide and oligo- d(T) affinity chromatography will be used to resolve the modified Gs from other membrane proteins. 2) to use the purified (modified) Gs peptide in the production of reagent antibodies to Gs; 3) to use these antibodies in the development and application of methods for assessing the immunologic and biologic properties of Gs from erythrocyte membranes of subjects with PHP; 4) to determine the applicability of the oligo d(T) chromatography methods to the resolution of other proteins (e.g., transducin, and the inhibitory guanine nucleotide binding protein) that are specific ADP-ribose substrates. These studies may help to elucidate molecular lesions(s) in Gs which impair adenylate cyclase hormone responsiveness, and may provide further insight into hormone resistance in PHP. Ultimately, it is hoped that an immunologic approach to the study of the mechanism(s) of action of Gs will help identify the domain or subunits of Gs that regulate the complex interactions of the regulatory protein with hormone receptors and the catalytic unit.

Parathyroid secretory protein (SP-I) is an acidic glycoprotein that is co-stored and co-secreted with parathyroid hormone (PTH) under calcium control. It has been shown to be chemically similar, if not identical to Chromogranin A, the major protein of the adrenal medulla chromaffin granule, that is co-stored and secreted with epinephrine and other granule contents. Moreover, SP-I has been found in every endocrine cell examined-- but never in an exocrine or epithelial cell. We and others speculate that SP- I (or chromogranin A) plays an important but yet-to-be-defined role in the processing, packaging or secretion of endocrine proteins, possibly by providing an intracellular "traffic signal". We propose two approaches to test this hypothesis. In the first approach we will construct recombinant genomes containing cDNA encoding either SP-I or PTH and insert them into the AR4- 2J exocrine pancreas cell and into the MDCK polarized epithelial kidney cell, neither line of which expresses SP-"I. We will then test the effect of the introduction of the SP-I cDNA on processing and secretion of endogenous protein(s) and on that of the exogenous pth. We will also evaluate posttranslational modifications to the expressed SP-I. In the second approach, we will attempt to block the synthesis of SP-I in an endocrine cell by introducing antisense cDNA to mRNA for SP-I. We will construct the suitable genome containing antisense SP-I and transfect the TT cell, a medullary thryoid carcinoma-derived line that contains expresses both SP-I and calcitonin. We will then evaluate the effect of this procedure on the processing and secretion of calcitonin. These approaches will provide alternative means to determine if and how SP-I affects the secretion of exported proteins.

The Research Center in Oral Biology will function to expand dental research at the State University of New York at Buffalo into three integrated projects: (I) Salivary Defense Factors; (II) Bacterial Virulence Factors; and (III) Host Response Systems. The scientific theme of this RCOB is the Modulation of Natural Defense in Oral Disease. It is our expectation that the RCOB will provide a mechanism whereby dental researchers at SUNY/Buffalo can combine their areas of scientific expertise with modern technology to begin our next generation of studies. The foundation for this RCOB is in place and is enhanced by: 1) a group of dedicated scientists with an established productive record in dental research; and 2) a commitment of support from the University and the School of Dental Medicine; and 3) the existence of a Periodontal Disease Clinical Research Center and Salivary Dysfunction Center which would utilize basic information gained from an RCOB to develop new treatment modalities for plaque mediated diseases and salivary dysfunctions. Project I (Salivary Defense Factors) represents a new generation of research aimed at enhancing the protective functions of saliva. This project utilizes modern technologies in protein and carbohydrate chemistry, biophysics, and computer modeling to design and synthesize salivary substances with enhanced functions relating to bacterial clearance/adherence mechanisms and lubrication. Information obtained could lead to the design of more effective artificial salivas which are custom-designed for a patient's individual needs. Project II (Bacterial Virulence Factors) is designed to identify characteristics of black-pigmenting Bacteroides which are determinants of virulence and to study the immunochemical, biochemical, and molecular biological mechanisms by which virulence is expressed. This group of organisms has been chosen for study since they are proposed to play a predominant role in odontogenic infections. Information obtained could provide a rationale for improving host defenses during the susceptible stages of Bacteroides infection. Project III (Host Response Systems) provides new initiatives to further our understanding of host response to oral bacterial infections. Host responses to be studied include the role of the neutrophil in host defense, cellular mechanism involved in the activation of bone resorption, mechanisms of bacterial mediated connective tissue invasion, and factors involved in the regeneration of the periodontal attachment apparatus. Information obtained should lead to innovative approaches to enhance host resistance to tissue destruction and regeneration of new tissues.

