An updated role of microRNA-124 in central nervous system disorders: a review

1The discovery of microRNAs (miRNAs) introduced an understanding of the novel type of regulatory control over gene expression during plant and animal development. Mature miRNAs are non-coding transcripts of 18–25 nucleotides, which imperfectly bind to complementary sequences in the 3′-untranslated regions (UTRs) of target mRNAs to negatively regulate target gene expression. It is predicted that one third of human genes are miRNA targets.

2MicroRNA-124 (miR-124) is highly and specifically expressed in all brain regions except for the pituitary gland, and at 100 times lower expression in other tissues. It was first identified in mice, and mature miR-124 is wholly homologous in mice, rats, and human. miR-124 has been reported to participate in chronic stress, neurodegeneration, alcohol/cocaine neuroadaptation, synapse morphology, neurotransmission long-term potentiation, neurodevelopment myeloid cell function, and hematopoiesis. In mammalian neurons, miR-124 suppresses the levels of 100s of non-neural genes, which contributes to the acquisition and maintenance of neuronal identity. Furthermore, when miR-124 is aberrantly expressed, it contributes to pathological conditions involving the central nervous system (CNS) system. It has also been shown to be useful as a diagnostic and prognostic indicator of CNS disorders, such as brain tumor and stroke. Here we review the role and potential therapy of miR-124 in CNS development and disorders.

3Central nervous system development refers to the processes that generate, shape, and reshape the nervous system, from the earliest stages of embryogenesis to death. There has been particular attention on miRNAs in the context of mammalian nervous system development. compared miRNAs expression in embryonic neuron and astrocyte cultures, and found miR-124, a brain-enriched miRNA, preferentially expressed in neurons. miR-124 expression gradually increased and accumulated in parallel to neuronal maturation during CNS development. Blocking miR-124 activity in mature neurons leads selectively to increased levels of non-neuronal transcripts, while increasing miR-124 activity in non-neuronal HeLa cells showed a shift of expression profile toward that of neuronal phenotype. demonstrated that miR-124 promoted this shift by triggering brain-specific alternative pre-mRNA splicing. In mouse embryonic development, miR-124 directly targets polypyrimidine-tract-binding protein (PTBP) mRNA, which encodes a global repressor of alternative pre-mRNA splicing in non-neuronal cells. During neuronal differentiation, miR-124 reduces PTBP levels, leading to the transition from non-nervous system to nervous system specific alternative splicing patterns. Interestingly, repression of PTBP is sufficient to induce trans-differentiation of fibroblasts into functional neurons.

4Moreover, microRNA-124 is a subventricular zone (SVZ) neuronal fate determinant. The SVZ is the largest neurogenic niche in the adult mammalian brain. Knockdown of endogenous miR-124 maintained purified SVZ stem cells as dividing precursors, whereas ectopic expression led to increased neuron formation. Sox9 was demonstrated to be the physiological target of miR-124 responsible for this role. Neurogenesis in embryonic neuroepithelial cells in the spinal cord is also regulated by miR-124 and its target Sox9. Several other targets of miR-124 have been identified in mediating the process of neurogenesis. For example, miR-124 in SVZ progenitor cells mediates stroke-induced neurogenesis by targeting the JAG-Notch signaling pathway in adult rats. miR-124 also ensures the transition from neural progenitors to neurons by repressing two endogenous targets, LAMC1 and ITGB1, which are highly expressed by neural progenitors, but are repressed upon neuronal differentiation in the chick embryos. SCP1 down-regulation is another critical factor for inducing neurogenesis during embryonic CNS development in both chick and mouse embryos, and miR-124 contributes to this process in part by down-regulating SCP1 expression.

5Besides effects on neuron fate, miR-124 also contributes to the control of neurite outgrowth during neuronal differentiation possibly by cytoskeleton regulation in mouse P19 cells, and affects dendritic differentiation by regulating RhoG. Overexpression of miR-124 could lead to reduced astrocytic lineage differentiation by inhibiting STAT3 signaling. miR-124 is essential for hippocampal axogenesis and retinal cone survival, as it represses Lhx2 translation in mice. In post-mitotic neurons, miR-124 represses BAF53a, which is essential for an evolutionarily conserved program of post-mitotic neural development and dendritic morphogenesis in mouse embryos.

