Background

Central nervous system (CNS) injuries and diseases, such as stroke, spinal cord injury (SCI), neurodegenerative diseases (NDDs), glioblastoma (GBM), and multiple sclerosis (MS), as well as the excessive neuroinflammation they cause, are the major causes of morbidity and mortality (Collaborators 2019; Qureshi et al. 2009). As beneficial treatments for these complex pathologies are lacking and there are obstacles in the development of effective therapies, successful treatments for CNS injuries and disorders are needed to minimize both acute and chronic cellular damage, prevent secondary damage caused by treatment, and construct and maintain a beneficial microenvironment. The ability of microRNAs (miRNAs) to regulate molecular function, influence cellular behavior, and modulate inflammatory signaling is an area of ongoing research and has gained much attention in recent years (Akerblom and Jakobsson 2014; Bhalala et al. 2013; Juzwik et al. 2019).

miRNAs are a new class of gene products, the discovery of which represents a novel insight into the regulatory network of gene expression during the development of plants and animals (Ambros 2004; Bartel 2004; Rodriguez et al. 2004). Mature miRNAs are small non-coding RNAs that are approximately 22 nucleotides in length. These small molecules are incorporated into the RNA-induced silencing complex (RISC) to silence gene expression through translational repression or mRNA degradation by binding to sites of antisense complementarity in the 3′ untranslated regions of target mRNAs (Lagos-Quintana et al. 2001). Generally, each miRNA can regulate hundreds of target genes, and the expression of more than one-third of all human genes is predicted to be mediated by miRNAs (Esquela-Kerscher and Slack 2006; Guarnieri and DiLeone 2008). In addition, miRNAs are implicated in various cellular processes, such as cell proliferation, cell differentiation, cellular metabolism, and immune responses, in both physiological and pathological conditions (Bi et al. 2009; Gauthier and Wollheim 2006; Hatfield and Ruohola-Baker 2008; Matsubara et al. 2007). Several miRNAs have been shown to play important roles in the regulation of CNS development (Saraiva et al. 2017), and the capacity of miRNAs to inhibit the action of hundreds of transcripts against CNS injuries and disorders makes them candidates for stabilizing the homeostatic state of the transcriptome (Manakov et al. 2012). The brain-specific miRNAs are predominantly miR-124, -101, -127, -128, -131, and -132 (Mishima et al. 2007). Additionally, miR-9, -21, -29b, -124, -137, 146a, and -155 have been shown to be involved in the modulation of CNS pathophysiology and are considered promising therapeutic targets (Bhalala et al. 2013; Su et al. 2016).

Among the highly expressed miRNAs, miR-124 accounts for 25–48% of all brain miRNAs (Lagos-Quintana et al. 2002). Moreover, miR-124 expression in the mouse CNS is more than 100 times higher than that in other organs. The expression ratios of miR-124 in the CNS are 60.7% for the cerebellum and 35.4% for the spinal cord (Mishima et al. 2007). Such abundant miR-124 expression in the CNS indicates that miR-124 may play an indispensable role in normal CNS functions, which has been attracting much attention for further research.

In general, miR-124 matures from three different precursor variants, called primary (pri) miRNA, located on human chromosomal positions 8p23.1 (miR-124-1), 8q12.3 (miR-124-2), and 20q13.33 (miR-124-3) (Gebauer et al. 2013; Lagos-Quintana et al. 2002; Mishima et al. 2007). Although the loci they are transcribed from are different, generally, it is accepted that most pre-miRNAs all cleave to the same mature miRNAs and thus possess the same characteristics once loaded into RISC. In addition, a study on miR-124 monitoring by a fluorescence tracer system revealed that it was significantly upregulated during the direct neuronal conversion of mouse embryonic fibroblasts reprogrammed by the ectopic expression of Ascl1, Brn2, and Myt1L (Sano et al. 2017). Moreover, miR-124a knockout mice exhibited CNS abnormalities, including small brain size and axonal mis-sprouting of dentate gyrus granule cells (Sanuki et al. 2011). Similarly, some miR-124 knockdown planarians displayed a reduction in overall brain size during their brain regeneration (Sasidharan et al. 2017). These studies suggest that miR-124 plays a significant role in neuronal wiring and neuronal maturation.

