MON-668 Leptin Resistance Alters Neuroplasticity-Linked Genes in Adult Hypothalamic Neurons

Abstract Disclosure: D.A. Zieba: None. E.A. Oclon: None. A. Gurgul: None. I. Jasielczuk: None. T. Szmatola: None. Leptin resistance impairs hypothalamic signaling, altering neuronal gene expression and neuroplasticity and contributing to hypothalamic dysfunction. SMLA, a superactive mouse leptin antagonist, competes with wild-type leptin for receptor binding with 60-fold greater efficiency, offering a unique approach to modulating leptin signaling. This study examines the transcriptomic effects of leptin resistance and SMLA treatment in hypothalamic neurons. The adult-derived mHypoA-2/12 hypothalamic cell line was cultured in DMEM supplemented with 10% FBS and antibiotics. Cells were plated in 24-well plates at 1.5 × 10⁵ cells/well and maintained at 37°C with 5% CO₂. Experimental groups included Ctrl, BA (50.6 nM leptin, 31.25 µM antagonist), and CA (12.8 nM leptin, 31.25 µM antagonist). Cells were incubated for 24 hours before RNA isolation. Total RNA was extracted, quality-checked, and quantified. Libraries were prepared using QuantSeq 3’ mRNA-Seq, pooled, and sequenced (150 bp, single-end) to obtain at least 6 million reads per sample. Sequencing quality was assessed with FastQC, trimming, and filtering with Flexbar. Reads were mapped to the mouse GRC39 genome (STAR aligner) and counted with Htseq-count. Data normalization, clustering, and differential expression analysis were performed using DESeq2 on iDEP.96. Gene Ontology and KEGG pathway enrichment were analyzed with FDR < 0.05. Statistical analyses were performed in JASP using one-way ANOVA with Tukey post hoc or Kruskal-Wallis with Dunn post hoc as appropriate. RNA-seq validation by qPCR showed strong correlation (r > 0.85, p < 0.05). Leptin resistance led to significant gene changes critical for neuroplasticity and neuronal stress responses. Txnip decreased (logFC = −2.77, p < 7.19 × 10⁻¹⁰), while Hdac7 (logFC = +1.38, p < 1.29 × 10⁻⁸) and Ctnnbip1 (logFC = +1.69, p < 4.71 × 10⁻¹⁰) increased, suggesting potential disruptions in oxidative protection and synaptic remodeling. SMLA treatment reversed these changes, increasing Txnip (logFC = +1.48, p < 0.003) and reducing Hdac7 and Ctnnbip1 expression (logFC = −1.03 and −1.22, respectively). Additionally, Casp3 expression normalized with SMLA (logFC = +1.13, p < 1.22 × 10⁻⁵), potentially restoring synaptic pruning. Results suggest that SMLA may mitigate leptin resistance-induced disruptions in neuroplasticity. In summary, our findings demonstrate that leptin resistance alters key gene networks involved in neuroplasticity and stress responses in hypothalamic neurons. SMLA treatment reverses these molecular changes, restoring gene expression profiles linked to oxidative protection and synaptic remodeling. Understanding the mechanisms of leptin signaling modulation may provide a foundation for developing strategies to address leptin-related disorders. This research was financially supported by the grant no. UMO-2023/51/B/NZ9/00651. Presentation: Monday, July 14, 2025