DNAJC12基因突变通过芳香族氨基酸羟化酶介导的智力障碍机制研究

近年发现DNAJC12基因突变可导致智力障碍，血苯丙氨酸升高，脑脊液多巴胺和5-羟色胺降低，现有治疗手段无法完全控制症状。研究提示DNAJC12基因突变可导致苯丙氨酸羟化酶活性下降，DNAJC12与酪氨酸羟化酶、色氨酸羟化酶、Hsp70可分别免疫共沉淀，因此我们推测DNAJC12基因突变通过分子伴侣方式影响Hsp70ATP酶活性，导致芳香族氨基酸羟化酶（AAAHs）稳态异常，影响神经递质代谢，进而导致智力障碍。在前期研究基础上，本项目将利用CRISPR/Cas9技术构建DNAJC12基因敲除细胞和动物模型，应用qRT-PCR、Western blot、HPLC、圆二色谱法、免疫共沉淀等技术研究DNAJC12基因突变对Hsp70ATP酶活性、AAAHs折叠和降解及神经发育的影响，明确DNAJC12基因突变是否通过影响Hsp70ATP酶活性间接导致AAAHs功能下降，为该病的靶向治疗提供依据。

Intellectual disability (ID) is a severe and common birth defect. Recently it is found that DNAJC12 mutation can cause an intellectual disability syndrome，but the mechanism still remain unclear. Patients with DNAJC12 mutations presented elevated blood phenylalanine concentration and low dopamine and serotonin in the cerebrospinal fluid indicating that the intellectual disability is related to the abnormal metabolism of aromatic amino acids. Further study demonstrate that DNAJC12 genetic mutations lead to lower phenylalanine hydroxylase activity. DNAJC12 protein binds to tyrosine hydroxylase or tryptophan hydroxylase, respectively, as shown by co-immunoprecipitation experiment. Hsp70 also interacts with DNAJC12 as detected in protein complex. The objective of this study is to investigate whether DNAJC12 mutations affect Hsp70 ATPase activity, the latter may lead to the unfolding and degradation of aromatic amino acid hydroxylases. Human cell lines and mouse models with DNAJC12 knockout and mutations will be established. The effect of DNAJC12 mutation on Hsp70 ATPase activity and steady state of aromatic amino acid hydroxylase will be investigated by using qRT-PCR, Western blot, Circular dichroism in these in vivo and in vitro models, Intellectual disability will be evaluated in mouse models as well as whether it is caused by abnormal aromatic amino acid metabolism. Our study would clarify the mechanism of how DNAJC12 gene mutations lead to mental intellectual disability and reveal possible intervention targets.