miR-182-5p Regulates Nogo-A Expression and Promotes Neurite Outgrowth of Hippocampal Neurons In Vitro

Nogo-A protein is a key myelin-associated inhibitor of axonal growth, regeneration, and plasticity in the central nervous system (CNS). Regulation of the Nogo-A/NgR1 pathway facilitates functional recovery and neural repair after spinal cord trauma and ischemic stroke. MicroRNAs are described as effective tools for the regulation of important processes in the CNS, such as neuronal differentiation, neuritogenesis, and plasticity. Our results show that miR-182-5p mimic specifically downregulates the expression of the luciferase reporter gene fused to the mouse Nogo-A 3′UTR, and Nogo-A protein expression in Neuro-2a and C6 cells. Finally, we observed that when rat primary hippocampal neurons are co-cultured with C6 cells transfected with miR-182-5p mimic, there is a promotion of the outgrowth of neuronal neurites in length. From all these data, we suggest that miR-182-5p may be a potential therapeutic tool for the promotion of axonal regeneration in different diseases of the CNS.


Introduction
During postnatal development, central nervous system (CNS) neurons lose their ability to regenerate, in part due to the presence of myelin-derived inhibitors of axonal outgrowth and neuroregeneration, such as MAG, OMGp, and Nogo-A [1]. The RTN4 gene (Nogo) belongs to the family of reticulon-encoding genes that produces three major protein variants, RTN4A (Nogo-A), RTN4B (Nogo-B), and RTN4C (Nogo-C), by alternative splicing, promoter usage, and alternative polyadenylation, respectively [2]. They all share a common 188-amino-acid C-terminal membrane-spanning domain known as a reticulon homology domain (RHD domain) consisting of two hydrophobic regions flanking a hydrophilic loop (Nogo-66), which is followed by a short C-terminal tail. Nogo-A is the largest of the Nogo isoforms, with two known neurite growth inhibitory domains, including amino-Nogo (Nogo-A-∆20) at the N terminus and the extracellular loop Nogo-66 [3].
Nogo function depends on the tissular expression of each isoform [4]. Nogo-A has been studied extensively in the CNS and is responsible for the inhibition of neurite outgrowth, acting as a myelin-associated inhibitor of axon regeneration. Nogo-A is mostly expressed in spinal motor, DRG, sympathetic, hippocampal, and Purkinje neurons, as well as oligodendrocytes. In the spinal cord, Nogo-A is mainly expressed in grey matter, especially by large motor neurons of the ventral horns [5][6][7]. The functions carried out by Nogo-A in neurons and oligodendrocytes appear to be quite different. Whereas neuronal Nogo-A seems to act as a direct local restrictor of synaptic and dendritic plasticity, oligodendrocytic Nogo-A may act as an inhibitor of axonal growth by binding to its receptor (Nogo receptor, NgR1) located on neurons. This binding transduces the inhibitory signal to the cell interior via transmembrane co-receptors LINGO-1 and p75NTR or TROY, by which the small GTPase RhoA is activated [1,3,[8][9][10]. Multiple studies have demonstrated the efficacy of targeting the Nogo-A/NgR1 pathway for functional recovery and neural repair after spinal cord trauma, ischemic stroke, optic nerve injury, and models of multiple sclerosis [11].
MicroRNAs (miRNAs) are an abundant class of small non-coding RNAs that operate as epigenetic modulators of gene expression in physiology but also in pathophysiological processes. They are involved in post-transcriptional gene silencing by base pairing to their target mRNAs of protein-coding genes, resulting in reduced translation of the protein by mRNA repression or degradation [12,13]. The flexibility and efficiency of miRNA function provide both spatial and temporal gene regulatory capacities that are essential for establishing neural networks. The expression of miRNAs is ubiquitous in neural tissues, and many regulate neuronal differentiation, neuritogenesis, excitation, synaptogenesis, and plasticity [13][14][15]. There is a close relationship between miRNAs and intrinsic determinants of axonal regeneration. Several miRNAs have been proven to regulate the expression levels of targets involved in neurite outgrowth and axonal regeneration after CNS injury. For example, miR-431 is nerve-injury-induced miRNA that stimulates regenerative axon growth by silencing Kremen1, an antagonist of Wnt/beta-catenin signaling [16] or miR-133b, that has also been shown to promote neurite outgrowth in primary cortical neurons and PC12 cells by targeting RhoA [17]. Other examples are miR-124, a well-conserved brain-specific miRNA that promotes neurite outgrowth of M17 cells by targeting ROCK1 GTPase [18], or miR-222, which regulates neurite outgrowth from DRG neurons by targeting PTEN [19].
However, a functionally validated miRNA that regulates the expression of Nogo-A has not been yet described. Although the reticulon (RTN) family isoforms mature through splicing and alternative polyadenylation processes, the RTN4 gene shares the highly conserved carboxy-terminal reticulon domain and 3 UTR. We thus seek functional validated miRNAs capable of inhibiting the activity of the Nogo family members, and only miR-182-5p was found to directly target Nogo-C 3 UTR and decrease Nogo-C protein levels in cardiomyocytes cells [20]. miR-182-5p is a member of the miR-183 family located on chromosome 7q31-34 and is described as an oncogenic miRNA due to its capacity to enhance cancer cell proliferation, survival, tumorigenesis, and drug resistance [21,22]. Although miR-182-5p roles are well known in cancer, little is known about its function in the CNS under normal and pathophysiological conditions. Wang and colleagues demonstrated that miR-182-5p promotes axonal growth and regulates neurite outgrowth via the PTEN/AKT pathway in cortical neurons [23]. Moreover, miR-182-5p is the most abundant miRNA in retinal ganglion cell axons, where it regulates Slit2-mediated axon guidance in vitro and in vivo [24]. Furthermore, Yu and colleagues showed that miR-182-5p inhibits Schwann cell proliferation and migration by targeting FGF9 and NTM in sciatic nerve injury [25].
Because all Nogo protein variants share the conserved carboxy-terminal reticulon domain and 3 UTR, we hypothesize that miR-182-5p could also regulate Nogo-A expression. In the present study, we perform a bioinformatic and validation characterization of the miR-182-5p site in the Nogo-A 3 UTR to demonstrate for the first time that miR-182-5p downregulates Nogo-A protein expression in Neuro-2a and C6 cells and promotes neurite outgrowth of rat primary hippocampal neurons in vitro.

