Implication of CLN8 in the spatial distribution of lysosomes

PESAOLA F; BISBAL M; NOHER DE HALAC I.; QUASSOLLO G,

Boston

Conferencia; The 15th International Conference on Neuronal Ceroid Lipofuscinosis (Batten Disease); 2016

BACKGROUND: CLN8- is a putative 286 aa, transmembrane protein encoded bythe CLN8 gene, whose mutations underlayCLN8 disease of NCL. This protein shuttles between Endoplasmic Reticulum (ER)and the ER-Golgi Intermediate Compartment (ERGIC). Its malfunction isassociated with the typical NCL aggregates of lipofuscin-like compounds inlysosomes in cells of peripheral tissues.   Theseaggregates are related to neuronal degeneration in the brain. CLN8 role, aswell as how mutations trigger lysosomal storage disorder and neurodegenerationis still unknown. Here, we evaluate how CLN8expression is involved in the spatial pattern of lysosomes. METHODS: HeLa cellswere transfected with one of following constructs: soluble GFP (control),GFP-CLN8wt, shCLN8 or shLuc (control sh). Lysosomes were marked with anti-LAMP1antibody by immunofluorescence. Images were taken with a Disk Scanning Unit(DSU) microscope and analyzed using ImageJ-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 Distribution Function (RDF) iscalculated by SpatTrack and refers to the number of particles surrounding eachparticle within a certain radius. This index showed differences (p < 0.0001)among all treatments. Finally, we measured distances of each particle to thenucleus that showed difference (p < 0.01) between control and shCLN8 but notamong the other treatments. DISCUSSION: Different levels of CLN8 expression cause changes in thespatial distribution of lysosomes in HeLa cells suggesting that lysosomesaggregate more and farther from nucleus at low levels of expression than in thenormal condition. Evidences showed altered lysosomal pattern in HeLa cells transfectedwith CLN3 and CLN5 constructs when these non-lysosomal proteins were mutated. Acommon modified lysosomal distribution pathway may be related with thephysiopathology of CLN3-, CLN5- and CLN8 diseases.