HIF prolyl hydroxylase 2/3 deletion disrupts astrocytic integrity and exacerbates neuroinflammation

Astrocytes constitute the parenchymal border of the blood-brain barrier (BBB), modulate the exchange of soluble and cellular elements, and are essential for neuronal metabolic support. Thus, astrocytes critically influence neuronal network integrity. In hypoxia, astrocytes upregulate a transcriptional program that has been shown to boost neuroprotection in several models of neurological diseases. We investigated transgenic mice with astrocyte-specific activation of the hypoxia-response program by deleting the oxygen sensors, HIF prolyl-hydroxylase domains 2 and 3 (Phd2/3). We induced Phd2/3 deletion in experimental autoimmune encephalomyelitis (EAE) in a therapeutic approach that led to an exacerbation of the disease mediated by massive immune cell infiltration. We found that Phd2/3-ko astrocytes, though expressing a neuroprotective signature, exhibited a gradual loss of gap-junctional Connexin-43 (Cx43), which was induced by vascular endothelial growth factor-alpha (Vegf-a) expression. These results provide mechanistic insights into astrocyte biology, their critical role in hypoxic states, and in chronic inflammatory CNS diseases.

Methods
By using TRAP (translating ribosome affinity purification) just actively translating mRNA from astrocytes of mice Aldh1l1-creERT2; Phd2fl/fl or wt/wt; Phd3fl/fl or wt/wt; L10a-eGFP was collected (spinal cord and brain stem), purified and sequenced at the HiSeq4000 system. For detailed protocol information please refer to Heiman et al., 2014 and Rosiewicz et al., 2020. Then raw fastq files were quality checked by FastQC, then rRNA content was filtered-out with SortMeRNA and finally aligned to mouse genome by Salmon.

星形胶质细胞（Astrocytes）构成血脑屏障（blood-brain barrier, BBB）的实质边界，可调节可溶性物质与细胞成分的跨屏障交换，同时为神经元提供关键的代谢支持。因此，星形胶质细胞对神经元网络的完整性具有决定性影响。在缺氧条件下，星形胶质细胞会激活一套转录程序，该程序已在多种神经系统疾病模型中被证实可增强神经保护效应。本研究通过敲除氧感受器——缺氧诱导因子脯氨酰羟化酶结构域2和3（HIF prolyl-hydroxylase domains 2 and 3, Phd2/3），构建了星形胶质细胞特异性激活缺氧应答程序的转基因小鼠模型。我们在实验性自身免疫性脑脊髓炎（experimental autoimmune encephalomyelitis, EAE）模型中实施Phd2/3敲除的治疗性干预，却发现该干预导致大量免疫细胞浸润，进而加重了疾病进程。研究发现，尽管Phd2/3基因敲除（Phd2/3-ko）的星形胶质细胞表达神经保护特征基因，但它们的间隙连接蛋白43（gap-junctional Connexin-43, Cx43）会逐渐丢失，这一现象由血管内皮生长因子-α（vascular endothelial growth factor-alpha, Vegf-a）的表达所介导。本研究结果为星形胶质细胞生物学、其在缺氧状态下的关键作用，以及慢性炎症性中枢神经系统疾病中的病理生理机制提供了新的机制性见解。

研究方法：本研究采用翻译核糖体亲和纯化（translating ribosome affinity purification, TRAP）技术，收集Aldh1l1-creERT2; Phd2fl/fl 或 wt/wt; Phd3fl/fl 或 wt/wt; L10a-eGFP小鼠星形胶质细胞中正在活跃翻译的mRNA（采集脊髓与脑干样本），对其进行纯化后，使用HiSeq4000系统完成高通量测序。详细实验流程请参照Heiman等（2014）与Rosiewicz等（2020）的研究报道。原始fastq文件首先通过FastQC进行质量质控，随后使用SortMeRNA过滤去除核糖体RNA（rRNA），最终通过Salmon软件将测序序列比对至小鼠参考基因组。