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Genetic suppression of loss of TPP1

TPP1 缺失的基因抑制

基本信息

批准号：
9372001
负责人：
Richard H Gomer
金额：
$ 21.8万
依托单位：
TEXAS A&M UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-05-01 至 2019-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9372001
关键词：
Ammonium Chloride Amoeba genus Animal Model Autophagocytosis Biological Models CLN2 gene Caenorhabditis elegans Cell Nucleus Cells Child Childhood Chloroquine Cholesterol Cleaved cell Code Complementary DNA DNA cassette Defect Deposition Development Dictyostelium Dictyostelium discoideum Drosophila genus Drug Targeting Enzymes Exhibits Fibroblasts Future Genes Genetic Genetic Screening Genetic Suppression Genetic Transcription Growth Human Hybrids In Vitro Insertional Mutagenesis Jansky-Bielschowsky Disease Knock-out Lysosomes Mediating Messenger RNA Methods Modeling Movement Mus Mutagenesis Mutate Mutation Names Neurodegenerative Disorders Neuronal Ceroid-Lipofuscinosis Neurons Nomenclature Orthologous Gene Patients Peptide Hydrolases Pharmacology Phenotype Proteins Psyche structure RNA Saccharomyces cerevisiae Shotguns Site Spielmeyer-Vogt Disease System Technology Testing Therapeutic Vertebrates Vision Work Yeasts cDNA Library cell transformation cell type disease-causing mutation effective therapy enzyme activity genetic approach high reward high risk homologous recombination knockout gene oxysterol binding protein restriction enzyme screening social tool tripeptidyl aminopeptidase vector

项目摘要

Neuronal ceroid lipofuscinosis (NCL) is the most common childhood-onset neurodegenerative disease. NCL is inevitably fatal, and there is no effective therapy. Children with NCL show a normal early growth but then exhibit a progressive decline in movement, vision and mental abilities, and an accumulation of autofluorescent deposits in neurons and other cell types. A subtype of NCL called Late-Infantile NCL (LINCL) is caused by mutations in the protease tripeptidyl peptidase 1 (TPP1; encoded by the CLN2 gene). Little is known about the normal function of TPP1, and an intriguing possibility is that an identification of genetic suppressors of a loss of TPP1 might identify pharmacological targets to ameliorate the effects of TPP1 loss. Although TPP1 is highly conserved among vertebrates, TPP1 orthologs have not been detected in Drosophila, C. elegans, or S. cerevisiae. In the genetically tractable social amoeba Dictyostelium discoideum, DdTpp1 is a TPP1 ortholog, and there are several similarities between Dictyostelium tpp1¯ cells and cells from children with LINCL. In a preliminary genetic screen for suppressors of the tpp1¯ phenotype, and screening for a reversion of just one of the phenotypes of tpp1¯ cells, we found that disruption of a protein with similarity to mammalian oxysterol-binding proteins suppresses some but not all of the tpp1¯ phenotypes. Preliminary work then indicated that fibroblasts from some children with LINCL have abnormally high levels of cholesterol. The existence of a partial genetic suppressor of tpp1¯, and the usefulness of this approach to guide work on cells from LINCL patients, suggests the exciting possibility that targeting specific proteins could be a viable way to suppress some of the effects of loss of TPP1 function. In this high risk/ high reward R21 proposal, we propose to use the power of Dictyostelium genetic screens to identify the genes, which, when disrupted, suppress tpp1¯ phenotypes. In Aim 1 we will use random insertional mutagenesis to complete the partial genetic screen for suppressors, and screen for a reversion of multiple phenotypes. In Aim 2 we will use a complementary genetic approach, shotgun antisense, to similarly screen for revertants. The sustained impact of the proposed studies will be the identification, in a genetically tractable system, of the key downstream effectors of TPP1. This work will impact our understanding of TPP1 in a model system, and will serve as a necessary basis for future work to test the hypothesis that, in a mammalian system, blocking the function of one or more proteins identified in the Dictyostelium genetic screen could be useful as a therapeutic for LINCL.

神经元蜡质脂褐质沉着症（NCL）是最常见的儿童期发病的神经退行性疾病。 NCL 不可避免地致命，并且没有有效的治疗方法。患有 NCL 的儿童表现出正常的早期生长，但然后表现出运动、视力和心理能力逐渐下降，并且神经元和其他细胞类型中的自发荧光沉积物。 NCL 的一个亚型称为晚期婴儿 NCL (LINCL) 是由蛋白酶三肽基肽酶 1（TPP1；由 CLN2 基因编码）突变引起的。小的是人们已经了解 TPP1 的正常功能，并且一个有趣的可能性是，鉴定出 TPP1 的遗传基因 TPP1 缺失的抑制剂可能会确定药理学靶点来改善 TPP1 缺失的影响。尽管 TPP1 在脊椎动物中高度保守，但在果蝇中尚未检测到 TPP1 直向同源物，线虫或酿酒酵母。在遗传上易驯化的社会性阿米巴盘基网柄菌中，DdTpp1 是一种 TPP1 直系同源物，盘基网柄菌 tpp1 细胞与来自儿童的细胞有一些相似之处林肯。在对 tpp1´ 表型抑制因子的初步遗传筛选中，并筛选回复仅针对 tpp1 细胞的一种表型，我们发现与哺乳动物相似的蛋白质的破坏氧甾醇结合蛋白抑制部分但不是全部 tpp1´ 表型。接下来就是前期工作表明一些 LINCL 儿童的成纤维细胞胆固醇水平异常高。这 tpp1 部分基因抑制因子的存在，以及这种方法指导细胞工作的有用性来自 LINCL 患者的研究表明，靶向特定蛋白质可能是一种可行的方法，这一点令人兴奋。抑制 TPP1 功能丧失的一些影响。在这个高风险/高回报的 R21 提案中，我们建议利用盘基网柄菌遗传筛选的力量来识别基因，这些基因在被破坏时会抑制 tpp1 ¯ 表型。在目标 1 中，我们将使用随机插入诱变来完成部分遗传筛选抑制子，并筛选多种表型的逆转。在目标 2 中，我们将使用互补遗传方法，鸟枪反义，以类似地筛选回复体。拟议研究的持续影响将在遗传易处理的系统中识别 TPP1 的关键下游效应子。这部作品将影响我们对模型系统中TPP1的理解，并将作为未来工作的必要基础检验以下假设：在哺乳动物系统中，阻断在盘基网柄菌基因筛选可用于治疗 LINCL。