Abstract
Purpose :
Prevention of retinal ganglion cell (RGC) death and optic nerve degeneration in glaucoma and other optic neuropathies remain challenging. Hence, we aimed to test the neuroprotective effects of small extracellular vesicles (sEVs) from a CRISPR-engineered human embryonic reporter cell line (BRN3B-H9) and disclose possible targets of their miRNAs as well as affected signaling pathways associated with the neuroprotection.
Methods :
sEVs were isolated from the conditioned medium of BRN3B-H9 cells and human dermal fibroblasts through buoyant density ultracentrifugation. The neuroprotective properties of sEVs were tested in mouse primary mixed retinal cell cultures by counting β-III tubulin- and DAPI-positive cells and in-depth morphometric analysis. miRNA sequencing data of sEVs were used to analyze differential expression (DE) patterns of miRNAs for identifying possible signaling mechanisms involved in the neuroprotection.
Results :
Among different tested concentrations, 3x109 sEVs from BRN3B-H9 per 1.25x105 mixed retinal cells showed the highest levels of RGC protection compared to the untreated group (2.5-fold, p<0.0001) along with improved neurite growth including number of axons (5.6-fold, p=0.008), axonal length (0.5-fold, p=0.0001), and number of axonal tree branches (2.2-fold, p<0.0001). DE of miRNA cargos in sEVs revealed a total of 70 miRNAs (44 upregulated and 26 downregulated) with a cut-off of >50 fold-change at FDRP ≤0.05. Among the top 5 differentially upregulated miRNAs, miR-519a-3p, and miR-291a-3p were suggested to directly regulate DKK1 in retinal cells that eventually activate neuroprotective mechanisms through a common WNT signaling pathway, while miR-204-5p may provide neuroprotection by directly regulating ADORA2A through CREB and other crucial signaling pathways. Likewise, among the top 5 downregulated miRNAs, 4 miRNAs, specifically miR-374c-5p may provide neuroprotection by directly regulating ADAM10 and CNR1 through axonal guidance along with EGFR, NOTCH, CREB, and other key neuronal signaling pathways.
Conclusions :
The sEVs generated from BRN3B-H9 cells can prevent RGC death through their specific miRNA cargos affecting key signaling mechanisms that provide a framework for further testing of selected miRNAs and identified pathways essential for neuroprotection.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.