Kendal Scot Broadie - US grants

DESCRIPTION (provided by applicant): This research program investigates interactive roles of secreted signals and extracellular matrix proteoglycans in the development of neuromuscular junction (NMJ) synapses. Our long-term strategy has been to use Drosophila forward genetic screens to identify new genes and characterize novel mechanisms driving embryonic synaptogenesis and the differentiation of synaptic function. Numerous genetic mutants discovered over the last five years provide the foundation of this proposal, by highlighting the importance of extracellular glycosylation and proteolytic mechanisms shaping trans-synaptic signaling. The multiple interacting proteins we focus on in the proposal include: 1) Heparan Sulfate (HS) 6-O-sulfotransferase Hs6st and 2) HS 6-O-sulfatase Sulf1, two functionally-paired enzymes acting on the same N-sulfoglucosamine C6 carbon; 3) the secreted Jelly Belly (Jeb) trans-synaptic signal binding to the 4) Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase, both of which are regulated by 5) GlcNAc Transferase 1 (Mgat1) and 6) secreted Mind-the-Gap (Mtg) lectin for N-acetylglucosamine (GlcNAc)-proteoglycans; and 7) secreted matrix metalloproteinase (MMP) and tissue inhibitor of MMP (TIMP), both of which genetically interact with 8) Fragile X Mental Retardation Protein (FMRP) in regulating synaptogenesis. From this fertile foundation, we propose to focus intensively on the extracellular glycobiology of intercellular interactions driving NMJ development. Our core hypothesis is that extracellular glycans function as staging platforms that bind and combinatorially modulate multiple trans-synaptic signals driving synaptogenesis. We propose three specific aims to test this hypothesis. In Aim I, we use sulf1/hs6st mutants as a tool to investigate the roles of Heparan Sulfate Proteoglycan (HSPG) mechanisms in WNT (Wingless, Wg)/TGFb/BMP (Glass Bottom Boat, Gbb) trans-synaptic signaling. In Aim II, we test extracellular N-glycan mechanisms in regulation of the newly-defined Jeb-Alk trans-synaptic signaling, as well as interactions with the Wg and Gbb pathways. In Aim III, we test roles of MMPs and TIMP in synaptic ECM remodeling, in conjunction with FMRP synaptogenic requirements. We will test how this mechanism regulates HSPGs to control the combinatorial trans-synaptic signaling of Jeb, Wg and Gbb ligands. This work has direct relevance for numerous neuromuscular and neurological diseases, including Congenital Disorders of Glycosylation (CDG) and Fragile X syndrome (FXS), the leading heritable cause of cognitive impairment and autism spectrum disorders. PUBLIC HEALTH RELEVANCE: Heavily glycosylated extracellular matrix, membrane-anchored and transmembrane proteins play critical roles in shaping intercellular signals driving synapse formation. Defects in these intercellular interactions are linked to neurological disorders caused by neuromuscular (muscular dystrophies) and cognitive (mental retardation) synaptic impairments. This research program utilizes the powerful Drosophila genetic system to identify new genes and molecular mechanisms critical for establishing the extracellular environment to control intercellular signaling events during synaptogenesis.

Description (Provided by applicant): The hypothesis driving this proposal is that presynaptic dysfunction is a common causative factor leading to cell death in multiple inherited neurodegenerative diseases. This hypothesis is based on the observations that 1) synaptic function mediates neuronal survival during development, 2) mutations which strongly impair presynaptic function result in massive, progressive neuronal degeneration, 3) a number of presynaptic proteins have been directly implicated in neurodegenerative diseases and 4) neuronal dysfunction/synapse loss is known to precede by a substantial period the manifestation of cell death in these diseases. To date, however, there is no established direct evidence of synaptic dysfunction mediating neuronal death during neurodegenerative disease states. The goal of this proposal is to assay synaptic maintenance in two genetic models of neurodegenerative diseases: Drosophila models of Parkinson's Disease (PD), a classic "protein storage" disease, and Niemann-Pick Type C (NP-C), a classic "lipid storage" disease. Drosophila was selected for its attractive properties as a new molecular genetic model of neurodegeneration, and its long history as the foremost genetic model for synaptic studies. PD and NP-C were selected as representative of a large number of related neurodegenerative disorders. The Drosophila PD model has been recently established through transgenic over-expression of human alpha-synuclein (a presynaptic protein) and shown to accurately recapitulate the diagnostic features of human PD. A Drosophila model of NP-C is being established through mutation (loss-of-function) of the endogenous NPC I gene, the known cause of human NP-C disease. Specifically, this proposal is to conduct age-progressive studies of synaptic mechanisms in Drosophila PD and NP-C models to correlate synaptic maintenance with the onset, progression and prevalence of neurodegeneration. The first aim is to improve Drosophila models by generating fluorescently tagged alpha-synuclein and NPCI proteins whose levels can be reversibly regulated through a temperature-dependent ubiquination strategy. Secondly, to confirm gross features of neurodegeneration in these models with behavioral assays and examination of nervous system/neuronal architecture. Third, and most importantly, to assay synaptic development, function and maintenance in these models. Assays will include electrophysiological measurements of neurotransmission, quantitative fluorescent optical imaging of protein and lipid dynamics in the presynaptic terminal and ultrastructural studies of presynaptic architecture. Together, these studies will allow a conclusive determination of whether synaptic maintenance is compromised in PD and NP-C, and is the causative factor that leads to neuronal cell death and neurodegeneration in these disease states.

