BDNF Modulation by microRNAs: An Update on the Experimental Evidence

MicroRNAs can interfere with protein function by suppressing their messenger RNA translation or the synthesis of its related factors. The function of brain-derived neurotrophic factor (BDNF) is essential to the proper formation and function of the nervous system and is seen to be regulated by many microRNAs. However, understanding how microRNAs influence BDNF actions within cells requires a wider comprehension of their integrative regulatory mechanisms. Aim: In this literature review, we have synthesized the evidence of microRNA regulation on BDNF in cells and tissues, and provided an analytical discussion about direct and indirect mechanisms that appeared to be involved in BDNF regulation by microRNAs. Methods: Searches were conducted on PubMed.gov using the terms “BDNF” AND “MicroRNA” and “brain-derived neurotrophic factor” AND “MicroRNA”, updated on 1 September 2023. Papers without open access were requested from the authors. One hundred and seventy-one papers were included for review and discussion. Results and Discussion: The local regulation of BDNF by microRNAs involves a complex interaction between a series of microRNAs with target proteins that can either inhibit or enhance BDNF expression, at the core of cell metabolism. Therefore, understanding this homeostatic balance provides resources for the future development of vector-delivery-based therapies for the neuroprotective effects of BDNF.


Introduction
MicroRNAs are a class of non-coding RNAs which do not code for proteins but carry out their biological function by regulating the cell proteome at the translational level.They can be expressed within the activation of a gene promoter or their own promoters [1,2], and play a regulatory role in protein synthesis by targeting and degrading RNA transcripts containing compatible nucleotide sequences [3][4][5][6][7].MicroRNAs are seen to participate in the functional regulation at the distant synaptic sites in neuronal cells, and to modulate inflammatory mechanisms that lead to neurological diseases [8,9].
As a main expressed neurotrophin, brain-derived neurotrophic factor (BDNF) plays essential roles in the development and maintenance of neural tissues [10,11].The signaling of the mature form of BDNF via tropomyosin receptor kinase (Trk) B participates in neuronal survival, dendritogenesis, synaptogenesis, axon growth, and synaptic function; meanwhile, the release of a pro-BDNF isoform can bind with a low affinity to p75 neurotrophin receptor (p75NTR) and lead to apoptosis, so that a tight regulation of BDNF activity is necessary for the proper functioning of the central nervous system (CNS) [12][13][14].
Analyses in silico estimate hundreds of microRNAs as possible regulators of BDNF.However, with a 10-20% variability detected in the predicted regulatory relationships between genes and microRNAs in the human RefSeq dataset, the effective regulation of BDNF mRNA transcripts by microRNAs in biological systems is much smaller [15].In addition, the microRNA affinity for multiple targets and microRNA-microRNA interactions in a cell milieu influence their regulation and cannot be predicted by computational logarithms.As the studies typically address only one or a few microRNAs in their experiments, it remains a challenge to design pre-clinical studies based on computational predictions [2,16].
Regarding the notion that microRNAs target and degrade RNA transcripts across different tissues and regulate protein function via direct and indirect mechanisms; a broader look at the possible scenarios where BDNF actions can potentially be affected by microR-NAs shall include all the available data reporting interactions between microRNAs and BDNF in biological experiments.Further, understanding how microRNAs effectively regulate BDNF actions provides the basis for the development of potential therapies against neurodegenerative conditions.Therefore, we have collected all the available data on the post-transcriptional regulation of BDNF by microRNAs evidenced in experimental studies, and provided a synthesis of the regulatory mechanisms currently demonstrated.

