👤 Dixie-Ann Persaud-Sawin

Juvenile neuronal ceroid lipofuscinosis (JNCL) belongs to the neuronal ceroid lipofuscinoses characterized by blindness/seizures/motor/cognitive decline and early death. JNCL is caused by CLN3 gene mutations that negatively modulate cell growth/apoptosis. CLN3 protein (CLN3p) localizes to Golgi/Rab4-/Rab11-positive endosomes and lipid rafts, and harbors a galactosylceramide (GalCer) lipid raft-binding domain. Goals are proving CLN3p participates in GalCer transport from Golgi to rafts, and GalCer deficits negatively affect cell growth/apoptosis. GalCer/mutant CLN3p are retained in Golgi, with CLN3p rescuing GalCer deficits in rafts. Diminishing GalCer in normal cells by GalCer synthase siRNA negatively affects cell growth/apoptosis. GalCer restores JNCL cell growth. WT CLN3p binds GalCer, but not mutant CLN3p. Sphingolipid content of rafts/Golgi is perturbed with diminished GalCer in rafts and accumulation in Golgi. CLN3-deficient raft vesicular structures are small by transmission electron microscopy, reflecting altered sphingolipid composition of rafts. CLN1/CLN2/CLN6 proteins bind to lysophosphatidic acid/sulfatide, CLN6/CLN8 proteins to GalCer, and CLN8 protein to ceramide. Sphingolipid composition/morphology of CLN1-/CLN2-/CLN6-/CLN8- and CLN9-deficient rafts are altered suggesting changes in raft structure/lipid stoichiometry could be common themes underlying these diseases. Show less

The neuronal ceroid lipofuscinoses are pediatric neurodegenerative diseases with common clinical features. Of the nine clinical variants (CLN1-CLN9), six have been genetically identified. Most variants manifest cell death and dysregulated sphingolipid metabolism, suggesting the proteins defective in these disorders may interact along one pathway. NCL patient-derived cell lines exhibit cell growth and apoptotic defects that reverse following transfection with the wild-type gene. The membrane-bound proteins CLN3, CLN6, and CLN8 complement each other, as do CLN1 and CLN2 proteins, with respect to growth and apoptosis. The CLN2 protein also corrects growth and apoptosis in CLN3-, CLN6-, and CLN8-deficient cell lines. Neither CLN1-deficient nor CLN2-deficient growth defects are corrected by CLN3, CLN6, and CLN8 proteins. CLN2, CLN3, CLN6, and CLN8 proteins co-immunoprecipitate and co-localize to early and/or recycling endosomes and lipid rafts. Additionally, CLN2p and CLN1p co-immunoprecipitate. The work presented supports interactions between NCL proteins occurring at multiple points along one pathway. Show less

Apoptosis, Golgi fragmentation and elevated ceramide levels occur in Juvenile Neuronal Ceroid Lipofuscinosis (JNCL) neurons, lymphoblasts and fibroblasts. Our purpose was to examine whether apoptosis is the mechanism of cell death in JNCL. This was tested by analyzing caspase-dependent/independent pathways and autophagy, and caspase effects on ceramide and Golgi fragmentation. zVAD prevented caspase activation, but not all cell death. Inhibiting caspase-8 suppressed caspases more than inhibition of any other caspase. Inhibiting caspase-8/6 was synergistic. zVAD suppressed autophagy. 3-methyladenine suppressed caspase activation less than zVAD did. Blocking autophagy/caspase-8/or-6 was synergistic. Blocking autophagy/caspase-3/or-9 was not. Inhibiting caspase-9/3 suppressed autophagy. Golgi fragmentation was suppressed by zVAD, and blocked by CLN3. CLN3, not zVAD, prevented ceramide elevation. caspase-dependent/independent apoptosis and autophagy occur caspase-dependent pathways initiate autophagy Golgi fragmentation results from apoptosis ceramide elevation is independent of caspases, and CLN3 blocks all cell death, prevents Golgi fragmentation and elevation of ceramide in JNCL. Show less

