背景:自噬体形成的保留为解决神经系统疾病提供了有希望的策略,如帕金森病(PD)。线粒体相关的内质网(ER)膜(MAM)不仅是与各种神经系统疾病有关的焦点,而且在支持自噬体的生物发生中起着至关重要的作用。
目的:本研究旨在阐明连翘苷抗PD的神经保护特性及其促进自噬体形成的潜在机制。
方法:通过荧光染色评估ER和线粒体共定位。采用膜联蛋白V-异硫氰酸荧光素(FITC)荧光来定量线粒体表面上可接近的心磷脂(CL)。使用CL探针测定试剂盒评估SH-SY5Y细胞的MAM部分内的CL水平。利用单烷基尸胺染色来检测SH-SY5Y细胞中的自噬体形成。在A53T-α-突触核蛋白(αSyn)诱导的PD小鼠模型中,连翘苷的抗PD特性采用开放场进行评估,爬杆,和旋转杆测试,以及脑切片中TH+神经元的免疫组织化学染色。
结果:在A53T-αSyn处理的SH-SY5Y细胞中,连翘苷通过抑制CL外化和恢复MAM完整性促进自噬体形成。连翘苷增强受体表达增强蛋白1(REEP1)在MAM和线粒体内的定位,支持MAM形成。线粒体中REEP1水平增加,归因于连翘苷,增强了REEP1和NDPK-D之间的相互作用,从而减少CL外部化。此外,连翘苷表现出小鼠运动功能的剂量依赖性增强,伴随着黑质内多巴胺能神经元的丰度增加。
结论:这些发现阐明了连翘苷通过上调MAM内REEP1表达来增强MAM形成的能力,同时通过REEP1-NDPK-D相互作用衰减CL外化。这些机制支持自噬体生物发生,提供抗A53T-αSyn诱导的PD的能力。因此,我们的研究促进了对连翘苷复杂机制的理解,并强调了其作为PD治疗方法的潜力,开辟天然产物药理学的新途径。
BACKGROUND: The preservation of autophagosome formation presents a promising strategy for tackling neurological disorders, such as Parkinson\'s disease (PD). Mitochondria-associated endoplasmic reticulum (ER) membranes (MAM) serve not only as a focal point linked to various neurological disorders but also play a crucial role in supporting the biogenesis of autophagosomes.
OBJECTIVE: This investigation aimed to elucidate the neuroprotective properties of phillyrin against PD and its underlying mechanisms in promoting autophagosome formation.
METHODS: ER and mitochondria co-localization was assessed via fluorescent staining. Annexin V-fluorescein isothiocyanate (FITC) fluorescence was employed to quantify accessible
cardiolipin (CL) on mitochondrial surfaces. The levels of CL within the MAM fraction of SH-SY5Y cells were evaluated using a CL probe assay kit. Monodansylcadaverine staining was utilized to detect autophagosome formation in SH-SY5Y cells. In an A53T-alpha-synuclein (αSyn)-induced PD mouse model, the anti-PD properties of phillyrin were assessed using open field, pole climbing, and rotarod tests, as well as immunohistochemistry staining of TH+ neurons in the brain sections.
RESULTS: In A53T-αSyn-treated SH-SY5Y cells, phillyrin facilitated autophagosome formation by suppressing CL externalization and restoring MAM integrity. Phillyrin enhanced the localization of receptor expression-enhancing protein 1 (REEP1) within MAM and mitochondria, bolstering MAM formation. Increased REEP1 levels in mitochondria, attributed to phillyrin, enhanced the interaction between REEP1 and NDPK-D, thereby reducing CL externalization. Furthermore, phillyrin exhibited a dose-dependent enhancement of motor function in mice, accompanied by an increase in the abundance of dopaminergic neurons within the substantia nigra.
CONCLUSIONS: These findings illuminate phillyrin\'s ability to enhance MAM formation through upregulation of REEP1 expression within MAM, while concurrently attenuating CL externalization via the REEP1-NDPK-D interaction. These mechanisms bolster autophagosome biogenesis, offering resilience against A53T-αSyn-induced PD. Thus, our study advances the understanding of phillyrin\'s complex mechanisms and underscores its potential as a therapeutic approach for PD, opening new avenues in natural product pharmacology.