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. Author manuscript; available in PMC: 2025 Feb 16.
Published in final edited form as: Neurosci Lett. 2024 Jan 28;823:137662. doi: 10.1016/j.neulet.2024.137662

Upregulation of developmentally-downregulated miR-1247-5p promotes neuroprotection and axon regeneration in vivo

Agnieszka Lukomska 1,#, William C Theune 1,#, Matthew P Frost 1, Jian Xing 1, Anja Kearney 1, Ephraim F Trakhtenberg 1,*
PMCID: PMC10923146  NIHMSID: NIHMS1962931  PMID: 38286398

Abstract

Numerous micro-RNAs (miRNAs) affect neurodevelopment and neuroprotection, but potential roles of many miRNAs in regulating these processes are still unknown. Here, we used the retinal ganglion cell (RGC) central nervous system (CNS) projection neuron and optic nerve crush (ONC) injury model, to optimize a mature miRNA arm-specific quantification method for characterizing the developmental regulation of miR-1247-5p in RGCs, investigated whether injury affects its expression, and tested whether upregulating miR-1247-5p-mimic in RGCs promotes neuroprotection and axon regeneration. We found that, miR-1247-5p is developmentally-downregulated in RGCs, and is further downregulated after ONC. Importantly, RGC-specific upregulation of miR-1247-5p promoted neuroprotection and axon regeneration after injury in vivo. To gain insight into the underlying mechanisms, we analyzed by bulk-mRNA-seq embryonic and adult RGCs, along with adult RGCs transduced by miR-1247-5p-expressing viral vector, and identified developmentally-regulated cilial and mitochondrial biological processes, which were reinstated to their embryonic levels in adult RGCs by upregulation of miR-1247-5p. Since axon growth is also a developmentally-regulated process, in which mitochondrial dynamics play important roles, it is possible that miR-1247-5p promoted neuroprotection and axon regeneration through regulating mitochondrial functions.

INTRODUCTION

MiRNAs can target multiple mRNAs and regulate various biological processes in the same cell1,2, but neuronal-specific roles of many miRNAs are still poorly understood. Examples of various miRNAs’ involvement in neurodevelopment, axon growth, and neuroprotection3–11 suggest that studying the roles of specific miRNAs in developmental processes such as axon growth and in neuroprotection after injury is important. Thus, here we focused on a specific miRNA, miR-1247, which is involved in physiological and pathophysiological processes by targeting different mRNAs in various tissues12–20, and within the CNS is associated with Huntington’s disease21, but whose potential roles in neurodevelopmental processes such as axon growth or in neuroprotection after injury were not explored yet. To address these questions, we used a specific type of prototypical CNS projection neuron, the RGC, which alters gene expression through postmitotic development22. Like other mammalian CNS projection neurons, RGCs lose their intrinsic axon growth capacity during maturation23,24, and do not spontaneously regenerate axons severed by injury25,26. Thus, here we used the RGC and ONC injury model for investigating whether (a) miR-1247 is developmentally-regulated and targets mRNAs associated with RGC maturation, (b) injury affects miR-1247 expression in RGCs, and (c) exogenous miR-1247 elicits RGC neuroprotection and promotes axon regeneration after CNS white matter injury in vivo. We answered these questions by analyzing miR-1247 expression in RGCs isolated from different developmental stages and after ONC, identifying developmentally-regulated mRNAs that were restored to embryonic levels in RGCs by miR-1247, bioinformatically characterizing biological processes regulated by miR-1247 in RGCs, and testing whether expressing miR-1247 in adult RGCs through AAV2 promotes neuroprotection and axon regeneration after ONC injury.

RESULTS

To investigate the neurodevelopmental regulation of miR-1247 in RGCs, we analyzed its expression using small-RNA-seq in RGCs purified from embryonic (E18) and postnatal (P5) developmental stages, as the RGC transcriptome is substantially altered between these stages of maturation22, and also because at E18 developmental axon growth capacity is robust but is substantially reduced by P524,27. A miRNA precursor produces two miRNAs from its respective 5p and 3p arms, each of which is a potential distinct mature miRNA28. We found that, while miR-1247–3p is not meaningfully expressed at either age, miR-1247-5p is expressed robustly in embryonic RGCs but its expression is developmentally-downregulated during maturation (Fig. 1A–B).

Figure 1. Regulation of miR-1247-5p expression in RGCs during development, after optic nerve injury, and experimentally through gene therapy.

Figure 1.

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(A) Visualization of normalized reads in IGV Viewer small-RNA-seq of purified RGCs aligned to the miR-1247 locus in chr12, shows that miR-1247-5p is highly expressed at E18 and downregulated by P5, whereas miR-1247–3p expression was only bordering noise at either age.

