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Abstract #108300 Published in IGR 23-4
Microfluidic Platforms Promote Polarization of Human-Derived Retinal Ganglion Cells That Model Axonopathy
Boal AM; McGrady NR; Chamling X; Kagitapalli BS; Zack DJ; Calkins DJ; Risner MLTranslational vision science & technology 2023; 12: 1
PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: Thus, microfluidic platforms promote polarized hRGCs that enable modeling of axonopathy. TRANSLATIONAL RELEVANCE: Microfluidic platforms may be used to assay compartmentalized degeneration that occurs during glaucoma.
Vanderbilt Eye Institute, Department of Ophthalmology & Visual Sciences, Vanderbilt University Medical Center, Nashville, TN, USA.
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