In this review, we provide an overview of miR-124, its roles in CNS development, and summarize recent data of its role in neuroinflammation, ischemic stroke, SCI, NDDs, GBM, MS, and the methods for the delivery of miR-124. The mechanisms underlying miR-124 actions are also discussed. Finally, the potential of miR-124 as a therapeutic target to ameliorate CNS injuries and diseases is highlighted.

MiR-124 in Neuroinflammation

Neuroinflammation acts as a central component of CNS injuries and diseases and appears to be responsible for their progression. Upon CNS damage, resident glial cells, including microglia and astrocytes, become activated and induce proinflammatory cytokine, chemokine, and matrix metalloproteinase production, contributing to blood–brain barrier (BBB) disruption, followed by the infiltration of macrophages from the peripheral blood into the damaged core, and the above processes have been extensively reviewed elsewhere (Burda and Sofroniew 2014). In the recovery outcome, the immune system plays a dual beneficial and detrimental role, which has also been well documented (Jacobs et al. 2012; Medzhitov 2008; Varley et al. 2015). An excessive inflammatory response may be more harmful than the original insult, leading to neurological dysfunction (Jayaraj et al. 2019). miR-124 acts as one of the key miRNAs regulating neuroinflammation in various CNS pathologies, which may shed light on novel therapies targeting miR-124 for treating CNS insults, and we will review that in this section.

Neuroinflammation is a process that involves omnidirectional communication of multiple cells and molecular interactions to mount an inflammatory response and disease pathogenesis or to maintain CNS functions by modifying inflammation-related neuroprotection. Therefore, to target neuroinflammation, reconstructing an appropriate immune microenvironment should be beneficial to enhance therapeutic effects, rather than the broad immune suppression that is typically perceived.

Among the cell types involved in neuroinflammation, microglia are critical tissue-specific immune cells acting as resident macrophages that influence brain development, maintain the neuronal environment, as well as respond to and induce CNS diseases (Orihuela et al. 2016; Prinz et al. 2019). In response to the different stages of CNS injuries, microglia and macrophages are activated to subsequently increase the release of inflammatory cytokines; however, miR-124 was demonstrated to maintain microglia in a quiescent state. After splenectomy, the secretion of proinflammatory cytokines, such as TNF-α and IL-6, predisposes individuals to mental or cognitive disorders (Wan et al. 2007). However, miR-124 was shown to reduce microglial activation-mediated neuroinflammation by targeting vesicle-associated membrane protein 3 (Chen et al. 2019). In addition, miR-124 inhibited TNF-α and IL-6 production by lipopolysaccharide (LPS)-stimulated macrophages by targeting signal transducer and activator of transcription 3 (STAT3) and TNF-α converting enzyme (TACE) (Sun et al. 2013). In a model of cocaine-induced neuroinflammation, intranasally delivered miR-124-loaded engineered extracellular vesicles (EVs) were detected in the CNS where they significantly reduced the expression of inflammatory markers, such as TLR4, MYD88, STAT3, NF-κB p65, and the microglial activation marker IBA1(Chivero et al. 2020). These data indicate that engineered EVs can deliver miR-124 into the CNS to alleviate cocaine-mediated microglial activation.

Activated microglia will be polarized toward different activation states, M1 and M2, which represent the two extremes. Generally, polarized M1 microglia and macrophages release destructive proinflammatory mediators, such as TNF-α, IL-6, and IL-1β, causing further damage to host tissue. Polarized M2 microglia and macrophages clear cellular debris and release neuroprotective trophic factors and anti-inflammatory mediators, such as IL-10, TGF-β, and IL-4 (Mosser and Edwards 2008; Orihuela et al. 2016). Modulating the beneficial polarization of M1/M2 is important for nerve recovery. The contribution of miR-124 in the polarization of macrophages to the M2 phenotype has been described in a recent review, and it has been well studied in various CNS diseases (Ponomarev et al. 2013). Ponomarev et al. found that miR-124 was expressed in microglia but not in peripheral monocytes or macrophages. They demonstrated that miR-124 induced microglial quiescence and suppressed experimental autoimmune encephalomyelitis (EAE) by deactivating macrophages through direct inhibition of C/EBP-α, one of the M1 polarization transcription factors (Ponomarev et al. 2011). When performing more detailed gene expression profiling in miR-124-transfected macrophages, they found that miR-124 not only downregulated the M1-associated markers, such as IL-6, TNF-α, and inducible nitric oxide synthase but also upregulated the expression of M2-associated markers, including TGF-β1, ARG1, and FIZZ1 (Ponomarev et al. 2011). Bone marrow mesenchymal stem cell-derived exosomal miR-124 attenuated neurological damage in a model of spinal cord ischemia–reperfusion injury by downregulating Ern1 and promoting M2 macrophage polarization (Li et al. 2020). Intracerebral miR-124 administration after stroke resulted in a significant increase in the M2 phenotype, marked by ARG1 upregulation, paralleled by a decrease in the M1 phenotype. This substantial decrease in the M1/M2 ratio improved neurological deficits after stroke and correlated strongly with functional outcomes (Hamzei Taj et al. 2016a, b).