(B) Plot of miR-1247–3p and miR-1247-5p expression levels (normalized counts) in E18 RGCs (on x-axis) versus expression fold-change from E18 to P5 RGCs on (y-axis), shows that miR-1247-5p is robustly expressed and significantly (p < 0.01) developmentally-downregulated in maturing RGCs, whereas miR-1247–3p expression only bordering noise and is not significantly (p > 0.01) regulated in maturing RGCs. Mean ± SEM of miRNAs’ normalized expression values are shown (n = 2). Statistical significance (indicated by * p-value < 0.0001) of developmental regulation (E18 relative to P5 RGCs) was determined using differential expression analysis by DESeq2 and featureCounts. N.S. = Not significant.

(C) Pipeline for validation of miRNA expression. Adaptors, which are biased towards binding selectively to the 3’ and 5’ end-modifications of mature miRNAs, are sequentially ligated to a sample of total RNA from RGCs. Small-RNA cDNA library is prepared using reverse transcription followed by a limited number of PCR cycles to produce a DNA library. Following purification of DNA library, PCR is performed using a miRNA-specific primer (for either miR-1247-5p or spike-in control) and a universal reverse primer specific to the 3’ library adaptor.

(D-E) Representative agarose gel images of amplified spike-in control ath-miR-775, using DNA libraries (which were prepared with “+” or without “-” the addition of ath-miR-775 spike-in RNA) as a template for PCR amplification, shows the specificity of ath-miR-775 signal (D), which is unaltered by varying annealing temperatures, as marked (E).

(F-G) Representative image of sqPCR analysis of miR-1247-5p expression at E18, P5 and adult stages of RGC development, which confirmed that the sequences detected by small-RNA-seq are mature miRNAs (which along with the adaptors expected to be ~85 bp) and not their precursors (that would have appeared higher on the gel), and thereby validated small-RNA-seq prediction of miR-1247-5p developmental downregulation (F). Quantitation of miR-1247-5p sqPCR gel band intensity in samples prepared from E18, P5 and adult RGCs (as marked) shows that the progressive decline of miR-1247-5p expression during RGC maturation was statistically significant. Mean ± S.E.M shown; n = 3 samples for each condition (each sample was prepped from different animals). Data analyzed using ANOVA, overall F = 292.8, p < 0.001, with p-values of pairwise comparisons determined by posthoc LSD. Significant differences (p < 0.001) indicated by an asterisk * (G).

(H) Sanger-sequencing of sqPCR products purified from the gel bands (shown in F), which corresponded to mature miR-1247-5p, aligned to the miR-1247-5p locus (visualized in the respective chromosomal region shown in the panel A). Each Sanger-sequencing result aligned to only 1 site in the mouse genome, and blasting against the mouse miRbase database confirmed specificity of the sequence (score 105, e-value 0.002).

(I-J) Representative image of sqPCR analysis of miR-1247-5p expression in uninjured and injured adult RGCs, shows that miR-1247-5p is further downregulated after ONC injury (I). Quantification (as above), shows that decline of miR-1247-5p expression in RGCs after injury was statistically significant. Mean ± S.E.M shown; n = 3 samples for each condition (each sample was prepped from different animals). Data analyzed using t-test 2-tailed, and significant difference (p < 0.003) is indicated by an asterisk * (J).

(K) Experimental timeline: AAV2 viral vectors expressing miR-1247-5p-mimic, or scrambled shRNA-control, and co-expressing an mCherry-reporter of transduction, were injected intravitreally in 8-week-old mice. 2 weeks later ONC was performed. Animals were sacrificed at 2 weeks after ONC.

(L-M) Representative confocal images of the retinal flatmounts immunostained with for βIII-Tubulin show that expression of an mCherry-reporter of transduction in βIII-Tubulin+ RGCs was at similar proportions in both treatment conditions, scrambled shRNA-control (L) and miR-1247-5p-mimic (M) respectively. Scale bar, 20 μm.

In order to validate small-RNA-seq-predicted developmental regulation of miR-1247-5p, we optimized a new semi-quantitative PCR (sqPCR) method for direct measurement of mature miRNA expression levels in primary cells. Adapters from a small-RNA-seq library kit, whose ligation is biased towards the 5’ phosphate and 3’ OH end-modifications unique to small-RNAs such as mature miRNAs, are used to generate a miRNA cDNA library. Then, using a miRNA-specific forward primer and a universal reverse primer for the 3’ adapter, a specific miRNA is PCR-amplified from the template (i.e., cDNA library). The current standard for validation of miRNA expression is qPCR29. For example, the stem-loop RT-PCR which is highly sensitive30–32 and can discriminate between highly similar miRNA sequences29,33. However, the stem-loop RT-PCR relies primarily on miRNA’s small size, and does not discriminate specific mature miRNAs from miRNA biogenesis intermediates and other small-RNA species, which can overlap in their respective size and sequence29. In order to circumvent such concerns, our method relies on the end-modifications specific to mature miRNAs (Fig. 1C). This approach limits the potential for nonspecific amplification of larger RNA species or capture of unwanted miRNA biogenesis intermediates, as they lack the 3’ and 5’ modifications of mature miRNAs. Although this method could also be used for qPCR such as TaqMan or SYBR Green, the sqPCR method provides validation of target-size-specific and target-sequence-specific amplification through visualization of the PCR band on a gel (~85 bp for mature miRNA with the ligated adaptors) and Sanger-sequencing of the PCR product, thereby eliminating confounding inappropriate size products from comparative analysis between conditions. For normalization and comparative analysis of relative expression across cDNA libraries, we utilized an arabidopisis thaliana miRNA (ath-miR-775)-mimic as a spike-in control. Arabidopsosis thaliana miRNA-mimics are used as spike-in controls for normalization of endogenous mammalian miRNAs, because these plant miRNAs do not share homology with endogenous RNAs of mammalian species34,35. We used ath-miR-775, because it had no matches in miRbase, and we determined it to be amplifiable by sqPCR across a range of annealing temperatures without confounding background in libraries without spike-in (Fig. 1D–E).

