MicroRNA-19a-PTEN axis is involved in the developmental decline of axon regenerative capacity in retinal ganglion cells

Irreversible blindness from glaucoma and optic neuropathies is attributed to retinal ganglion cells (RGCs) losing the ability to regenerate axons. While several transcription factors and proteins have demonstrated enhancement of axon regeneration after optic nerve injury, mechanisms contributing to the age-related decline in axon regenerative capacity remains elusive. Here, we show that microRNAs are differentially expressed during RGC development, and identify microRNA-19a (miR-19a) as a heterochronic marker; developmental decline of miR-19a relieves suppression of PTEN, a key regulator of axon regeneration, and serves as a temporal indicator of decreasing axon regenerative capacity. Intravitreal injection of miR-19a promotes axon regeneration after optic nerve crush in adult mice, and increases axon extension in RGCs isolated from aged human donors. This uncovers a previously unrecognized involvement of the miR-19a-PTEN axis in RGC axon regeneration, and demonstrates therapeutic potential of microRNA-mediated restoration of axon regenerative capacity via intravitreal injection in patients with optic neuropathies. Freshly purified retinal ganglion cells (RGCs) from embryonic day 21 (E21), post-natal day 6 (P6), and post-natal day 30 (P30) Sprague Dawley rats using Neural Tissue Dissociation Kit Postnatal Neurons (Miltenyi Biotec, #130-094-802) and Retinal Ganglion Cell Isolation Kit (Miltenyi Biotec #130-096-209) were collected for detection of age-related changes in microRNA expression using microarray (SurePrint G3 Rat v16.0 miRNA Array Kit, Agilent). There were three biological replicates for E21 and P6 RGC samples each, and two biological replicates for P30 RGC samples. Expression values were extracted using Agilent Feature Extraction Software (v9.5.3.1) with the default protocols and settings for background subtraction and signal intensity processing. Non-uniform outliers were excluded. Data was further processed using GeneSpring GX Software (v11.5). The data set was normalized using a 75th percentile shift normalization method, and p-values were obtained using a moderated t-test with a Benjamini-Hochberg correction.