Methods
In order to retire all the scientific publications possibly reporting data from the analysis of microRNAs and BDNF expression in a same biological system, a systematic search was conducted on PubMed.govusing the following combination of terms: ["BDNF" AND "MicroRNA"] OR ["brain-derived neurotrophic factor" AND "MicroRNA"].All the available publications were retrieved and screened by abstract.Papers published without open access were requested from the authors via email or ResearchGate.Studies containing data from BDNF and microRNA analyses in vivo or in vitro were considered for inclusion.The studies reporting data of microRNAs that did not influence BDNF regulation, reviews, articles not written in English, high-throughput profiles, and computational predictor studies, as well as those not made available by the authors, were excluded from discussion during full-text assessment.The last search update, performed in 1 September 2023, launched 314 papers published from 2006 to 2023 indexed in PubMed (see Figure 1).The studies selection was performed using the software Mendeley 1.19.8.Two-hundred and ninety-seven articles were sought for retrieval according to the inclusion criteria of containing data from BDNF and microRNA analyses, after removal of duplicates.Two hundred and sixty-one papers were assessed by full text.A total of 171 were found to include the analyses of BDNF and diverse microRNAs and were included in the qualitative synthesis, after exclusion criteria (Figure 1).A list of the studies and microRNAs involved in BDNF regulation was displayed in Table 1.
miR-584, miR-744, miR-26a-1, and miR-26a-2 subtypes [24,28,58,60,72,75,78,88,93,94,96,102,112,125,126,128,132,133,137,190].The evidence that miR-206 is able to suppress BDNF synthesis in diverse tissues such as the cardiac muscles, the skeletal muscles, and the endothelial tissue elucidates a role for microRNAs in tissuetissue communication, although their actions might be locally regulated [30,43,45,48,61,92,102,115,120,139,142,169,171,177,191].MicroRNAs play crucial roles in immunoinflammatory reactions.In normal conditions, the CNS parenchyma is not exposed to peripheral immune cells or robust inflammatory responses, and the microglia and astrocytes remain quiescent.However, upon stress, the astrocytes and microglia transiently activate and produce chemokines and cytokines, and other small-molecule messengers (prostaglandins, nitric oxide, and reactive oxygen species-ROS) which contribute to the inflammatory response and subsequent restoration of CNS homeostasis [195].The study by Kynast and colleagues [38] identified that miR-124 is constitutively expressed in neurons of the dorsal horn in the spinal cord, where its elevation is associated with a decrease in BDNF levels, while a decrease in miR-124 levels leads to the elevation in methyl CpG binding protein 2 (MeCP2) and BDNF expression levels.From a different perspective, the studies by [129,196] demonstrated that miR-124 is able to attenuate an acute increase in pro-inflammatory factors in the CNS by suppressing the early growth response 1 (EGR1) and preventing a decline in BDNF expression.Conversely, Yu et al. [180] showed that BDNF administration increased the expression levels of miR-3168, and suppressed the secretion of interleukin (IL)-1β, TNF-α, and IL-6 in the activated macrophage.
Another mechanism by which microRNAs indirectly modulate BDNF synthesis in inflammatory conditions involve the Let-7 miRNA family [197], which include let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, let-7i, miR-98, and miR-202 [198].The dysregulation of let-7 leads to a less differentiated cellular state and cell-based diseases such as cancer.Cho and colleagues [53] investigation in neural tissue reported that let-7a levels increase in microglia following the accumulation of ROS and pro-inflammatory cytokines.The data indicated that let-7a participates in reducing nitrite production while increasing the levels of inducible NO synthase and IL-6.Anti-inflammatory events accompanied an upregulation in BDNF expression levels.Alternatively, Nguyen and colleagues [111] detected that miRlet-7i suppresses the synthesis of progesterone receptor membrane component 1, reducing the progesterone-inducible release of BDNF by astrocytes.Such a reduction has a negative effect on neuronal tissue recovery.These findings show that Let-7 members might exert specific roles that positively or negatively affect BDNF function in the CNS parenchyma.
Although the regulation of protein function by microRNAs mostly always depends on their nucleotide sequence to target mRNA transcripts present in the same micro en-vironment, in silico predictions of BDNF-target microRNAs are not always confirmed in biological systems.Meanwhile, some experiments have pointed out that the microRNA targeting of BDNF mRNA is selectively guided by their prime untranslated region (3 ′ -UTR) [28,51,127,130].Having noted the presence of two variants of 3 ′ UTR regions in the mRNA transcripts of BDNF, which exert an influence on their cellular trafficking/localization [199], the mechanistic regulation of BDNF by microRNAs within a cell might as well occur in a local specificity manner, a least in cells that express the two BDNF mRNA 3 ′ UTR isoforms.