6In general, miR-124 has been shown to promote cell differentiation and repress cell proliferation. In neuroblasts, miR-124 was highly expressed in cells at G0/G1 phase, which results in the repression of Cdk6, a protein mediating cell-cycle progression from G0/G1. miR-124 is expressed from the beginning of eye development in Xenopus, and has been shown to repress cell proliferation in the optic cup. However, this is not true during earlier development. It was recently reported that miR-124 is both necessary and sufficient to promote cell proliferation and repress neurogenesis at the optic vesicle stage which precedes optic cup formation, showing an anti-neural role by negatively regulating the expression of the pro-neural marker NeuroD1, and revealing a novel regulatory role of miR-124 in neural development.

7Memory storage and memory-related synaptic plasticity rely on precise spatiotemporal regulation of gene expression. miR-124 plays a critical role in the regulation of signaling molecules underlying synaptic plasticity and memory. miR-124 was exclusively present presynaptically in a sensory-motor synapse where it constrains serotonin-induced synaptic facilitation through regulation of the transcriptional factor CREB in Aplysia, suggesting a role for miR-124 in long-term plasticity of synapses in the mature nervous system. miR-124 regulates cocaine-induced plasticity by targeting BDNF in rats, which is well implicated in synaptic plasticity and plays a central role in reward and memory. Thus, miR-124 may be involved in neurodegeneration diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD).

8Alzheimer’s disease is an age-related chronic neurodegenerative disease and the most prevalent type of dementia in elderly people. miR-124 level is down-regulated (human-originated, n = 5; ), while the expression of β-site APP cleaving enzyme 1 (BACE1) is up-regulated, in AD patients’ brain, indicating a possible inverse relationship between them. further demonstrated that miR-124 overexpression or knockdown could decrease or increase the expression of BACE1, and found that miR-124 may work as an important regulating factor to alleviate cell death in the process of AD by targeting BACE1 in rat PC12 cells, which is considered to participate in the rate-limiting step in the production of neurotoxic Aβ. Melatonin levels are decreased in the serum of AD patients, and its supplementation is able to reverse AD pathology and memory deficits. demonstrated that melatonin rescues the EPACs/miR-124/Egrl signaling pathway in rats, which is important in learning and memory.

9Non-coding miRNAs are necessary for the survival of post-mitotic cells such as neurons that die in PD and other brain diseases. showed that mice lacking Dicer in specific dopamine neurons develop a progressive loss of neurons later in life, displaying a Parkinson’s-like disease. Thus, miRNAs are essential for maintaining dopaminergic neurons in the brain, and participate in the pathogenesis of PD. Furthermore, a recent review has shown that one–fourth (49 out of 202, MIRECORDS database) of “validated” targets of miR-124 are de-regulated in PD, indicating an important role for miR-124 in the regulation of this disease.

10A number of miRNAs are shown to have important roles in the regulation of stress responses, and indeed miRNAs are shown to impart robustness to stress responses. The capacity of miRNAs to inhibit 100s of transcripts that are activated by stress makes them candidates for stabilizers of the homeostatic state of the transcriptome.

11Mental stress modifies both cholinergic neuro-transmission and alternative splicing in the brain. reported that stress changes rat brain miRNA profiles detected by microarray, and some of these stress-regulated miRNAs, including miR-124, regulate alternative splicing. Also, miR-124 is up-regulated in the rat hippocampus after acute immobilization stress. Another study examined the effect of maternal separation stress on miRNA expression and found the miR-124 level is elevated in the prefrontal cortex of stressed mice at P14. Paraventricular nucleus (PVN) neurons are affected by psychological stress through immune response activation. However, miR-124 was not affected by stress in mouse PVN. In vitro, miR-124 from primary cultured neurons was significantly increased in response to all four neuronal challenge response sets (transfection, KCl, kainite, aging; ).