miR-182-5p Is Predicted to Regulate Mouse, Rat, and Human Nogo-A 3 UTRs
A bioinformatics-based prediction of the potential targets of miR-182-5p in mouse mRNAs was performed. Because the various available programs can yield rather different predictions, we combined miRmap, miRanda 3.3a, TargetScan 8.0, and miRWalk 3.0 programs to search for mouse miR-182-5p gene targets ( Figure 1A). A total of 191 common genes were identified by the four prediction programs, including Nogo (RTN4 gene). miR-182-5p has a canonical site at the human Nogo-A 3′UTR, with a hybridization energy (ΔGhybrid) of −27.10 kcal/mol; therefore, the interaction between miR-182-5p and its seed region in the human Nogo-A 3′UTR is thermodynamically stable.
Taken together, the bioinformatics approach supports the potential of miR-182-5p as a site in the sequence of Nogo-A 3′UTR in the three species studied; therefore, miR-182-5p can play a biologically relevant role in regulating their expression. Description and main data of the process of identification of mouse RTN4 gene (Nogo-A) as miR-182-5p-predicted target by miRmap, miRanda 3.3a, TargetScan 8.0, and miRWalk 3.0. The table shows the algorithm prediction scores, binding probability, free energy gained by transitioning from the state in which the miRNA and the target are unbound (∆G open) (kcal/mol) and the state in which the miRNA binds its target (∆G binding), according to each algorithm. (B) Alignment of the seed region of miR-182-5p with the Nogo-A 3′UTR in mouse (Mmu), rat (Rno), and human (Hsa) mRNAs. MiR-182-5p sequence appears in blue type, and miRNA seed regions appear in bold type. (C) Main data of the target site accessibility and hybrid diagram seed site of miR-182-5p on human Nogo-A 3′UTR by STarMir tool. MiR-182-5p sequence appears in blue type, and miRNA seed region appears underlined. , rat (Rno), and human (Hsa) mRNAs. miR-182-5p sequence appears in blue type, and miRNA seed regions appear in bold type. (C) Main data of the target site accessibility and hybrid diagram seed site of miR-182-5p on human Nogo-A 3 UTR by STarMir tool. miR-182-5p sequence appears in blue type, and miRNA seed region appears underlined.
According to the employed prediction programs, the Nogo-A 3 UTR of all species has one binding site for miR-182-5p. Alignment of this putative site in rat, mouse, and human sequences demonstrates the evolutionary conservation of the Nogo-A site among mammalian species ( Figure 1B). Because the three Nogo protein variants share the same 3 UTR region, we observe that this site matches with the already validated miR-182-5p site in the Nogo-C 3 UTR [20]. Analyses of target site accessibility of the mRNA secondary structure by STarMir tool further support miR-182-5p targeting on the human Nogo-A 3 UTR. The logistic probability (LogitProb) of the Nogo-A 3 UTR site being a miR-182-5p binding site is 0.635 ( Figure 1C). In general, a LogitProb of 0.5 indicates a fairly good chance of miRNA binding [26]. The hybrid diagram of the seed site ( Figure 1C) shows that miR-182-5p has a canonical site at the human Nogo-A 3 UTR, with a hybridization energy (∆Ghybrid) of −27.10 kcal/mol; therefore, the interaction between miR-182-5p and its seed region in the human Nogo-A 3 UTR is thermodynamically stable.
Taken together, the bioinformatics approach supports the potential of miR-182-5p as a site in the sequence of Nogo-A 3 UTR in the three species studied; therefore, miR-182-5p can play a biologically relevant role in regulating their expression.