The research in this proposal will elucidate mechanisms underlying changes that occur in the functional properties of neurons due to the process of aging. We will focus on a major part of the basal ganglia, the neostriatum, because this ares of the brain is in- volved in pathological conditions associated with aging. We will determine the nature and time-course of the age-related changes in the dopaminergic nigrostriatal connections and their interactions with corticostriatal afferents. In one set of experiments, the effects of the aging process will be determined on the ability of the putative neurotransmitters glutamate, gamma-aminobutyric acid, and dopamine to alter neurophysiological activity of caudate neurons. These experiments will concentrate on demonstrating that dopamine's ability to modulate corticostriatal responses is compromised during aging. The changes in membrane properties of striatal neurons during aging will also be determined. In a second series of experiments immunohistochemical procedures will be used to identify age-related changes in inputs to the striatum from the substantia nigra that contain dopamine and inputs that contain glutamate. The age-related changes of the relationship between dopamine containing synapses and synapses of corticostriatal inputs will be assessed in a final experiment. The studies outlined in this proposal will indicate how neurotransmitter changes during aging are reflected in neuronal function and how they relate to morphological alterations. The information obtained from these studies is important because pathologies in the neostriatum and/or the substantia nigra are involved in age-related neurological disorders exemplified by Parkinson's Disease and Huntington's Disease.

DESCRIPTION (adapted from application): This is an application to continue his investigations of complex loci of Drosophila to understand the regulation of promoter-enhancer interactions. He made seminal contributions in this area by elucidating how localized patterns of the gene even-skipped (eve) are established along the anteroposterior axis of the precellular Drosophila embryo. The regulation of the segmentation stripes is due to the combined action of broadly distributed activators and narrowly-expressed, short-range repressors. Short range repressors act at distance of less than 100 bp and repress the action of specific activators while long range repression regulates specific enhancers that lie >1000 bp away and are thought to act through repression of general machinery. The present application extends beyond this earlier work focusing on two general goals: 1) The mechanism of action of repressors, both short-range and long-range, and 2) The mechanism by which long-range enhancer-promoter interactions are modulated, in both a positive sense via facilitator proteins and in a negative direction via the use of insulator DNA or competition. There are 3 specific aims: (i) Identify repressors as either short-range or long-range and investigate the mechanism of repression by identifying co-repressors. (ii) Identify proteins required for the enhancer-blocking activity of the specific attenuator Fab-7 and the gypsy insulator. (iii) Investigate the role of promoter competition in the regulation of specific enhancer-promoter interactions in the ANT-C and BX-C gene complexes. The general approach in all of these aims is to use transgenic reporters whose expression is modulated by precisely engineered promoter and enhancers. For example, the initial experiments use a fly strain with a beta-galactosidase reporter transgene. The expression is controlled by enhancers from the eve loci, known as stripe 2 and stripe 3 enhancers since they are responsible for eve expression in these localized region of the precellular embryo. Repressor binding sites positioned close to stripe 2 enhancer and at a distance from stripe 3 can reconstruct the two modes of repression. Repression activity is monitored by the disappearance of blue stripes in the embryo. Other transgenes use Drosophila eye color gene white as the reporter.

