Recent studies have consistently demonstrated that the regulation of chromatin and gene transcription plays a pivotal role in the pathogenesis of neurodevelopmental disorders. Among many genes involved in these pathways, KMT2C, encoding one of the six known histone H3 lysine 4 (H3K4) methyltransferases in humans and rodents, was identified as a gene whose heterozygous loss-of-function variants are causally associated with autism spectrum disorder (ASD) and the Kleefstra syndrome phenotypic spectrum. However, little is known about how KMT2C haploinsufficiency causes neurodevelopmental deficits and how these conditions can be treated. To address this, we developed and analyzed genetically engineered mice with a heterozygous frameshift mutation of Kmt2c (Kmt2c^+/fs mice) as a disease model with high etiological validity. In a series of behavioral analyses, the mutant mice exhibit autistic-like behaviors such as impairments in sociality, flexibility, and working memory, demonstrating their face validity as an ASD model. To investigate the molecular basis of the observed abnormalities, we performed a transcriptomic analysis of their bulk adult brains and found that ASD risk genes were specifically enriched in the upregulated differentially expressed genes (DEGs), whereas KMT2C peaks detected by ChIP-seq were significantly co-localized with the downregulated genes, suggesting an important role of putative indirect effects of Kmt2c haploinsufficiency. We further performed single-cell RNA sequencing of newborn mouse brains to obtain cell type-resolved insights at an earlier stage. By integrating findings from ASD exome sequencing, genome-wide association, and postmortem brain studies to characterize DEGs in each cell cluster, we found strong ASD-associated transcriptomic changes in radial glia and immature neurons with no obvious bias toward upregulated or downregulated DEGs. On the other hand, there was no significant gross change in the cellular composition. Lastly, we explored potential therapeutic agents and demonstrate that vafidemstat, a lysine-specific histone demethylase 1 (LSD1) inhibitor that was effective in other models of neuropsychiatric/neurodevelopmental disorders, ameliorates impairments in sociality but not working memory in adult Kmt2c^+/fs mice. Intriguingly, the administration of vafidemstat was shown to alter the vast majority of DEGs in the direction to normalize the transcriptomic abnormalities in the mutant mice (94.3 and 82.5% of the significant upregulated and downregulated DEGs, respectively, P < 2.2 × 10⁻¹⁶, binomial test), which could be the molecular mechanism underlying the behavioral rescuing. In summary, our study expands the repertoire of ASD models with high etiological and face validity, elucidates the cell-type resolved molecular alterations due to Kmt2c haploinsufficiency, and demonstrates the efficacy of an LSD1 inhibitor that might be generalizable to multiple categories of psychiatric disorders along with a better understanding of its presumed mechanisms of action.

最近的研究一致表明，染色质和基因转录的调节在神经发育障碍的发病机制中发挥着关键作用。在参与这些途径的众多基因中，KMT2C编码人类和啮齿动物中六种已知的组蛋白 H3 赖氨酸 4 (H3K4) 甲基转移酶之一，被确定为其杂合功能丧失变异与自闭症谱系障碍有因果关系的基因。 ASD）和 Kleefstra 综合征表型谱。然而，人们对KMT2C单倍体不足如何导致神经发育缺陷以及如何治疗这些疾病知之甚少。为了解决这个问题，我们开发并分析了带有Kmt2c杂合移码突变的基因工程小鼠（Kmt2c ^+/fs小鼠），作为具有高病因学有效性的疾病模型。在一系列行为分析中，突变小鼠表现出类似自闭症的行为，例如社交性、灵活性和工作记忆障碍，证明了它们作为自闭症谱系障碍模型的表面有效性。为了研究观察到的异常的分子基础，我们对它们的成年大脑进行了转录组分析，发现 ASD 风险基因在上调的差异表达基因 (DEG) 中特别富集，而 ChIP-seq 检测到的 KMT2C 峰显着富集在上调的差异表达基因 (DEG) 中。 -定位于下调基因，表明Kmt2c单倍体不足的假定间接效应的重要作用。我们进一步对新生小鼠大脑进行了单细胞 RNA 测序，以便在早期阶段获得细胞类型解析的见解。通过整合 ASD 外显子组测序、全基因组关联和死后大脑研究的结果来表征每个细胞簇中的 DEG，我们发现放射状胶质细胞和未成熟神经元中与 ASD 相关的强烈转录组变化，并且对上调或下调 DEG 没有明显的偏向。另一方面，细胞组成没有显着的总体变化。最后，我们探索了潜在的治疗药物，并证明 Vafidemstat（一种赖氨酸特异性组蛋白去甲基酶 1 (LSD1) 抑制剂）对其他神经精神/神经发育障碍模型有效，可改善成年 Kmt2c +/fs 小鼠的社交障碍，但不能改善^工作记忆。有趣的是，vafidemstat 的施用被证明可以改变绝大多数 DEG，使突变小鼠的转录组异常正常化（显着上调和下调的 DEG 分别为 94.3% 和 82.5%，P  < 2.2 × 10 ^-16，二项式检验），这可能是行为救援背后的分子机制。总之，我们的研究扩展了具有高病因学和表面有效性的 ASD 模型库，阐明了Kmt2c单倍体不足导致的细胞类型解析分子改变，并证明了 LSD1 抑制剂的功效，该抑制剂可能适用于多种类型的精神疾病更好地理解其假定的作用机制。