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Heather Kayew Mak, Xiu Juan Zhang, Shuk Han Ng, Christopher Kai-Shun Leung; Inhibition of let-7 microRNA promotes axon regeneration in retinal ganglion cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4498.
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Vision loss is irreversible in part because retinal ganglion cells (RGCs) lose capacity to regenerate axons at an early age. We investigated this loss with age-related change in microRNA (miRNA) expression and found that inhibition of the let-7 family enhances axon extension after injury.
Microarray was performed on RGCs of Sprague Dawley (SD) rats at embryonic day 21 (E21), postnatal day 6 (P6), and postnatal day 30 (P30). RGC axon lengths from P6 SD rats and human adult eye donors were measured by microfluidic chambers (MFCs) and single-cell analysis, respectively, after adeno-associated viral (AAV) transduction of a let-7 tough decoy (TuD) inhibitor (AAV-let-7TuD-mCherry) or control. C57BL/6 mice were intravitreally injected with AAV-let-7TuD-mCherry or control 3 weeks before optic nerve crush and the number of regenerating axons was measured 4 weeks after crush.
Microarray revealed that 76 miRNAs had ≥4-fold difference in expression between E21 and P30 RGCs; 32 (42%) were up- and 44 (58%) were down-regulated. Specifically, let-7a, let-7b, let-7c, let-7d, and let-7f increased by (mean±SEM) 4.2±0.2-, 14.1±0.6-, 9.3±0.4-, 7.3±0.3-, and 4.6±0.4-fold from E21 to P30 (p≤0.028). Inhibition of the let-7 family (let-7abcdefgi) using AAV-let-7TuD-mCherry suppressed let-7 levels by 6.3±1.1-fold, and increased RGC axon length in MFCs by 1.62±0.57-fold on day 14 (p=0.034). Human adult RGCs with AAV-let-7TuD-mCherry had longer axon and total neurite lengths by 59.3±12.3% (p=0.003) and 50.6±16.2% (p=0.002), respectively. C57BL/6 mice with AAV-let-7TuD-mCherry had on average 51.3±36.2% more regenerating axons after optic nerve crush compared with control (p=0.020) (axons were measured at 0.2mm and at every 0.5mm from the optic nerve crush site up to 4mm).
Differential expression of miRNAs coincides with age-related decline in RGC axon growth potential. We showed that this loss can be partially restored by inhibition of let-7 in vitro and in vivo, suggesting a therapeutic potential of miRNAs for optic nerve regeneration.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
In situ hybridization of let-7a in (a) E21, P6, and P30 SD rat retinas and (b) human fetal and adult RGCs. Scale bar: 25μm
P6 RGC axons in MFCs (a) were longer with AAV-let-7TuD-mCherry (p=0.034), than control (b). Scale bar: 100μm. (c) Number of regenerating axons after optic nerve crush was greater with AAV-let-7TuD-mCherry (p=0.020), than control.
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