1-磷酸鞘氨醇（S1P）与孤独症谱系障碍的发病关联及机制研究

Autism Spectrum Disorders(ASD)is a severe developmental disorder at the early stage of infancy and early childhood ,for which the etiology is unknown. The current study suggeststhat abnormal neurological development and dysfunctional synaptic transmission might be an important factor of this disease.Our early metabolism study found that Sphingosine1phosphate(S1P)level, which maintains neurons functions,increased significantly inserum of children with autism and correlated with its clinical phenotype, indicating that S1P may have a certain association with ASD.The proposed project will use autistic children and valproicacid-induced rat autism model as the research objects,to analyze the S1P level of ASD children using the case-control study design, and to detect the changes in the levels of S1P and Sph in serum and brain tissue of VPA-induced autism model. Furthermore, we will observe their key enzyme's expressions and the change of activity in S1P metabolic pathways, and observe the cognitive and behavioral changes of VPA-treated offspring before and after sphingosine kinase blocking in order to explore complex association of the S1P metabolic abnormalities and ASD. Patch clamp technique will be used to study the regulation of S1P, its receptors and signal transduction pathways to the activity of potassium channels in hippocampal neurons, to illustrate the mechanism by which abnormal S1P is associated with ASD. To clarify the role of S1P in autism etiology, development and its possible mechanism at whole, cellular and molecular levels.

孤独症谱系障碍（ASD）是起始于婴幼儿时期的严重发育障碍性疾病，病因不明。研究提示神经发育及突触传导异常可能是导致发病的重要因素。本课题组前期代谢组学研究也发现ASD患儿血清中维持神经元功能的1-磷酸鞘氨醇（S1P）明显升高并与其临床表型相关，提示S1P异常可能与ASD有一定关联。本项目以ASD患儿和VPA诱导的孤独症模型鼠为研究对象，进行ASD患儿S1P及前体Sph水平的病例-对照研究；检测孤独症模型鼠血清和脑组织中S1P、Sph水平及S1P代谢通路中关键酶的表达和活性变化，观察鞘氨醇激酶阻断前后孤独症模型鼠认知及行为改变，探讨S1P代谢异常与孤独症发病的复杂关联。此外，采用膜片钳技术，以维持神经元及突触传导的基本功能单位钾通道为靶点，研究S1P及其受体和信号转导通路对海马神经元钾通道活性的影响及调控，从细胞和分子水平深入探讨S1P异常与ASD关联的途径及其可能机制。

孤独症谱系障碍（ASD）的神经发育及突触传导异常可能是导致发病的重要因素。基于课题组前期研究发现ASD患儿血清中1-磷酸鞘氨醇（S1P）明显升高并与其临床表型相关，提示S1P异常可能与ASD有一定关联。本研究通过进一步扩大样本量，采集ASD和对照组儿童的静脉血，采用LC-MS检测血清中S1P含量，证实ASD患儿血清中的S1P水平明显增高。成功建立VPA诱导的ASD模型鼠，并证实其海马组织中SphK2/S1P水平显著升高，且表现出明显的学习和记忆功能损害；SphK阻断干预可有效降低S1P水平，通过减少线粒体 cytochrome c释放进而缓解海马神经元凋亡。SphK阻断干预通过降低VPA鼠海马组织中 S1P的水平，减少海马神经元的凋亡，同时增强其自噬水平进而改善VPA鼠学习和记忆损害。FTY720干预明显改善VPA鼠的认知和行为损害，减少海马CA1区神经元的损伤；FTY720通过干预VPA鼠海马组织S1P/S1Pr1信号减轻小胶质细胞介导的神经炎症反应；同时提高海马组织抗氧化能力，减轻氧化应激损伤，增强其抗凋亡能力，进而改善VPA鼠认知和行为损害。提示S1P水平与ASD的认知行为损伤存在明确关联，降低S1P水平可改善孤独症模型鼠认知行为损伤，可能通过抑制cytochrome c释放，减少神经元凋亡，增强神经元自噬，减轻氧化应激损伤，减轻小胶质细胞介导的神经炎症反应而发挥作用。同时项目又增加另一种ASD模型鼠BTBR鼠的研究，通过检测其血清及海马组织S1P水平，明确BTBR鼠血清和海马组织中S1P水平显著增加，给予SphK阻断剂干预，证实干预后ASD的认知社交行为及相关蛋白有明显改善，并采用分子生物及膜片钳技术，通过局部改变S1P水平，证实S1P由其S1PR介导通过MAPK及AC/cAMP/PKA信号通路调控Kv4.2通道的活性，进一步阐明S1P在ASD发病机制的可能作用途径。此外，通过对BTBR和对照组鼠海马组织泛素化等6种蛋白质翻译后修饰初筛，明确BTBR鼠海马组织巴豆酰化与二羟基异丁酰化表现显著差异，为后续S1P在ASD发病机制研究提供新的方向。本项目从人群研究到动物模型的机制验证，在整体、细胞及分子水平上，综合阐明了S1P与ASD发病的复杂关联及可能途径，为临床靶向干预提供新思路。