Vanderbilt Eye Institute, Department of Ophthalmology & Visual Sciences, Vanderbilt University Medical Center, Nashville, TN, USA.
Wilmer Eye Institute, Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Transl Vis Sci Technol. 2023 Apr 3;12(4):1. doi: 10.1167/tvst.12.4.1.
Axons depend on long-range transport of proteins and organelles which increases susceptibility to metabolic stress in disease. The axon initial segment (AIS) is particularly vulnerable due to the high bioenergetic demand of action potential generation. Here, we prepared retinal ganglion cells derived from human embryonic stem cells (hRGCs) to probe how axonal stress alters AIS morphology.
hRGCs were cultured on coverslips or microfluidic platforms. We assayed AIS specification and morphology by immunolabeling against ankyrin G (ankG), an axon-specific protein, and postsynaptic density 95 (PSD-95), a dendrite-specific protein. Using microfluidic platforms that enable fluidic isolation, we added colchicine to the axon compartment to lesion axons. We verified axonopathy by measuring the anterograde axon transport of cholera toxin subunit B and immunolabeling against cleaved caspase 3 (CC3) and phosphorylated neurofilament H (SMI-34). We determined the influence of axon injury on AIS morphology by immunolabeling samples against ankG and measuring AIS distance from soma and length.
Based on measurements of ankG and PSD-95 immunolabeling, microfluidic platforms promote the formation and separation of distinct somatic-dendritic versus axonal compartments in hRGCs compared to coverslip cultures. Chemical lesioning of axons by colchicine reduced hRGC anterograde axon transport, increased varicosity density, and enhanced expression of CC3 and SMI-34. Interestingly, we found that colchicine selectively affected hRGCs with axon-carrying dendrites by reducing AIS distance from somas and increasing length, thus suggesting reduced capacity to maintain excitability.
Thus, microfluidic platforms promote polarized hRGCs that enable modeling of axonopathy.
Microfluidic platforms may be used to assay compartmentalized degeneration that occurs during glaucoma.
轴突依赖于蛋白质和细胞器的长距离运输，这会增加疾病中代谢应激的易感性。轴突起始段（AIS）由于动作电位产生的高生物能量需求而特别脆弱。在这里，我们制备了源自人类胚胎干细胞的视网膜神经节细胞（hRGC），以探究轴突应激如何改变 AIS 形态。
将 hRGC 培养在盖玻片或微流控平台上。我们通过针对轴突特异性蛋白锚蛋白 G（ankG）和树突特异性蛋白突触后密度蛋白 95（PSD-95）的免疫标记来测定 AIS 的特异性和形态。使用能够进行流体隔离的微流控平台，我们将秋水仙碱添加到轴突隔室中以损伤轴突。我们通过测量霍乱毒素亚基 B 的顺行轴突运输和针对裂解型半胱天冬酶 3（CC3）和磷酸化神经丝 H（SMI-34）的免疫标记来验证轴突病变。我们通过针对 ankG 进行免疫标记并测量 AIS 与体的距离和长度来确定轴突损伤对 AIS 形态的影响。
基于 ankG 和 PSD-95 免疫标记的测量，与盖玻片培养相比，微流控平台在 hRGC 中促进了明显的体-树突与轴突隔室的形成和分离。秋水仙碱化学损伤轴突会降低 hRGC 的顺行轴突运输，增加突变量密度，并增强 CC3 和 SMI-34 的表达。有趣的是，我们发现秋水仙碱通过减少 AIS 与体的距离和增加长度，选择性地影响带有轴突的树突的 hRGC，这表明维持兴奋性的能力降低。
因此，微流控平台促进了极化的 hRGC，从而能够模拟轴突病。
微流控平台可用于检测青光眼期间发生的分隔性变性。
