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The neuronal ceroid lipofuscinoses (NCLs) are autosomal recessive diseases with progressive dementia, motor disturbance, epilepsy, visual loss and early death, affecting mainly children but also adults. Mutations in 13 distinct genes cause NCLs with marked phenotypic heterogeneity and undefined pathomechanism. CLN8 gene shows wide complexity with 31 different mutations leading to late-infantile NCL variants (vLINCL) and progressive epilepsy with mental retardation (EPMR) (www.ucl.ac.uk/ncl/mutation.shtml). CLN8 encodes an ER-resident transmembrane protein with an ER-ERGIC retrieval signal, a TLC domain normally related to lipid sensing/trafficking, and possible multiple roles (1, 2). We proposed to study the functional meaning of CLN8-VAPA interaction previously characterized (Passantino et al., 2013), being VAPA an ER-docking-site for cytosolic lipid-binding-proteins responsible of ceramide transport, sphingolipid (SL), phospholipid and cholesterol synthesis/trafficking. We expect to define CLN8 implication in SL, and pinpoint the molecular bases of the phenotypic heterogeneity. In the frame of the current project, using recombinant proteins and mammalian expression vectors (mono/bicistronic) encoding flCLN8, its subregions and flVAPA, and in vitro and in vivo assays, we identified a putative CLN8 region interacting with VAPA, that is highly conserved in vertebrates and bears some vLINCL mutations. In vitro SphingoStrips and TLC overlay assay revealed no direct binding of flCLN8 and subregions to ceramides;whereas, metabolic labeling studies showed kinetic changes in the ceramide pathway of HeLa cells overexpressing CLN8 and VAPA. Interestingly, CLN8 (presumably the N-terminus) was able to bind sulfatide, which regulates myelination; considering the myelin defects in NCLs (3), further studies are ongoing. Throughout site-directed-mutagenesis, we created a model of EPMR-type mutation. In cells expressing both EPMR and VAPA, we found a strong EPMR-VAPA interaction and higher sphingomyelin and cholesterol levels. We also generated CRISPR/Cas9 CLN8 KO clones that show to maintain survival and migration capacities similar to native HeLa cell line but grow as autophagy predisposed cells. In CLN8-KO clones, even though the high VAPA levels, the only difference observed in sphingolipid was a marked change in the ganglioside patterns, with a shift toward more complex species. The overall results indicate a multifaceted and at least in part novel role of CLN8 alone and with VAPA toward SL trafficking, also supported by the finding that flCLN8 and subregions were able to bind different phosphoinositides in vitro. We are underway using the newly created CRISPR-CERT KO clones to analyze this ceramide transporter as part of CLN8-VAPA complex and possible target of CLN8 dysfunction. Fibroblast cells of patients with CLN8-vLINCL and EPMR are being used to correlate phenotype/genotype divergences to the CLN8-VAPA complex. |
