CCNK基因变异通过CDK(12/13)/CCNK复合物导致智力障碍的分子机制研究

Intellectual disability (ID) is one of the most common birth defects, the research on genetic etiologies and pathogenesis is a major issue of public health. In our previous studies, we found that variations in the CCNK gene were associated with syndromic intellectual disability, so we firstly demonstrated that reduced expression of CCNK would cause neurodysplasia in CCNK knockdown zebrafish, but the pathogenesis remained unknown. Further study found that down-regulation of CCNK might affect CDK(12/13)/CCNK complexes. In consideration of the important roles of CDK12 and CDK13 in regulating RNA polymerase II, we propose that CCNK variations might reduce the level of Pol II transcriptional regulation by affecting CDK/CCNK complexes, leading to down-regulated expression of CDK5 and DDR related genes associated with neurodevelopment, resulting in abnormity of axon extension, synaptic development and neuronal migration, and promoting neuronal apoptosis. We intend to investigate the influences on the CDK/CCNK complexes and Pol II transcriptional regulation caused by CCNK variations in vitro and vivo. Moreover, rescue experiments will be performed on important targets. The study will illuminate the underlying mechanism of ID caused by CCNK variations, and provide possible therapeutic targets.

智力障碍是儿童最常见的出生缺陷之一，研究其病因及发病机制是关乎民生的重大问题。我们前期首次发现CCNK基因变异与患儿综合征性智力障碍密切相关，并在ccnk敲减斑马鱼模型中证实其表达下调会导致神经发育异常，但其致病机制不明。进一步研究发现CCNK表达下调会影响CDK(12/13)/CCNK复合物，鉴于CDK12、CDK13在调控Pol II中的重要作用，我们推测CCNK基因变异可能通过影响CDK/CCNK复合物，降低Pol II转录调控水平，从而引起下游神经发育关键基因CDK5及DDR相关基因表达下调，最终导致轴突延伸、突触发育和神经元迁移异常，同时促进神经细胞凋亡，从而导致智力障碍。本项目拟在体内外模型中探索CCNK基因变异对CDK/CCNK复合物、Pol II转录调控水平及神经发育等方面的影响及机制，并对所发现的重要靶点进行干预，以明确CCNK基因变异导致智力障碍的机制及可能的干预靶点。

CCNK基因（编码细胞周期蛋白K）致病变异可导致CCNK相关神经发育障碍综合征, 该综合征的基因型-表型谱和潜在的致病机制仍然不清楚,是亟待解决的临床和科学问题。为了探索CCNK基因变异影响神经发育的潜在机制，我们建立了患者(携带CCNK基因致病变异)来源的多能干细胞(induced pluripotent stem cells , iPSCs)和神经祖细胞(neural progenitor cells ,NPC)模型。此外，我们构建了NPC特异性的Ccnk敲除(knockout, KO)小鼠模型，并进行了分子和组织形态学分析。我们发现患者来源的NPC模型和Ccnk KO小鼠模型均出现NPC增殖受损和凋亡细胞增多的现象。随后，我们将上述体内外模型进行转录组学分析，揭示了CCNK相关综合征特有的转录组特征，其中包括对NPC增殖和凋亡可能至关重要的WNT5A基因。进一步，为了证实WNT5A的可能作用，我们体内外模型中均进行了挽救实验。我们发现Wnt5a抑制剂可显著改善患者来源NPC的增殖受损和胚鼠发育皮层来源NPC细胞的凋亡增加，以上结果表明Wnt5a可能是一种很有前途的疾病干预靶点。