葡糖脑苷脂酶转运障碍在脑衰老及帕金森病中的作用及机制研究

Brain aging is a serious challenge caused by society aging. To fight against brain aging, we need to clarify the pathophysiological mechanism involved first. As well known, aging is a high risk factor for neurodegenerative diseases, such as Parkinson's disease. Thus, researches based on Parkinson's disease may provide an effective entry point for elucidating the mechanisms of aging and exploring possible corresponding interventions. This application will examine the enzymatic activity of glucocerebrosidase (GCase) in elderly populations, patients with Parkinson's disease, and patients with GBA mutations first, and evaluate its correlation with aging process, brain metabolism, and alpha-synuclein oligomerization. Furthermore, the potential binding molecules to GCase will also be screened. In which, the molecules related to GCase trafficking will be focused on. After that, we will also detect the molecules targeting the regulation of GCase transport and localization, in both animal models and cell models of aging respectively, and then verify the above molecules (not limited to lysosomal integral membrane protein type 2，LIMP-2) involved in the mechanism of alpha-synuclein degradation. These efforts may make therapeutic interventions based on glucocerebrosidase possible to improve brain aging, not limited in Parkinson's disease.

脑衰老是老龄化所带来的严峻挑战，对抗脑衰老需要首先阐明其病理生理机制，方能有的放矢。衰老是帕金森病等神经退行性疾病发生和发展的高危因素，基于帕金森病等疾病的研究或将为阐明衰老的机制和开展相应的干预提供有效的切入点。本申请将检测老龄人群、散发帕金森病患者人群、GBA基因突变人群中葡糖脑苷脂酶（GCase）的酶活性，评估其和衰老、脑代谢功能、以及α-突触核蛋白寡聚体表达的相关性，并筛选潜在的GCase靶向结合分子。重点关注与GCase转运功能障碍相关的分子。继而，本申请将在衰老的动物模型和细胞模型中，分别预测靶向调控GCase转运与定位的分子（如清道夫受体蛋白LIMP-2等），并验证上述分子参与α-突触核蛋白降解的机制。这些工作使得基于葡糖脑苷脂酶改善脑衰老（并不局限于帕金森病）成为可能。

葡糖脑苷脂酶基因（glucocerebrosidase, GBA）是最为人所知的PD发病的风险基因，GBA编码的蛋白产物为葡糖脑苷脂酶（β-glucosidase, GCase），是一种溶酶体水解酶，该酶的功能缺陷可导致其底物葡萄糖神经酰胺的异常沉积。然而，GBA突变与帕金森病(Parkinson's disease, PD)发生发展之间的机制尚不完全清楚。细胞内新合成GCase被转运至内质网经过加工后，需要与溶酶体整合膜蛋白2（lysosomal integral membrane protein type 2, LIMP-2）结合后，转运至溶酶体后方可发挥其水解酶活性进而降解α-syn。有研究显示，LIMP-2基因敲除小鼠脑中，GCase酶活性显著降低，α-syn大量聚集，并伴随着脂质堆积、自噬-溶酶体功能的障碍。提示LIMP-2在GCase发挥溶酶体水解酶功能方面具有重要作用。然而，在GBA-PD脑中，GCase酶活性改变、自噬-溶酶体功能障碍的发生是否也与LIMP-2相关的研究目前鲜有报道。.本研究结果显示，在GBA L483P点突变的细胞系中，GCase酶活性降低，GCase与LIMP-2结合水平降低，并伴随着GCase在内质网上大量潴留。提示GCase转运障碍将会影响GCase活性，进一步导致多巴胺能神经元功能障碍。进一步构建了LIMP-2上调表达载体并转入到SNCA A53T/GBA L483P细胞中，结果显示上调LIMP-2可以促进LIMP-2与突变GCase结合，逆转L483P突变的GCase在内质网的滞留水平，缓解了内质网应激水平。LIMP-2促进GCase在溶酶体定位，增加GCase酶活性，并改善了由于GBA L483P突变导致的自噬-溶酶体功能障碍、促进α-syn的降解并改善多巴胺能神经元的功能。.综上，GBA L483P突变导致GCase转运功能障碍是GCase酶活性降低的主要原因。突变GCase与LIMP-2结合水平降低，大量滞留在ER引发内质网应激，同时自噬-溶酶体功能降低，进而引发α-syn大量聚集，多巴胺能神经元功能受损。而LIMP-2可以促进GCase转运并定位至溶酶体，缓解了内质网应激水平，改善了溶酶体功能，加剧α-syn的降解，最终发挥多巴胺能神经元的保护作用。以上提示，上调LIMP-2表达是治疗GBA-PD有前途的策略。