General hallmarks of microRNAs in brain evolution and development

Abstract

MicroRNAs (miRNAs) are endogenous, small non-coding RNA molecules that mediate post-transcriptional gene suppression by incomplete matches with their host mRNAs. In the central nervous system, miRNAs that functionally interact with their target genes constitute a flexible, robust and buffered regulatory network, exerting diverse roles in brain evolution and development. However, distinct variation either in hub miRNA expression levels or patterns may initiate and/or progress various adult-onset nerve-related diseases. In this review, we will summarize the current knowledge about the general hallmarks of brain miRNAs that act as vital determinants in increasingly complicated neural activities. We endeavor to provide a constructive insight into the neuroscience research in the quest to comprehend molecular underpinnings of physiological functions and pathological disorders in central nervous system.

Keywords:

• central nervous system
• development
• evolution
• small non-coding RNA

Introduction

miRNAs constitute a large class of short (∼22nt) endogenous noncoding RNA molecules, which were initially discovered in Caenorhabditis elegans and perceived as the products of heterochronic genes.¹ To data, more than 28000 miRNAs have been preserved in the latest miRNA registry² (Release 21.0, http://www.mirbase.org). The genomic sources of novel miRNA genes, for example, exons, introns, transposable elements and repeats, have been summarized in greater detail by others, yet the appearance and expansion of miRNAs in animal genomes is still in flux.^3-9 Exploring the ultimate origin may contribute enormously to understanding the relationship between evolutionary adaptability of miRNAs and biological complexity. The formation and functional mechanism of mature miRNA have been extensively reported^3,10-14 (Fig. 1). Generally speaking, miRNAs are originated from long primary transcripts (pri-miRNA) into ∼60–90 nt hairpin-loop precursors (pre-miRNAs) by Drosha-DGCR8 complex in the nucleus, exported by exportin-5 (Exp-5) and further cleaved by the ribonuclease III family member Dicer into mature sequences in the cytoplasm.^10-13 The mature miRNA is then incorporated into the miRNA-induced silencing complex (miRISC) that typically guide it to mRNA targets, negatively regulating gene expression at the post-transcriptional level by imperfect matches with the 3-end untranslated regions (3′UTRs) of target mRNAs.^3,14

Figure 1. Overview of the generation and functional mechanism of mature miRNA. The sequence of the entire process is ordered by the arrow.

In the last decade, miRNAs have been confirmed to be major gene regulators in animal genomes, functioning by fine-tuning a broad range of biological processes and regulating the expression of at least one-third of all human genes.^15-17 Furthermore, miRNAs have been reported to be ubiquitously expressed in midbrain, hippocampus, frontal cortex and cerebellum and ever increasing evidence shows that miRNAs exert diverse functions in the central nervous system^18–21 (Fig. 2). These include neural genesis, differentiation and development as well as the process of synapse formation. Previous studies have also revealed that miRNAs gradually increases or decreases at the expression level during brain growth, highlighting a role of miRNA in brain morphogenesis.^16,22-24 Considering the pervasiveness and functionality of brain miRNAs, it is likely that distinct variation either in related miRNA expression levels or patterns may initiate and/or progress various adult-onset nerve-related diseases.^25-27

Figure 2. Percentage of miRNAs expressed in different areas of the brain and the related neuro-physiological functions.

The human brain, with an estimated 10000 different cell types, contains about 100–200 billion neurons that constitute a sophisticated neural network.^26,28 The complexity of information processed exceeds by far any other human system. Understanding the general principles of brain miRNAs related to the constant neural adaptation to environmental cues may be particularly meaningful for illuminating how the brain works through the intricate regulation of miRNAs. In earlier studies, research on miRNA functional features has been hampered due to the limitations of massively parallel detection technology. However, with the rapid development of microarray and high-throughput sequencing technologies, it is possible for investigators to simultaneously detect the sequence and expression change of all the miRNAs in a given sample under a particular condition. In the current review, we will systematically summarize the general features of miRNAs in the central nervous system, in order to shed light on the versatile roles of these small non-coding RNA molecules in brain evolution, development and even disorders.