In neurons, miR-124 overexpression was shown to improve the adverse effects of oxygen–glucose deprivation (OGD) and reperfusion-induced PC12 cellular injuries, and significantly repress NF-κB signaling activation and proinflammatory cytokine production by regulating NOX2 (Wu et al. 2020). In addition, microglial exosomal miR-124 treatment in traumatic brain injury suppressed neuronal inflammation in scratch-injured neurons by suppressing the activity of mTOR signaling, mediated via PDE4B; this effect contributed to neurite outgrowth when microglial exosomal miR-124 was transported into neurons (Huang et al. 2018). Consistently, a recent study detected that M2-exosomes could improve the outcome of middle cerebral artery occlusion (MCAO). The associated mechanism in neurons might be partly related to exosomal miR-124 and its downstream target USP14 (Song et al. 2019b). These results suggest that M2 microglia and macrophages not only modulate the microenvironment via anti-inflammatory or neurotrophic factors but also deliver exosomal miR-124 to neurons and other cell types to achieve a more beneficial crosstalk.

Pro/anti-inflammatory mediators act not only on microglia and neurons but also on astrocytes, which presents another opportunity for therapeutic miR-124-mediated modulation of the neuroinflammatory microenvironment. In response to a wide variety of CNS insults of varying nature and severity, astrocytes undergo reactive astrogliosis and become reactive astrocytes (Mori et al. 2008; Sofroniew 2009). Similar to the M1/M2 microglia and macrophages, there are different subtypes of reactive astrocytes, termed A1 and A2. Transcriptome analysis of reactive astrocytes reveals that LPS-induced A1 reactive astrocytes upregulate many genes that have been previously shown to be destructive to the synapses. In contrast, ischemia-induced A2 reactive astrocytes were shown to upregulate many neurotrophic factors, which promoted synapse repair (Zamanian et al. 2012). Indeed, A1 astrocytes are toxic and accumulate in many CNS diseases, such as amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), and MS (Liddelow and Barres 2017; Liddelow et al. 2017). Therefore, preventing the formation of A1 astrocytes, promoting A1/A2 astrocyte shift, or blocking the A1 neurotoxin hold great potential for treating CNS diseases (Yun et al. 2018). Activated microglia induce A1 astrocytes by secreting IL-1α, TNF-α, and C1q, and A1 astrocytes lose the ability to promote neuronal survival, outgrowth, synaptogenesis, and phagocytosis and induce the death of neurons and oligodendrocytes (Liddelow et al. 2017). In vivo inhibition of microglia-mediated formation of A1 astrocytes prevented the death of axotomized CNS neurons (Liddelow et al. 2017; Yun et al. 2018). Neuron-derived exosomes enriched with miR-124-3p promoted function recovery by suppressing myosin heavy chain 9 activity, which led to the downregulation of M1 microglia and A1 astrocytes in vitro and in vivo (Jiang et al. 2020). In addition, it has been reported that the Notch-STAT3 axis and the NF-κB/C3/C3aR pathway are involved in A1 astrocyte activation (Lian et al. 2015; Qian et al. 2019). Coincidently, Notch and NF-κB are extensively studied targets of miR-124, although additional experimental data are required to fill this knowledge gap (Gan et al. 2019; Jiang et al. 2016). The roles and potential mechanisms of miR-124 in neuroinflammation are summarized in Fig. 1. Emerging evidence indicates that during neuroinflammation, there must be crosstalk among neurons, microglia, and astrocytes through the release of diverse signaling molecules that determine the fates of astrocytes and microglia (Jha et al. 2019). Thus, an indirect approach for modulating the neuroinflammatory microenvironment could involve miR-124-mediated downregulation of proinflammatory factors, such as TNF-α and IL-1, that typically induce M1 microglia polarization and subsequent microglia-induced A1 astrocyte formation (Fig. 2).

