脆性X综合征(FXS)的症状,由Fmr1的单个基因突变引起的,越来越多地与大脑皮层内星形胶质细胞信号的紊乱有关。我们最近证明了嘌呤能信号通路,利用核苷三磷酸及其代谢产物促进双向神经胶质和神经胶质-神经元相互作用,在源自FXS的Fmr1敲除(KO)小鼠模型的皮质星形胶质细胞中上调。提高Fmr1KOP2Y嘌呤能受体水平与细胞内钙释放延长相关,突触蛋白分泌升高,和发育电路的过度活跃。然而,由于相对缺乏可用于测量嘌呤和嘧啶的灵敏和可重现的定量方法,确定Fmr1KO星形胶质细胞中这些因子的丰度是有限的.因此,我们开发了一种与质谱联用的亲水相互作用液相色谱方案,以比较野生型和Fmr1KO小鼠星形胶质细胞之间细胞内和细胞外嘌呤能分子的丰度。UDP浓度的显著差异,ATP,AMP,相对于WT,在Fmr1KO星形胶质细胞内发现了腺苷细胞内储存。与从WT星形胶质细胞收集的培养基相比,Fmr1KO星形胶质细胞条件培养基中腺苷的细胞外水平也显着升高。星形细胞膜结合的CD39外核苷酸酶的糖基化,促进突触释放后的配体分解,在Fmr1KO星形胶质细胞培养物中也升高。一起,这些差异表明Fmr1KO皮质星形胶质细胞内嘌呤能信号系统的进一步失调,可能导致FXS嘌呤能受体激活和细胞病理学的显着改变。
The symptoms of fragile X syndrome (FXS), caused by a single gene mutation to Fmr1, have been increasingly linked to disordered astrocyte signalling within the cerebral cortex. We have recently demonstrated that the purinergic signalling pathway, which utilizes nucleoside triphosphates and their metabolites to facilitate bidirectional glial and glial-neuronal interactions, is upregulated in cortical astrocytes derived from the Fmr1 knockout (KO) mouse model of FXS. Heightened Fmr1 KO P2Y purinergic receptor levels were correlated with prolonged intracellular calcium release, elevated synaptogenic protein secretion, and hyperactivity of developing circuits. However, due to the relative lack of sensitive and reproducible quantification methods available for measuring purines and pyrimidines, determining the abundance of these factors in Fmr1 KO astrocytes was limited. We therefore developed a hydrophilic interaction liquid chromatography protocol coupled with mass spectrometry to compare the abundance of intracellular and extracellular purinergic molecules between wildtype and Fmr1 KO mouse astrocytes. Significant differences in the concentrations of UDP, ATP, AMP, and adenosine intracellular stores were found within Fmr1 KO astrocytes relative to WT. The extracellular level of adenosine was also significantly elevated in Fmr1 KO astrocyte-conditioned media in comparison to media collected from WT astrocytes. Glycosylation of the astrocyte membrane-bound CD39 ectonucleotidase, which facilitates ligand breakdown following synaptic release, was also elevated in Fmr1 KO astrocyte cultures. Together, these differences demonstrated further dysregulation of the purinergic signalling system within Fmr1 KO cortical astrocytes, potentially leading to significant alterations in FXS purinergic receptor activation and cellular pathology.