背景:细胞间的通讯对于组织的反应至关重要,适应,并在盛行的环境中茁壮成长。几种机制介导细胞间信号,包括隧道纳米管,间隙连接,和细胞外囊泡(EV)。根据当地和系统条件,电动汽车可能含有促进生存的货物,神经保护,或病理学。我们对病理性细胞间信号传导的理解已经通过使用源自人类多能干细胞(hPSC)的神经元的疾病模型得到了支持。
方法:这里,我们使用hPSC衍生的视网膜神经节细胞(hRGC)和小鼠视觉系统来研究调节EV产生对细胞间运输和细胞存活的影响。我们通过抑制中性鞘磷脂酶(nSMase)降低鞘磷脂分解代谢成神经酰胺,探讨了EV调节对细胞存活的影响,使用GW4869。我们通过探测膜联蛋白A5,磷脂酰丝氨酸,可行的线粒体,和线粒体活性氧。在体内,我们进行了GW4869眼内注射,并测量了RGC和上丘神经元密度以及RGC顺行轴突转运。
结果:用GW4869给药hRGC24小时后,我们发现nSMase的抑制降低了神经酰胺并增强了GM1神经节苷脂的积累。这种抑制作用也降低了小型电动汽车的密度,增加了大型电动汽车的密度,富集了促凋亡蛋白,附件A5。降低nSMase活性增加hRGC凋亡起始,由于凋亡颗粒的密度和摄取增加,由膜联蛋白A5结合磷脂鉴定,磷脂酰丝氨酸。我们通过开发GW4869处理和未处理的hRGC的共培养系统来测定线粒体的细胞间运输。在处理过的细胞中,抑制nSMase减少了活线粒体的数量,不仅在治疗中驱动线粒体活性氧,而且在共培养中添加了幼稚的hRGC。在老鼠身上,在玻璃体内单次注射GW4869后20天,我们发现上丘中RGC及其轴突受体神经元的显著损失。随后,顺行RGC轴突向丘的运输急剧减少。
结论:总体而言,我们的数据表明,通过抑制nSMase来扰乱鞘磷脂的生理分解代谢重组质膜相关鞘脂,改变神经元生成的电动汽车的轮廓,并通过改变促存活与变性EV的平衡来促进体外和体内神经变性。视频摘要。
Cell-to-cell communication is vital for tissues to respond, adapt, and thrive in the prevailing milieu. Several mechanisms mediate intercellular signaling, including tunneling nanotubes, gap junctions, and extracellular vesicles (EV). Depending on local and systemic conditions, EVs may contain cargoes that promote survival, neuroprotection, or pathology. Our understanding of pathologic intercellular signaling has been bolstered by disease models using neurons derived from human pluripotent stems cells (hPSC).
Here, we used hPSC-derived retinal ganglion cells (hRGC) and the mouse visual system to investigate the influence of modulating EV generation on intercellular trafficking and cell survival. We probed the impact of EV modulation on cell survival by decreasing the catabolism of sphingomyelin into ceramide through inhibition of neutral sphingomyelinase (nSMase), using GW4869. We assayed for cell survival in vitro by probing for annexin A5, phosphatidylserine, viable mitochondria, and mitochondrial reactive oxygen species. In vivo, we performed intraocular injections of GW4869 and measured RGC and superior colliculus neuron density and RGC anterograde axon transport.
Following twenty-four hours of dosing hRGCs with GW4869, we found that inhibition of nSMase decreased ceramide and enhanced GM1 ganglioside accumulation. This inhibition also reduced the density of small EVs, increased the density of large EVs, and enriched the pro-apoptotic protein, annexin A5. Reducing nSMase activity increased hRGC apoptosis initiation due to enhanced density and uptake of apoptotic particles, as identified by the annexin A5 binding phospholipid, phosphatidylserine. We assayed intercellular trafficking of mitochondria by developing a coculture system of GW4869-treated and naïve hRGCs. In treated cells, inhibition of nSMase reduced the number of viable mitochondria, while driving mitochondrial reactive oxygen species not only in treated, but also in naive hRGCs added in coculture. In mice, 20 days following a single intravitreal injection of GW4869, we found a significant loss of RGCs and their axonal recipient neurons in the superior colliculus. This followed a more dramatic reduction in anterograde RGC axon transport to the colliculus.
Overall, our data suggest that perturbing the physiologic catabolism of sphingomyelin by inhibiting nSMase reorganizes plasma membrane associated sphingolipids, alters the profile of neuron-generated EVs, and promotes neurodegeneration in vitro and in vivo by shifting the balance of pro-survival versus -degenerative EVs. Video Abstract.