Late infantile neuronal ceroid lipofusinosis (LINCL), caused by mutations in the CLN2 gene, is a progressive fatal childhood neurodegenerative disease. The CLN2 gene encodes a lysosomal protease with tripeptidyl peptidase activity, designated as CLN2p/TPPI. The overall goal of this proposal is to better understand the biological function of CLN2p/TPPI as well as to develop systems and reagents that can be used for prevention and treatment of the disease. There are four specific aims.
SPECIFIC AIM 1 is the production and characterization of CLN2-deficient mice. These mice are expected to recapitulate many of the clinical and pathological features of LINCL and thus provide a valuable baseline for the future testing of potential therapies. In addition, comparison of the phenotypes of a CLN2-nulI, a CLN2- hypomorph, and a compound heterozygote between the two should provide insights into the levels of CLN2p/TPPI required for normal cellular function, which will be useful in designing strategies for enzyme replacement and gene therapies.
SPECIFIC AIM 2 is to use the mice created in Specific Aim 1 to identify the primary substrates for CLN2p/TPPI as well as material that accumulates as a secondary response to the enzyme deficiency. This will provide important information for understanding the biological function of CLN2p/TPPI and may shed light on the cellular processes that underlie pathogenesis, potentially leading to novel approaches to therapy.
SPECIFIC AIM 3 is to determine the substrate specificity of CLN2p/TPPI using combinatorial peptide libraries and to use this information to develop an improved enzyme assay. Such an assay may facilitate carrier screening, more efficient prediction of disease outcome, evaluation of the efficacy of potential therapies, and improve screening for small molecule therapeutic agents.
SPECIFIC AIM 4 is to elucidate the three dimensional structure of the CLN2p/TPPI proenzyme. CLN2p/TPPI is synthesized as a zymogen that is proteolytically processed and converted to an active form upon reaching the lysosome. Detailed structural analysis will be crucial for understanding the mechanism of activation and for designing CLN2p/TPPI derivatives for therapeutic applications.
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