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

PESAOLA F; VENIER AC; NOHER DE HALAC I; BISBAL M

Mar del Plata

Congreso; REUNIÓN ANUAL DE SOCIEDADES DE BIOCIENCIA 2019; 2019

SAIC-SAFE-SAP-SAB-NANOMED-ar-AACyTAL

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<0.01). Respect to protein distribution in neurons, the index of LAMP1 in control cells was 1.8 ± 0.2 and 1.5 ± 0.1 in CLN8-deficient cells. For TfR, the index in control neurons was 4.5 ± 0.4, and 2.5 ± 0.2 in CLN8-deficient cells (p<0.001). For TMEM106b, the index in control neurons was 1.4 ± 0.1 and 1.5 ± 0.1 in CLN8-deficient cells. CLN8 deficiency increases the lysosomal pH, which affects the normal development of lysosomal metabolism. We suggest that this effect indirectly alters the distribution of some proteins, as we observed for TfR, possibly also affecting iron metabolism, observed in CLN8 disease. Further studies are needed to understand the pathophysiology of CLN8 disorder.