CLN8p deficiency impairs lysosomal motility and protein distribution in hippocampal neuronal model

NOHER DE HALAC, RITA INÉS; BISBAL, MARIANO; PESAOLA, FAVIO

Londres

Congreso; 16th Conference on Neuronal Ceroid Lipofuscinosis; 2018

Society for the Study of Neuronal Ceroid Lipofuscinosis

Background: CLN8 protein, which mutations cause CLN8 disease, is an Endoplasmic Reticulum (ER)-resident transmembrane protein that shuttles between ER and Golgi Apparatus. Its role, still poorly known, was related to sphingolipids biosynthesis. We aim to study the effects of CLN8p deficiency on lysosomal dynamics and protein distribution in neuronal model. Methods: Hippocampal rat neurons of 7 d.i.v. were transfected with pYFP, pCLN8wt (overesxpression) or pshCLN8 plasmid to modulate CLN8 expression. Dendritic ramification was evaluated by Sholl analysis. For kymographs, lysosomes were marked with Lysotracker. For protein distribution, LAMP1, transferrin receptor (TfR) and TMEM106b were overexpressed and specifically marked. Distribution was expressed as polarity index (Idendrites/Iaxon). Images were analysed by ImageJ-Fiji. Results: Sholl analysis revealed a dendritic shortening and diminished ramification in deficient cells. Kymographs of dendritic segments showed that the number of moving lysosomes is increased both in CLN8 overexpressed and deficient neurons; however, direction was not altered. Moreover, trajectory and speed of lysosomes were significantly increased in dendrites of deficient cells (p