Table_2_Valproic Acid Induces Autism-Like Synaptic and Behavioral Deficits by Disrupting Histone Acetylation of Prefrontal Cortex ALDH1A1 in Rats.DOCX

ObjectivesThis study aimed to investigate the impact of valproic acid (VPA) on the histone acetylation of acetaldehyde dehydrogenase 1A1 (ALDH1A1) and the mechanism underlying VPA-induced autism-like behavior.
MethodsFemale Sprague-Dawley rats were intraperitoneally injected with VPA during gestation to establish an autism model in their offspring. Some offspring prenatally exposed to VPA were randomly treated with MS-275, one histone deacetylase (HDAC) inhibitor, or retinoic acid (RA) after birth. Behavioral tests were conducted on the offspring 6 weeks after birth. Electrophysiological experiments were performed to investigate long-term potentiation (LTP) in the prefrontal cortex (PFC). The expression levels of AMPA receptors (GluA1 and 2), NMDA receptors (GluN1 and 2), synapsin 1 (SYN1), HDAC, acetylated histone 3 (AcH3), RA receptor alpha (RARα), and ALDH1A1 in the PFC were measured by Western blotting and quantitative polymerase chain reaction. ALDH enzyme activity in PFC tissue was detected using a Micro ALDH Assay Kit. The RA level in the PFC was measured using ultrahigh-performance liquid chromatography/tandem mass spectrometry. A chromatin immunoprecipitation (ChIP) experiment explored the interaction between the ALDH1A1 gene and AcH3.
ResultsOffspring prenatally exposed to VPA showed autism-like behavior, upregulated the levels of LTP and GluN2A, GluA1, and SYN1 proteins relevant to synaptic plasticity in the PFC. The expression levels of HDAC3 mRNA and protein were increased. On the other hand, there was a significant reduction in the levels of AcH3, RARα, RA, ALDH1A1 mRNA and protein, the level of ALDH activity and AcH3 enrichment in the ALDH1A1 promoter region in VPA-induced offspring. Administration of MS-275 in VPA offspring significantly elevated the levels of AcH3, ALDH1A1 mRNA and protein, ALDH activity, RA, the level of RARα protein and the binding of AcH3 to the ALDH1A1 promoter. In addition, the GluA1 protein level and LTP were reduced, and most behavioral deficits were reversed. After RA supplementation in the VPA-treated offspring, the RA and RARα protein levels were significantly upregulated, GluA1 protein and LTP were downregulated, and most autism-like behavioral deficits were effectively reversed.
ConclusionThese findings suggest that VPA impairs histoneacetylation of ALDH1A1 and downregulates the RA-RARα pathway. Such epigenetic modification of ALDH1A1 by VPA leads to autism-like synaptic and behavioral deficits.

研究目标
本研究旨在探讨丙戊酸（valproic acid, VPA）对乙醛脱氢酶1A1（acetaldehyde dehydrogenase 1A1, ALDH1A1）组蛋白乙酰化的影响，以及丙戊酸诱导自闭症样行为的潜在机制。

研究方法
选用雌性Sprague-Dawley大鼠，于妊娠期腹腔注射丙戊酸以构建子代自闭症模型。将部分产前暴露于VPA的子代大鼠于出生后随机给予组蛋白去乙酰化酶（histone deacetylase, HDAC）抑制剂MS-275或视黄酸（retinoic acid, RA）干预。于子代大鼠出生6周后开展行为学测试；采用电生理实验检测前额叶皮层（prefrontal cortex, PFC）的长时程增强（long-term potentiation, LTP）。通过蛋白质印迹法与定量聚合酶链反应检测前额叶皮层中AMPA受体（AMPA receptors, GluA1、GluA2）、NMDA受体（NMDA receptors, GluN1、GluN2）、突触素1（synapsin 1, SYN1）、HDAC、乙酰化组蛋白3（acetylated histone 3, AcH3）、视黄酸受体α（retinoic acid receptor alpha, RARα）以及ALDH1A1的表达水平。使用Micro ALDH检测试剂盒检测前额叶皮层组织中的ALDH酶活性；采用超高效液相色谱-串联质谱法检测前额叶皮层中的RA水平。通过染色质免疫沉淀（chromatin immunoprecipitation, ChIP）实验探究ALDH1A1基因与AcH3的相互作用。

研究结果
产前暴露于VPA的子代大鼠表现出自闭症样行为，其前额叶皮层内与突触可塑性相关的LTP水平，以及GluN2A、GluA1、SYN1蛋白表达均上调。HDAC3的mRNA与蛋白表达水平升高；与此同时，VPA诱导的子代大鼠体内AcH3、RARα、RA、ALDH1A1的mRNA与蛋白水平、ALDH活性水平，以及ALDH1A1启动子区域的AcH3富集程度均显著降低。向VPA暴露子代大鼠给予MS-275干预后，AcH3、ALDH1A1的mRNA与蛋白水平、ALDH活性、RA水平、RARα蛋白水平，以及AcH3与ALDH1A1启动子的结合水平均显著升高；此外，GluA1蛋白水平与LTP水平降低，多数行为学缺陷得以改善。在VPA处理的子代大鼠中补充RA后，RA与RARα蛋白水平显著上调，GluA1蛋白与LTP水平下调，多数自闭症样行为缺陷得到有效逆转。

研究结论
本研究结果表明，VPA可损害ALDH1A1的组蛋白乙酰化，并下调RA-RARα信号通路。VPA对ALDH1A1的此类表观遗传修饰，可导致自闭症样突触与行为缺陷。