结旁区静止线粒体通过线粒体-内质网桥接影响血管性脱髓鞘的机制探索

The major pathological changes of cerebral white matter lesions (WML) induced by small vascular disease and chronic hypoperfusion is demyelination. Different with autoimmune demyelination, The WML demyelination might be the secondary change to axonal damage. Our previous study showed that chronic hypoperfusion can increase axonal mitochondrial docking accompanied with demyelination, however the causal relationship between the two phenomena and the underlying molecular mechanism need to be further studied. Therefore, we put forward the hypothesis: under WML condition, increased docking mitochondria in axonal paranode area is the early change of demyelination, and dysfunction of mitochondria may occur due to swollen and abnormal metablism over time. Then the abnormal mitochondria lead to a reduction in the number of Mitochondria-associated ER membrane(MAM). The acetylation state of Caspr is necessary for maintaining stability of Caspr and Contactin, sustaining stability of myline. Under WML condition, the reduction of MAM induced the activation of EndoH, which then increase deacetylation of Caspr by EndoH, eventually lead to dissociation of Caspr and Contactin and myelin instability. To test this hypothesis, we will use in vitro DRG cells culture and bilateral common carotid artery stenosis (BCAS) animal model. By molecular modulation of mitochondria docking proteins, we will investigate the important role of axonal docking mitochondria in maintenance of myelin stability. From the novel point of view that axonal mitochondria may regulate myelin stability, we will study the mechanism of WML induced demyelination. This will help us interpret the neural function defect caused by the WML and provide a new sight for clinical intervention.

慢性颅内低灌注所致的脱髓鞘是脑白质损伤(WML)的主要病理改变。和免疫性脱髓鞘不同，WML髓鞘脱失可能是轴突损害的继发结果。我们发现WML模型中，伴随着髓鞘脱失，神经元轴突结旁区线粒体存在异常堆积和结构异常，但二者的因果关系和机制不明。我们提出假说: 慢性低灌注状态下，神经元轴突内线粒体轴浆流发生异常，线粒体异常锚定增多。郎飞氏结结旁区堆积的线粒体功能障碍将导致线粒体-内质网桥接减少，进而导致内质网功能障碍。分布于内质网的糖苷酶Endo H进而被激活，介导Caspr蛋白质N端脱糖基化，引起结旁区髓鞘稳定复合物Capsr和Contactin相互黏附作用下降，继而出现血管性脱髓鞘现象。为验证假说，拟采用体外有髓背根神经培养和BCAS动物模型，通过分子手段干预线粒体锚定蛋白，探讨线粒体锚定在维持髓鞘结构稳定性中的作用。本研究将从轴突内线粒体锚定影响脱髓鞘这一新视角展开研究，阐释WML病理机制。

慢性颅内低灌注所致的脱髓鞘是脑白质损伤(WML)的主要病理改变，我们发现WML模型中，伴随着髓鞘脱失，神经元轴突内线粒体存在异常堆积和结构异常，但二者的因果关系和机制不明。本研究采用体外小脑脑片培养和BCAS动物模型，通过分子生物学手段，探讨轴突内线粒体在维持髓鞘结构稳定性中的作用。研究发现慢性低灌注状态下，神经元轴突内线粒体轴浆流发生异常，线粒体锚定增多，运动减少。轴突内堆积的线粒体形态异常，长度增加，直径减小，线粒体功能障碍释放大量ROS。轴突内大量ROS引起髓鞘复合物稳定性下降，继而出现血管性脱髓鞘。在BCAS模型利用SNPH-shRNA AAV下调SNPH表达或利用SNPH-shRNA转染脑片下调SNPH表达，发现下调SNPH表达后轴突内线粒体锚定减少，ROS减少，髓鞘结构相关蛋白表达增加。而利用慢病毒过表达SNPH，则可增加轴突内静止线粒体数量，加剧脱髓鞘。为进一步证实ROS介导了线粒体锚定对脱髓鞘的影响，实验进一步采用ROS抑制剂 MitoQ抑制ROS水平，结果发现MitoQ可部分逆转SNPH过表达引起的髓鞘结构破坏。为促进结果的应用和推广，研究进一步探索了丁苯酞在WML中的治疗作用。结果发现，丁苯酞可通过抑制SNPH水平，继而减轻慢性颅内低灌注状态下神经元轴突内线粒体堆积，减轻ROS水平，发挥神经保护作用。本研究从轴突内线粒体锚定影响脱髓鞘这一视角阐释了WML病理新机制。