Nogo-A and miR-182-5p Expression in Neural Cell Lines
We chose Neuro-2a and C6 cell lines to study Nogo-A regulation by miR-182-5p mimic in vitro due to their Nogo-A protein expression levels ( Figure 2A) and endogenous  Figure 2B). Conversely, SH-SY5Y cell line was discarded because Nogo-A protein is undetected in its extracts.

Nogo-A and miR-182-5p Expression in Neural Cell Lines
We chose Neuro-2a and C6 cell lines to study Nogo-A regulation by miR-182-5p mimic in vitro due to their Nogo-A protein expression levels ( Figure 2A) and endogenous expression of miR-182-5p ( Figure 2B). Conversely, SH-SY5Y cell line was discarded because Nogo-A protein is undetected in its extracts.

MiR-182-5p Targets the Mouse Nogo-A 3′UTR and Downregulates Its Protein Expression
The luciferase activity of the pmiRGLO plasmid in the presence of miR-182-5p mimic was evaluated to rule out any effect of the miRNA on plasmid expression, and no significant effects were detected ( Figure 3A) (109.1 ± 21.71% vs. 100% empty pmiRGLO without mimic co-transfection; two-tailed paired t-test, T4 = 0.4203, n.s. p = 0.6958).
Finally, to evaluate the modulation of Nogo-A protein expression levels by miR-182-5p, Neuro-2a and C6 cells were transfected with either miR-182-5p or negative control mimics for 24 h, and protein expression levels were detected by immunoblot assay. Transfection with miR-182-5p mimic significantly downregulated the endogenous Nogo-A protein levels compared to negative control mimic transfection in both the Neuro-2a cell line (70.98 ± 3.98% vs. 100% Negative control mimic; two-tailed paired t-test, T2 = 7.291, p =