Neuroplasticity and BDNF Regulation by microRNAs
The expression of BDNF is present in progenitor cells from the early embryonic phase and in neural tissue throughout the whole lifespan.Its participation in essential processes such as dendritogenesis, axonal innervation and synaptogenesis, neuronal growth, and survival guarantees the maintenance and proper functioning of the neuronal tissue [200][201][202].
Some studies' evidence shows that Sonic hedgehog (Shh), a basic protein expressed in the mid-line CNS as an inductive signal in the patterning of the ventral neural tube, the anterior-posterior limb axis, and the ventral somites [212], is able to relieve the suppression of miR-206 on BDNF mRNA translation, which, in turn, enhances BDNF-TrkB signaling during the differentiation and innervation processes, as seen in muscle cells [26,34].Shh is a key signaling molecule in the embryonic morphogenesis and organization of the nervous system.Its signaling via the receptor patched-mediated-smoothened receptor complex is putative to the development of the neural tube, while the abnormal activation of Shh signaling implicates various types of cancers [212].This indirect and positive effect of Shh on BDNF activity seems to be involved in a complex and phasic destabilization of cell homeostasis during differentiation in mesenchymal cells.
BDNF binding to TrkB receptors at the neuronal cells' surface leads to the dimerization and transphosphorylation of a critical regulator of actin dynamics in the axons and dendrites named LIM domain kinase 1 (LIMK1).This occurs independently of TrkB kinase activity.The LIMK1 mRNA transcript is a target for miR-134 in the axon and dendrite cell compartments, and is able to annul BDNF/TrkB-induced protein synthesis during the synaptic activity, whenever TrkB activation is not sufficient to surpass miR-134 suppression on LIMK1.This suggests that miR-134 actively participates in competitive synapses formation, and establishes a role for this microRNA in the fine-tuned regulation of neuroplasticity processes [17,23,25,62,80,213,214].
Several microRNAs were found to target different components of BDNF-TrkB signaling intracellular cascades, consequently decreasing the activity of the cAMP response element binding (CREB) protein, leading to a decrease in BDNF gene expression.The investigation by Thomas et al. [90] identified that miR-137 regulates the levels of various proteins within the PI3K-Akt-mTOR pathway in neurons, namely, p55g, PTEN, Akt2, GSK3b, mTOR, and rictor.And this negatively affects the BDNF-induced dendritic outgrowth.In addition, miR-221, miR-383, and miR-199a-5p were shown to suppress the synthesis of Wnt2, which is a glycoprotein with essential roles in embryonic development and dendrite development [215].The neuronal activity enhances the CREB-dependent transcription of Wnt2, which, in turn, stimulates dendritic arborization.Both Wnt2 and BDNF are CREB-responsive genes, and, thus, Wn2t suppression results in a decrease in BDNF expression possibly via the Wnt2/CREB/BDNF axis [108,216,217].Additionally, some microRNAs were reported to be negatively correlated with the levels of BDNF in studies, i.e., miR-183/96 [44,54], miR-134 [105,122,138], and miR-182-5p [174,182].

