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.
Late Infantile Neuronal Ceroid Lipofuscinosis (LINCL) is a fatal childhood-onset neurodegenerative disease caused by mutations that result in loss of activity of an enzyme called tripeptidyl peptidase 1 (TPP1). In a model system, we found that mutation of a different gene can partially reverse the effects of loss of TPP1. This project will use this model system to identify potential drug targets that could reverse some of the effects of the loss of TPP1, and thus potential drug targets to help children with LINCL.