Dorsal is a maternal regulatory factor that initiates dorsoventral patterning of the precellular Drosophila embryo. It is a member of the Rel family of transcription factors that is initially distributed throughout the cytoplasm of unfertilized eggs, but shortly after fertilization it translocates into nuclei. This nuclear transport process is regulated by the Toll signaling pathway, which is employed in a variety of processes, including insect immunity and acute/inflammatory responses in mammals. The resulting Dorsal nuclear gradient specifies the embryonic mesoderm, neurogenic ectoderm, and dorsal ectoderm, through the differential regulation of target genes such as snail, sog, and dpp. The snail gene encodes a zinc finger repressor that establishes the boundary between the presumptive mesoderm and neurogenic ectoderm. It is a short-range repressor that must bind within 100 bp of either upstream activators or the core promoter in order to inhibit gene expression. Dpp is a member of the TGF-beta family, and a Dpp activity gradient subdivides the dorsal ectoderm into the amnioserosa and dorsal epidermis. The Dpp gradient depends on sog, which encodes a secreted inhibitory protein that binds Dpp. High levels of Sog are thought to inhibit Dpp signaling, while low levels enhance signaling. The proposed study represents a continuation of our efforts to determine how the Toll-Dorsal signaling pathway controls the dorsoventral patterning of the early Drosophila embryo. The research plan includes three specific aims: (i) determine how Snail functions as a repressor; (ii) identify the components of the Toll signaling pathway that diffuse in precellular embryos to create the Dorsal nuclear gradient; and, (iii) determine how Sog- Dpp interactions create a peak Dpp signaling threshold that specifies the amnioserosa.

The primary goal of the RCOB at State University of New York at Buffalo is to expand the scientific base that underlies the nation's capability to prevent and control oral diseases and disorders and to improve oral health. Our scientific expertise and facilities were greatly expanded during the previous granting period through a combination of RCOB and University resources. This enables RCOB scientists to address scientific problems in an integrated fashion using protein chemistry, molecular biology, biophysics, immunology, microbiology and cell biology. The secondary goal of the RCOB is to maintain a center of excellence that will attract investigators of high quality to dental research, continue our long- standing tradition of research training at all levels of career development, and serve as a focal point to encourage productive research- related collaborations with other institutions. The Center uses the combined efforts of a team of dentist-scientists, microbiologists, biochemists, biophysicists, molecular biologists and cell biologists from both the School of Dental Medicine and the School of Medicine and Biomedical Sciences at the University at Buffalo. Additionally, scientists from Roswell Park Cancer Institute and the University of Minnesota have joined the Center to augment research efforts. Projects 1, 2 and 3 propose to enhance the protective qualities of saliva by the design and development of salivary substitutes to combat plaque mediated disease in subjects with normal salivary flow as well as those with xerostomia. A multidisciplinary approach involving protein chemistry, molecular biology, biophysics and bioengineering will design and synthesize salivary substances with enhanced functions relating to bacterial clearance/adherence mechanisms and lubrication. Projects 4, 5 and 8 are designed to characterize virulence factors of Porphyromonas gingivalis that might be determinants of disease and to study the immunochemical, biochemical and molecular biological mechanisms by which these factors are expressed and subsequently modulated. Studies will focus on the role of fimbriae in adhesion and outer membrane proteins in bacterial coaggregation and iron regulation.

DESCRIPTION (adapted from applicant's abstract) The experiments in this proposal are designed to continue investigations into neuronal processes controlling dopamine modulation of responses mediated by activation of glutamate and GABA receptors in the striatum. The first two aims are a direct continuation of studies begun in the presently funded proposal and will examine the hypothesis that the direction of DA modulation of responses mediated by activation of glutamate receptors is a function of the subtype of glutamate receptor activated and the subtype of DA receptor activated. The third aim is new and will examine the hypothesis that the direction of DA modulation of responses mediated by activation of GABA-A receptors is a function of the DA receptor subtype activated, its site of location on pre- and/or postsynaptic elements and the type of neuron.

