癫痫持续状态后树突内F-actin杆状聚集的成因及细胞骨架-内体-溶酶体途径引发神经元死亡的机制

After status epilepticus, some neurons in the brain are destined for death after a silent period, but the underlying mechanisms are still unknown. Before death, the dendritic spines of impaired neurons are seriously reduced, and the F-actin cytoskeleton within the spines becomes progressively depolymerized. We recently found that F-actin was transiently aggregated into rod-like structures in the dendritic shafts after its disappearance from the dendritic spines. Since F-actin is anchored to the microtubules within the dendritic shafts, we propose that the depolymerization and rod-like aggregation of F-actin may be due to the decontruction of dendritic microtubules. Moreover, the excitotoxic damage due to status epilepticus results in abnormally enhancement of endocytosis, and the rod-like aggregation of F-actin may be partially related to the translocation of endosome-associated F-actin into dendrites. Since the formation and transport of endosomes rely highly on the intact cytoskeletons, the depolymerization of F-actin and microtubules may result in damages to the endosome-lysosome system, and ultimately lead to neuronal death. This project is aimed to investigate the origins of the rod-like accumulation of dendritic F-actin and make clear the mechanisms of neuronal death by the cytoskeleton-endosome-lysosome pathway after status epilepticus, by which we hope to provide a new theoretical basis for epilepsy treatment.

癫痫持续状态后脑内神经元出现迟发性死亡，其机制尚不清楚。神经元死亡之前，树突棘广泛缺失，树突棘内F-actin骨架发生不可逆性解聚。我们发现F-actin从树突棘消失后会短暂聚集在树突干内形成特殊的杆状结构。以此为线索，我们注意到癫痫脑内F-actin损伤、微管解聚和内体-溶酶体异常三种现象共存并相互影响。树突干内微管解聚使F-actin丧失了锚定和调节位点，可能是树突棘塌陷和F-actin杆状聚集的主要原因；另一方面，癫痫放电后突触后膜内吞活动增强，F-actin随着内吞小泡进入树突内也会导致树突干F-actin聚集现象。更重要的是，内吞物质从内体到溶酶体的转运过程高度依赖F-actin和微管，后两者解聚势必会导致内体-溶酶体功能紊乱引发神经元死亡。因此，本项目拟采用体内外实验阐明树突干内F-actin杆状聚集的成因，探讨F-actin和微管解聚通过内体-溶酶体途径引起神经元死亡的机制。

癫痫持续状态和全脑缺血均会在敏感脑区引起神经元迟发性死亡，本项目提出假说，认为迟发性神经元死亡内F-actin损伤、微管解聚和内体-溶酶体系统异常共存并互相影响。该项目通过两种动物模型及一种体外神经元死亡模型，借助共聚焦显微镜、电子显微镜、共定位分析、蛋白质分离、以及脂类分析等技术，对上述假说进行了验证，并探讨稳定细胞骨架防治神经元死亡的可能性。结果发现1）迟发性神经元死亡过程中F-actin短暂性聚集形成的杆状结构发生在树突干内，树突棘F-actin损伤与树突干内微管解聚密切相关；2）神经元发生迟发性死亡过程中树突棘内F-actin降解，导致对应的突触前成分发生代偿性增大。因此尽管突触前成分数目相对下降，但是突触前成分的面积分数变化并不显著；3）神经元死亡与细胞骨架损伤密切相关，增强细胞骨架稳定性在一定程度上可以阻止或延缓神经元死亡，但是过度稳定细胞骨架会干扰神经元正常功能，加重神经元损伤；4）神经元内膜性细胞器官的分布和定位与细胞骨架的完整密切相关，神经元迟发性死亡过程中细胞骨架降解伴随着内体-自噬体-溶酶体系统发生紊乱，神经元的细胞体和近端树突内膜性成分相应增多；5）由于内体-自噬体-溶酶体系统功能异常，神经元的内膜系统结构的结构发生破坏，磷脂分解，释放出大量游离脂肪酸。这导致濒死或死亡神经元对质膜探针的亲和性显著增加。上述结果显示迟发性神经元死亡过程中细胞骨架损伤、内体-溶酶体系统功能紊乱以及质膜系统异常之间关系密切，为治疗和检测癫痫或脑缺血诱导的神经元损伤提供新的线索。