SUN-021 Lipotoxicity-Induced Alteration of Hypothalamic Neuronal Protein Turnover

Abstract Disclosure: E.A. Oclon: None. A. Gurgul: None. I. Jasielczuk: None. T. Szmatola: None. D.A. Zieba-Przybylska: None. Accumulated toxic lipids induce ER stress, oxidative stress, and mitochondrial dysfunction, leading to cell death. Despite a growing emphasis on the role of lipotoxicity in neurons, the mechanisms behind these disruptions, particularly those affecting protein turnover, remain unclear. This study aimed to investigate the effects of lipotoxicity on protein turnover-related pathways in hypothalamic neurons using RNA-seq to analyze gene expression changes. The mHypoA-2/12 hypothalamic cell line was cultured in high-glucose DMEM with 10% FBS and antibiotics. Cells were seeded in 24-well plates and incubated at 37°C with 5% CO₂. Palmitic acid (PA) was dissolved in serum-free DMEM with 5% fatty acid-free BSA for treatment, while controls received serum-free DMEM with 5% BSA. Cellular parameters were assessed, including viability, caspase-3/7 activity, and calcium levels. Total RNA was extracted, quantified, and analyzed using RNA-seq (QuantSeq 3’ mRNA-Seq). Sequencing was performed with a minimum depth of 6 million reads per sample. Differential expression, clustering, and pathway enrichment were analyzed with DESeq2 and iDEP.96. Statistical analyses were performed in JASP using one-way ANOVA with Tukey post hoc or Kruskal-Wallis with Dunn post hoc as appropriate. PA treatment (5 mM, 24 h) reduced cell survival by 60.8% (p < 0.001), increased caspase-3/7 activity (2.1-fold, p < 0.001), and elevated calcium levels (p < 0.01), indicating cellular stress and apoptosis activation. Pathway enrichment analysis revealed upregulation of processes involved in protein turnover and stress responses, including protein folding (GO:0006457; p = 7e-14), ER stress response (GO:0034976; p = 1.08e-08), and ER-associated degradation (ERAD; GO:0036503; p = 2.32e-07). Key genes related to protein turnover, such as Hspa5 (logFC: 1.38, p < 0.001), Hspa1b (logFC: 4.69, p = 0.005), and Hsp90aa1 (logFC: 1.21, p < 0.001), were significantly upregulated, indicating a robust cellular response to manage protein misfolding under lipotoxic stress. Our findings show that lipotoxicity in hypothalamic neurons activates processes related to protein turnover, mainly through the upregulation of chaperone-mediated protein folding and quality control. This suggests a cellular attempt to manage protein misfolding under lipotoxic stress. Increased apoptosis, decreased cell viability, and elevated calcium levels further support the cytotoxic effects of certain lipids, indicating that the stress may overwhelm the protective mechanisms to manage protein misfolding, ultimately leading to neuronal dysfunction. The activation of ER stress-related pathways, including protein folding, ER-associated degradation (ERAD), and the unfolded protein response, highlights the significant cellular effort to manage misfolded proteins. This research was financially supported by the Grant no. UMO-2023/51/B/NZ9/00651. Presentation: Sunday, July 13, 2025