Using this miRNA sqPCR method, we proceeded to validate small-RNA-seq-predicted regulation of miR-1247-5p during RGC maturation, and also tested its expression in fully mature adult RGCs. We found that, miR-1247-5p was expressed robustly in embryonic RGCs, and then progressively downregulated through postnatal and adult stages of development (Fig. 1F–G). The specificity of the mature miRNA amplification by sqPCR was confirmed by genome-alignment of the amplified sequence (determined by Sanger-sequencing), which matched reads obtained with small-RNA-seq (Fig. 1H). Next, we tested whether miR-1247-5p expression in RGCs changes after injury, and found that miR-1247-5p was further reduced by 2 weeks after ONC (Fig. 1I–J).

In order to investigate whether re-upregulating miR-1247-5p will promote neuroprotection and/or optic nerve axon regeneration, we first validated that the AAV2 vectors expressing miR-1247-5p-mimic transduce injured RGCs. The viruses expressing miR-1247-5p-mimic or scrambled shRNA-control, and co-expressing an mCherry reporter of transduction, were injected intravitreally in adult mice, and 2 weeks later ONC was performed (experimental timeline in Fig. 1K). At 2 weeks after ONC, the retinas were immunostained for an anti-βIII-Tubulin antibody (a neuronal-specific marker which selectively labels the RGCs within the retina), and then analyzed for co-expression of an mCherry-reporter. RGC transduction efficiency of ~30% was comparable to transduction with the control vector (mCherry reporter-labeled RGCs), see Methods for more details (Fig. 1L–M). Then, we tested miR-1247-5p-mimic in an established 2-weeks after ONC axon regeneration assay26,36–40, in which the optic nerve is injured by crush in order to severe all RGC axons, and axon regeneration is assayed 2 weeks later (experimental timeline in Fig. 2A). ONC was performed at 2 weeks after AAV2 vectors expressing miR-1247-5p-mimic or scrambled shRNA-control were injected intravitreally in adult mice. To visualize the regenerating axons or their absence, axonal tracer CTB was injected prior to sacrifice at 2 weeks after ONC. The number of regenerating axons was quantified in longitudinal sections of the optic nerve (no spared axons were detected in either group), and RGC survival was quantified in retinal flatmounts. We found that, miR-1247-5p-mimic promoted sparse but long-distance (approximately ~2 mm past the ONC site) axon regeneration, relative to only minor axonal sprouting (as expected) in control animals (Fig. 2B–D). Expression of miR-1247-5p-mimic also promoted RGC survival compared to the injured control group (Fig. 2E–F).

Figure 2. miR-1247-5p expression promotes axon regeneration and RGC survival after optic nerve injury.

Figure 2.

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(A) Experimental timeline: 8-week-old mice were pre-treated with AAV2 vectors expressing miR-1247-5p-mimic or scrambled shRNA-control. ONC injury was performed 2 weeks later. Axonal tracer CTB was injected intravitreally 1 day prior to sacrifice at 2 weeks after ONC.

(B-C) Representative images of the optic nerve longitudinal sections with CTB-labeled axons after ONC from the animals pre-treated with AAV2 expressing miR-1247-5p-mimic or scrambled shRNA-control, as marked (B). Insets: Optic nerve regions proximal and distal to the injury site are magnified for better visualization of the axons or their absences (C). Scale bars, 500 μm (main panels), 50 μm (insets).

(D) Quantitation of CTB-labeled regenerating axons at 2 weeks after ONC, at increasing distances from the injury site, after pre-treatment with AAV2 expressing miR-1247-5p-mimic or scrambled shRNA-control, as marked. Data analyzed using repeated measures ANOVA and posthoc LSD, sphericity assumed, overall F = 69.8, p < 0.02 (significant difference between the conditions indicated by an asterisk *). Mean ± SEM shown; n = 4 optic nerves per group.

