Anne C. Hart - US grants

DESCRIPTION (Adapted from the applicant's abstract): Despite the critical role mechanical stimuli play in hearing and touch, the molecular basis of mechanosensation in vertebrates remains elusive. The proposed research uses genetic, cellular, and molecular techniques in C. elegans to identify receptor proteins, signal transduction pathways and synaptic pathways involved in the detection of noxious mechanical and chemical stimuli. The C. elegans ASH neurons respond to nose touch, high osmolarity, and volatile repellents. Mutations have been identified which perturb responses to specific ASH stimuli. The genes involved will be molecularly characterized. They will address the role of the OSM-10 protein in osmosensation, and examine newly identified peptidergic neurotransmitters in the ASH circuit.

DESCRIPTION (From the Applicant's Abstract): Eight neurodegenerative diseases are caused by expansion of glutamine tracts in proteins, including spinal cerebellar ataxias, DRPLA, Huntington's disease and Kennedy's disease. It is unclear why expanded polyglutamine tracts are toxic and there is no treatment for these diseases. We have developed a C. elegans overexpression model for polyglutamine neurotoxicity by expressing huntingtin protein fragments in neurons and we will assess the toxic activity of mutant ataxin-1 and ataxin-3 in these same C. elegans neurons. Mutation of pqe-1 specifically and strongly enhances polyQ toxicity in C. elegans. We will clone and characterize pqe-1. We will characterize and clone additional enhancer genes and will identify, characterize and clone suppressor genes. The corresponding proteins are candidate modifiers of polyglutamines toxicity in Huntington's and other polyglutamine diseases. We will generate mutations in C. elegans homologs of huntintin, ataxin-1 and ataxin-3 to address their role in the nervous system and in neurodegeneration. The relationship between these genes, polyglutamine toxicity, the apoptotic pathway and other candidate genes will be assessed. We will identify human homologs of enhancer and suppressor genes identified in our genetic screens and assess their role in polyglutamine toxicity in other model organisms to address the relevance of these candidate genes to human disease.

[unreadable] DESCRIPTION (provided by applicant): The conserved Notch signaling pathway is critical for cell fate specification and lateral inhibition during development in vertebrates and invertebrates. Notch receptors bind extracellular ligands (e.g. Delta, Serrate and LAG-2) containing a conserved DSL domain that is critical for receptor activation. We have identified a new family of putative Notch ligands in C. elegans with homologs in vertebrates. Unlike canonical Notch ligands, these proteins lack the conserved DSL domain but they share a conserved sequence motif with classical Notch ligands. The first aim of this proposal examines in detail the function of one of these new putative ligands. SEL-14 is required for normal development and preliminary studies suggest that it activates Notch signaling. The role of SEL-14 in Notch signaling will be examined at a molecular and cellular level using genetics, lineage analysis and immunohistochemistry. The developmental role of C. elegans SEL-14 homologs will also be addressed by identification of deletion alleles, expression analysis, characterization of mutant phenotypes, and genetic analysis. The second aim tests the hypothesis that SEL-14 directly interacts with Notch receptors and cooperates with classical DSL ligands in receptor activation. This model suggested by in vivo studies and will be tested in vitro using two-hybrid studies and tissue culture experiments. We will also examine the role of a previously uncharacterized, conserved sequence motif that is found only in SEL-14, C. elegans homologs and classical Notch ligands. Structure/function studies will address the function of this conserved motif in SEL-14 and in classical Notch ligands. Understanding Notch ligands is critical given the essential role of Notch signaling in development, stem cell maintenance, cancer and memory. Defects in Notch signaling and presenilin processing cause numerous diseases. Deltas has been implicated in spondylocostal dysostosis. Mutations in NotchS and Jaggedl cause CADASIL and Alagille syndromes, respectively. These are dominantly inherited disorders associated with stroke and dementia. Notch and APR not only share common processing pathways like presenilins, but recent studies suggest that Notch and APR may both contribute to Alzheimer's disease pathology like presenilins. All of these disorders are poorly understood and no effective treatment is available. We anticipate that understanding ligand regulation of Notch signaling will be a first step in the development of therapies for these and other disorders. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Notch signaling plays well described roles in developmental cell fate decisions, in the regulation of stem cell proliferation and in numerous diseases. However, Notch signaling is also critical for the function of adult neurons. Notch plays a role in memory retention in mice and Drosophila, yet the targets of Notch signaling in neurons are unclear. We have demonstrated that the C. elegans lin-12 Notch receptor acts in adult animals to modulate behavior. In the studies proposed here, we will identify the molecular pathways by which Notch signaling alters neuronal function and behavior using the powerful genetic techniques available in C. elegans. In a pilot screen, we identified five genes expressed in the nervous system that are likely direct targets of Notch. All five genes encode proteins that are likely critical for Notch signaling in vertebrate neurons as well. In this proposal, we address the mechanisms and pathways by which these Notch target genes act in the adult nervous system to regulate neuronal activity and behavior. PUBLIC HEALTH RELEVANCE: Notch signaling is critical for normal function of the human nervous system. Mutations in Notch3 and Jagged1 cause CADASIL and Alagille syndromes, respectively. These are dominantly inherited disorders associated with stroke and dementia. Combined, their incidence is at least 1 in 50,000, although CADASIL is likely under-diagnosed. Recent evidence also suggests that Notch signaling is up-regulated in Down's syndrome patients. Interestingly, Notch and amyloid precursor proteins directly interact suggesting that Notch signaling may be important in the memory defects associated with Alzheimer's disease. There is no effective treatment for these disorders. Given the conservation across species of Notch regulatory mechanisms and targets, we anticipate that identifying the targets of Notch signaling in C. elegans will reveal critical targets of Notch modulation in humans that are relevant in both normal and pathological conditions.

Project 3 "Genetic Analysis of SMN Pathways in C. elegans" Summary Spinal Muscular Atrophy (SMA) is one of the most common inherited genefic disordersV The corresponding SMN (Spinal Motor Neuron) protein is essential in all cells as SMN is a critical component of the Gemin complex that assembles protein/RNA complexes critical for mRNA translation and processing^. However, recent studies suggest that the SMN protein may also act in stress granules and axonal mRNA transport^. We do not understand why decreased function of SMN causes selective neuromuscular degeneration nor is there consensus on which molecular pathways are critical for SMA pathology. The C. e/egans genome contains only one SMN gene {smn-1 or Cesmn-1) and loss of Cesmn-1 function causes defective development, progressive behavioral defects, and premature death. C. elegans and Drosophila are ideal for the identification and validation of molecular pathways relevant to biological processes;the genefic dissecfion of apoptosis is an excellent example of the power of harnessing invertebrate models. In the studies proposed here, we will identify conserved modifier genes and cellular pathways that are critical for SMN loss of function neuromuscular defects. We utilize C. elegans assays to identify and characterize genes that act as modifiers of SMN loss of function defects.