Fig. 1
figure 1

The role and potential mechanism of miR-124 in neuroinflammation. miR-124 regulates its targets and the signaling pathways of microglia, astrocytes, and neurons to participate in the activation of microglia and astrocytes, as well as neuroinflammation response. (Created with BioRender.com)

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Fig. 2
figure 2

Proposed communication of multicellular model regulated by miR-124 in neuroinflammation after CNS injuries/diseases. Under pathological condition of the CNS, miR-124 maintains microglia in a quiescent state or inhibits M1 microglia polarization. Polarized M1 microglia and macrophages release destructive proinflammatory mediators to further damage host tissues. Additionally, miR-124 promotes M2 microglia/macrophage polarization and induces neuroprotective trophic factors and the release of anti-inflammatory mediators. Activated microglia further induce A1 astrocytes by secreting Il-1α, TNF, and C1q. Exosomes loaded with miR-124 secreted by M2 microglia can promote the reparation of damaged neurons. Analogously, exosomes enriched with miR-124 promote function recovery by suppressing the activation of M1 microglia and A1 astrocytes. (Created with BioRender.com.)

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Neuroinflammation is now recognized as the hallmark of virtually all neurological disorders (Glass et al. 2010). It is a complex and well-orchestrated process by various groups of neurons and glial cells in nervous systems. In addition, it is not considered synonymous with poor CNS outcomes anymore. As discussed above, there are now multiple examples of the significant benefits of inflammatory responses to the injured CNS for neuroprotection and for regenerative responses. A major strategy is to employ existing medications that polarize immune subsets into those beneficial phenotypes (Yong et al. 2019). According to the studies reviewed above, miR-124 has a comprehensive anti-inflammatory and neuroprotective effect when it acts on groups of cells within the CNS in the process of the neuroinflammatory response; thus, it is promising for the treatment of CNS injuries and diseases.

miR-124 in Cerebral Stroke and Brain Injury

Cerebral stroke is a major cause of mortality and morbidity (Feigin et al. 2017). Cerebral ischemia (ischemic stroke) accounts for about 80–90% of all strokes, and intracerebral hemorrhage (ICH) accounts for approximately 10–20% of strokes (Feigin et al. 2009; Sacco et al. 2009). Cerebral stroke involves a complex pathology, which triggers a cascade of cellular responses, and ultimately results in focal neurological deficits as well as long-term cognitive and motor impairment. miRNAs are involved in regulating many aspects of the pathogenic mechanisms following a stroke, thereby providing insights into new therapeutic avenues. Continuing research on the brain-specific miR-124 has accelerated efforts to explore whether it is an effective target for stroke treatment.

Ischemic Stroke

Multiple reports have shown that miR-124 levels increased after an acute ischemic stroke in both experimental MCAO models and in patients. Jeyaseelan et al. (2008) first reported an increased level of miR-124 in brain samples from a rat model following MCAO and 24-h reperfusion. Consistently, miR-124 concentration increased in rat plasma after MCAO induction (Laterza et al. 2009; Weng et al. 2011). miR-124 expression levels were significantly high in the plasma, serum, and cerebrospinal fluid of stroke patients as well as in the brain samples of men who died of brain stroke (Ji et al. 2016; Leung et al. 2014; Sessa et al. 2019; Sorensen et al. 2017). In addition, studies suggest that a higher concentration of miR-124 was related to a larger infarct volume after stroke and more unfavorable outcomes after stroke treatment (He et al. 2019; Sorensen et al. 2017). The mechanisms that result in changes in circulating miR-124 levels after stroke are still unclear. Possibly, it could be the result of increased miR-124 released from damaged brain tissue into circulation, which may be explained by the significant correlation between plasma miR-124 and lesion size (Rainer et al. 2016). In summary, miR-124 is not only a promising candidate biomarker after stroke but also an early prediction and risk stratification indicator for stroke treatment.

