CLN8 deficiency impairs dendritic development in hippocampal neuronal model

PESAOLA FAVIO; QUASSOLLO GONZALO; BISBAL MARIANO

Rio de Janeiro

Congreso; 13 International Congress of Inborn Errors of Metabolism; 2017

ICIEM

CLN8 (one of 13 diseases grouped as Neuronal Ceroid Lipofuscinosis, NCL) iscaused by mutations in CLN8, which encodes for a putative 286 aa,transmembrane protein CLN8p. This protein shuttles between EndoplasmicReticulum (ER) and the ER-Golgi Intermediate Compartment (ERGIC). As inall NCL, its malfunction causes aggregates of lipofuscin-like compounds intolysosomes of different cell types, affecting neurons to a large extent,causing neurodegeneration. CLN8p role as well as how its mutations triggerlysosomal disorder, is still unknown. Here, we evaluate how CLN8expression levels affect neuronal morphology. Embryonic hippocampal rat neurons of 2 and 9 d.i.v. were transiently transfected with pYFP (control) alone or co-transfected with pCLN8wt(overexpression) or pshCLN8 (silencing). 2 d.i.v. neurons were marked withanti-Tau and anti-Tubulin by immunostaining for axonal length measure. 9d.i.v. neurons were used for dendritic branching study by Sholl analysis. Forkymographs, lysosomes were stained with Lysotracker. Images were taken inan epifluorescence microscope and in a Disc Scanning Unit microscope formovies, and analyzed with ImageJ-Fiji software. Statistics were done by one-way or two-way ANOVA (with or without repeated measures) depending onexperiment.