Long term goals are the development of rational therapies for the juvenile form of Batten disease and cancer based on the specifics of molecular, subcellular and biochemical determinants of the impact of CLN3 on apoptosis and ceramide generation. Batten disease or the Neuronal Ceroid Lipofuscinosis are a group of fatal disorders characterized by progressive cognitive and motor impairment, blindness, intractable seizures, and accelerated neuronal and photoreceptor loss. Multiple lines of evidence have converged to suggest that the defect in CLN3 causing JNCL results in apoptotic neuronal death. These are: presence of nuclear chromatin condensation by EM and evidence of DNA fragmentation by TUNEL staining of JNCL neurons, upregulation of Bcl-2 in surviving, nonapoptotic JNCL neurons, and increased ceramide levels in JNCL brain. At a cellular level, overexpression of CLN3 protein results in resistance to apoptosis induced by serum deprivation and chemotherapeutic agents, inhibition of caspase-3 activation, and a modulation of ceramide levels. Blocking CLN3 expression in human post-mitotic neurons results in spontaneous apoptosis, which was prevented by the antiapoptotic drug, flupirtine. Importantly, these antiapoptotic mechanisms also operate in cancer. There is significant over-expression of CLN3 in human and mouse cancer cell lines, and solid human colon cancer. An antisense- CLN3 adeno-virus blocks CLN3 expression in these lines, causes inhibition of cancer cell growth, increased apoptosis with loss of potential across the mitochondrial membrane, and an increase in ceramide levels. Cells derived from JNCL patients are an excellent model for the study of effects of CLN3 on apoptosis and ceramide. ? ? Overall hypothesis: the CLN3 protein impacts apoptotic pathways has a negative regulatory role in apoptosis. Specific hypotheses: a) subcellular localization(s) for this membrane protein impacts its role in apoptosis; b) motifs within CLN3 contribute to its regulatory effects on apoptosis; c) CLN3 modulates one of the steps in ceramide synthesis clearance pathways; and d) motifs within CLN3 and or CLN3 subcellular localization(s) may have an effect on ceramide levels.
Specific aims i nclude: 1) to establish the impact of CLN3 on apoptotic pathways by analyzing global gene expression; 2) to determine the determinants of CLN3 for regulating apoptosis by defining subcellular localization and CLN3 amino acids and motifs resulting in this regulation; and 3) To determine whether a step in ceramide synthesis/clearance is modulated by CLN3 by scrutinizing ceramide synthesis/clearing enzymes and metabolism in CLN3-deficient cells. ? ?
| Persaud-Sawin, Dixie-Ann; McNamara 2nd, James O; Rylova, Svetlana et al. (2004) A galactosylceramide binding domain is involved in trafficking of CLN3 from Golgi to rafts via recycling endosomes. Pediatr Res 56:449-63 |
