Genetic suppression of loss of TPP1

神经元蜡样脂褐素沉积症(NCL)是最常见的儿童期起病的神经退行性疾病。 NCL不可避免地是致命的，目前还没有有效的治疗方法。NCL儿童表现出正常的早期发育，但 然后表现出运动、视力和智力的进行性下降，并积累了 神经元和其他类型的细胞中有自发荧光沉积。NCL的一种亚型，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。小才是 已知TPP1的正常功能，一个耐人寻味的可能性是，鉴定基因 TPP1缺失的抑制子可能会识别药理靶点以改善TPP1缺失的影响。 尽管TPP1在脊椎动物中高度保守，但在果蝇中尚未发现TPP1的同源基因， 秀丽隐孢子虫，或酿酒酵母。DdTpp1是一种遗传易感性的盘状网囊阿米巴。 Tpp1同源基因，Dictyostelialtpp1‘细胞与儿童的Tpp1细胞有几个相似之处。 LINCL。在对TPP1‘表型抑制基因的初步遗传筛选中，以及对逆转的筛选中 仅在TPP1细胞的一种表型上，我们发现一种与哺乳动物相似的蛋白质的破坏 氧固醇结合蛋白抑制一些但不是全部的TPP1‘表型。然后做好前期工作 表明一些LINCL儿童的成纤维细胞具有异常高的胆固醇水平。这个 TPP1的部分遗传抑制因子的存在及其对指导细胞工作的有效性 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 抑制TPP1功能丧失的部分影响。在这份高风险/高回报的R21提案中，我们建议 使用Dictyostelius基因筛查的能力来识别基因，这些基因在被干扰时抑制TPP1. 表型。在目标1中，我们将使用随机插入突变来完成对 抑制子，并筛选多个表型的逆转。在目标2中，我们将使用一种互补基因 方法，鸟枪反义，以类似的方式筛选回复基因。拟议研究的持续影响 将是在一个遗传上易于处理的系统中，识别TPP1的关键下游效应因子。这部作品 将影响我们在模型系统中对TPP1的理解，并将作为今后工作的必要基础 为了检验这一假设，在哺乳动物系统中，阻断一种或多种在 Dictyostelius基因筛查可作为LINCL的治疗手段。

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.