B. ABSTRACT. The overall goal of the proposed study is to determine the molecular mechanisms underlying localized patterns of gene expression in the early embryos of sea urchins and ascidians. We anticipate that a detailed comparison of the two systems will provide basic insights into the gene networks responsible for directing lineage-specific patterns of gene expression in metazoan embryos. In addition, these studies should provide information regarding the evolutionary origins of the chordate body plan since sea urchins and ascidians are members of sister clades even though they possess radically divergent organizations. The Levine component of the research plan includes four specific aims. First, we will identical minimal cis-regulatory modules in the promoter regions of Ci-sna and Ci- fkh that mediate expression in the muscle, gut, and epidermal lineages at the time when these tissues are specified in 32-cell stge embryos. Second, minimal cis elements cis elements will be used to isolate maternal regulatory factors from crude nuclear extracts of Ciona and sea urchin embryos. The goal of these experiments is to identify and characterize regulatory factors that interact with AC-core E box elements, which appear to play a key role in the activation of Ci-sna expression during the specification of the primary muscles. Third, the expression and activities of maternal determinants will be investigated using a variety of techniques, including in situ hybridization and immunolocalization assays, and ectopic expression assays using heterologous promoters in transgenic embryos. Fourth, we will investigate the relationship of the sea urchin and ascidian body plans using inter-phyletic comparisons of cis regulatory modules. For example, Ci-sna and Ci-fkh elements will be microinjected into sea urchin embryos, and the corresponding sea urchin elements will be expressed in Ciona embryos via electroporation.

[unreadable] DESCRIPTION (provided by applicant): This proposal will examine cellular mechanisms underlying the dysfunctions detected in Huntington's disease (HD) using four different murine models. The lethal mutation in HD produces an expanded trinucleotide (GAG) repeat within the protein huntingtin. It causes selective neurodegeneration especially in the striatum and cortex, by an unidentified mechanism. Each of the HD models we will examine exhibits a different phenotype produced by unique transgene constructs or 'knocked-in" GAG repeat lengths. By evaluating multiple models we will be able to examine the dysfunctions in more detail and understand the specificity and sequence of physiological changes common to HD and the models. Based on our preliminary studies, we have uncovered several common cellular deficits in two models. These are enhanced responsiveness of N-methyl-D-aspartate (NMDA) receptors in the striatum associated with increased Ca2+ flux, a marked decrease in K+ conductances and a change in the corticostriatal synaptic response. A third model also displays the enhanced response to NMDA. Some of these changes potentially predispose striatal medium-sized spiny neurons to excitotoxic damage. Using a physiological approach, we will examine four hypotheses concerning the cellular mechanisms of dysfunction in HD: 1) alterations in ionotropic glutamate receptor function and changes in evoked and spontaneous excitatory synaptic inputs to striatal neurons 2) alterations in metabotropic glutamate and dopaminergic receptor modulation of ionotropic glutamate receptor function, 3) alterations in K+ conductances and 4) alterations in Ca2+ conductances. The precise onset of changes will be investigated in relationship to the expression of behavioral deficits by using animals that are presymptomatic or after development of overt motor signs. We will examine striatal and corticostriatal neurons, visualized in the slice preparation or acutely dissociated cells, to characterize basic functions by current- and voltage-clamp analyses. Because HD destroys so many different capabilities - intellectual, physical and emotional - the insights gained from this research elucidating the cellular malfunctions in HD are relevant to understanding other GAG repeat disorders and neurological diseases associated with protein aggregate pathologies like Alzheimer's and Parkinson's disease.

The proposed research plan represents a continuation of our efforts to unravel the complex gene regulation networks responsible for the specification and differentiation of endomesoderm lineages in the ascidian, Ciona intestinalis. Particular efforts will focus on two such lineages: the tail muscles and notochord. The research plan includes three specific aims. First, a combination of bioinformatics methods and experimental assays will be used to identify new enhancers that mediate either notochord-specific or muscle-specific gene expression in electroporated Ciona embryos. Conserved sequence motifs will be identified within coordinately regulated enhancers, and efforts will be made to identify the specific transcription factors that interact with these newly identified motifs. Second, a variety of methods will be used to determine whether the elegant endomesoderm gene regulation network defined in sea urchins is conserved in Ciona. Most of our efforts will focus on the cis- and trans- regulation of two key endomesoderm patterning genes: Krox1 and GataE. Third, newly established GFP visualization methods will be used to characterize the mutant phenotypes produced by overexpressing endomesoderm patterning genes such as Krox1, FoxA, and Brachyury. The successful execution of the proposed research plan will provide the first systematic comparision of a well-defined gene regulation network in two related, but morphologically distinct, animals.

