利用斑马鱼模型研究人类腺苷酸琥珀酸裂解酶缺陷疾病

Adenylosuccinate lyase (ADSL) deficiency is a rare recessive neurological disorder with psychomotor retardation, features of autism, and epilepsy, leading to early death of the patients. ADSL is the causative gene of this disease. Due to the fact that ADSL is a bi-functional enzyme which catalyzes two steps of de novo purine synthesis, two intermediate products, SAICAR and S-AMP, would theoretically accumulate as their de-phosphate form SAICAr and S-Ado in the CSF and urine of patients. Two hypotheses were proposed to explain the onset of the symptoms, i.e., lacking of normal level of purine which could result in a shortage of ATP and low levels of basic ribonucleoside and ribonucleotide and impair basic cellular activities such as DNA duplication and mRNA transcription, or accumulation of the two intermediates which could exert toxic effects on embryonic development. However, neither of these two hypotheses has been supported by clinical observations or laboratory experimental results. Measurement of ATP levels in cultured fibroblast cells from patients showed no difference comparing with normal people, and there seems no direct correlation between the severity of the syndrome and concentration of the intermediates. Unfortunately, there is no animal disease model available to further dissect this kind of disease and zebrafish is an ideal vertebrate model for this purpose. Here, we will first generate ADSL deficiency model in zebrafish by using both morpholino knock-down and TALEN mediated gene targeting approaches and then investigate the embryonic developmental defects and mechanisms of ADSL deficiency as well as attempt to screen for potential molecules for effective therapeutic treatment for this disease.

腺苷酸琥珀酸裂解酶缺陷（ADSL deficiency）是一种罕见的单基因隐性遗传病，患者往往出现一系列机能紊乱，包括运动神经发育迟滞、癫痫、自闭及肌肉无力等，最终导致患者在新生儿到青少年期间死亡。ADSL催化嘌呤从头合成途径中的两步关键反应，该酶缺失会造成AMP缺乏以及SAICAr和S-Ado两种中间产物累积等两方面的后果，但是，目前并不清楚其详细致病机制，也缺乏有效的治疗手段，原因之一是缺乏ADSL deficiency的动物模型。本项目拟通过morpholino knock down和TALEN定点突变等方法建立模拟人类ADSL缺陷的斑马鱼疾病模型，并通过跟其上游基因腺苷酸琥珀酸合成酶的斑马鱼突变体相结合，研究ADSL deficiency的致病机理，并进一步通过一系列神经系统GFP荧光标记的斑马鱼，从发育的角度剖析引发该病症的生理机制，从而为改进临床治疗的思路和手段提供指导与依据。

腺苷酸琥珀酸裂解酶缺乏症（ADSL deficiency）是腺苷酸琥珀酸裂解酶（adenylosuccinate lyase，ADSL）基因发生突变所导致的一种罕见的单基因隐性遗传病，主要症状表现为智力发育迟滞、癫痫和自闭。根据ADSL酶的功能，人们推测其致病机理可能有如下两种，一是由于嘌呤合成受阻导致腺嘌呤单磷酸核苷酸（AMP）缺乏，另一种是由于代谢中间产物（SAICAr和S-Ado）积累导致的毒性反应。但是，这两种猜测均与已有的实验结果存在一定的矛盾，目前并不清楚其详细致病机制，也缺乏有效的治疗手段，原因之一是缺乏ADSL deficiency的动物模型。本项目利用adssl的突变体以及抑制adssl或adsl表达的吗啉环反义寡核苷酸（morpholino，MO），建立了模拟人类ADSL缺乏症的斑马鱼模型，并分析了其发病机制。敲低adsl后，胚胎在48 hpf（hours post fertilization）出现头面部发育迟滞，并且围心腔出现水肿。这与敲低adssl后或adssl突变体的表型类似，并可以被注射AMP部分拯救。LC-MS和HPLC检测结果显示，adsl 的下调导致AMP的浓度显著下降。行为检测发现adsl和adssl 的基因表达下调均导致胚胎出现平时不活跃和短时间过度活跃的行为异常。通过转基因标记鱼系，我们发现在adsl和adssl 缺失的胚胎中单胺类神经元和GABA能神经元的发育有缺陷。与Adsl相比，Adss活性缺失仅会影响AMP的合成，不会有中间产物的累积。这两个基因表达下调后出现相似的表型，说明ADSL deficiency的症状很有可能是由于AMP在胚胎早期发育中阶段性供应不足所导致，而非中间产物的积累造成。据此推测，ADSL deficiency很可能是由于胚胎早期发育阶段缺乏AMP而造成神经系统的发育缺损，自闭和癫痫等症状是神经系统发育缺陷的病理反映。根据本项目的研究结果，我们认为ADSL deficiency患者发病时已经造成了神经损伤，此时已很难设计药物来逆转这些损伤，因此，对ADSL deficiency疾病宜采取预防为主的策略，最好是通过产前诊断筛查潜在的突变胚胎，尽早采取适当措施，或者终止妊娠，或者尽早干预，尽量在早期胚胎发育过程中适量补充AMP，阻止由于AMP缺乏所造成的神经损伤。