12Hundreds of transcripts endogenously expressed in neurons with target sites for miR-124 are coordinately upregulated in a variety of neuronal stresses. Overexpression of miR-124 indeed significantly inhibits expression of 100s of stress-induced transcripts detected by microarray. Glucocorticoid and its receptor (glucocorticoid receptor, GR) exert profound effects on a variety of physiological processes, including adaptation to stress. Acute or chronic stress decreased GR mRNA in the PVN. found that miR-124 decreased GR protein level and GR-mediated events. miR-124 expression also varies over time during the stress hyporesponsive period, a neonatal period when GC signaling is modulated. By regulating GRs, miR-124 can affect a variety of systemic stress responses.

13MicroRNAs have unique expression profiles in cells of the innate and adaptive immune systems and have pivotal roles in the regulation of both cell development and function. designated miRNAs which notably affect both immune and neuronal functions as NeurimmiRs. NeurimmiRs may act as ‘negotiators’ between the nervous and immune systems. The cholinergic anti-inflammatory pathway is a link between the brain and the immune system of the host in response to an immune challenge. This pathway controls the inflammatory response through interaction with peripheral α7 subunit–containing nicotinic acetylcholine receptors (α7AChR) expressed on macrophages. Our group found miR-124 is necessary for the cholinergic anti-inflammatory action by inhibiting the production of pro-inflammatory cytokines, and miR-124 may act as an NeurimmiRs.

14The p38 mitogen-activated protein kinases (MAPKs) are central regulatory nodes coordinating acute stress and inflammatory responses. reported that expression of the p38α protein is suppressed in the brain by two neuron-selective miRNAs, miR-124, and -128. miR-124 may influence neuroimmunity by affecting p38α -mediated signaling.

15Microglial cells are macrophages that are resident in the brain and spinal cord and form the frontline defense of the innate immune system. Utilizing a mouse model of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), showed that microglia miR-124 expression decreased by ~70% during the course of the disease, while overexpression of miR-124 could promote microglia quiescence and suppress EAE by deactivating macrophages via the C/EBP-α-PU.1 pathway. Treatment of mice with miR-124 at the onset of EAE substantially ameliorated clinical symptoms and enhanced recovery in mice.

16The ability to prevent and attenuate EAE has implications for miR-124 in the treatment of neurodegenerative diseases such as MS and AD, where microglial cells are thought to play an integral role in the inflammatory process. Studies have already documented inflammation in AD, PD, amyotrophic lateral sclerosis (ALS), MS, and a growing number of other nervous system pathologies. However, a major question is whether pharmacological inhibition of inflammation pathways will be able to safely reverse or slow the course of disease, which needs further investigation.

17Plasma miRNAs have been investigated as biomarkers for various diseases, including stroke. Recently, microarray analyses were done to characterize the miRNA expression profile in various stroke models. The abundance of miR-124 in the CNS has accelerated efforts to determine if it can be used as an effective stroke treatment.

18first reported an elevated level of miR-124 in the brain samples from rats with middle cerebral artery occlusion (MCAO) followed by 24 h reperfusion. One of its predicted targets, the VSNL1 gene, which is a neuronal calcium sensor protein identified as a specific and promising plasma biomarker of stroke patients, was decreased in parallel with the increased miR-124 under the same conditions. Our previous study found miR-124 is significantly increased in ischemic penumbra as compared with that in the non-ischemic area in MCAO mice. Accordingly, brain tissues from stroke-prone spontaneously hypertensive rats (SHR-SP) showed higher miR-124 levels than in spontaneously hypertensive rats (SHRs; ). Markedly increased plasma miR-124 was also observed at 24 h after stroke for both transient and permanent occlusions in rats. Consistently, miR-124 was predicted to suppress acetylcholinesterase (AChE; ), whose plasma level was reduced in patients post ischemic stroke, indicating a physiological relevance between miR-124 and AChE. The above suggests that plasma miR-124 released from the infracted brain may be a promising candidate biomarker for stroke identification.

