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.