Under ischemic stroke conditions, the availability of oxygen and glucose to tissues is delayed, which elicits multifaceted responses and triggers primary and secondary insults. It involves a complex pathology, including multiple types of cellular responses, such as astrogliosis, microgliosis, and peripheral source of macrophage activation, as well as excitotoxicity, oxidative stress, inflammation, neuronal apoptosis, neurodegeneration, and BBB destruction. Ultimately, ischemic stroke leads to the death of brain tissue and causes focal neurological deficits (Burda and Sofroniew 2014; Tobin et al. 2014). The mechanisms of miR-124 as therapeutic targets for ischemic stroke should be based on its actions against stroke pathology; some of these mechanisms are discussed here.

Neurogenesis after stroke occurs even in adult rodents and humans (Arvidsson et al. 2002; Jin et al. 2006). However, the self-repair of injured brain through neuronal replacement from the differentiation of neuronal precursors is insufficient (Adamczak et al. 2017; Shimada et al. 2010). For efficient repair, a promising avenue is the optimization of this neurogenesis process to promote the self-repair mechanism. miR-124 has been shown to play an important role in promoting neurogenesis under physiological and pathological conditions. Some evidence has also demonstrated that miR-124 promoted the survival and neuronal differentiation of neuronal stem cells (NSCs), partly via direct suppression of paired box 3 expression or enhanced proliferation (Wei et al. 2018). It also induced the differentiation of NSCs into neurons by activation of the Wnt/β-catenin pathway through direct targeting of the disheveled binding antagonist of β-catenin 1 (Jiao et al. 2018). In addition, miR-124 also contributed to the regulation of neurite outgrowth during neuronal differentiation. Functional enrichment analysis revealed that miR-124 influenced axon regeneration mainly by regulating cellular component organization, axonogenesis, and cell morphogenesis (Su et al. 2018). Suppression of miR-124 led to reciprocal increases in mRNA levels of target genes that inhibited axonal and dendritic projections (Morris et al. 2015). Studies have also shown that miR-124 directly targeted and downregulated the endogenous expression of oxysterol-binding protein to promote neurite outgrowth and elongation during the development of the C57BL/6 mouse cortex (Gu et al. 2016). In addition, miR-124 directly targeted CBX2, a negative regulator of neuronal differentiation, to stimulate neurite development (Gu et al. 2018). According to its multiple functions in promoting neuronal differentiation and neurite outgrowth, miR-124 is a potential therapeutic target after ischemic stroke. Under ischemic conditions, miR-124 was reported to inhibit neuronal progenitor cell proliferation and promote neuronal differentiation by targeting JAG1 (Liu et al. 2011). Modified exosomes loaded with miR-124 crossed the BBB and were efficiently delivered to the infarct site, where they induced robust cortical neurogenesis to protect against ischemic injury (Yang et al. 2017). Despite the evidence supporting the neurogenesis-promoting function of miR-124, its in vivo application is less straight forward; although it has been shown that miR-124-loaded nanoparticles increased survival and neuronal differentiation of NSCs in vitro, it did not contribute to stroke outcomes in vivo (Saraiva et al. 2018). However, before the substantial use of miR-124 in the therapeutic promotion of neurogenesis, the dosage, mode of administration, time frame, and therapeutic window need to be clarified.