miRNAs are continuously being added into the genome during brain evolution

Differential gene expressions among species evolutionary lineages have been suggested to be vital for phenotypic differences such as human-specific features, e.g., language and tool-making.²⁹ Protein-coding RNAs, mutations in regulatory elements proximal to genes, changes in expression or sequence of distal regulators and transcription factors have all been reported for differences in cognitive functions.^30-34 These unambiguous genetic variations are not enough to explain the differences in brain morphological complexity across Metazoa. However, some noncoding components such as miRNAs may be a supplementary explanation.^32,35

miRNAs are continuously being added into metazoan genomes. Several waves of miRNA repertoire expansion have been observed to coincide with the advent of bilaterians, vertebrates, and placental mammals.^5,36-38 Meunier et al., based on high-throughput RNA sequencing data, have confirmed increased synthesis and diversity of miRNAs in mammals than in birds and nearly the same number of miRNAs in closely related species through systematic comparative analysis among 5 major organs, including brain and cerebellum, from 6 species.³⁸ Massively parallel sequencing of brain miRNAs between human and chimpanzee or rhesus macaque highlights a cluster of human brain-specific miRNAs.^39,40 However, ˜30% of the total human miRNA pool has no detectable expression, implying their recent origin.^4,41 It seems that recent miRNA molecules have few conserved targets and low expression and will not be preserved by purifying selection over long evolutionary time periods.^42,43 Thus, it appears that human brain-specific miRNA genes are not necessarily associated with human special cognitive function. Dramatically, a recent report found a human brain-specific miRNA termed miR-941 whose target genes are involved in neurotransmitter signaling.⁴⁴ Another primate-specific and human brain-enriched miRNA is miR-1202, which has be reported to be associated with the pathophysiology of depression.⁴⁵ The main biological pathways related to neural activities of the 2 miRNAs above are shown in Figure 3. Based on these discoveries, we can speculate that newborn miRNAs in the central nervous system have important implications in the evolution and increased complexity of animal brain and an increasing number of important new miRNAs will be discovered with the rapid development of better miRNA detecting systems and molecular biotechnology.

Figure 3. Principal mechanisms of miR-941 and miR-1202 in neural activities. (A) miR-941 inhibits the expression of its host gene DNAJC5. DNAJC5, coupled with its directly interacted gene RAB3A and indirectly interacted gene WDR7, is associated with neurotransmitter release.⁴⁴ (B) miR-1202 expression is decreased in the donor brain with depression compared with the normal control. While the host gene expression of miR-1202, GRM4, is increased in the donor brain with depression. GRM4 is expressed throughout the brain and is associated with glutamatergic, dopaminergic, GABAergic and serotonergic neurotransmission.⁴⁵

Non-conservatively expressed miRNAs are involved in brain evolution

The primary sequence of the mature miRNA is highly conserved and rarely mutates across species.³⁶ Comparing the homologous hairpins of the miRNAs cloned from human brain among 17 animal species, 75% of known human brain miRNAs are found to be conserved in vertebrates and mammals, 14% are conserved in invertebrates, and the rest are species-specific.⁴⁰ Sequence conserved miRNAs are usually more broadly and robustly expressed than non-conserved ones.^46,47 Nevertheless, it appears that miRNA sequence conservation does not imply expression conservation. Although 325 miRNAs expressed in the prefrontal cortex are found to have high sequence conservation between human and chimpanzee, 11% are expressed at significantly different levels in these 2 species.³⁹ Comparisons of brain miRNAs among different primates reveal 19 developmentally regulated miRNAs that are 24-fold more divergent in human than in chimpanzee prefrontal cortex, which are found to be evolving far more rapidly than other classes of miRNA genes, indicating the crucial role of miRNAs in metazoan evolution.¹⁷ Human-specific expressed miRNAs (miR-184, miR-487a, miR-383, miR-34c-5p and miR-299-3p) located in neurons are found to be inversely related with their target gene expressions.³⁹ Interestingly, the 5 miRNAs, with their host mRNAs, are found to be conserved at the primary sequence level. Most of the target genes of the 5 miRNAs are significantly enriched in neural functions associated with learning and memory pathways (Table 1). Thus, the expression divergence of the 5 miRNAs between human and chimpanzee prefrontal cortex may have a crucial role in human lineage evolution. By fine tuning gene expression, non-conservatively expressed miRNAs, including brain species-specific expressed miRNAs, may to a large extent, be involved in species specific phenotypic adaptations. However, the direct evidence remains to be established.