(E-F) Quantitation of RGC survival in retinal flatmounts immunostained for an RGC marker βIII-Tubulin, pretreated with miR-1247-5p-mimic or scrambled shRNA-control, as marked. Data analyzed by independent samples t-test, 2-tailed; significant difference (p < 0.04) indicated by an asterisk *; mean ± SEM shown, n = 3–4 retinas per group (E). Representative images of the retinal flatmounts immunostained for an RGC marker βIII-Tubulin, as marked; sale bar, 20 μm (F).

Since miR-1247-5p is developmentally-downregulated in RGCs, we then tested whether it targets developmentally-regulated mRNAs associated with RGC maturation. In order to characterize the genes that are directly or indirectly regulated by miR-1247-5p in RGCs, AAV2 vectors expressing miR-1247-5p-mimic or scrambled shRNA-control were injected intravitreally in adult mice, 2 weeks later RGCs were isolated through immunopanning followed by FACS for mCherry reporter-labeled transduced RGCs and analyzed by mRNA-seq (experimental timeline in Fig. 3A). Then, we analyzed whether miR-1247-5p regulates the expression of genes that are developmentally-regulated in RGCs. We found a subset of inversely-related developmentally-upregulated mRNAs (E18 to adult RGCs) and the overlapping mRNAs that were downregulated in adult RGCs by the treatment with miR-1247-5p-mimic (relative to control-treated adult RGCs), as well as inversely-related developmentally-downregulated mRNAs and the overlapping mRNAs that were upregulated in adult RGCs by the miR-1247-5p-mimic treatment. Next, we analyzed whether these developmentally-regulated genes, whose expression was reverted to embryonic RGC levels by miR-1247-5p, are co-associated with specific biological processes. Using enrichment and functional clustering analysis of gene annotation/ontology41,42, we identified several developmentally-regulated biological processes that reverted to embryonic RGC levels of enrichment by the treatment with miR-1247-5p-mimic (Fig. 3B). The specific differentially expressed and developmentally-regulated genes that were co-associated with the identified biological processes are shown (Fig. 3C–D). These data suggest that, miR-1247-5p regulates a subset of genes and biological processes associated with RGC development that could be reverted towards their embryonic state by experimental upregulation of miR-1247-5p in adult RGCs.

Figure 3. Expressing miR-1247 in adult RGCs reverses certain biological processes towards embryonic state of immature neuron.

Figure 3.

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(A) Experimental timeline: Both eyes of five 8-week-old mice per condition were pre-treated with AAV2 vectors expressing miR-1247-5p-mimic or scrambled shRNA-control. 2 weeks later, approximately 5,000 mCherry+ RGCs per condition were isolated. Then, scramble shRNA-control and miR-1247-5p-mimic-treated RGCs were analyzed by mRNA-seq (see methods for more technical details).

(B) Functional annotation clustering analysis of enriched terms, performed using DAVID, identified biological processes enriched within the differentially expressed (at least 1.75 FC) genes, which were inversely regulated between RGC development and miR-1247-5p-mimic treatment. Bar plot of the biological processes unenriched/enriched in adult RGCs by the treatment with miR-1247-5p-mimic, shown rank-ordered by the enrichment score, and the direction of their respective inverse developmental regulation is color-coded, as indicated.

(C-D) Differentially expressed genes comprising functional annotation clusters of the biological processes (shown in panel B) that are developmentally-upregulated but downregulated by the miR-1247-5p-mimic treatment (C) and developmentally-downregulated but upregulated by the miR-1247-5p-mimic treatment (D) in RGCs.

Although we found that experimentally expressing the developmentally-downregulated miR-1247-5p in adult RGCs reinstates a subset of embryonic RGC gene network, it also affected expression of non-developmentally-regulated genes, which may have contributed to axon regeneration and neuroprotection promoted by miR-1247-5p-mimic. Therefore, to further explore how miR-1247-5p-mimic regulates RGC axon regeneration and neuroprotection, we used functional annotation clustering (as above) for analyzing all the genes that are differentially expressed in adult RGCs by miR-1247-5p-mimic treatment (even if they are not developmentally-regulated). However, we found that a number of non-developmentally-regulated differentially expressed genes were also associated with the same biological processes that we identified above as developmentally-regulated (Fig. 4A), thereby expanding the number of genes affected by miR-1247-5p-mimic that are associated with the same targeted biological processes. Furthermore, the only two substantially enriched terms (‘development’ and ‘electron transport chain’) added by functional annotation clustering analysis of all differentially expressed genes (as opposed to only those that are also developmentally-regulated) were linked to the related developmentally-regulated terms by gene network analysis (Fig. 4B–C), as follows: The term ‘development’ was linked to the term ‘primary cilia’, thus expanding the interpretation that both terms relate to the same biological process (primary cilia development), which was regulated by miR-1247-5p-mimic. Also, the term ‘electron transport chain’ was linked to the term ‘mitochondrial function”, thus indicating that mitochondrial function associated with its electron transport chain was regulated by miR-1247-5p-mimic. Moreover, the term ‘transcription regulation’ was linked to the term ‘mitochondrial function’, suggesting that a component of the transcriptional mechanism activated downstream of miR-1247-5p targets is specific to regulation of mitochondria-related processes.

