Brown Christopher R, Foster James D
Department of Biomedical Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202-9037.
bioRxiv. 2025 Mar 13:2025.03.12.642908. doi: 10.1101/2025.03.12.642908.
Autism spectrum disorder (ASD) is a developmental disorder of the nervous system characterized by a deficiency in interpersonal communication skills, a pathologic tendency for repetitive behaviors, and highly restrictive interests. The spectrum is a gradient-based construct used to categorize the widely varying degrees of ASD phenotypes, and has been linked to a genetic etiology in 25% of cases. Prior studies have revealed that 30% of ASD patients exhibit hyperserotonemia, or elevated whole blood serotonin, implicating the serotonergic system in the pathogenesis of ASD. Likewise, escitalopram, a selective-serotonin reuptake inhibitor (SSRI), has been demonstrated to improve aberrant behavior and irritability in ASD patients, potentially by modulating abnormal brain activation. Prior studies have uncovered proband patients with rare mutations in the human serotonin transporter (hSERT) that manifest enhanced surface expression and transport capacity, suggesting that abnormal enhancement of hSERT function may be involved in the pathogenesis of ASD.
HEK-293 cells stably expressing WT, C109A, I425L, F465L, L550V, or K605N hSERT were subject to analysis for palmitoylation via Acyl-Biotin Exchange followed with hSERT immunoblotting. F465L functional enhancement was confirmed by surface analysis via biotinylation and saturation analysis via 5HT transport. F465L palmitoylation, surface expression and transport capacity were then assessed following treatment with 2-bromopalmitate or escitalopram.
Here, we reveal that palmitoylation is enhanced in the ASD hSERT F465L and L550V coding variants, and confirm prior reports of enhanced kinetic activity and surface expression of F465L. Subsequently, treatment of F465L with the irreversible palmitoyl acyl-transferase inhibitor, 2-bromopalmitate (2BP), or escitalopram, rectified enhanced F465L palmitoylation, surface expression, and transport capacity to basal WT levels.
Tests assessing L550V for surface expression, transport capacity, and reactivity to inhibition of palmitoylation was not assessed. In addition, further characterization is necessary for internalization rates, degradative mechanisms, the impact of cysteine-mediated substitutions, and other SSRIs on these processes.
Overall, our results implicate disordered hSERT palmitoylation in the pathogenesis of serotonergic ASD subtypes, with basal recovery of these processes following escitalopram providing insight into its molecular utility as an ASD therapeutic.
自闭症谱系障碍（ASD）是一种神经系统发育障碍，其特征在于人际沟通技能缺陷、重复行为的病理倾向以及高度受限的兴趣。该谱系是一种基于梯度的结构，用于对ASD表型的广泛不同程度进行分类，并且在25%的病例中与遗传病因相关。先前的研究表明，30%的ASD患者表现出高血清素血症，即全血血清素升高，这表明血清素能系统参与了ASD的发病机制。同样，艾司西酞普兰，一种选择性血清素再摄取抑制剂（SSRI），已被证明可以改善ASD患者的异常行为和易怒情绪，可能是通过调节异常的大脑激活来实现的。先前的研究发现，携带人类血清素转运体（hSERT）罕见突变的先证者患者表现出增强的表面表达和转运能力，这表明hSERT功能的异常增强可能参与了ASD的发病机制。
稳定表达野生型（WT）、C109A、I425L、F465L、L550V或K605N hSERT的HEK-293细胞通过酰基生物素交换进行棕榈酰化分析，随后进行hSERT免疫印迹。通过生物素化进行表面分析和通过5-羟色胺（5HT）转运进行饱和分析来确认F465L的功能增强。然后在用2-溴棕榈酸酯或艾司西酞普兰处理后评估F465L的棕榈酰化、表面表达和转运能力。
在此，我们发现ASD的hSERT F465L和L550V编码变体中的棕榈酰化增强，并证实了先前关于F465L动力学活性和表面表达增强的报道。随后，用不可逆的棕榈酰酰基转移酶抑制剂2-溴棕榈酸酯（2BP）或艾司西酞普兰处理F465L，将增强的F465L棕榈酰化、表面表达和转运能力纠正至基础野生型水平。
未评估针对L550V的表面表达、转运能力以及对棕榈酰化抑制反应性的测试。此外，内化速率、降解机制、半胱氨酸介导的替代物的影响以及其他SSRI对这些过程的影响还需要进一步表征。
总体而言，我们的结果表明hSERT棕榈酰化紊乱参与了血清素能ASD亚型的发病机制，艾司西酞普兰处理后这些过程的基础恢复为其作为ASD治疗药物的分子效用提供了见解。
