Disruption of the intellectual disability-linked gene Hs6st2 in mice decreases heparan sulfate 6-O-sulfation in the brain and impairs memory

Heparan sulfate (HS) is a linear polysaccharide that plays a key role in cellular signaling networks. HS functions are regulated by its 6-O-sulfation, which is catalyzed by HS 6-O-sulfotransferases (HS6STs). Although mutations in HS6ST2 cause intellectual disability in human patients, the role and molecular mechanisms of HS6ST2 in the adult mammalian brain remain unknown. Here we found that the brain specifically expresses the long isoform of Hs6st2, which encodes a protein that retains the sulfotransferase domain and acquires a novel sub-structure. To determine the role of Hs6st2 in the brain, we carried out a series of behavioral and molecular assessments on the Hs6st2 knockout mice. We found that Hs6st2 knockout mice exhibit high body weight, hyperactivity, and memory-related deficits. To determine the molecular mechanisms underlying these deficits, we carried out strong anion exchange-high performance liquid chromatography and RNA sequencing. We found that knockout of Hs6st2 decreases HS 6-O-sulfation levels in the brain and impairs transcriptome in the hippocampus. We also found that the transcriptome changes are enriched in genes involved in ribosome and protein translation pathways, which are likely due to the downregulation of the fibroblast growth factor signaling. Together, our study demonstrates the role and molecular mechanisms of Hs6st2 in the adult mammalian brain, which provides new insights into the role of HS in brain health and disease. In this study, we analyzed the splicing patterns of Hs6st2 in different mouse tissues and investigated the protein structures of the long and short Hs6st2 isoforms using machine learning-based predictions. We also used cryo-recovery approach to re-generate the Hs6st2 knockout mice (KO) and carried out a series of behavioral and molecular assessments, including behavioral tests, strong anion exchange-high performance liquid chromatography (SAX-HPLC), and RNA sequencing (RNA-seq).硫酸乙酰肝素（Heparan sulfate, HS）是一类线性多糖，在细胞信号网络中发挥关键作用。HS的功能受其6-O-硫酸化修饰调控，该过程由HS 6-O-硫酸基转移酶（HS 6-O-sulfotransferases, HS6STs）催化。尽管HS6ST2的突变会导致人类患者出现智力障碍，但HS6ST2在成年哺乳动物大脑中的作用与分子机制仍不明晰。本研究发现，大脑特异性表达Hs6st2的长亚型，该亚型编码保留硫酸基转移酶结构域并获得新型亚结构的蛋白质。为明确Hs6st2在大脑中的作用，我们对Hs6st2基因敲除小鼠开展了一系列行为学与分子生物学评估。结果显示，Hs6st2敲除小鼠表现出体重偏高、活动过度以及记忆相关认知缺陷。为探究这些缺陷背后的分子机制，我们采用强阴离子交换高效液相色谱法与RNA测序进行了分析。研究发现，Hs6st2敲除会降低大脑中HS的6-O-硫酸化修饰水平，并损伤海马体的转录组。同时，转录组变化富集于核糖体与蛋白质翻译通路相关基因，这一现象可能源于成纤维细胞生长因子信号通路的下调。综上，本研究阐明了Hs6st2在成年哺乳动物大脑中的作用与分子机制，为理解HS在脑健康与疾病中的功能提供了新视角。此外，本研究分析了Hs6st2在不同小鼠组织中的剪接模式，并基于机器学习预测方法探究了Hs6st2长短两种亚型的蛋白质结构。我们还采用冷冻复苏法构建并重获Hs6st2基因敲除小鼠（KO），并开展了一系列行为学与分子生物学实验，包括行为学测试、强阴离子交换高效液相色谱（strong anion exchange-high performance liquid chromatography, SAX-HPLC）以及RNA测序（RNA sequencing, RNA-seq）。