Figure 4. Expressing miR-1247 in adult RGCs affects primarily developmentally-regulated biological processes.

Figure 4.

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(A) Functional annotation clustering analysis of the enriched terms, performed using DAVID, identified biological processes enriched within the differentially expressed (at least 1.75 FC) genes after miR-1247-5p-mimic treatment (irrespective of whether or not these genes are also developmentally-regulated in RGCs, as in Fig. 3). Bar plot of the biological processes unenriched/enriched in adult RGCs by the treatment with miR-1247-5p-mimic, shown rank-ordered by the enrichment score, and the direction of their respective regulation is color-coded, as indicated.

(B-C) Gene-concept network plots of the top 3 downregulated (B) and top 3 upregulated (C) biological processes (rank-ordered in panel A by enrichment score) after miR-1247-5p-mimic treatment in adult RGCs. Functional annotation clustering terms, as marked, shown as gray circles (i.e., nodes), with circle size corresponding to the number of genes within that node. Links between genes are based on interconnected nodes sharing the same genes. Color-coded scale bar indicates each gene’s expression fold-change after miR-1247-5p-mimic treatment in adult RGCs.

(D) Heatmap of positive and negative gene regulators of apoptosis in control and miR-1247-5p-mimic-treated adult RGCs, analyzed by mRNA-seq. Genes did not show significant changes in expression after the treatment, consistent with no statistically significant differences (p > 0.05) in gene expression identified by CuffDiff analysis. N.S. = Not significant.

Because mitochondrial dynamics are involved in axon growth and regeneration40,43–48, it is possible that mitochondria-associated genes are indirectly upregulated downstream of the miR-1247-5p treatment facilitated axon regeneration. However, mitochondria-related genes also could be enriched in cells progressing towards apoptosis49–51. Therefore, we analyzed whether apoptosis-related genes52–57 were affected by miR-1247-5p treatment, but found no significant change in expression of these genes (Fig. 4D). Thus, it is possible that miR-1247-5p-mimic selectively promoted neuroprotective/regenerative mitochondria-related processes, without co-activating apoptotic pathways. Also, because RGCs primary cilia are affected by ONC injury58 and experimental stabilization of primary cilia in injured neurons was reported to be neuroprotective59, it is possible that selective regulation of ciliogenesis processes by miR-1247-5p treatment contributed to neuroprotection of RGCs.

DISCUSSION

An inherent limitation of bioinformatics analyses is that bioinformatic predictions require experimental validations in the next steps of investigation. For example, it would be important to investigate in future studies which mitochondrial functions are regulated by miR-1247-5p in RGCs, and to test whether miR-1247-5p promotes neuroprotection and axon regeneration through specific mitochondrial functions. Also, as bioinformatics analysis identified the ‘metal ion binding’ term amongst the most downregulated by miR-1247-5p treatment biological processes, it would be important to investigate whether miR-1247-5p regulates metal ions such as zinc, iron, and calcium, which were previously established to play various roles in neuronal survival and axon regeneration60–62. Furthermore, as not all genes downregulated by miR-1247-5p treatment are necessarily direct mRNA targets of miR-1247-5p, it would be important to identify in future studies miR-1247-5p’s direct targets and how they lead to differential expression of multiple downstream genes, specifically those that are directly involved in promoting neuroprotection and axon regeneration. Also, as the identified indirectly upregulated downstream genes and biological processes may be the key effectors of neuroprotection and axon regeneration promoted by miR-1247-5p, it would be important to investigate them further. Because miR-1247-5p itself is developmentally-downregulated in RGCs during maturation, it would also be important to investigate whether miR-1247-5p plays a role in regulation of normal developmental axon growth, potentially through the developmentally-regulated genes and biological processes affected by miR-1247-5p. Finally, as therapeutically expressing miR-1247-5p-mimic in RGCs promotes neuroprotection and axon regeneration, our findings could help develop treatments for glaucoma and optic neuropathies, as well as other CNS injuries and diseases. Although the number of regenerated axons (shown in Figure 2) is modest, transduction efficiency by the viral vector we used was also limited (approximately 30%, see Methods), thus, it is possible that utilization of improved viral vectors63,64 and/or more efficient delivery methods may result in a higher extent of neuroprotection and axon regeneration. However, any significant extent of functional neuroprotection and axon regeneration that could be achieved therapeutically would be important, considering that medical need for such therapeutics is largely unmet.

METHODS

Animal use, surgeries, intraocular injections.