OGD, as well as injury-induced cell death, including apoptosis, contribute to the cellular damage that occurs in ischemic stroke (Puyal et al. 2013; Zaiman et al. 2011). Increasing neuronal survival and anti-apoptosis could attenuate infarction and improve functional outcomes after stroke. miR-124 has been shown to inhibit neuronal apoptosis in the cerebral ischemic region of rats by activating the Wnt/β-catenin signaling pathway (Che et al. 2019). Injection of miR-124 mimetics significantly reduced cerebral infarction-induced neurological deficits and infarction areas (Che et al. 2019). miR-124 was demonstrated to have anti-apoptotic and neuroprotective effects. Moreover, in a recent study, using RISC immunoprecipitation, researchers identified 98 high-confidence miR-124 targets, some of which directly led to decreased viability and higher apoptotic rates in miR-124-deleted neurons, both of which could be rescued by miR-124 overexpression. In addition, the study demonstrated that miR-124 regulation was essential for the long-term survival of terminally differentiated neurons by targeting a cascade of apoptosis-relevant genes (Kutsche et al. 2018). Thus, these qualities could contribute to the following hypothetical mechanism: induction of K63-linked RIP1 ubiquitination, mediated by UBXN1 repression and inhibition of USP14-dependent RE1 silencing transcriptional factor (REST) degradation (Doeppner et al. 2013; Song et al. 2019c). However, conflicting results of miR-124 on apoptosis after stroke have also been reported in other cell types. By targeting the PI3K/AKT signaling pathway and promoting reactive oxygen species (ROS) production, miR-124 potentially induced apoptosis in brain vascular endothelial cells (Wang et al. 2018b). A recent study also demonstrated that ischemic postconditioning exerts its neuroprotective effect by negatively regulating the PI3K/AKT2 signaling pathway by miR-124. miR-124 significantly decreased the expression of Caspase-3 and BAX and increased the expression of the anti-apoptotic protein Bcl-2. Similarly, the inhibition of miR-124 also inhibits cellular apoptosis and autophagy by increasing the PI3K/AKT/mTOR signaling pathway (Miao et al. 2020). With more research, it is possible that the comprehensive effects of miR-124 on apoptosis and stroke treatment could be clinically applied.

Excessive excitatory glutamate (Glu) neurotransmission is toxic and leads to neuronal cell death. After an ischemic stroke, Glu is released in high levels, while Glu transporters (GLT-1) and reuptake is widely suppressed. miR-124 was reported to attenuate excitotoxicity by upregulating the expression of GLT-1 via the AKT and mTOR pathways in astrocytes injured by OGD/reperfusion (Huang et al. 2019). Angiogenesis can also contribute to recovery after stroke. miR-124 increased angiogenesis in the ischemic striatum 56 days after MCAO, as shown by CD31 immunohistochemistry (Doeppner et al. 2013). In addition to the abovementioned findings, miR-124 also plays an important role in regulating inflammation.

ICH

ICH is usually caused by the rupture of small blood vessels secondary to chronic hypertension or other vasculopathy (Qureshi et al. 2009). Compared to ischemic stroke, ICH has a higher mortality and leads to more severe disability (An et al. 2017b). Primary and secondary ICH have similar underlying pathological changes induced by the pooling of blood in the brain parenchyma and hematoma formation (Steiner et al. 2006). These changes are associated with multiple biological processes, including oxidative damage, inflammation, and edema formation, all of which contribute to an extensive cascade of cellular and molecular alterations in the brain that add to further destruction of brain tissue (Keep et al. 2012). Studies on ICH patients and rodent models have demonstrated that plasma miR-124 was induced by collagenase during the acute injury phase, suggesting that miR-124 is derived from brain injuries. In rodents, these levels are further decreased during the delayed recovery phase, which suggests healing of the brain injury, and it finally returned to baseline levels when the rats fully recover (Wang et al. 2018d). This pattern of miR-124 plasma concentration is a promising candidate biomarker for the early detection and predictive prognosis of human ICH. Given that the cascade of cellular and molecular changes, such as excitotoxicity, inflammation, neuronal apoptosis, and neurodegeneration, are similar to observed in ischemic stroke, miR-124 probably plays a similar function in ICH as that observed in ischemic stroke. Unfortunately, there are limited studies showing that miR-124 ameliorates ICH-induced inflammatory injury, though data indicate that this is achieved by modulating microglial polarization toward the M2 phenotype via C/EBP-α, as well as by significantly attenuating neuronal apoptosis via the Bcl-2/Bcl-xl pathway in vivo and in vitro (Yu et al. 2017). We look forward to more studies that may reveal whether miR-124 plays an important role in ICH.