Juvenile neuronal ceroid lipofuscinosis (JNCL) is due to mutations in the CLN3 gene. We previously determined that CLN3 protein harbors a highly conserved motif, VYFAE, necessary for its impact on cell growth and apoptosis. Using molecular modeling we demonstrated that this motif is embedded in a stretch of amino acids that is homologous to and structurally compatible with a galactosylceramide (GalCer) binding domain. This domain is present in the V3 loop of the HIV-1 gp120 envelope protein, beta-amyloid protein, and the infectious form of prionic protein, and defines a binding site for lipid rafts. We determined the subcellular localization of CLN3 in different cell systems including human neurons, primary rat hippocampal neurons, normal human fibroblasts, and JNCL fibroblasts homozygous for the 1.02 kb deletion in genomic DNA. Wild-type CLN3 protein was present within Golgi, lipid rafts in the plasma membrane, and early recycling endosomes, but not late endosomes/lysosomes. Wild-type CLN3 internalized from the plasma membrane to the Golgi via Rab4- and Rab11-positive recycling endosomes. Wild-type CLN3 co-localized with GalCer in the Golgi and in lipid rafts at the plasma membrane in normal cells. Neither mutant CLN3 protein nor GalCer were found at the plasma membrane in JNCL fibroblasts. Mutant CLN3p was retained within the Golgi and partially mis-localized to lysosomes, failing to reach recycling endosomes, plasma membrane, or lipid rafts. These studies identify a novel CLN3 domain that may dictate localization and function of CLN3. Show less

Juvenile Batten disease (JNCL) is an autosomal recessive disease that results from mutations in the CLN3 gene. The wild-type CLN3 gene coding sequence has 15 exons, and the translated protein consists of 438 amino acids. The most commonly observed mutation is a 1.02 kb deletion in the genomic DNA. This deletion results in a truncated protein due to the loss of amino acids 154-438, and the introduction of 28 novel amino acids at the c-terminus. We demonstrate that, compared to normal controls, CLN3-deficient immortalization of lymphoblasts homozygous for this deletion grow at a slower rate, and show increased sensitivity to etoposide-induced apoptosis, supporting the notion that CLN3 may negatively regulate apoptosis. Using immortalized JNCL lymphoblast cell lines as a model system, we assess the effects of specific CLN3 mutations on cell growth rates and protection from etoposide-induced apoptosis. Protection from etoposide-induced apoptosis occurs and the cell growth rate is restored following transfection of JNCL lymphoblasts with mutant CLN3 cDNA that includes exons 11 or 13. We show that deletion of the glycosylation sites 71NQSH74 and 310NTSL313, and also mutations within the highly conserved amino acid stretches 184WSSGTGGAGLLG195, 291VYFAE295 and 330VFASRSSL337, result in slowed growth and susceptibility to apoptosis. Show less

Juvenile Batten disease is a neurodegenerative disease caused by accelerated apoptotic death of photoreceptors and neurons attributable to defects in the CLN3 gene. CLN3 is antiapoptotic when overexpressed in NT2 neuronal precursor cells. CLN3 negatively modulates endogenous ceramide levels in NT2 cells and acts upstream of ceramide generation. Because defects in regulation of apoptosis are involved in the development of cancer, we evaluated the expression of CLN3 on both mRNA and protein levels in a variety of cancer cell lines and solid colon cancer tissue. We also observed the effect of the blocking of CLN3 protein expression on cancer cell growth, survival, ceramide production, and apoptosis by using an adenovirus-bearing antisense CLN3 construct. We show that CLN3 mRNA and protein are overexpressed in glioblastoma (U-373G and T98g), neuroblastoma (IMR-32 and SK-N-MC), prostate (Du145, PC-3, and LNCaP), ovarian (SK-OV-3, SW626, and PA-1), breast (BT-20, BT-549, and BT-474), and colon (SW1116, SW480, and HCT 116) cancer cell lines but not in pancreatic (CAPAN and As-PC-1) or lung (A-549 and NCI-H520) cancer cell lines. CLN3 is also up-regulated in mouse melanoma and breast carcinoma cancer cell lines. We found CLN3 expression is 22-330% higher than in corresponding normal colon control tissue in 8 of 10 solid colon tumors. An adenovirus-expressing antisense CLN3 (Ad-AS-CLN3) blocks CLN3 protein expression in DU-145, BT-20, SW1116, and T98g cancer cell lines as seen by Western blot. Blocking of CLN3 expression using Ad-AS-CLN3 inhibits growth and viability of cancer cells. It also causes elevation in endogenous ceramide production through de novo ceramide synthesis and results in increased apoptosis as shown by propidium iodide and JC-1 staining. This suggests that Ad-AS-CLN3 may be an option for therapy in some cancers. More importantly these results suggest that CLN3 is a novel molecular target for cancer drug discovery. Show less