The Neuroscience Services Core exists to meet three interrelated objectives: (1) to provide KC investigators with core facilities and services to support their ongohag biomedical research programs; (2) to facilitate new, interdisciplinary neuroscience collaborations amongst KC investigators; and (3) to provide broader access to emerging technologies throughout the KC that have been embraced by a sufficient number of KC investigators, through pilot research, to warrant integration at the level of a core. We designate a core activity a service when the KC supports core staff to provide investigators with specific services. We designate a core activity as a facility when the KC provides the equipment, space, and technical training, but laboratory personnel are specifically responsible for performing the studies. Note that some of the resources provide both kinds of opportunities for KC investigators. The Neurosciences Service core includes: Confocal and Light Microscopy Imaging Facility; Neurochemistry Service; Tissue Culture Facility; Scientific Instrumentation Service; Mouse Behavioral Phenotyping Facility; Mouse Gene Targeting Services; Molecular Neuroanatomy Facility; Human Psychophysiology Service/Facility; Human and Animal Imaging Facility and Service

DESCRIPTION (provided by applicant): Recent advances suggest that cholesterol and sphingolipids, components of membrane lipid raft domains, play critical roles in both presynaptic function and neurodegenerative disorders. We propose to investigate the roles of sphingolipid metabolizing enzymes/transporters in the coincident maintenance of neurotransmission and neuronal viability. In the last funding cycle, we identified the Drosophila slug-a-bed (slab) gene, which encodes the ceramidase enzyme at the heart of sphingolipid metabolism. SLAB ceramidase facilitates vesicular trafficking and fusion mediating neurotransmitter release. We also established a Drosophila model of Niemann Pick C (NPC) disease, a lipid-storage neurodegenerative disorder characterized by mistrafficking and accumulation of sphingolipids and cholesterol. 95% of human NPC cases are caused by mutation of the NPC1 gene, which encodes a putative sphingolipid transporter in endosomal-ike organelles. Two Drosophila NPC1 proteins, dNPC1a/b, similarly reside in presynaptic organelles and are independently essential. dNPC1 mutants impair vesicular and protein trafficking in the presynaptic terminal and cause age-progressive neurodegeneration. Thus, the hypothesis driving this proposal is that maintenance of sphingolipid domains is essential for protein and vesicle trafficking underlying presynaptic function, and that disruption of this pathway triggers synaptic dysfunction causative to neurodegeneration. We propose four Specific Aims to test this hypothesis. First, to use confocal imaging and subcellular fractionation to investigate the role of SLAB ceramidase and dNPC1a/b in lipid/protein trafficking regulation in neurons. Second, to use electrophysiology, dye imaging and electron microscopy to assay roles of sphingolipid turnover in regulating presynaptic function. Third, to use clonal techniques to generate SLAB ceramidase and dNPC1a/b deficient populations of neurons to assay the consequence on cell viability and the progression of neurodegeneration. Finally, to employ genetic and molecular interaction studies to probe the molecular mechanisms by which these proteins mediate presynaptic function or, in their absence, cause neuronal death. Our approach uniquely combines sphingolipid-pathway mutants, sophisticated cell biological approaches and Drosophila genetics to probe the emerging role of sphingolipid-dependent pathways in neuronal function. The proposed work promises to substantially increase our understanding of protein and vesicle trafficking mechanisms critical to neurotransmission, and to reveal defects of common causality to a number of disparate neurodegenerative diseases.