[unreadable] DESCRIPTION (provided by applicant): This application requests funding for the 2005 Gordon Research Conference on CAG Triplet Repeat Disorders to be held at Mount Holyoke College, South Hadley, Massachusetts from July 24-29, 2005. This will be the third Gordon Research Conference on CAG repeat disorders, the first was held in 2001 at Mount Holyoke College and the second was held in II Ciocco, Barga, Italy in May 2003. During the last decade, the mutation that causes a major group of inherited neurological disorders was found to be a CAG triplet repeat expansion. So far, this group of diseases includes Huntington's disease, the spinocerebellar ataxias 1, 2, 3, 6, 7 and 17, spinal and bulbar muscular atrophy and dentatorubral pallidoluysian atrophy. In each case, the CAG repeat lies within the coding region of the gene and results in an abnormally long polyglutamine tract in the mutant protein. Similarities in the underlying genetics and neuropathology suggest that the mechanisms of pathogenesis share common features. However, these diseases result in different anatomical distributions of the selective loss of neurons in the brain and spinal cord, and therefore the factors that distinguish them also need to be unraveled. Since the identification of the genetic defects, significant insights have been gained into the pathogenesis of these diseases. The field has progressed to the extent that the development of rational therapeutics is not only on the horizon but occurring already. In order to increase the pace of the basic research, and at the same time set in place the contacts and clinical resources necessary to move the basic science into the clinic, a multidisciplinary research effort is required. It is essential that collaborative projects between scientists from diverse specialties ranging from organic chemistry, fruit fly genetics to clinical neurology can be established. This conference on CAG triplet repeat disorders will gather together young investigators and established senior scientists to deliver provoking lectures on the cutting-edge of science. In keeping with the Gordon Research Conference format, there will be generous time allocated for both structured discussions led by peers and for informal discussions and social interactions to facilitate collaborations. Strong emphasis is placed on training and mentoring of young scientists, and time will be devoted to career issues. All participants will be required to present posters. Priority will be given to women, minorities, and persons with disabilities when selecting participants. [unreadable] [unreadable]