19Although there is no correlation between the infarct size and plasma miR-124 level in rats after MCAO introduction, efforts have been made to explore whether miR-124 treatment is effective in stroke. showed that exogenous miR-124 reduced brain injury and functional impairment, enhanced neurovascular remodeling, and increased angioneurogenesis 8 weeks post-stroke in mice with MCAO, which is possible via the pathway involving Usp14-dependent REST degradation. Similarly, our previous study showed that miR-124 overexpression decreased the infarct area of MCAO mice. The anti-apoptosis proteins Bcl-2 and Bcl-xL, key regulators in attenuating stroke-induced apoptotic cell death, are found to be the targets of miR-124 in this protective role for stroke. miR-124 was also found to inhibit stroke-induced neurogenesis by targeting the JAG1/Notch signaling pathway.

20However, conflicting miR-124 stroke therapy results have also been reported. For example, showed that knockdown of cerebral miR-124 reduced cell death and infarct size, and improved neurological outcomes by negatively regulating Ku70. Additionally, found that although a miR-124 mimic did not affect the infarct volume at 24 h after ischemia, inhibition of miR-124 effectively reduced the ischemic injury due to iASPP expression up-regulation. Therefore, the use of miR-124 as an effective stroke treatment necessitates further research.

21Distinct patterns of miR-124 expression have been observed in many cancers including glioblastomas. miR-124 expression is significantly decreased in anaplastic astrocytoma and glioblastoma relative in human patients to non-neoplastic brain tissue, and is expressed at different levels in glioblastoma compared with normal brain. Overexpression of miR-124 induced morphological changes and neural differentiation in mouse neural stem cells and oligodendroglioma, accompanied by reduced self-renewal and tumorigenicity. In addition, the ectopic expression of miR-124 in a glioblastoma cell line resulted in significant inhibition of migration and invasion, suggesting that miR-124 may be a novel inhibitor of glioblastoma invasion.

22Several targets of miR-124 mediating this process have been identified. It was reported that miR-124 controls self-renewal and tumorigenic competence of human glioblastoma cells by targeting SCP1 and PTPN12 phosphatases. demonstrated that the tumor suppressor activity of miR-124 could by partly due to its inhibitory effects on glioma stem – like traits and invasiveness through down-regulation of SNAI2 in human. Others reported that miR-124 inhibits glioma cells migration and invasion by down-regulation of ROCK1, and induced glioma differentiation by suppressing Twist and SNAI2.

23A clinical investigation also showed a negative correlation between miR-124 expression and a hypoxic gene signature in glioblastoma patient samples. Increased miR-124 expression affects the ability of tumor cells to survive under O2 and/or nutrient deprivation, while miR-124 re-expression increases cell death in vivo and enhances the survival of mice bearing intracranial xenograft tumors. miR-124 exerts this phenotype in part by directly regulating TEAD1, MAPK14/p38α, and SERP1, which are factors involved in cell proliferation and survival under stress. Besides glioblastoma, neuroblastoma is an embryonic tumor derived from the autonomic nervous system neural-crest tissues, and is the most common extracranial solid tumor in children. showed that miR-124 plays a pivotal role in neuroblastoma by targeting aryl hydrocarbon receptor (AHR), which may promote neuroblastoma cell differentiation. These studies indicate that miR-124 is a potential therapeutic target in brain tumor treatment.

24We have discussed the importance of miR-124 in neuronal development and function of the brain. Changes of miR-124 levels can serve as biomarkers that indicate the functional status of a normal brain, as well as progression of CNS diseases. Interestingly, it seems inverse effect of miR-124 in tumorogenic events and neurodegenerative processes. For example, miR-124 alleviates cell death in the process of AD by targeting BACE1, while increases cell death in glioblastoma by regulating TEAD1, MAPK14/p38α, and SERP1. This might be relevant to different targets involved depending on the context of diseases, cells and surroundings. Moreover, its regulation of numerous targets (summarized in Table 1) classifies it as a suitable treatment for the complexity of pathophysiological events launched by multifactorial CNS diseases, which require therapies that are able to trigger a large set of orchestrated genes. Therefore, we believe miR-124 and other miRNAs that fulfill this criterion will replace single gene therapies for the treatment of these kinds of diseases in the near future.

25The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.