All animal studies were performed at the University of Connecticut Health Center with approval of the Institutional Animal Care and Use Committee and of the Institutional Biosafety Committee, and performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research. Mice were housed in the animal facility with a 12-h light/12-h dark cycle (lights on from 7:00 AM to 7:00 PM) and a maximum of five adult mice per cage. Food and water were available ad libitum. The study used wild-type 129S1/SvImJ mice (The Jackson Laboratory). Optic nerve surgeries and intravitreal injections were carried out on mice of both sexes 8–12 weeks of age (average body weight 20–26 g) under general anesthesia, as described previously26,38,39,65. The viruses included adeno-associated virus serotype 2 (AAV2) vectors expressing miR-1247-5p-mimic (target sequences: 5′-ACCCGTCCCGTTCGTCCCCGGA-3′), and scrambled control (sequences: 5’-TCGAGGGCGACTTAACCTTAGG-3’) shRNAs (titers ~1 × 1013 GC/mL; VectorBuilder, Inc.). All viral vectors co-expressed an mCherry reporter of transduction. Viruses (2 μl per eye) were injected intravitreally, avoiding injury to the lens, in 8-week-old mice, which were randomly assigned to experimental or control conditions, 2 weeks prior to ONC surgery. It is standard protocol to inject AAV2 two weeks prior to ONC, because this lead time allows sufficient time for transduction and expression of the shRNAs in RGCs at the time of ONC37,66 (the timings of treatment and injury are shown in the experimental timelines in Figs. 1K, 2A, 3A). Transduction efficiency was approximately 30%. AAV2 RGC transduction efficiency varies between studies, with some viral vectors achieving a higher transduction efficiency26,37–39,67,68. Cholera toxin subunit B (CTB) conjugated to Alexa Fluor 488 dye (C34775, ThermoFisher Scientific) was injected (1% in 3 μl PBS) intravitreally one day prior to sacrifice, at 2 weeks after ONC, in order to visualize the regenerating axons.

Tissue processing.

Standard histological procedures were used, as described previously26,38,39,65,69. Briefly, anesthetized mice were transcardially perfused with isotonic saline followed by 4% paraformaldehyde (PFA) at 2 weeks after ONC, the eyes and optic nerves were dissected, postfixed 2 hours, the retinas were dissected-out, and optic nerves were transferred to 30% sucrose overnight at 4 °C. The optic nerves were then embedded in OCT Tissue Tek Medium (Sakura Finetek), frozen, cryosectioned longitudinally at 14 μm, and then mounted for imaging on coated glass slides. For analyzing RGC transduction and survival, resected (into PBS at 4 °C) free-floating retinas were immunostained in 24-well plate wells and, after making 4 symmetrical slits, flat-mounted on coated glass slides for imaging. For immunostaining, the retinal tissues were blocked with appropriate sera, incubated overnight at 4 °C with primary antibody against βIII-Tubulin (1:500; rabbit polyclonal, Ab18207, Abcam), then washed 3 times, incubated with appropriate Alexa Fluor 594 dye-conjugated secondary antibody (1:500; A11012, Thermo Fisher Scientific) overnight at 4 °C, washed 3 times again, and mounted for imaging.

Quantification of regenerated axons and RGC survival.

To visualize the regenerating axons or their absence after treating with the AAV2 vectors expressing miR-1247-5p-mimic or scrambled control, axonal tracer (Alexa Fluor 488-conjugated CTB 1% in 3 μl PBS) was intravitreally injected one day before animals were euthanized 2 weeks following ONC. Longitudinal sections of the optic nerve were examined for possible axon sparing26. No spared axons were found in control, and no evidence of axon sparing was found in experimental conditions (i.e., at 2 weeks after injury, no axons were found at the most distal from the injury region of the optic nerve). Regenerated axons (defined as continuous fibers, which are absent in controls and are discernible from background puncta and artefactual structures) were counted manually using a fluorescent microscope (40x/1.2 C-Apochromat W; AxioObserver.Z1, Zeiss) in at least 4 longitudinal sections per optic nerve at 0.5 mm, 1 mm, 1.5 mm, 2 mm, and 2.5 mm distances from the injury site (identified by the abrupt disruption of the densely packed axons near the optic nerve head, as marked by a rhombus in Fig. 2B), and these values were used to estimate the total number of regenerating axons per nerve, as described26,38,39,65. For representative images, serial fields of view along the longitudinal optic nerve tissue section were imaged as above; z-stacks with 5 planes at 0.5 μm intervals were deconvoluted, merged, and stitched (ZEN software, Zeiss). Then, processed images of 2 tissue sections from the same optic nerve were superimposed over each other and merged using Photoshop CS6 (Adobe). The edges of the tissue were optically trimmed (i.e., cropped-out) due to artefactual autofluorescence that is common at tissue edges. RGC survival was quantified in retinal flatmounts as described26,38,39,65,69, by immunostaining with an antibody to βIII-Tubulin (neuronal marker Tuj1), taking advantage of the selective expression (within the retina) of βIII-tubulin in RGCs. ImageJ software was used to count βIII-Tubulin positive cells from images acquired (using a fluorescent microscope, 20x LD; Zeiss, AxioObserver.Z1, Zeiss) at 1–2 mm from the optic nerve head in four directions, then averaged to estimate overall RGC survival per mm2 of the retina. For demonstration of transduction efficiency (representative images in Fig. 1K–M), the flat-mounted retinas were immunostained for βIII-Tubulin, and then ganglion cell layer was imaged using a confocal microscope (63x Oil; LSM800, Zeiss), showing a subset of βIII-Tubulin+ RGCs that were also mCherry+ (mCherry is a reporter of transduction with AAV2). Investigators performing the surgeries and quantifications were masked to the group identity by another researcher until the end of the experiment.