Genetic factors recently have been implicated in rare, familial forms of Parkinson=s disease (PD) and these factors may also function in the more common sporadic form of the disorder. Different genes appear to be involved including alpha-synuclein, parkin, UCH-L1, NR3A2 (NURR1) and DJ-1. Discovery of specific genes involved in the disease is extremely useful in understanding the progression of PD, basic cellular mechanisms, risk factors and provides opportunities to test out new treatment paradigms to halt or delay the progress of the disorder. The general working hypothesis is that mutations in genes involved in PD can be utilized in mouse models to identify early underlying neurotransmission alterations in PD both in the classical nigrostriatal pathway as well as other brain regions critical for the progression of this disorder. The areas studied will be the striatum and the substantia nigra pars compacta [which contains the dopamine (DA) neurons that project to the striatum]. In addition, we hypothesize that areas outside of the striatum and substantia nigra also will display a progression of neuronal dysfunctions that may be linked to PD behaviors. Thus, we also will examine the progression of electrophysiological changes in sensorimotor cortical areas. It is within multiple areas of the brain that physiological changes lead to many of the abnormalities that ultimately cause the symptoms of PD. There are two specific aims: 1) To determine how genetic manipulations known to cause PD in humans when replicated in mice alter the synaptic responses of medium-sized spiny striatal neurons, cortical pyramidal neurons of sensorimotor cortex and their modulation by DA and how the basic electrophysiological properties of these neurons and SN DA neurons are altered; and 2) To determine how knock-out of parkin or over-expressionof alpha-synuclein alters amino acid receptor function and its modulation by activation of DA receptors in medium-sized striatal neurons and cortical pyramidal neurons of sensorimotor cortex. We will examine basic neuronal communication and electrophysiological characteristics and then the changes in the functional properties of glutamate, _- aminobutyric acid (GABA) and DA receptors in the striatum, sensorimotor cortical pyramidal neurons and substantia nigra DA neurons in multiple genetic mouse models. We will use existing mouse models (parkin knock-out and alpha-synuclein over expressing mice) that we have obtained through collaborations as well as the new mouse models generated in the Core and tested in Projects 1 and 2. This project is closely integrated with Project 1 which will examine the progression of behavioral and neuropathological changes in the same mouse models (we will obtain time points for electrophysiological analyses based on data from that project) and Project 2 which will concentrate on the progression of neurochemical changes (we will obtain information on pharmacological approaches from that project). This project also will benefit from information obtained on the association of parkin and alpha-synuclein with components of synaptic vesicles obtained in Project 4. In a similar manner the information we provide will benefit Projects 1, 2, and 4 (by providing specific functional information about synaptic changes) and could provide a bridge for the clinical data obtained in Project 5 by indicating the progression of the types of synaptic changes that occur. Thus, the analyses of these models provides our Center with unique opportunities in a highly interactive, multidisciplinary environment aimed at understanding the progressive mechanisms of cellular dysfunction causing PD. Of primary clinical significance, such analyses can provide novel tools for preclinical testing of neuroprotective strategies.

DESCRIPTION (provided by applicant): The fatal mutation in Huntington's disease (HD) leads to an expanded glutamine repeat within the huntingtin protein which causes neuronal dysfunction typically followed by selective neurodegeneration especially within the striatum and cortex. These dysfunctions in neurons and circuits occur during the development of the disease phenotype, well before there is significant cell loss. The experiments in this application are designed to understand the functional changes that occur in specific populations of neurons during the progression of the HD phenotype and to uncover new targets and approaches for therapies. Our working hypothesis is that the most conspicuous cellular dysfunctions leading to pathology in HD result from a combination of cell- autonomous changes and cell-cell interactions. This two-hit hypothesis implies that mutation of the gene in the cell alone may not be sufficient to cause significant dysfunction; other changes have to occur to cause symptoms of the disease, and some of these include altered intercellular synaptic interactions. Previously, we examined changes in the striatum, the cortex and corticostriatal interactions, as the cortical input is one of the two major excitatory inputs to the striatum. However, the excitatory thalamic input to the striatum may be as important as the cortical input in the HD phenotype. It is presently unclear if both thalamostriatal and corticostriatal pathways contribute equally or differentially to alterations in striatal neurons. Aim 1 will use optogenetics to specifically and separately activate striatal glutamatergic inputs to identified subpopulations of striatal neurons and determine their relative contribution to cellular alterations. Medium-sized spiny neurons of the direct and indirect striatal output pathways also display unique, selective and complex alterations as the HD phenotype progresses. These will affect their targets in globus pallidus and substantia nigra. To our knowledge, striatal outputs in HD have not been studied in any detail, especially in mouse models, yet they are extremely important because they determine how the basal ganglia influence the thalamus and cortex. Aim 2 will specifically examine alterations in striatal output target structures while Aim 3 will manipulate striatal output pathwas differentially in an attempt to counter the imbalance of direct and indirect pathways as the disease progresses. Our studies use state-of-the-art optogenetic techniques to specifically activate or inhibit subclasses of neurons as well as genetic techniques to remove expression of the mutant huntingtin gene in subclasses of neurons. Together, the studies will provide the basis for novel and rational treatments for HD by delineating more restricted targets spatially and temporally and will be relevant for understanding other CAG triplet repeat diseases and neurodegenerative disorders.