DESCRIPTION (provided by applicant): Fragile X Syndrome (FraX) is a developmental brain disorder with abnormal neuron architecture development and functional plasticity of the developing brain, causing mental retardation, learning disabilities and autism. My lab established the Drosophila FraX model and proved it provides direct insights into molecular and cellular bases of the human disease state. In this revised proposal, I ask your support to take advantage of this model, and the relative simplicity of the Drosophila brain, to test core hypotheses of FraX and interventions to correct brain defects. First, I will test the temporal requirements for the Fragile X Mental Retardation Protein (FMRP) in defined brain neural circuit formation and the maintained manifestation of normal behaviors from these circuits. I target two extremely well-defined brain circuits;1) the circadian clock circuit that regulates regular motor activity cycles, and 2) the mushroom body (MB) circuit that mediates olfactory learning/memory consolidation. This aim will characterize the development of these circuits and their synaptic connectivity in the presence vs. absence of FMRP. A transgenic conditional FMRP expression system will be used to test the hypothesis that FMRP is required specifically during a transient period of neural circuit development to establish maintenance of normal behaviors. Transgenic animals expressing FMRP during discrete developmental windows will be tested for anatomical neural circuit formation, synaptic structure and function, and the behavior outputs of circadian activity and olfactory learning and memory consolidation. Second, I will test the hypothesized role of FMRP in sensory modality, electrical and synaptic neurotransmission activity-dependent changes in brain neural circuit development. The interaction between FMRP function and brain circuit activity will be assayed in double mutant combinations and with an array of proven transgenic tools that either increase or decrease electrical activity in targeted brain regions. The specific role of metabotropic neurotransmitter receptor signaling will be assayed in both cholinergic and glutamatergic brain regions using a combination of genetic mutants and pharmacological studies. The temporal roles of these pathways will be assessed with timed application of drugs during defined stages of brain development to define temporal windows for therapeutic intervention. The role of FMRP in local translation control downstream of a neurotransmission activity-induced phosphorylation will be tested by making transgenic constitutively phosphorylated or desphosphorylated mimic proteins. It is imperative to appreciate that these hypothesized functions have never been tested in vivo, in an animal model. Third, I will use new proteomic technologies to screen for brain protein changes occurring during specific periods of brain development, in the presence and absence of FMRP. In parallel, systematic forward genetic screens will be pursued to directly identify dfmr1 genetic interactors. Together, these aims are designed to make maximal use of the proven Drosophila FraX genetic model. I am the only one poised to pursue this work and truly believe that I can aid enormously in the understanding and treatment of Fragile X Syndrome. PUBLIC HEALTH RELEVANCE: Fragile X Syndrome is a developmental brain disorder that is the most commonly inherited form of both mental retardation and autism spectrum disorders. This proposal studies the molecular and cellular basis of this disease in the developing brain and directly tests therapeutic interventions aimed at correcting the brain developmental abnormalities.

Project Summary Fragile X Mental Retardation Protein (FMRP) is an mRNA-binding translational repressor whose loss causes Fragile X syndrome (FXS), which we fight with a well-established Drosophila model. A revolution in transgenic technology (FlyLight) in this last funding cycle provides unprecedented ability to target individual neurons within brain learning/memory circuitry. We target a single projection neuron (mPN2) with olfactory dendritic input and two synaptic outputs: 1) biased-stereotyped connection in Lateral Horn (innate behavior), 2) activity-dependent probabilistic connection in Mushroom Body (learned behavior). Cross-comparing these synapse classes in one neuron is a phenomenal advantage. We hypothesize simultaneous introduction of sensory activity and FMRP activity-sensor defines the critical period (CP), a restricted developmental time window when early-use activity refines synaptic connectivity and circuit function. In this renewal, we focus on CP activity-dependent translation control in the refinement of synaptic connectivity and circuit function to optimize the mature behavioral output. In Aim I, we test FMRP RNA-binding translational control of critical period structural/functional development at all 3 mPN2 synaptic sites: dendritic input and dual axonal outputs. We use a range of targeted transgenic tools to assay synapse architecture, ultrastructure and molecular assembly, in normal animals compared to the FXS model in staged developmental studies. We introduce targeted optogenetic channels driven throughout the circuit to bidirectionally manipulate activity during CP development, monitoring changes with GCaMP Ca2+ reporters, ArcLight voltage reporters and patch-clamp electrophysiology. In mPN2 developmental studies, we test activity-dependent FMRP roles in CP synaptic connectivity and function. In Aim 2, we test the partnership of FMRP with other RNA-binding proteins (RBPs) in this activity-dependent translation repression mechanism. RBPs rarely act alone, and our preliminary data suggest RBPs Staufen and Pumilio partner with FMRP, with their coupled expression jointly delineating the critical period. We will test formation of FMRP/Staufen/Pumilio complexes and combinatorial roles in mPN2 translation repression. Using double mutant combinations, we will test combinatorial functions in remodeling of CP synaptic connectivity, circuit function and learning/memory behavioral outputs. In Aim 3, we test 3 novel translational targets; 1) phosphatase Corkscrew, 2) ESCRTiii core protein Shrub and 3) Neurobeachin homolog Rugose. Preliminary data show all 3 mRNAs bind FMRP, proteins transiently elevated in the CP in the FXS model and over-expression phenocopies FXS. We hypothesize FXS elevation of 1) Corkscrew increases synaptic signal transduction, 2) Shrub impairs synapse elimination via a local pruning module, and 3) Rugose accelerates synaptic trafficking to impair CP synaptic remodeling. We will test these roles individually with characterized mutants, and test whether CP restoration of protein levels corrects FMRP/Staufen/Pumilio loss phenotypes. FXS is a primary heritable cause of intellectual disability (ID) and autism spectrum disorder (ASD), and targeted interactors are likewise causally linked to ID/ASD states.