miR-182-5p Targets the Mouse Nogo-A 3 UTR and Downregulates Its Protein Expression
The luciferase activity of the pmiRGLO plasmid in the presence of miR-182-5p mimic was evaluated to rule out any effect of the miRNA on plasmid expression, and no significant effects were detected ( Figure 3A) (109.1 ± 21.71% vs. 100% empty pmiRGLO without mimic co-transfection; two-tailed paired t-test, T 4 = 0.4203, n.s. p = 0.6958).
Differences in cell density were observed in the transfected cultures. Transfection of both mimics significantly reduced the C6 cell density in the co-cultures compared to nontransfected C6 cell co-culture (control co-culture) (two-way ANOVA, F2,12 = 25.52, p <
Differences in cell density were observed in the transfected cultures. Transfection of both mimics significantly reduced the C6 cell density in the co-cultures compared to non-transfected C6 cell co-culture (control co-culture) (two-way ANOVA, F 2,12 = 25.52, p < 0.0001; Tukey's multiple comparison post hoc test, p < 0.0001), although hippocampal neuronal densities were not significantly changed ( Figure 4C).

Discussion
Amongst many factors, one of the major inhibitory signals of the CNS environment to regrowth is the myelin-associated Nogo pathway, which plays an important role in regeneration [27][28][29]. Oligodendrocytic Nogo-A binds to its neuronal receptor (NgR1) and co-receptors (LINGO-1 and p75NTR or TROY), inhibiting neurite outgrowth of the neurons [8][9][10]. In the present study, we describe and validate, for the first time, Nogo-A posttranscriptional regulation by a miRNA in two murine neural cell lines, Neuro-2a and C6, which have been extensively used to study neuronal differentiation and axonal growth [30,31]. We demonstrate that miR-182-5p downregulates Nogo-A expression, promoting neurite outgrowth of primary hippocampal neurons in vitro.
The involvement of Nogo-A in neurodegeneration has been described in diverse CNS diseases, such as ocular diseases, multiple sclerosis, Alzheimer's disease, and amyotrophic lateral sclerosis, as well as spinal cord injury (SCI) and traumatic brain injury. However, the role of Nogo-A is not restricted to the CNS; Nogo-A also inhibits the spread and migration of non-neuronal cell, such as fibroblasts and vascular endothelial cells [32,33].

