CLN8p is involved in spatial distribution of lysosomes

Roma

Simposio; SSIEM; 2016

Society for the Study of Inborn Errors of Metabolism

Background: CLN8 disease (one of 13 diseases grouped as NeuronalCeroid Lipofuscinosis, NCL) is caused by mutations in CLN8, whichencodes for a putative 286 aa, transmembrane protein CLN8p. Thisprotein shuttles between Endoplasmic Reticulum (ER) and the ERGolgiIntermediate Compartment (ERGIC). As in all NCL, its malfunctioncauses aggregates of lipofuscin-like compounds into lysosomesof different cell types, affecting in most extent neurons, causingneurodegeneration. CLN8p role as well as how its mutations triggerlysosomal storage disorder, is still unknown. Here, we evaluate howCLN8 expression is involved in the spatial distribution of lysosomes.Methods: HeLa cells were transfected with one of following constructs: solubleGFP (control), GFP-CLN8wt, shCLN8 or shLuc (sh control). Lysosomeswere marked with anti-LAMP1 antibody by immunofluorescence. Imageswere taken with a Disk Scanning Unit (DSU) microscope and analyzed usingImageJ-Fiji and SpatTrack softwares.Results: Four indexes were calculated to analyze spatial pattern of lysosomes.Nearest Neighborhood (NN) and Clark?s Aggregate Index (CAI) refer to distancesbetween particles. Both indexes showed a significant difference(p < 0.05) between CLN8wt and shCLN8 or control. Radial DistributionFunction (RDF) is calculated by SpatTrack and refers to the number of particlessurrounding each particle within a certain radius. This index showeddifferences (p < 0.0001) among all treatments. Finally, we measured distancesof each particle to the nucleus that showed difference (p < 0.01) between controland shCLN8 but not among the other treatments.Discussion: Ddifferent levels of CLN8 expression cause changes in the spatialdistribution of lysosomes in HeLa cells. Evidences in CLN3 and CLN5showed altered lysosomal pattern in HeLa cells when these non-lysosomalproteins were mutated. A common modified lysosomal distribution pathwaymay be related with the physiopathology of CLN3-, CLN5- and CLN8diseases.