Restoration of lysosomal membrane integrity in cell models of Pompe disease depends on fatty acid synthase and its product palmitic acid.

backgroundLysosomal storage disorders (LSDs) are rare inherited diseases caused by mutations in genes encoding lysosomal proteins, leading to substrate accumulation and lysosomal dysfunction. Whether this build-up compromises lysosomal membrane integrity remains an open question. In this study, we aimed to investigate the presence of lysosomal membrane damage in LSD cells and to identify mechanisms that could reveal potential strategies for improvement of lysosomal function.

methodsPrimary skin fibroblasts from patients with LSDs were used to evaluate lysosomal integrity and function. Lysosomal immunopurification enabled lipidomic and proteomic profiling, complemented by viral transduction for targeted protein expression at lysosomal membranes. Pharmacologic and metabolite treatments were applied in loss-of-function and rescue analyses. Microscopy-based approaches were used to assess lysosomal activity, membrane damage, and integrity. As a complementary model, we employed conditional mouse embryonic fibroblasts (MEFs) lacking class 3 phosphoinositide 3-kinase (PI3K-3), which exhibit lysosomal dysfunction and autophagy defects characteristic of LSDs.

resultsIn Pompe disease, caused by mutations in the acid alpha-glucosidase gene, glycogen accumulation aggravates persistent lysosomal membrane damage. Moreover, using loss- and gain-of-function models of PI3K-3, we showed that its product, phosphoinositide-3-phosphate (PI3P), was essential for preserving lysosomal membrane integrity. Notably, Pompe patient fibroblasts exhibited chronic lysosomal damage, marked by increased galectin-3 (GAL-3) recruitment and reduced levels of lysosome-associated PI3K-3. Targeted restoration of PI3K-3 at lysosomal membranes rescued this defect and stabilized membrane integrity. Proteomic analyses following genetic or pharmacologic inhibition of PI3K-3 revealed a striking downregulation of fatty acid metabolism, including depletion of fatty acid synthase (FASN). We further demonstrated that PI3K-3 and FASN functionally interacted to maintain lysosomal integrity in Pompe cells. Supplementation with the FASN product palmitate reproduced the protective effect of PI3K-3 targeting, enhancing lysosomal proteolysis and restoring autophagic flux. Importantly, palmitate treatment also improved lysosomal function in fibroblasts from patients with Gaucher disease and type 2 neuronal ceroid lipofuscinosis, extending the relevance of this mechanism across LSDs.

conclusionsThese findings in cell models of LSDs uncover a link between PI3K-3, fatty acid metabolism, and lysosomal membrane integrity. They provide a basis for future in vivo validation of PI3K-3 activation and metabolic modulation as adjuvant treatments to improve lysosomal function in LSDs.