Discussion
Amongst many factors, one of the major inhibitory signals of the CNS environment to regrowth is the myelin-associated Nogo pathway, which plays an important role in regeneration [27][28][29]. Oligodendrocytic Nogo-A binds to its neuronal receptor (NgR1) and co-receptors (LINGO-1 and p75NTR or TROY), inhibiting neurite outgrowth of the neurons [8][9][10]. In the present study, we describe and validate, for the first time, Nogo-A post-transcriptional regulation by a miRNA in two murine neural cell lines, Neuro-2a and C6, which have been extensively used to study neuronal differentiation and axonal growth [30,31]. We demonstrate that miR-182-5p downregulates Nogo-A expression, promoting neurite outgrowth of primary hippocampal neurons in vitro.
The involvement of Nogo-A in neurodegeneration has been described in diverse CNS diseases, such as ocular diseases, multiple sclerosis, Alzheimer's disease, and amyotrophic lateral sclerosis, as well as spinal cord injury (SCI) and traumatic brain injury. However, the role of Nogo-A is not restricted to the CNS; Nogo-A also inhibits the spread and migration of non-neuronal cell, such as fibroblasts and vascular endothelial cells [32,33].
The dysregulation of Nogo-A following CNS injury, in particular SCI, is in line with the expression changes of numerous genes that play vital roles in the pathogenesis of secondary CNS damage or axonal regeneration [34,35]. Most of these genes are regulated by the post-transcriptional regulator miRNAs [36], which showed an altered expression following injury [37][38][39][40][41]. Both in vitro and in vivo evidence has demonstrated that these miRNAs participate in crosstalk with key genes involving processes of neuronal plasticity, neuronal degeneration, axonal regeneration, remyelination, and glial scar formation after SCI through translational repression or mRNA degradation [42][43][44][45]. Thus, miR-133b, miR-135a-5p, and miR-29a regulate neurite outgrowth and axon regeneration by targeting RhoA, ROCK1/2, and PTEN genes, respectively. Moreover, the overexpression of these miRNAs has been shown to contribute to spinal cord regeneration and functional recovery in murine SCI models [17,[46][47][48]. Similarly, miR-182-5p has been found to be involved in secondary damage of CNS processes and neuronal regeneration. According to miRNATissueAtlas2 (https://ccb-web.cs.uni-saarland.de/tissueatlas2, accessed on 13 January 2022; [49]), miR-182-5p is mainly expressed in blood vessels, the epididymis, and the CNS, especially in the spinal cord.
Previous analysis from our laboratory [38] and others [37,50,51] revealed miR-182-5p as one of the most downregulated miRNAs after injury, in agreement with recently described time-course miR-182-5p expression results in SCI [40]. In this study, the highest downregulation point of miR-182-5p expression was observed at 7 days post injury, with expression recovery at 28 days, which interestingly parallels both Nogo-A protein and mRNA expressions, which rapidly rose to a peak after 7 days and then gradually declined again after 14 days [52]. However, to dare, a validated miRNA targeting Nogo-A has not been reported. In accordance with our studies, the modulatory role of miR-182-5p on Nogo-A, which was validated by our luciferase and immunoblot results (Figure 3), could explain this dynamic of expression changes following injury. The validity of the miR-182-5p and Nogo-A interaction is supported by the miR-182-5p regulation of another member of the RTN4 family, namely Nogo-C, which shares 3 UTR with Nogo-A.
Overexpression of miR-182-5p targets the Nogo-C 3 UTR and decreases its protein levels, protecting cardiomyocytes from apoptosis and preserving cardiac function after myocardial infarction [20]. However, single genes may produce a variety of mRNA isoforms by mRNA modification, such as alternative polyadenylation or splicing, which could alter the selective recruitment of miRNAs to the 3 UTR [53,54]. It has been observed that nearly all genes have multiple alternative polyadenylation signals located at different positions in the 3 UTR [55]. Thus, we approached the validation of the miR-182-5p as a regulator of Nogo-A, as in both mouse and human RTN4 gene, it has been described at more than one putative poly(A) signal site downstream of the stop codon, and the miR-182-5p site is located between these polyadenylation signals. Our bioinformatics analyses confirmed that miR-182-5p binding site on Nogo-A 3 UTR is conserved across different mammalian species, including humans. This miR-182-5p binding site in human Nogo-A 3 UTR has been confirmed by the STarMir CLIP-based tool, supporting the possibility of Nogo-A regulation by miR-182-5p in human cells. Our experimental data concerning reporter gene regulation and Nogo-A endogenous expression levels after overexpression of miR-182-5p in cell cultures validate this microRNA response element in the Nogo-A 3 UTR.
Although the role of miR-182-5p as a regulator of neurite outgrowth has been described in cortical and midbrain neurons through activation of the PTEN/AKT pathway [23,56], our results could provide a broader implication with regards to axonal regeneration. Our functional assays (Figure 4) showed that the downregulation of Nogo-A due to miR-182-5p overexpression in neural cells eased the neurite outgrowth of primary hippocampal neurons. However, a better understanding of miR-182-5p regulation on Nogo-A, including exploring non-canonical mechanisms (e.g., paracrine regulation by miR-182-5p expressing cells), is needed to establish its precise role following CNS injury. Analyses would greatly benefit from gain-and loss-of-function assays employing stable and conditional cell lines.
Neutralizing Nogo-A by function-blocking antibodies or genetic knockout (KO) has been shown to improve axonal sprouting and regeneration in the injured spinal cord and brain [11,57,58], and the clinical potential of anti-Nogo-A antibodies for managing SCI is currently being investigated in two clinical trials (ClinicalTrials.gov accessed on 25 January 2022|Identifiers: NCT03935321 and NCT03989440). Therefore, Nogo-A downregulation by overexpression of miR-182-5p could be a potential treatment for different diseases and conditions that implicate axonal degeneration.
miR-182-5p binding site accessibility on the human Nogo-A 3 UTR (3 UTR-Nogo-A) was analyzed using the STarMir tool [26], an implementation of logistic prediction models developed with miRNA binding data from crosslinking immunoprecipitation (CLIP) studies. In the STarMir web (https://sfold.wadsworth.org/cgi-bin/starmirtest2.pl, accessed on 5 November 2021), we input hsa-miR-182-5p into the option of "microRNA sequence(s), microRNA ID(s)". The human 3 UTR-Nogo-A sequence was input into the option of "single target sequence, manual sequence entry". With the choice of "V-CLIP based model (human)", "Human (homo sapiens)", and "3 UTR", a set of parameters (described in http://sfold.wadsworth.org/data/STarMir_manual.pdf accessed on 5 November 2021) was displayed in the output window for further analysis.