Table 1. Functional enrichment analysis of human brain-specific expressed miRNAs

miRNA ID	KEGG pathway	Biological process
has-miR-299-3p	Axon guidance
	Neurodegenerative Diseases	Synaptic transmission
has-miR-184	Long-term potentiation
	Phosphatidylinositol signaling system	Signal transduction
	Melanogenesis	Cell surface receptor mediated signal transduction
has-miR-487a	Adherens junction	Neuronal activities
	Tight junction	mRNA transcription
has-miR-34c-5p	Colorectal cancer	Other mRNA transcription
	Prostate cancer
has-miR-383	N.A.

Note: KEGG pathway is determined by each target gene set; biological process is determined by the union set of all the target genes. N.A. represents unknown.

miRNAs constitute more flexible, robust and buffered brain transcriptional regulatory networks

The brain is the most complicated organ system.^26,28 It is thought that some characteristics of brain miRNAs; their abundant expression, co-expressed as clusters and more target sites, promote more flexibility, robustness or buffering of miRNA-mRNA regulatory networks. The result is increased combinatorial control of the evolution of brain morphological and functional complexity. But the abnormality of hub miRNAs in the regulatory network may lead to vulnerable regulatory circuits that will cause dysfunctional brain processes.

Brain-enriched miRNAs

It has been reported that approximately 70% of all miRNAs are expressed in brain regions.^48,49 This is not surprising given the cellular and transcriptional complexity of the central nervous system. According to previous studies, 65 miRNAs are found to be abundantly expressed in mouse brain based on massively parallel sequencing or chip techniques followed with traditional miRNA detection methods.^50-53 We used the miRWalk2.0 database⁵⁴ (http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/) Prediction Programs (miRanda, miRDB, miRWalk, RNA22 and TargetScan) to predict target genes and obtained 423 putative host mRNAs. Specifically, based on a functional analysis using DAVID functional analysis tool⁵⁵ (Release 6.7, http://david.abcc.ncifcrf.gov/), the targets of brain-enriched miRNAs are significantly enriched in various developmental and other basic biological processes (Table 2). Some brain-enriched miRNAs have been reported with important roles in normal brain development and aberrant expressions of these miRNAs are related to cancer or nerve-related diseases. For example, let-7 family members are found highly expressed in the developing mammalian brain.^23,56 As a spatiotemporal code for cell fate, let-7 is associated with embryonic and adult neurogenesis.^57,58 In addition, let-7 itself has no effect on the proliferation of normal human astrocytes but exerts an anti-tumorigenic effect on glioblastoma cells, abnormal expression of which will cause glioblastoma or neurocytoma.^59,60 miR-9, highly expressed in hippocampus, controls not only the timing of neurogenesis, but dendritic development and microglial activation as well as neural apoptosis.^52,61-65 miR-9 can also attenuate Aβ-induced synaptotoxicity, appearing to play an important role in the development of Alzheimer's disease.⁶⁶ Another brain-enriched miRNA is miR-124 which has been confirmed as a neuronal fate determinant, controlling the choice between neuronal and astrocyte differentiation by fine-tuning Ezh2 expression.^67,68 Downregulation of miR-124 may be linked to malignant tumor progression and poor prognosis in patients with gliomas.⁶⁹ Taken together, paying more attention to the potential role of brain-enriched miRNAs may contribute to further unveiling the potential physiological and pathological mechanisms of neuronal regulation.

