Genetic suppression of loss of TPP1

神经元蜡样质脂褐质沉积症（NCL）是儿童期最常见的神经退行性疾病。 NCL不可避免地是致命的，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， 然后表现出运动、视力和心理能力的逐渐下降， 在神经元和其他细胞类型中的自体荧光沉积物。NCL的一种亚型称为晚期婴儿NCL（LINCL） 由蛋白酶三肽基肽酶1（TPP 1;由CLN 2基因编码）的突变引起。之甚少 关于TPP 1的正常功能，一个有趣的可能性是， TPP 1缺失的抑制剂可能会确定药理学靶点，以改善TPP 1缺失的影响。 虽然TPP 1在脊椎动物中高度保守，但在果蝇中尚未检测到TPP 1直系同源物， C. elegans或S.酿酒。在遗传上易处理的社会性阿米巴Dictyosteelicdiscoideum中，DdTpp 1是一种 TPP 1直系同源物，并且在网骨藻tpp 1细胞和患有 LINCL。在对tpp 1 <$表型的抑制基因进行初步遗传筛选和对回复突变进行筛选时， 我们发现，对一种与哺乳动物细胞相似的蛋白质的破坏， 氧固醇结合蛋白抑制某些但不是全部的tpp 1 ′表型。那么前期工作 表明来自LINCL患儿的成纤维细胞胆固醇水平异常高。的 存在tpp 1 ′的部分遗传抑制基因，以及这种方法在指导细胞研究方面的有用性 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， 抑制TPP 1功能丧失的一些影响。在这个高风险/高回报的R21建议中，我们建议 利用网囊藻基因筛选的力量来鉴定基因，当这些基因被破坏时， 表型在目标1中，我们将使用随机插入诱变来完成以下基因的部分遗传筛选： 抑制子，并筛选多种表型的逆转。在目标2中，我们将使用互补的基因 方法，鸟枪反义，以类似的筛选回复突变体。拟议研究的持续影响 将是识别，在一个遗传学上易于处理的系统，关键下游效应的TPP 1。这项工作 将影响我们在模型系统中对TPP 1的理解，并将作为未来工作的必要基础 为了验证这一假设，在哺乳动物系统中，阻断一种或多种在哺乳动物中鉴定的蛋白质的功能， Dictyosteoblastoma基因筛选可用作LINCL治疗剂。

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.