RNA Extraction and Quantitative Real-Time PCR (RT-qPCR)
Total RNA was isolated from Neuro-2a, C6, and SH-SY5Y cells with an miRNeasy Kit (Qiagen) and was analyzed with a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific) to determine its concentration and purity (260/280 and 260/230 ratios).
To determine cellular miR-182-5p expression, 10 ng of total RNA was reverse-transcribed and amplified using TaqMan miRNA gene expression assay (TaqMan ® MicroRNA assay #002284, Applied Biosystems) following the manufacturer's protocols. The U6 small nuclear RNA served as an internal control (TaqMan ® MicroRNA assay #001973, Applied Biosystems). The abundance of the miRNA was measured in a thermocycler ABI Prism 7900 fast (Applied Biosystems) applying 40 cycles of a two-step protocol: 15 s at 95 • C plus Pharmaceuticals 2022, 15, 529 9 of 13 1 min at 60 • C. The ∆Ct value was defined as the difference between the cycle threshold of amplification kinetics (Ct) of the target miRNA and its U6 loading control [59].
Neurite outgrowth lengths were assessed using the method described by Rønn and colleagues [60]. Briefly, the absolute neurite length (L) per neuron was estimated by counting the number of intersections (I) between neurites and test lines of a grid superimposed on the co-culture images and the equation L = (πd/2)I, where d is the vertical distance between the test lines of the grid. The neurite length increase per neuron was calculated using the control co-culture (with non-transfected C6 cells) as reference, that is, subtracting the mean neurite length per neuron of the control co-culture from the mean neurite length per neuron of the mimic-transfected co-cultures. C6 cells and hippocampal neuronal density were analyzed by calculating the total number of each type of cell per mm 2 in the different co-cultures (a total of nine images of 0.27 mm 2 per condition were analyzed).

Statistical Analysis
Statistical significance of the transfection effects was tested using paired Student's t-test or two-way ANOVA with Tukey's multiple comparison post hoc tests. Data are expressed as mean ± SEM, as indicated in the figure legends. Statistical analyses and graphic design were conducted using GraphPad Prism version 8.0.0 (GraphPad Software). Differences were considered statistically significant when the p-value was below 0.05.

Conclusions
This study provides novel information about the regulatory action of miR-182-5p on Nogo-A axonal regeneration inhibition. We are the first to describe and functionally validate Nogo-A regulation by miR-182-5p, which targets Nogo-A 3 UTR, downregulates Nogo-A protein expression levels, and promotes neurite outgrowth in murine neural cell lines. Thus, miR-182-5p could be a promising therapeutic tool for diseases or conditions that implicate axonal pathology, such as SCI, brain injury, and Parkinson's or Alzheimer's diseases, among others. Funding: This research was supported by Fundación Tatiana Pérez de Guzmán el Bueno (Neurociencia 2016) and the Council of Education, Culture and Sports of the Regional Government of Castilla La Mancha (Spain) and co-financed by the European Union (FEDER), "A way to make Europe" (SBPLY/17/180501/000376). Altea Soto was funded by the Council of Education, Culture and Sports of the Regional Government of Castilla La Mancha (Spain). M. Asunción Barreda-Manso is funded by the Council of Health of the Regional Government of Castilla La Mancha (Spain).

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
Data is contained within the article.