Genetic Suppression of Loss of TPP1 Grant uri icon

abstract

  • 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 butthen exhibit a progressive decline in movement, vision and mental abilities, and an accumulation ofautofluorescent 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 isknown about the normal function of TPP1, and an intriguing possibility is that an identification of geneticsuppressors 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 aTPP1 ortholog, and there are several similarities between Dictyostelium tpp1¯ cells and cells from children withLINCL. In a preliminary genetic screen for suppressors of the tpp1¯ phenotype, and screening for a reversionof just one of the phenotypes of tpp1¯ cells, we found that disruption of a protein with similarity to mammalianoxysterol-binding proteins suppresses some but not all of the tpp1¯ phenotypes. Preliminary work thenindicated that fibroblasts from some children with LINCL have abnormally high levels of cholesterol. Theexistence of a partial genetic suppressor of tpp1¯, and the usefulness of this approach to guide work on cellsfrom LINCL patients, suggests the exciting possibility that targeting specific proteins could be a viable way tosuppress some of the effects of loss of TPP1 function. In this high risk/ high reward R21 proposal, we proposeto 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 forsuppressors, and screen for a reversion of multiple phenotypes. In Aim 2 we will use a complementary geneticapproach, shotgun antisense, to similarly screen for revertants. The sustained impact of the proposed studieswill be the identification, in a genetically tractable system, of the key downstream effectors of TPP1. This workwill impact our understanding of TPP1 in a model system, and will serve as a necessary basis for future workto test the hypothesis that, in a mammalian system, blocking the function of one or more proteins identified inthe Dictyostelium genetic screen could be useful as a therapeutic for LINCL.

date/time interval

  • 2017 - 2020