Primary neuron isolation, semi-quantitative PCR (sqPCR), and Sanger sequencing.

Retinas were resected from both sexes of 5 mice of the respective ages: Embryonic day 18 (E18), postnatal day 5 (P5), or 10–12-week adult. RGCs were isolated from retinal single cell suspension by Thy1 immunopanning (CD90, MCA02R, Serotec) after depletion of macrophages (anti-mouse macrophage antibody, AIA31240, Accurate Chemical) and washing off the nonadherent cells, as previously described67,70–72, with the following modifications for adult tissue: digestion in papain decreased to 10 minutes, amount of papain increased by 25%, only one macrophage depletion plate used for 20 minutes, and Thy1 panning dish shaken more stringently, also as we previously described40,73. RNA was isolated from pelleted cells using the Quick RNA Microprep Kit (ZR1050, Zymo Research). RNA concentration was measured with NanoPhotometer NP80 (Implen). Equivalent concentrations and quantities of total RNA were used as input for small-RNA cDNA library prep kit (E7330S, NEB). For spike-in control, ath-miR-775 miRNA-mimic (5’-UUCGAUGUCUAGCAGUGCCA-3’) was used. Blast alignment of ath-miR-775 against mature mouse miRNAs using miRbase yielded no matches (mirbase.org/search, search sequences: mature miRNAs, search method: BLASTN, E-value cutoff: 10, organism: mouse). Spike-in control was used in preparation of small-RNA cDNA library for miRNA sqPCR: 5.5 μl of total RNA (100 ng total RNA) was mixed with 0.5 μl of a of ath-miR775 miRNA-mimic (at 0.625 ng/μl) concentration after 1:800 dilution from custom RNA stock solution (10620310, ThermoFisher Scientific) for spike-in control. Spike-in with ath-miR775 miRNA-mimic was used for normalization of expression across library replicates and timepoints/conditions for comparative analysis. All subsequent steps of small-RNA library prep were performed according to the manufacturer protocol (E7330S, NEB), through purification of the DNA library using the Monarch PCR and DNA Cleanup Kit (T1030S, NEB). Libraries were eluted in 30 μl of nuclease-free water. For sqPCR, 2 μl of library was used as input in a 50 μl PCR reaction using Q5 Polymerase (M0491S, NEB). All samples were prepared using a single PCR master mix (buffer, water, reverse primer, dNTPs and polymerase). Then, 2 μl of template along with 2.5 μl of miRNA-specific primer (either for miR-1247-5p, 5’-CCGTCCCGTTCGTCCC-3’ or spike-in control ath-miR-775, 5’-TTCGATGTCTAGCAGTGCCA-3’) were added to each sample, and PCR was run for 34 cycles on a BioRad T100 Thermal Cycler. After PCR, samples were electrophoresed on a 1.2% agarose TAE gel. The gels were imaged using a BioRad Universal Hood II, and images were exported in tiff format. Gel band intensities in the images were quantified using ImageJ software. 3 biological replicates per condition were used. Intensity of the gel band, matching the expected size of the amplified target miRNA with adaptors, was normalized to spike-in control band intensity, and then normalized by sum across replicates. Gel bands matching the expected size of the amplified target miRNA with adaptor were excised, sqPCR products were purified using the Zymoclean Gel DNA Recovery Kit (D4001, Zymo Research) and Sanger sequencing was performed (QuintaraBio, Farmington, CT). The identified sequences were aligned to the genome (mm39) and blasted against the mouse miRbase database.

RGC small-RNA-seq and bulk-mRNA-seq datasets.

