Pereira Maria da Conceição, Morais Sara, Sequeiros Jorge, Alonso Isabel
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.
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.
ASN Neuro. 2016 Mar 22;8(2). doi: 10.1177/1759091416637025. Print 2016 Mar-Apr.
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.
编码人类电压门控性 Cav2.1（P/Q 型）钙离子通道孔形成α1 亚基的 CACNA1A 基因变异会导致多种常染色体显性神经系统疾病，包括 1 型家族性偏瘫性偏头痛、2 型发作性共济失调和 6 型脊髓小脑共济失调。为了确定运动障碍中不协调症状的调节因子，我们使用秀丽隐杆线虫品系 CB55 进行了大规模功能性 RNAi 筛选，该品系在 unc-2 基因中携带截短突变，unc-2 基因是人类 CACNA1A 的线虫直系同源基因。筛选通过在 96 孔液体培养板中采用喂食法进行，使用 ORFeome v1.1 喂食文库，并利用液体培养中线虫的延时成像来评估甩尾行为的变化。我们寻找那些沉默后能改善 unc-2 的缓慢和不协调表型，或相互作用产生更严重表型的基因。在初次筛选得到的 350 个假定命中基因中，有 37 个基因始终显示出可重复的结果。其中至少 75%在秀丽隐杆线虫神经元中特异性表达。功能网络分析和基因本体论揭示，参与发育、生长、运动、信号转导和囊泡介导运输的基因出现了过度富集。我们扩展了与 unc-2/CACNA1A 相关的导致小脑共济失调的神经退行性变相关基因的功能网络，进一步证实了转化生长因子β通路的参与，并增加了一个新的信号级联——Notch 通路。