Cell Metabolism and BDNF Regulation by microRNAs
The post-transcriptional regulation of proteins elicits compensatory mechanisms to maintain the transcriptional activity of essential proteins involved in cell energy homeostasis.The integrative regulation of a number of proteins in the core of cell metabolism homeostasis affects the BDNF gene expression by various means, including its self-regulation via autocrine and/or paracrine TrkB signaling.BDNF/TrkB activation leads to the activation of several small G proteins in addition to the pathways regulated by mitogen-activated protein kinase (MAPK), PI 3-kinase (PI-3K), and phospholipase-Cγ (PLCγ) [218].Meanwhile, as miR-101 suppresses MAPK phosphatases 1 (which dephosphorylates p38, JNK, and ERK), it has a positive effect on ERK phosphorylation and the downstream activation of BDNF expression in cortical neurons [98].
The activity of AMP-activated protein kinase (AMPK) and CREB represents the axis of cell energy metabolism.A compensatory increase in CREB activity following a decrease in the concentrations of BDNF and MeCP2 was evidenced in the brain of 132/212 KO mice [18,65].While MeCP2 is a nuclear protein that may function as both a transcriptional activator or repressor, it works as a stabilizer of BDNF expression patterns and cell homeostasis [157,219,220].Another compensatory effect is seen for BDNF in dendritogenesis when the inhibition of miR-15a, and the consequent relief of BDNF suppression, can rescue dendritic maturation deficits in MeCP2-deficient neurons [70].Further, an upregulation in BDNF gene expression accompanies an increase in the expression of the miR-132/212 cluster, both of which target and suppress MeCP2 mRNA translation.The suppression of miR-132 and miR-212 on MeCP2 relieves its repression on BDNF expression.By this manner, the expression of BDNF and miR-132 and miR-212 represents a self-regulatory homeostatic mechanism that involves the nuclear protein MeCP2 at the core of cell metabolism [18,22,24,32,33,47,49,64,81,85,91].
The enzymatic activity of the histone deacetylase Sirtuin 1 (SIRT1) in the nicotinamide adenine dinucleotide (NAD)-dependent deacetylation of histones is crucial in protecting cells from oxidative stressors.SIRT1 activates the expression of mitochondrial DNA genes related to mitochondrial biogenesis, ATP generation, and cell proliferation.It was detected in experiments that SIRT1 is able to inhibit miR-134 expression by directly binding to its inhibitory elements, whereas SIRT1 deficiency and high levels of miR-134 result in a downregulation of CREB and BDNF expression, and a negative effect on neuronal survival/plasticity, another indirect mechanism by which miR-134 negatively affects BDNF function in the core of cell metabolism [37,71,72,149,160].Finally, the study by Oikawa and colleagues [67] showed that the guanine nucleotide binding protein alpha inhibitor 1 (GNAI1), an adenylate cyclase inhibitor which regulates the ATP conversion to cAMP, is a target of miR-124.In physiological conditions, the suppression of GNAI1 by miR-124 increases in cAMP activity and leads to an upregulation of BDNF expression via the cAMP/PKA/CREB pathway.Indeed, alterations in cell metabolism and the microRNA environment reflect the regulation of BDNF.
MiR-124 suppression on BDNF activity negatively influences neuronal plasticity in various brain regions such as the hippocampus and striatum [21,35,46,77,100].More recently, [148] identified that miR-124 targets CREB mRNA, consequently downregulating the BDNF expression, and alters BDNF function via targeting various gene transcripts' downstream TrkB signaling, e.g., PI3K, Akt3, and Ras [181].Likewise, the miR-124 negatively influences BDNF activity by targeting and degrading mRNA transcripts of glucocorticoid receptors [103,110].Since signaling through glucocorticoid receptors potentiate BDNF actions via common intracellular pathways' downstream TrkB receptors activation [221], such a suppression on glucocorticoid receptor synthesis might reflect a negative modulation of BDNF function.From another perspective, by testing different exercise intensities, Mojtahedi and colleagues [42] showed that miR-124 levels increase with the intensity, and this increase is amplified in strenuous intensity.BDNF and TrkB also increased with intensity, but not in the strenuous intensity exercise.The findings indicate a threshold beyond which the changes in metabolic demands evoke an acute rise in miR-124 levels and its suppressive effect overcoming that of BDNF.
Amongst the indirect effects seen for microRNA on BDNF [40], the study registered that miR-9 upregulates BDNF expression in retinal ganglion cells by suppressing the restrictive silencer factor/RE1-silencing transcription factor (REST), a transcription repressor whose suppression is required for neuronal cell differentiation.Similarly, miR-29c has a positive effect on BDNF expression levels by targeting DNA methyltransferase 3 [56].The miR-705 was also found in a positive correlation with BDNF levels in an ischemic injured brain [106].Further, BDNF administration increases miR-214 expression during embryonic stem cell differentiation into endothelial cells [119], and it is found to promote vascular endothelial growth factor-C-dependent lymph angiogenesis by suppressing miR-624-3p in human chondrosarcoma cells [97].

Conclusions
As a conservative protein with essential roles in the core of cell functioning, the secretion of BDNF, which modulates the expression and signal through TrkB receptors, is regulated by several systems, which, ultimately, protect cell growth and differentiation processes from oncogenicity.This means that precise control, whether endogenous or exogenous, in the availability of BDNF is necessary in order to achieve positive outcomes in neuroprotection and recovery.In this sense, our data collection indicates that multiple microRNAs co-operatively regulate BDNF and several core proteins responsible for cell growth and metabolism homeostasis, in neuronal survival and recovery.This provides a prospective insight for the development of vector-derived therapies that can potentially address and modulate BDNF locally and favor tissue damage recovery with a lower risk of oncogenesis.

Limitations
The regulation of BDNF by microRNAs involves a complex and dynamic regulation of basic proteins at the core of neuronal cell homeostasis.Further, the increasing evidence of the participation of different microRNAs in the regulation of BDNF leads us to assume that there shall be much to be uncovered about such integrated regulatory mechanisms involved in the direct and indirect regulation of BDNF by microRNAs.This complexity represents a relevant limitation to future research towards the development of therapeutic strategies.

Figure 2 .
Figure 2. Experimental evidence of direct and indirect regulation of BDNF by microRNAs.Figure created by BioRender.com.

Table 1 .
Main findings in BDNF regulation by microRNAs.