DESCRIPTION (provided by applicant): Huntington's disease (HD) is a genetic autosomal neurodegenerative disorder that is always fatal and for which there are no effective treatments or cures. Patients carrying the mutation display motor dysfunction, cognitive impairment and psychiatric disturbances. Neuropathologically, HD is characterized by neuronal loss in the striatum and cortex and a progressive disconnection between cortex and striatum, interrupting the flow of information from the cortex to the basal ganglia. We have shown that imbalances in synaptic activity in the direct and indirect striatal output pathways differ during early and late stages of the disease and contribute to motor symptoms in two full-length transgenic mouse models of HD. In early stage HD, there is increased glutamate and GABA release onto direct pathway medium-sized spiny neurons (MSNs) while GABA release is increased in the late stage but only onto indirect pathway MSNs. Changes in synaptic activity are associated with increased repetitive behaviors in early stage HD mice and with decreased locomotion in late stage mice. Early stage changes may be mediated by elevated striatal dopamine (DA), because depletion of endogenous DA reduced repetitive behaviors and reversed some of the electrophysiological alterations. In contrast, decreased locomotion in late stage HD might be mediated by decreased DA function. The goal of this application is to employ novel optogenetic approaches, using light stimulation to activate and/or inhibit DA terminals in a mouse model of HD, to better understand the electrophysiological and behavioral dysfunctions. In Aim 1 we will selectively inhibit DA release in the striatum in early stage HD, using optogenetics by expressing halorhodopsin (which inhibits firing when activated by yellow light) in DA neurons. In Aim 2 we will selectively increase DA release in the striatum in late stage HD using optogenetics by expressing channel rhodopsin (which increases firing when activated with blue light). We hypothesize that reducing striatal DA release in early stage HD will restore synaptic activity of MSNs and will have beneficial effects on abnormal repetitive movements. In late stage HD, increasing DA release will restore some of the balance in MSN activity and will alleviate motor symptoms. PUBLIC HEALTH RELEVANCE: In Huntington's disease, abnormal striatal dopamine transmission induces time-dependent alterations in excitatory and inhibitory synaptic transmission that contribute to imbalances in activity of the direct and indirect striatal output pathways leading to motor and cognitive disturbances. In order to modify the differential symptoms in early and late stages of Huntington's disease, this application will alter dopamine release using novel optogenetic approaches to uncover new targets to alleviate symptoms and slow the progression of this devastating genetic disorder.

? DESCRIPTION (provided by applicant): One of the most fundamental problems in modern biology is to understand dynamic gene activity in time and space in the context of native chromosomes in living cells. The goal of the proposed study is to measure the levels of transcription produced by defined long-range chromosomal interactions in living cells. Traditional live imaging methods lack the spatial resolution to accurately determine the dynamics of gene activity, while bulk assays using fixed material strongly limit investigation of temporal dynamics. Here we propose to overcome these limitations by developing new methods of microscopy and computational analysis. Most of the studies will exploit the unique advantages of the early Drosophila embryo for the development of quantitative live cell imaging methods. Previous studies have identified hundreds of such interactions, and we will sample several of these to provide a titration of varying distances, from tens to hundreds of kilobases, as seen in mammalian systems. There are two specific aims: 1. Develop high-resolution imaging methods and associated computational algorithms for the visualization and quantification of dynamic enhancer-promoter interactions at select endogenous loci in living embryos. 2. Label regulatory regions and associated transcription units of individual genetic loci exhibiting long-range interactions, including trans-homolog associations during transvection at Hox loci, to measure in vivo the effect of chromosome topology on transcriptional activity. We plan to extend this approach to include the visualization of several hundred fluorescent DNA foci in a library of genetically engineered fly lines to establish a general overview of the dynamics of an entire chromosome in a living embryo and its impact on transcription. The successful realization of the proposed studies will greatly augment our current capacity to superimpose whole-genome maps based on fixed tissues onto the dynamic chromosomes of living cells. The resulting technologies will be immediately applied to the visualization of chromosome dynamics in mammalian tissues, particularly multipotent progenitor cells such as mouse hepatoblasts.