Table 2. Significantly enriched biological processes of mouse brain enriched miRNAs

Biological process	Gene numbers	P-value	Benjamini
nervous system development	46	6.00E-07	1.10E-03
negative regulation of biological process	59	5.60E-06	5.10E-03
embryonic organ development	20	6.20E-06	3.80E-03
negative regulation of cellular process	54	9.50E-06	4.30E-03
anatomical structure morphogenesis	51	1.40E-05	5.00E-03
localization	97	1.40E-05	4.30E-03
blood vessel development	19	2.70E-05	7.00E-03
cellular process	259	2.70E-05	6.30E-03
embryonic development	35	3.40E-05	6.90E-03
vasculature development	19	3.70E-05	6.80E-03
protein transport	34	4.60E-05	7.70E-03
establishment of protein localization	34	5.40E-05	8.20E-03
regulation of cell differentiation	24	6.40E-05	9.00E-03
protein localization	37	6.90E-05	9.00E-03
system development	76	7.60E-05	9.30E-03

Note: Fisher's exact test, p < 0.001; Benjamini-Hochberg corrected, p < 0.01.

Co-expressed as clusters

Previous studies suggest that miRNAs are separated by intervals as short as a few nucleotides in the genome. These are defined as miRNA clusters which tend to be co-expressed presumably due to sharing common cis-regulatory elements or being derived from polycistronic precursors.^53,70 Hence, it seems that miRNAs from the same cluster show a similar expression level and nearly the same host mRNAs and the similar functionalities. A recent study shows that miR-17-92 cluster expressed in the distal axons of primary cortical neurons can enhance axonal outgrowth by local modulation of PTEN protein levels.⁷¹ In the developing neocortex, miR-17-92 cluster can facilitate neural stem cells proliferation and differentiation.⁷² Two members of miR-17-92 cluster, miR-17 and miR-20a, have been validated to act as a vital role in the self-renewal of postnatal neural stem cells by targeting Trp53inp1—a downstream component of the p53 pathway.⁷³ Aberrant expression of miR-17-92 cluster members, such as miR-92, miR-19a, and miR-20, is involved in medulloblastoma and retinoblastoma progressions.^74–76 Another interesting miRNA cluster is miR-183-96-182, which exerts pleiotropic effects on cell survival, proliferation and migration in medulloblastoma.^77,78 Increased expression of miR-183-96-182 cluster members, miR-183, 96 and 182, are implicated in glioma carcinogenesis.⁷⁹ Members of the miR-29a/b-1 cluster are brain-enriched non-coding RNA molecules, loss of which may contribute to increased BACE1/β-secretase expression in sporadic Alzheimer's disease and locomotor impairment and ataxia.^80,81 miR-132-212 cluster, whose transcriptional activation depends on the neuronal activity-induced transcription factor, CREB, has been confirmed to be critical for the formation of ocular dominance in 2 studies.^82-84 In addition, mature miR-212 and miR-132 transcripts upregulate simultaneously in response to the induction of long-term potentiation in the dentate gyrus of adult rats, suggesting the role in synaptic plasticity modulation.⁸⁵ To sum up, miRNA clusters are closely related to the occurrence and progression of brain tumors or neurological disorders. Thus, they are vital for uncovering the pathogenesis of these challenging lesions and have the prospect of being effective therapeutic targets.

More target sites for miRNAs in brain

The 3′UTRs of mRNAs contain numerous functional motifs, and nearly one-half are miRNA recognition sites.⁸⁶ It is likely that the length of 3′UTR is correlated with the number of miRNA families that potentially target the mRNA, in other words, it is a vital factor in miRNA-target co-evolution.^87-90 With the rapid development of RNA-seq technology followed by bioinformatics analysis and experimental verification, the length of 3′UTRs has been confirmed to possess an unexpected diversity and expression specificity.⁹¹ It has been observed that proliferating cells like murine CD4+ T lymphocytes and tumor cells express mRNAs with shortened 3′UTRs and fewer miRNA target sites.^92,93 On the contrary, mRNAs expressed in neural tissues, especially in hippocampus, have evidently biased usage of longer 3′UTRs and more miRNA target sites.^94,95 This phenomenon together with the 2 hallmarks of brain miRNAs described above—enrichment in brain tissues and co-expressed as clusters—indicate additional possibilities for miRNA mediated regulation; promotion of a more flexible, robust and buffered functional miRNA-mRNA regulatory network in the central nervous system. Furthermore, the sequence of 3′UTR, under much less evolutionary pressure, is not conserved. Changing evolvability of miRNA targets implies that the miRNA-mRNA regulatory network may exert a far-reaching influence on the evolution of organismal diversity.⁹⁶ The physiological and pathological roles of long 3′UTRs in neural tissues have been previously reviewed.⁹¹ Whether mutations in 3′UTRs in specific neural tissues have an impact on the stability of the miRNA-mRNA regulatory network and further cause certain nerve-related diseases is still unclear.

