The impact of sphingosine-1-phosphate (S1P)-lyase deficiency on astrocyte physiology and on epigenetic regulation

鞘脂醇-1-磷酸（S1P）是一种进化上保守的鞘脂代谢分解代谢中间体，调节大脑中多种生物过程，包括神经发育、分化和生存。另一个有趣的发现是它在组蛋白乙酰化调节中的作用。S1P特异性结合组蛋白去乙酰化酶HDAC1和HDAC2，从而抑制它们的酶活性，阻止组蛋白尾部赖氨酸残基上乙酰基的去除。S1P裂解酶（SGPL1）在鞘脂分解代谢的最后一步不可逆地裂解S1P，生成乙醇胺磷酸和长链醛。毫无疑问，S1P在大脑发育中的重要作用。然而，关于S1P参与神经退行性疾病病理的报道是相当矛盾的。为了阐明S1P在大脑中的功能，我们建立了SGPL1在神经细胞中特异性失活的小鼠模型（SGPL1fl/fl/Nes）。正如预期的那样，SGPL1消融导致S1P在大脑中积累，导致神经元内质网应激，细胞内钙增加，突触前结构和功能受损，并伴有小鼠认知缺陷。此外，我们还证明，由于磷脂酰乙醇胺（PE）的产生减少，SGPL1缺乏症会在早期阶段阻断神经元自噬。因此，在SGPL1缺失的大脑中，S1P的积累和PE的同时下降会导致相当大的神经元损伤。由于神经元损伤可能引起炎症，我们研究了神经细胞中S1P积累导致的小胶质细胞改变。我们通过形态学和生化标记证明了SGPL1fl/fl/Nes小鼠大脑中小胶质细胞激活增加。此外，在facs分类和培养的SGPL1fl/fl/Nes小鼠的小胶质细胞中，我们注意到促炎细胞因子的分泌增加。鉴于编码SGPL1的基因突变导致越来越多的人类神经病变，我们决定扩展我们的研究，一方面分析SGPL1fl/fl/Nes小鼠中星形胶质细胞的命运，另一方面分析SGPL1缺失的大脑中S1P积累的表观遗传后果。因此，本项目旨在回答两个核心问题：1)神经靶向消融SGPL1如何诱导星形胶质细胞增生？II) S1P依赖性组蛋白乙酰化调控的分子机制是什么？具体目标是：1。探索星形胶质细胞增生的原因及其后果。b.评价原代培养星形胶质细胞的自噬作用。验证我们关于SGPL1活性、星形细胞激活和自噬之间联系的分子机制的假设。研究SGPL1缺陷脑中的组蛋白乙酰化。IIb：研究钙在组蛋白乙酰化中的作用。初步结果强烈鼓励实施这一项目。

Sphingosine-1-phosphate (S1P), an evolutionarily conserved catabolic intermediate of sphingolipid metabolism regulates diverse biological processes in the brain including neural development, differentiation, and survival. Another interesting finding is its function in the regulation of histone acetylation. S1P specifically binds to histone deacetylases HDAC1 and HDAC2 thus inhibiting their enzymatic activity and preventing the removal of acetyl groups from lysine residues within histone tails. S1P-lyase (SGPL1) irreversibly cleaves S1P in the final step of sphingolipid catabolism generating ethanolamine phosphate and a long-chain aldehyde. There is no doubt on the essential role of S1P in brain development. Yet, reports on the involvement of S1P in the pathology of neurodegenerative diseases are rather conflicting. In an attempt to clarify the function of S1P in the brain, we generated a mouse model in which SGPL1 was inactivated specifically in neural cells (SGPL1fl/fl/Nes). As expected, SGPL1 ablation leads to S1P accumulation in the brain causing neuronal ER-stress, an increase in intracellular calcium, impairment of presynaptic architecture and function, accompanied by cognitive deficits in mice. In addition, we demonstrated that SGPL1 deficiency blocks neuronal autophagy at its early stages because of reduced phosphatidylethanolamine (PE) production. Thus accumulation of S1P and the simultaneous decline of PE in SGPL1 deficient brains cause considerable neuronal damage. As neuronal damage might induce inflammation, we investigated the microglial alterations as a result of S1P accumulation in neural cells. We evidenced increased microglial activation in the brains of SGPL1fl/fl/Nes mice as shown by morphological as well as biochemical markers. Also, an increased secretion of pro-inflammatory cytokines in FACS-sorted and cultured microglia from SGPL1fl/fl/Nes mice was noticed. Given the increasing number of human neuropathalogies as a result of mutations in the gene encoding SGPL1, we decided to extend our studies and analyse on the one hand the fate of astrocytes in SGPL1fl/fl/Nes mice and on the other hand the epigenetic consequences of S1P accumulation in SGPL1 depleted brains. The present project thus aims to answer two central questions:I) How does neural-targeted ablation of SGPL1 induce astrogliosis?II) Which molecular mechanism underlies S1P dependent regulation of histone acetylation?Specific goals are:Ia. To explore the causes of astrogliosis and of its consequences. Ib. To assess autophagy in primary cultured astrocytes.Ic. To verify our hypothesis regarding the molecular mechanism that links SGPL1 activity, activation of astrocytes and autophagy.IIa. To investigate histone acetylation in SGPL1 deficient brains.IIb: To study the involvement of calcium regarding histone acetylation.Preliminary results strongly encourage implementation of this project.