All bulk small-RNA-seq and mRNA-seq samples from different age RGCs were isolated by immunopanning for Thy1 from within retinal single cell suspension (after immunopanning depletion of macrophages, as described above). For bulk-mRNA-seq of adult RGCs transduced with AAV2 (~30% transduction efficiency) expressing miR-1247-5p mimic or scramble shRNA mimic, RGCs were Thy1-immunopanned from single cell retinal suspension (from 10 retinas per condition) and FACS’ed for mCherry+ cells (as we described previously40). Approximately, 5,000 RGCs (per condition) were collected by FACS and RNA was isolated immediately using the Zymo QuickRNA microprep kit. Total RNA with RNA Integrity Number (RIN) ≥ 9 (by Bioanalyzer 2100 using the Nano 6000 kit, Agilent) was extracted using Direct-zol RNA MiniPrep kit (R2050, Zymo Research). For mRNA-seq, cDNA libraries were prepared using polyA-selected RNA (TruSeq RNA Library Prep Kit, Illumina), paired reads sequenced 100 bp from each end on HiSeq 2000 Sequencer (Illumina), passed QC filters, mapped to the mm39 genome and transcriptome by Hisat2, gene expression was normalized as fragments per kilobase of transcript per million mapped reads (FPKM), and analyzed by Cufflinks/CuffDiff74,75, as we previously described72,76. For small-RNA-seq, approximately 200,000 of Thy1-immunopanned RGCs were collected (from each condition) and RNA was isolated immediately for preparing cDNA libraries using adaptors that ligate selectively to the endogenously modified 5’ and 3’ ends of processed small RNAs, thereby enriching specifically for mature small RNAs, including miRNAs, and excluding degraded RNA fragments, precursors, and other RNA species (TruSeq Small RNA Kit, Illumina). Small RNA cDNA library products in the range of 140–160 base pairs (including 5’ and 3’ adapters and an insert corresponding to a small RNA) were sequenced as paired reads 100 bp from each end on HiSeq 2000 Sequencer (Illumina), passed QC filters, mapped to the mm39 mouse genome and transcriptome by Bowtie, and quantified/analyzed using featureCounts from the subread package77 and DESeq278. Alignments visualized using IGV viewer (Broad Institute)79.

Data availability.

E18 and P5 RGC bulk small-RNA-seq raw reads and processed data for miR-1247 locus analyzed in this study are available through the NCBI GEO under accession number GSE252517. E18 RGC and miR-1247-5p-mimic or scramble shRNA-mimic transduced adult RGC bulk mRNA-seq raw reads and processed data used in this study are available through the NCBI GEO under accession number GSE252517.

Gene annotation/ontology enrichment analysis and functional clustering.

Gene annotation/ontology enrichment analysis was performed on genes differentially expressed (fold-change >= 1.75) in bulk-mRNA-seq-profiled adult RGCs after miR-1247-5p treatment, with the higher expression condition expressing a gene at least 1 FPKM (or above). For the subset of genes differentially expressed after miR-1247-5p treatment that also recapitulated developmental expression in RGCs, genes developmentally-regulated <= −1.75 fold-change from E18 to adult were analyzed. Gene annotation/ontology enrichment analysis (with minimum enrichment score threshold set to 0.1) was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID)41,42, gprofiler2, enrichplot, clusterprofiler, and GOSemSim R packages80–82. DAVID functional annotation clusters (which group similar annotation terms into more meaningful clusters of enriched annotations) were plotted as a barplot using ggplot280–82. Genes comprising annotation/ontology terms that were found to be significantly enriched by DAVID, were also analyzed by a gene network plot (in Fig. 4B–C), using enrichplot R package80–82, which shows the terms that shared the same genes within the genes regulated by miR-1247-5p treatment.

Statistical analyses.

All tissue processing, quantification, and data analysis were done masked throughout the study. Sample sizes were based on accepted standards in the literature and our prior experiences. Sample size (n) represents total number of biological replicates in each condition. All experiments included appropriate controls. No cases were excluded in our data analysis, although a few animals that developed a cataract in the injured eye were excluded from the study, and their tissues were not processed. The data are presented as means ± SEM and was analyzed (as specified in the applicable Figure legends), where appropriate, by independent samples t-test 2-tailed, or one-way ANOVA with or without Repeated Measures, and a posthoc LSD test (SPSS), as indicated. All differences were considered significant at p < 0.05.

Highlights.

  • Novel role of micro-RNA miR-1247-5p in neuroprotection and axonal regeneration after optic nerve injury in vivo.

  • Developmental expression of miR-1247-5p in retinal ganglion cells is downregulated during maturation.

  • Experimental expression of miR-1247-5p in adult retinal ganglion cells regulates various biological processes.

ACKNOWLEDGMENTS

This work was supported by a grant from the National Institutes of Health (NIH) (Grant R01-EY029739, to E.F.T.). Portions of this research were conducted at the High Performance Computing Facility, University of Connecticut. We are grateful to Sophan Iv and Vijender Singh (Research IT Services, University of Connecticut), and Stephen King (High Performance Computing Facility, University of Connecticut), for assistance with bioinformatics tools. We thank Ashiti Damania and Mahit Gupta (University of Connecticut School of Medicine) for technical assistance.

Footnotes

DECLARATION OF INTERESTS

The authors declare no competing interests.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

E18 and P5 RGC bulk small-RNA-seq raw reads and processed data for miR-1247 locus analyzed in this study are available through the NCBI GEO under accession number GSE252517. E18 RGC and miR-1247-5p-mimic or scramble shRNA-mimic transduced adult RGC bulk mRNA-seq raw reads and processed data used in this study are available through the NCBI GEO under accession number GSE252517.

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