DDOD综合征小鼠模型听觉功能补偿和学习记忆障碍分子机制及治疗研究

DDOD (Dominant deafness-onychodystrophy，MIM 124480) syndrome is a kind of rare disease. The pathogenetic gene was identified as ATP6V1B2 by the applicant’s team. The common c.1516C>T mutation in ATP6V1B2 was reported in patients diagnosed DDOD syndrome worldwide. Through a 7-year-follow-up after cochlear implantation operation and systematic phenotype analysis in DDOD syndrome patients, the dysfunctions of learning and memory were discovered. Our previous work in zebrafish showed that atp6v1b2 is a housekeeping gene which plays important roles in the development. Knockdown of atp6v1b2 resulted in hair cell loss, pericardial oedema, a non-inflated swim bladder, shorter pectoral fins and impairment of angiogenic vessel growth. We then constructed the Atp6v1b2 c.1516C>T mutation knockin mouse model. The major phenotypes of the knockin mouse model include dysfunctions of learning and memory, idiopathic epilepsy and normal hearing. In this study, we will investigate the mechanism of hearing compensation, the pathological basis of dysfunctions of learning and memory in the knockin mouse model to clarify the regulatory factors and its threshold effect of Atp6v1b2 in different organs. The iPS cells derived from skin mechanocytes of DDOD syndrome patients will be constructed, mutation corrected and directionally differentiated. The biotherapy for DDOD syndrome will then be explored. The meaning of this study lies in two aspects, the phenotype expansions and pathogenesis illumination for DDOD syndrome. The results will provide theoretical basis for precise treatment and prognosis evaluation.

DDOD综合征又称显性遗传耳聋-甲发育不全综合征，其致病基因ATP6V1B2 由申请人团队鉴定，已报道的国内外所有患者均携带该基因c.1516C>T突变。对DDOD综合征患者进行人工耳蜗术后7年随访，发现其言语康复效果欠佳，经过系统表型再评估证实患者存在学习记忆障碍。我们前期在斑马鱼中研究了atp6v1b2基因对发育的影响，明确其与听觉、脑发育、四肢、血管生成等有关，为管家基因；构建了Atp6v1b2 c.1516C>T突变knockin小鼠模型，该模型表现为听力正常、学习记忆障碍和自发性癫痫。本课题拟研究该模型小鼠听觉补偿机制、学习记忆障碍的病理基础，分析管家基因ATP6V1B2异常在不同器官的功能调控和阈值效应；构建患者皮肤成纤维细胞来源的iPS细胞并进行突变修复和定向分化，探讨生物学治疗方案。本研究意义在于拓展DDOD综合征表型特征、明确其发病机制，为精准治疗、预后评估提供理论依据。

通过构建耳聋甲发育不全（DDOD）综合征基因突变敲入小鼠模型Atp6v1b2 Arg506X/ Arg506X并对其进行表型评估及功能性磁共振检查，发现小鼠模型学习记忆能力低下且合并大脑结构异常，在海马、嗅球、杏仁核等与学习记忆密切相关的脑区存在功能连接异常。进一步研究显示：模型小鼠海马CA1区神经元个数显著降低，上述突变引起组成V-ATPase的B2与V1E2亚基间相互作用减弱，影响V-ATPase的组装，进而导致蛋白功能异常。.在药物治疗方面，通过解析致病通路筛选治疗药物。借助听觉功能及溶酶体功能研究，申请人首先将Atp6v1b2 Arg506X/ Arg506X小鼠耳聋病理部位定位于内耳螺旋神经节，随后发现c.1516 C＞T突变导致螺旋神经元的溶酶体pH值升高，溶酶体降解功能降低，细胞内代谢产生的自噬小体无法被正常消化而出现堆积，即自噬流阻滞。自噬流阻滞进一步造成抗凋亡因子Bcl-2降解，促凋亡蛋白Bax被激活，活化的Bax结合到线粒体使线粒体膜的通透性增大，细胞色素C从线粒体释放到细胞质内，促进了凋亡复合体的形成。后者激活Caspase-3，启动细胞凋亡，最终导致听觉功能异常。针对这一通路，本团队筛选出抑制凋亡药物BIP-V5（Bax抑制蛋白V5型）。Atp6v1b2 Arg506X/ Arg506X小鼠自4周龄开始接受BIP-V5腹腔注射治疗，40周时无论听力表型还是病理改变都得到了改善。该研究为DDOD综合征的药物治疗提供新的视角。.在干细胞治疗方面，本课题通过使用非整合质粒递送系统，从DDOD综合征患者外周血单核细胞中制备了ATP6V1B2 p.Arg506X诱导多能干细胞系（iPSC，CPGHi002-A），该细胞系具有正常的核型及分化多能性。随后运用CRISPR/Cas9技术对该细胞系进行了基因矫正（CPGHi002-A-1），为综合征型耳聋患者的基因治疗奠定了基础。

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