CLN8 deficiency alters the motility of lysosomes in a hippocampal neuronal model

BISBAL M; NOHER DE HALAC I.; PESAOLA F

Atenas

Simposio; SSIEM 2018: Annual Symposium of the Society for the Study of Inborn Errors of Metabolism; 2018

Society for the Study of Inborn Errors of Metabolism

Background: CLN8p is an Endoplasmic Reticulum (ER) transmembraneprotein, in which mutations cause CLN8 disease, one of thirteen forms ofNeuronal Ceroid Lipofuscinosis. CLN8p shuttles between the ER and GolgiApparatus and is related to sphingolipid biosynthesis, in spite of its roleremaining unknown. We aim to study the effects of CLN8 deficiency onlysosomal dynamics and protein distribution in a neuronal model.Methods: Hippocampal rat neurons of 7 d.i.v. were transfected withpYFP, pCLN8wt (overesxpression) or pshCLN8 plasmids to modulateCLN8 expression. Dendritic morphology was evaluated by Sholl analysis.For kymographs, lysosomes were marked with Lysotracker. For proteindistribution, LAMP1, transferrin receptor (TfR) and TMEM106bwere overexpressed and specifically marked. Distribution was expressedas polarity index (Idendrites/Iaxon). Images were analysed by ImageJ-Fiji.Results: Sholl analysis revealed dendritic shortening and diminished ramificationin deficient cells. Kymographs of dendritic segments showedthat the number of moving lysosomes is increased both in CLN8overexpressed and deficient neurons; however, direction was not altered.Moreover, trajectory and speed of lysosomes were significantly increasedin dendrites of deficient cells (p< 0.001). Regarding protein distribution,both LAMP1 and TfR polarity indexes tend to decrease in deficient cells,not so for TMEM106b.Discussion: CLN8p deficiency alters dendritic pathways involved in lysosomalmotility, increasing their movement regardless of direction. This couldexplain dendritic shortening in CLN8 silenced neurons. One hypothesis suggeststhat movement impairment may be caused by mis-sorting of proteins.The results of protein distribution showed that traffic of proteins could bealtered, but it may not be the only explanation (TMEM106b, involved inlysosome motility, is not affected). Further analyses is needed to uncoverthe CLN8p involvement on lysosomal motility in neurons.