Gene defects responsible for the three major forms of the neuronal ceroid lipofuscinoses (NCLs), autosomal-recessive, neurodegenerative disorders of childhood, have been identified recently. Two of these genes, associated with the infantile (CLN1) and classical late infantile (CLN2) forms of NCL, code for lysosomal enzymes: palmitoyl-protein thioesterase and carboxypeptidase, respectively. The CNL3 gene, associated with juvenile NCL, the most common form of NCL in the United States, encodes a protein of predicted 438 amino acid residues. Until the present, 23 disease-associated mutations have been found in the CLN3 gene, including the 1.02-kb common deletion. The function of the CLN3 gene product is still unknown. Hydrophobicity plots indicate that CLN3 protein, in contrast to soluble lysosomal enzymes, is a highly hydrophobic transmembrane protein. Our preliminary date indicate that this protein is a resident of lysosomal membrane, has a long turnover rate, is phosphorylated, and shows extensive N-glycosylation of the complex type that demonstrates cell-type-specific differences. The selective vulnerability of particular cells to the CLN3 gene defect in spite of the widespread distribution of the storage suggests that CLN3 is vital for the metabolism of only specific cell types. Thus, this project proposal focuses on experimental verification of two working hypotheses predicting that i) the selective vulnerability of cells to the CLN3 gene defect is related to the different metabolism of CLN3 protein in particular cell types and ii) the selective vulnerability to the CLN3 gene defect is caused by the specific metabolic requirements of susceptible cells. As a first approach to elucidate these phenomena, we intend to address the following issues: i) whether the metabolism of CLN3 protein differs in various cell types and how disease-associated mutations affect this process and ii) how disease-associated mutations affect the function of lysosomes. The properties of CLN3 protein will be exploited in mammalian expression systems by expressing CLN3 protein alone, and CLN3 protein tagged with the green fluorescent protein and FLAG epitope. The biological consequences of disease-associated mutations for the biogenesis and integrity of lysosomes will be evaluated by using comparative morphological and biochemical studies of lymphocytic cell lines and cultured skin fibroblasts from healthy subjects and affected individuals also by experimentally modeling the lysosomal storage. These studies will be done by using immunoprecipitation, Western blotting, immunofluorescence, immunoelectron microscopy, protein sequencing, and subcellular fractionation techniques. We expect that the results of these studies will allow us to gain insight into the role of CLN3 protein in lysosomal function and to define how disease-associated mutations of CLN3 protein affect this process. These data also will help to clarify the role of the endosomal-lysosomal membrane in cell biology, in general.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS038988-03
Application #
6394185
Study Section
Special Emphasis Panel (ZRG1-BDCN-3 (01))
Program Officer
Murray, Gary
Project Start
1999-09-25
Project End
2002-08-31
Budget Start
2001-09-01
Budget End
2002-08-31
Support Year
3
Fiscal Year
2001
Total Cost
$81,002
Indirect Cost
Name
Institute for Basic Research in Dev Disabil
Department
Type
DUNS #
167205090
City
Staten Island
State
NY
Country
United States
Zip Code
10314
Zhang, Z; Butler, J D; Levin, S W et al. (2001) Lysosomal ceroid depletion by drugs: therapeutic implications for a hereditary neurodegenerative disease of childhood. Nat Med 7:478-84
Wisniewski, K E; Kida, E; Walus, M et al. (2001) Tripeptidyl-peptidase I in neuronal ceroid lipofuscinoses and other lysosomal storage disorders. Eur J Paediatr Neurol 5 Suppl A:73-9
Wisniewski, K E; Zhong, N; Philippart, M (2001) Pheno/genotypic correlations of neuronal ceroid lipofuscinoses. Neurology 57:576-81
Wisniewski, K E; Kida, E; Golabek, A A et al. (2001) Neuronal ceroid lipofuscinoses: classification and diagnosis. Adv Genet 45:1-34
Golabek, A A; Kida, E; Walus, M et al. (2001) CLN3 disease process: missense point mutations and protein depletion in vitro. Eur J Paediatr Neurol 5 Suppl A:81-8
Kida, E; Golabek, A A; Walus, M et al. (2001) Distribution of tripeptidyl peptidase I in human tissues under normal and pathological conditions. J Neuropathol Exp Neurol 60:280-92
Kida, E; Golabek, A A; Wisniewski, K E (2001) Cellular pathology and pathogenic aspects of neuronal ceroid lipofuscinoses. Adv Genet 45:35-68
Golabek, A A; Kida, E; Walus, M et al. (2000) CLN3 protein regulates lysosomal pH and alters intracellular processing of Alzheimer's amyloid-beta protein precursor and cathepsin D in human cells. Mol Genet Metab 70:203-13