CLN8 deficiency, associated with CLN8 disease of Neuronal Ceroid Lipofuscinosis, increases lysosomal pH and alters the Transferrin Receptor distribution in hippocampal neuronal model

PESAOLA FAVIO; VENIER ANA CLARA; NOHER INES; BISBAL MARIANO

Mar del Plata

Congreso; Reunión Anual de Sociedades de Biociencia SAIC, SAFE, SAB, SAP, AACYTAL, NANOMED-ar, HCS; 2019

CLN8 protein, whose mutations cause CLN8 disease, is an Endoplasmic Reticulum (ER)-resident transmembrane protein that travels between ER and Golgi Apparatus. It carries soluble lysosomal proteins and regulates the activity of I2PP2A, a PP2A phosphatase inhibitor. In this work, we aim to study the effects of CLN8 deficiency on lysosomal pH and protein distribution. HeLa cells and rat hippocampal neurons of 7 d.i.v. were transfected with pYFP, pCLN8wt or pshCLN8 plasmid to modulate the expression of CLN8. To study lysosomal pH, HeLa cells were co-transfected with the plasmid pALP (mApple-LAMP1-pHluorin), which locates the pHluorin (whose fluorescence is sensitive to pH) and the mApple protein on the inner and outer side of the lysosomal membrane. respectively. The intensity ratio (IpHluorin/ImApple) was taken as indicative of the luminal pH. To study protein distribution, neurons were co-transfected with pLAMP1, pTfR (transferrin receptor) or pTMEM106b. The protein distribution was expressed as polarity index (Idendrites/Iaxon). Images were taken by confocal microscopy and analyzed using ImageJ-Fiji and Motion Tracking softwares. The intensity ratio in CLN8-deficient HeLa cells was 1.3 ± 0.1 (mean ± SEM), in CLN8wt cells 1.01 ± 0.05, and in control cells 0.9 ± 0.1 (p