miRNA acts as a spatiotemporal code for brain development

The nervous system enables animals to adapt to environmental stimulus, which relies on a well-conserved toolkit of different molecular mechanisms.^97,98 The compensatory responses to a changing world need exquisite spatiotemporal regulation throughout a protracted period of time (Fig. 4). Previous studies based on large numbers of samples consisting of different brain regions or developmental time points describe the spatiotemporal dynamics of gene expression during brain development.^{22-24,52,58,99-103} That miRNA system controls, in part, both levels and translation of mRNA, shapes the deployment and robust regulation of gene networks during the construction and the remodeling of the brain.⁹⁷ Rapid growth of sequencing and chip techniques make it conceivable to detect all expressed miRNAs in a given brain region with a wide quantitative range, and highlights that miRNA acts as a spatiotemporal code for brain morphogenesis and neuronal fate.¹⁰⁴

Figure 4. Sketch map of the spatiotemporal regulation pattern of brain miRNAs. The thickness of the arrow is positively correlated with the number of differentially expressed miRNAs during the transition from one period to another. The rightmost diagram with different color areas represents a region-specific expression pattern of brain miRNAs.

Differential miRNA expressions in different brain regions are found. For example, Juhila et al. systematically compared the miRNA expression profiles between mouse frontal cortex and hippocampus and unveiled 39 miRNAs overexpressed in frontal cortex and 40 miRNAs overexpressed in hippocampus.¹⁰² Bak et al. dissected 13 different areas of the male Balb/c mouse brain and found 63 differentially expressed miRNAs within the central nervous system, among which 44 miRNAs showed more than threefold enrichment in specific regions.⁵² Across developmental time within human brain regions, 75 differentially expressed miRNAs were discovered.²⁴ With the exception of spatio-specific expression pattern, miRNA also exerts a temporal-specific expression pattern. The marked change of miRNA expression, for human beings, occurs during the transition from infancy to early childhood and, for mice, occurs within the first postnatal month, suggesting roles in early development.^22-24 Only a few differentially expressed miRNAs are found in later developing stages ranging from juvenile to adult.¹⁹ Nevertheless, the number of differentially expressed miRNAs between brain regions increases over developmental time; this is in contrast to mRNA expression which is globally similar between brain regions over development.^24,100 The spatiotemporal restricted expression pattern indicates the regionalization and dynamism of miRNA regulation. It is likely that understanding the biological roles of the differentially expressed miRNAs in specific regions or times may help to uncover the underlying mechanisms of the proper specialization and interconnectivity among different brain areas or developmental time points.

Conclusion

As post-transcriptional regulators of gene expression, miRNAs in the central nervous system have been regarded as vital triggers for brain development and neurological or psychiatric disease. Dramatic expansions of the brain miRNA repertoire also seems to be related to the emergence of mental and behavioral variation in closely related species during animal evolution. A recent study shows that human-specific glutamate dehydrogenase 2 can promote growth of IDH1^R132H glioma, which is considered as an inevitable cost of human evolution and development.¹⁰⁵ Ever-increasing miRNA repertoires, in some degree, are conducive to the complexity of the nervous system; on the other hand, it is possible to result in higher animal's susceptibility to nerve-related disorders. It appears that the non-conservatively expressed miRNAs, the robust miRNA-mRNA regulatory network and the particular spatiotemporal expression pattern can guarantee that the supreme commander, brain, can execute the intricate and fabulously complicated behaviors. The challenge is to explore what consequences may result from aberrant happenings to these hallmarks? It is these miRNAs and corresponding host mRNAs with general features that will be the hotspots of neuroscience research in the quest to comprehend the molecular underpinnings of physiological function and pathological disorder in the central nervous system.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

In particular, we thank Dr. Robert A. Haas (California Department of Public Health, USA) for helpful discussions and editing of the manuscript.

Funding

We gratefully thank the support provided by PUMC Youth Fund and the Fundamental Research Funds for the Central Universities (333210140149).