Disrupted extracellular matrix and cell cycle genes in autism-associated Shank3 deficiency can be rescued with Lithium

The Shank3 gene encodes the major postsynaptic scaffolding protein SHANK3. Its mutation causes a syndromic form of autism spectrum disorder (ASD): Phelan-McDermid Syndrome (PMDS). It is characterized by global developmental delay, intellectual disorders (ID), ASD behavior, affective symptoms, as well as extra-cerebral symptoms. Although Shank3 deficiency causes a variety of molecular alterations, they do not suffice to explain all clinical aspects of this heterogenic syndrome. Since global gene expression alterations in Shank3 deficiency remain inadequately studied, we explored the transcriptome in vitro in primary hippocampal cells from Shank3∆11(-/-) mice, under control and lithium (Li) treatment conditions, and confirmed the findings in vivo. The Shank3∆11(-/-) genotype affected the overall transcriptome. Remarkably, extracellular matrix (ECM) and cell cycle transcriptional programs were disrupted. Accordingly, in the hippocampi of adolescent Shank3∆11(-/-) mice we found proteins of the collagen family and core cell cycle proteins downregulated. In vitro Li treatment of Shank3∆11(-/-) cells had a rescue-like effect on the ECM and cell cycle gene sets. Reversed ECM gene sets were part of a network, regulated by common transcription factors (TF) such as cAMP responsive element binding protein 1 (CREB1) and β-Catenin (CTNNB1), which are known downstream effectors of synaptic activity and targets of Li. These TFs were less abundant and/or hypo-phosphorylated in hippocampi of Shank3∆11(-/-) mice and could be rescued with Li in vitro and in vivo. Our investigations suggest the ECM compartment and cell cycle genes as new players in the pathophysiology of Shank3 deficiency, and imply involvement of transcriptional regulators, which can be modulated by Li. This work supports Li as potential drug in the management of PMDS symptoms, where a Phase II study is ongoing. Primary hippocampal cells from P1-2 mice of WT and KO genotype were culutered for 14 days and received either vehicle or lithium treatment at day 9. The experiment was replicated 4 times. Sequencing was performed in 2 batches.

Shank3基因编码主要的突触后支架蛋白SHANK3。该基因的突变会引发一种综合征型自闭症谱系障碍（Autism Spectrum Disorder, ASD）——费伦-麦克德米德综合征（Phelan-McDermid Syndrome, PMDS）。该综合征以全面发育迟缓、智力障碍（Intellectual Disorders, ID）、自闭症谱系行为、情感症状以及脑外症状为特征。尽管Shank3缺失会引发多种分子层面的改变，但这些改变尚不足以解释该异质性综合征的全部临床表型。鉴于目前对Shank3缺失状态下的全基因表达改变研究仍不充分，本研究在对照及锂盐（Lithium, Li）处理条件下，对Shank3∆11(-/-)小鼠的原代海马细胞进行了体外转录组分析，并在体内验证了相关发现。Shank3∆11(-/-)基因型会整体影响转录组谱。值得注意的是，细胞外基质（Extracellular Matrix, ECM）与细胞周期的转录程序均遭到破坏。相应地，在青春期Shank3∆11(-/-)小鼠的海马组织中，我们发现胶原蛋白家族蛋白与核心细胞周期蛋白的表达均出现下调。对Shank3∆11(-/-)细胞进行体外锂盐处理后，其对细胞外基质与细胞周期基因集呈现出类似挽救的调控效果。发生表达逆转的细胞外基质基因集隶属于一个由共同转录因子（Transcription Factor, TF）调控的基因网络，这些转录因子包括cAMP应答元件结合蛋白1（cAMP responsive element binding protein 1, CREB1）与β-连环蛋白（β-Catenin, CTNNB1）——二者均为已知的突触活动下游效应因子，同时也是锂盐的作用靶点。在Shank3∆11(-/-)小鼠的海马组织中，这些转录因子的表达丰度较低，且/或存在磷酸化不足的情况，而体外及体内的锂盐处理均可挽救这一异常。本研究表明，细胞外基质区域与细胞周期基因是Shank3缺失病理生理学机制中的新参与因子，同时提示可被锂盐调控的转录调控因子参与了该过程。本研究支持锂盐可作为治疗PMDS症状的潜在药物，目前相关II期临床试验正在进行中。本研究获取P1-2龄野生型（Wild Type, WT）与敲除型（Knock Out, KO）小鼠的原代海马细胞，体外培养14天，并于第9天时分别给予溶剂对照或锂盐处理。本实验重复4次，测序分为2批进行。