? DESCRIPTION (provided by applicant): The fatal mutation in Huntington's disease (HD) leads to an expanded glutamine repeat within the huntingtin protein which causes neuronal dysfunction typically followed by selective neurodegeneration especially within the striatum and cortex. These dysfunctions in neurons and circuits occur during the development of the disease phenotype, well before there is significant cell loss. Recent studies in animal models have emphasized that synaptic cell-cell interactions play a role in the pathophysiology of this disease. For example, removing mutant huntingtin from the cerebral cortex ameliorates some HD symptoms. The experiments in this application are designed to understand the functional changes that occur in specific populations of cortical neurons during the progression of the HD phenotype and to uncover new targets and approaches for therapies. However, little is known about functional changes in cortical neurons, although these neurons also degenerate in HD. Before motor symptoms become apparent, sensory, cognitive and emotional disturbances occur and these seem to depend on aberrant communication in the cortex that probably involves thalamocortical pathways. These pathways have never been examined in HD. Our overarching hypothesis is that sensory and motor cortical areas are differentially and asynchronously affected during HD progression. We propose that sensory thalamocortical pathways are downregulated early leading to faulty integration and interpretation of sensory signals. In turn, the motor cortex becomes upregulated and disorganized, leading to altered corticostriatal communication and motor symptoms. In this grant proposal we will use state-of-the-art techniques in three different laboratories at UCLA. Aim 1 uses optogenetics and slice electrophysiology to examine mechanistically altered synaptic communication between thalamic sensory and motor nuclei and their corresponding cortical projection areas. Aim 2 uses high-density silicon microprobes to record firing of hundreds of neurons simultaneously in sensory and motor cortical areas as well as thalamic nuclei. Aim 3 uses genetically encoded calcium indicators to visualize neuronal activity in sensory and motor cortical areas. Together, the studies will provide new and important mechanistic insights into the understudied cortical dysfunction and will provide the basis for novel and rational treatments for HD by delineating more restricted targets spatially and temporally. These studies also will be relevant for understanding other CAG triplet repeat diseases and neurodegenerative disorders.

Summary One of the grand challenges of modern biology is to understand how gene activity is controlled in space and time, in the context of native chromosomes and in individual living cells. The goal of this proposal is to tackle exactly this challenge: we will develop new approaches to measure and manipulate long-range chromosomal interactions and quantify their effects on gene expression, in real-time and in living cells and tissues. By quantitatively mapping the relationship between transcription factor assembly (e.g. formation of biomolecular condensates), chromosome organization and transcription kinetics, our study will define how gene expression is controlled at unprecedented resolution. Transcriptional regulation forms the basis of cellular differentiation during organismal development, and its defects underlie a variety of disease states, from developmental disorders to cancer. Yet current methods are limited: traditional live-imaging lacks the spatial resolution to accurately define chromosome organization at the scale of individual genes, while bulk assays using fixed material are ill-suited for studying temporal dynamics. In addition, membrane-less nuclear condensates, which form through liquid-liquid phase separation, are thought to play key but as-yet-undefined roles in regulating transcription. To address these challenges, we will develop new imaging methods to measure chromosomal distances in living cells and build optogenetic tools to assemble/disassemble chromosome loops and nuclear condensates. We will deploy these tools to examine regulatory interactions at genomic scales characteristic of enhancer? promoter interactions in flies and mammals (from tens to hundreds of kilobases), and study their implications in the context of cell fate specification in the developing Drosophila embryo. The resulting technologies will be applied to analogous transcriptional loci in mouse embryonic stem cells and organoids derived from these cells. Together, the proposed studies will help reveal how robust mechanisms of cell type specification emerge from stochastic processes such as transcriptional bursts, fluctuations in the size and stability of biomolecular condensates, and dynamic instability of chromatin architecture. The overall goal of this project is to establish a quantitative link between chromatin architecture and transcriptional activity, which will ultimately allow us to take control of gene activity by re-engineering the transcriptional programs underlying developmental and disease processes.