This is a renewal of an application whose broad, long-term objectives are to understand the genetic and biochemical basis of Batten disease (Neuronal Ceroid Liofuscinosis; NCL). Particular emphasis will be placed on understanding the late-infantile (NCL2) and juvenile (NCL3) forms of the disease since these are common forms of inherited blindness, seizures and dementia in the U.S. We will test the hypothesis that NCL results from the inability to transport and degrade a normal mitochondrial protein (the DCCD-binding, proteolipid subunit c of mitochondrial ATP synthase (c- peptide for short). The lysosomal storage of this peptide, predominantly in neurons, retinal pigmentary epithelial cells and pancreas, is then hypothesized to give rise to clinical symptoms common to many lysosomal storage diseases. We will purify the c-peptide from NCL tissue, label it with [14C]DCCD or [125I], identify it by specific polyclonal antibody, check for any abnormal post-translational modifications such as trimethyl- lysine residues, and follow its catabolism after loading of normal and NCL fibroblasts and lymphoblastoid cell lines. This will only give indirect evidence so we have obtained cDNA for sheep c-peptide by PCR, spliced into a plasmid vector which contains a cytomegalovirus promoter etc. and stably overexpressed the gene in neuroblastoma cell line F-11. These and related lines are a model for the CNS. We will also transfect both normal and NCL lymphoblastoid cell lines. We will attempt to create autofluorescent pigment storage and then show that this also results in dolichol sugar accumulation, lysosomal phospholipase A1 inhibition.and other biochemical characteristics of NCL. We will determine if the ubiquitin pathway is normally involved in c-peptide disposal, look for a c-peptide transport protein by c-peptide affinity chromatography of brain extracts and look for c-peptide in lipoprotein complexes by their flotation densities in the range of 1.063-1.21g/ml on cesium chloride gradients in the ultracentrifuge. Any mutation will be assayed in the large number of cell lines available from patients, obligate heterozygotes (50% of normal activity) and a reliable diagnostic test will be devised. Understanding the defect in NCL will clearly open up a, whole new area of cell biology dealing with the transport and disposal of hydrophobic peptides. Finally, the, ability to detect carriers will have tremendous value in combatting this devastating neurodegenerative disorder of children.
| Cho, S; Dawson, G (1998) Enzymatic and molecular biological analysis of palmitoyl protein thioesterase deficiency in infantile neuronal ceroid lipofuscinosis. J Neurochem 71:323-9 |
| Dawson, G; Kilkus, J; Siakotos, A N et al. (1996) Mitochondrial abnormalities in CLN2 and CLN3 forms of Batten disease. Mol Chem Neuropathol 29:227-35 |
| Dawson, G; Kilkus, J; Siakotos, A N (1993) Reduced phospholipase activity, peptide storage and the pathogenesis of canine neuronal ceroid-lipofuscinosis. J Inherit Metab Dis 16:330-4 |
