title: Large-Scale Functional RNAi Screen in C. elegans Identifies TGF-β and Notch Signaling Pathways as Modifiers of CACNA1A.
abstract: Variants in CACNA1A that encodes the pore-forming α1-subunit of human voltage-gated Cav2.1 (P/Q-type) Ca(2+)channels cause several autosomal-dominant neurologic disorders, including familial hemiplegic migraine type 1, episodic ataxia type 2, and spinocerebellar ataxia type 6. To identify modifiers of incoordination in movement disorders, we performed a large-scale functional RNAi screen, using the Caenorhabditis elegansstrain CB55, which carries a truncating mutation in the unc-2gene, the worm ortholog for the human CACNA1A The screen was carried out by the feeding method in 96-well liquid culture format, using the ORFeome v1.1 feeding library, and time-lapse imaging of worms in liquid culture was used to assess changes in thrashing behavior. We looked for genes that, when silenced, either ameliorated the slow and uncoordinated phenotype of unc-2, or interacted to produce a more severe phenotype. Of the 350 putative hits from the primary screen, 37 genes consistently showed reproducible results. At least 75% of these are specifically expressed in the C. elegansneurons. Functional network analysis and gene ontology revealed overrepresentation of genes involved in development, growth, locomotion, signal transduction, and vesicle-mediated transport. We have expanded the functional network of genes involved in neurodegeneration leading to cerebellar ataxia related to unc-2/CACNA1A, further confirming the involvement of the transforming growth factor β pathway and adding a novel signaling cascade, the Notch pathway.
authors.affiliation: UnIGENe, Institute for Molecular and Cell Biology (IBMC), Instituto de Investigação e Inovação em Saúde (i3S), University of Porto, Portugal Abel Salazar Institute for the Biomedical Sciences (ICBAS), University of Porto, Portugal.
authors.forename: Maria da Conceição
authors.initials: Mda C
authors.lastname: Pereira
authors.affiliation: UnIGENe, Institute for Molecular and Cell Biology (IBMC), Instituto de Investigação e Inovação em Saúde (i3S), University of Porto, Portugal Abel Salazar Institute for the Biomedical Sciences (ICBAS), University of Porto, Portugal.
authors.forename: Sara
authors.initials: S
authors.lastname: Morais
authors.affiliation: UnIGENe, Institute for Molecular and Cell Biology (IBMC), Instituto de Investigação e Inovação em Saúde (i3S), University of Porto, Portugal Abel Salazar Institute for the Biomedical Sciences (ICBAS), University of Porto, Portugal CGPP, Institute for Molecular and Cell Biology (IBMC), Instituto de Investigação e Inovação em Saúde (i3S), University of Porto, Portugal.
authors.forename: Jorge
authors.initials: J
authors.lastname: Sequeiros
authors.affiliation: UnIGENe, Institute for Molecular and Cell Biology (IBMC), Instituto de Investigação e Inovação em Saúde (i3S), University of Porto, Portugal Abel Salazar Institute for the Biomedical Sciences (ICBAS), University of Porto, Portugal CGPP, Institute for Molecular and Cell Biology (IBMC), Instituto de Investigação e Inovação em Saúde (i3S), University of Porto, Portugal ialonso@ibmc.up.pt.
authors.forename: Isabel
authors.initials: I
authors.lastname: Alonso
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D017173:Caenorhabditis elegans; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D053263:Gene Regulatory Networks / Q000235:genetics; D006801:Humans; D009154:Mutation / Q000235:genetics; D034622:RNA Interference / Q000502:physiology*; D051880:Receptors, Notch / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics*; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D020864:Calcium Channels, N-Type
chemical_list: D015220:Calcium Channels; D051880:Receptors, Notch; D016212:Transforming Growth Factor beta; C105656:CACNA1A protein, human; D020864:Calcium Channels, N-Type
keywords: RNAi screen; disease modifiers; enhancer/suppressor; unc-2