Impairment of lipophagy by PNPLA1 mutations causes lipid droplet accumulation in primary fibroblasts of Autosomal Recessive Congenital Ichthyosis patients.

Autosomal Recessive Congenital Ichthyosis (ARCI) is a group of epidermal keratinization disorders. One of the disease-associated proteins, patatin-like phospholipase domain-containing protein-1 (PNPLA1), plays a key role in the epidermal omega-O-acylceramide synthesis and localizes on the surface of lipid droplets (LDs). Previously, routine clinical test results showed abnormal LD accumulation in blood smear samples of our ARCI patients with PNPLA1 mutations. To investigate the abnormal accumulation of LDs, we analyzed primary fibroblast cells of ARCI patients with PNPLA1 mutations (p.Y245del and p.D172N). We hypothesized that PNPLA1 mutations might affect lipophagy-mediated regulation of LDs and cause intracellular lipid accumulation in ARCI patients. LD accumulation was analyzed by fluorescence staining with BODIPY®493/503 in the fibroblasts of patient cells and PNPLA1 siRNA transfected control fibroblast cells. The expression of PNPLA1 and its effects on the lipophagy-mediated degradation of LDs were analyzed by immunocytochemistry and immunoblotting. Our results showed that mutant or downregulated PNPLA1 protein causes abnormal intracellular LD accumulation. We found that PNPLA1 mutations affect neither the cellular localization nor the expression levels of the protein in fibroblast cells. When we analyzed lipophagic degradation process, LC3 expression and the number of autophagosomes were significantly decreased in fibroblast cells of the patients. In addition, co-localization of LDs with autophagosomes and lysosomes were markedly less than that of the control group. PNPLA1 mutations caused disturbances in both autophagosome formation and fusion of autophagosomes with lysosomes. Our results indicate a possible role for PNPLA1 protein in LD regulation via lipophagy-mediated degradation.