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. 2022 Jan 20;42(1):101-107.
doi: 10.12122/j.issn.1673-4254.2022.01.12.

[Mechanism of valproic acid-induced dendritic spine and synaptic impairment in the prefrontal cortex for causing core autistic symptoms in mice]

[Article in Chinese]
F Wang  1 , L Wang  1 , Y Xiong  1 , J Deng  1 , M Lü  2 , B Tang  3 , X Zhang  3 , Y Li  1
Affiliations

[Mechanism of valproic acid-induced dendritic spine and synaptic impairment in the prefrontal cortex for causing core autistic symptoms in mice]

[Article in Chinese]
F Wang et al. Nan Fang Yi Ke Da Xue Xue Bao. .

Abstract
in English, Chinese

Objective: To investigate the mechanism of valproic acid (VPA) -induced impairment of the dendritic spines and synapses in the prefrontal cortex (PFC) for causing core symptoms of autism spectrum disorder (ASD) in mice.

Methods: Female C57 mice were subjected to injections of saline or VPA on gestational days 10 and 12, and the male offspring mice in the two groups were used as the normal control group and ASD model group (n=10), respectively. Another 20 male mice with fetal exposure to VPA were randomized into two groups for stereotactic injection of DMSO or Wortmannin into the PFC (n=10). Open field test, juvenile play test and 3-chamber test were used to evaluate autistic behaviors of the mice. The density of dendrite spines in the PFC was observed with Golgi staining. Western blotting and immunofluorescence staining were used to detect the expressions of p-PI3K, PI3K, p-AKT, AKT, p-mTOR, mTOR and the synaptic proteins PSD95, p-Syn, and Syn in the PFC of the mice.

Results: Compared with the normal control mice, the mice with fetal exposure to VPA exhibited obvious autism-like behaviors with significantly decreased density of total, mushroom and stubby dendritic spines (P < 0.05) and increased filopodia dendritic spines (P < 0.05) in the PFC. The VPA-exposed mice also showed significantly increased expressions of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR (P < 0.01) and lowered expressions of PSD95 and p-Syn/Syn in the PFC (P < 0.05 or 0.001). Wortmannin injection into the PFC obviously improved the ASD-like phenotype and dendritic spine development, down-regulated PI3K/Akt/mTOR signaling pathway and up-regulated the synaptic proteins in VPA-exposed mice.

Conclusion: In male mice with fetal exposure to VPA, excessive activation of PI3K/Akt/mTOR signaling pathway and decreased expressions of the synaptic proteins PSD95 and p-Syn cause dendritic spine damage and synaptic development disturbance in the PFC, which eventually leads to ASD-like phenotype.

目的: 探讨前额叶皮层（PFC）树突棘及突触发育障碍，诱发自闭症（ASD）小鼠核心症状的机制。

方法: 将C57小鼠按照完全随机设计，分为正常对照组（CON组），ASD模型组（VPA组），溶剂对照组（DMSO组）和Wortmannin抑制剂组（Wortmannin组），10只/组。旷场实验，青年玩耍实验和三箱社交实验评价小鼠的ASD样行为；高尔基染色观察小鼠PFC树突棘密度的变化；Western blot检测小鼠PFC信号通路相关蛋白p-PI3K、PI3K、p-AKT、AKT、p-mTOR、mTOR以及突触相关蛋白PSD95、pSyn、Syn的表达；免疫荧光染色观察PSD95、p-Syn的变化。

结果: 与CON组相比，VPA组小鼠出现了ASD样表型，PFC总的、成熟型树突棘密度减少（P < 0.05），未成熟型树突棘密度增加（P < 0.01），信号通路相关蛋白p-PI3K/PI3K、p-AKT/AKT和p-mTOR/ mTOR表达上调（P < 0.05），突触相关蛋白PSD95和p-Syn/Syn表达下调（P < 0.01或P < 0.001）。Wortmannin能改善小鼠ASD样表型，改善树突棘发育，下调PI3K/Akt/mTOR信号通路及上调突触相关蛋白的表达。

结论: PI3K/Akt/mTOR信号通路的过度激活和突触相关蛋白PSD95、p-Syn表达降低可能导致VPA诱导的ASD小鼠PFC树突棘损伤和突触发育障碍，最终导致ASD样表型。

Keywords: PI3K/Akt/mTOR; PSD95; Syn; autism; dendritic spine; synaptic.

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Figures

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1
小鼠旷场实验结果 Open-field test in the 4 groups (n=6-8). A: Open-field test. B: Time of self-grooming. C: Numbers of cross center grid. D: Numbers of cross grid. E: Numbers of climbing. F: Numbers of Vertical. *P < 0.05, ***P < 0.001 vs CON group; #P < 0.05 vs VPA group.
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小鼠青年玩耍实验结果 Juvenile play test in the 4 groups (n=6-8). A: Juvenile play test. B: Times of social interaction. C: Times of digging. ***P < 0.001 vs CON group; #P < 0.05 vs VPA group.
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小鼠三箱社交实验结果 Three-chamber sociability test in the 4 groups (n=6-8). A: Social interaction test. B: Novelty preference test. C: Time spent in A cage, B cage and interaction time with stranger 1 mice and object. D: Interaction time with stranger 1 mice and stranger 2 mice. *P < 0.05, **P < 0.001 vs CON group; #P < 0.05 vs VPA group.
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小鼠PFC高尔基染色结果 Results of Golgi-Cox staining in the PFC neurons in the 4 groups (30 dendrites from 3 mice). A: Microscopic images of the dendritic spines (Scale bar: 10 μm). B-E: Spine density of the total and each subtype. *P < 0.05, **P < 0.01, ***P < 0.001 vs CON group. ##P < 0.01, ###P < 0.001 vs VPA group.
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小鼠PFC中PI3K/AKT/mTOR信号通路相关蛋白表达水平 Expression level of proteins related to PI3K/AKT/mTOR signaling pathway (n=3). A: Western blots of p-PI3K, PI3K, p-AKT, AKT, p-mTOR, and mTOR. B-D: Relative expression levels of p-PI3K, p-AKT, and p-mTOR. *P < 0.05, ***P < 0.001 vs CON group. #P < 0.05, ##P < 0.01 vs VPA group.
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小鼠PFC突触相关蛋白表达水平 Expression levels of synaptic proteins (n=3). A: Western blots of PSD95, p-Syn, and Syn. B, C: Relative expression levels of PSD95 and p-Syn. D: Immunofluorescence staining of PSD95 (Original magnification: ×1000). E: Immunofluorescence staining of p-Syn (×400). **P < 0.01, ***P < 0.001 vs CON group, #P < 0.05, ##P < 0.01 vs VPA group.
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