miR-124 in SCI and Spinal Cord-Associated Neuropathic Pain

Promoting Neuronal Differentiation in SCI

SCI results in the loss of motion and sensory function below the damage plane, with devastating physical, psychosocial, and vocational implications for patients. Epidemiological studies by the World Health Organization (WHO) have shown that the incidence of SCI worldwide averages 10–40 people per million. China and the United States have a high incidence of SCI (more than 40 people per million a year) (Ahuja et al. 2017; Lee et al. 2014; Singh et al. 2014; Spinal Cord Injury (SCI) 2016 Facts and Figures at a Glance 2016; Witiw and Fehlings 2015). The pathological process of SCI includes primary and secondary mechanisms, which eventually form glial scars composed of microglia, reactive astrocytes, and secreted inhibitory proteins, such as chondroitin sulfate proteoglycan in the injured area to inhibit axon regeneration and myelination (Okada et al. 2018). A recent study indicated that miRNAs function as gene expression switches in key processes of SCI (Nieto-Diaz et al. 2014). Therefore, understanding how an injury affects miRNA expression and the meaning of these changes in the SCI pathological process will help explore the potential application of miRNAs. Among them, the link between the regulation of miR-124, which is highly expressed in the mammalian CNS, and SCI has attracted much attention (Song et al. 2019a).

A miRNA microarray (miCHIP) study revealed that miR-124 is one of the most highly expressed miRNAs in the rat spinal cord (Brandenburger et al. 2012). Moreover, changes in miR-124 expression are observed in neurons in the peri-lesion area of mice with SCI (Zhao et al. 2015b). In one study, the expression of miR-124 was significantly decreased within 7 days after SCI (Zhao et al. 2015b). Several neurons in the peri-lesion area were NeuN+/miR-124−, but the neurons distal to the peri-lesion area were NeuN+/miR-124+, indicating that miR-124 expression was downregulated after SCI. To date, no direct connection has been shown between miR-124 expression and the severity of SCI, although efforts have been made to explore the specific roles of miR-124 in SCI. Studies have shown that the upregulation of miR-124 dramatically increased the differentiation of NSCs into NeuN+ cells in vitro but reduced the percentage of GFAP+ cells in vivo in rats with SCI (Xu et al. 2012). Moreover, transplantation of bone marrow mesenchymal stem cells overexpressing miR-124 also promoted neuronal differentiation and, thus, accelerated the repair of SCI compared relative to control cells (Song et al. 2017; Zhao et al. 2015a; Zou et al. 2014). This effect may be attributed to miR-124, which was shown to directly target pyridoxal kinase (PDXK) to increase the expression of SCI repair-related proteins, such as TRH, PGI2, and GM (Song et al. 2017).

Attenuating Spinal Cord-Associated Neuropathic Pain

Neuropathic pain, or pain caused by a lesion or disease of the somatosensory nervous system, usually occurs in 40–50% of patients with SCI within the first year following SCI (Chi et al. 2019). miR-124 has been reported to play vital roles in spinal cord-associated neuropathic pain (Willemen et al. 2012). A recent study using microarray-based approaches has demonstrated that miR-124 is involved in pain processing and that it can attenuate inflammatory pain induced by complete Freund’s adjuvant via the inhibition of IL-6R expression in the spinal cord (Liu et al. 2017). Moreover, intrathecal administration of miR-124 completely prevented the transition from acute to persistent IL-1β-induced hyperalgesia in LysM-GRK2+/− mice, presenting evidence for miR-124 to treat persistent inflammatory (Willemen et al. 2012). This is consistent with the finding that miR-124 is a key negative regulator of neuroinflammation, which has been reviewed in another section of this article (MiR-124 in neuroinflammation).

Taken together, the above findings suggest that the neuroprotective and anti-inflammatory effects of miR-124 after SCI or spinal cord disorder make it a promising therapeutic candidate for targeting SCI.

miR-124 in NDDs

NDDs, which include Alzheimer’s disease (AD), PD, ALS, and frontotemporal dementia, are characterized by dysfunction and death of specific neuronal subtypes (Giuliani et al. 2017; Haston and Finkbeiner 2016). Along with the increasing human population, NDDs are becoming a challenge to healthcare systems. Unfortunately, there are still no effective treatment options, as current therapies only relieve symptoms (Mason et al. 2014; Jucker 2010; Mitsumoto et al. 2014; Socias et al. 2018).

In addition to progressive deterioration of neuronal structure and function (Bredesen et al. 2006), evidence derived from genetic, neuropathological, and in vitro or in vivo studies shows that NDDs often display common mechanisms and pathological features (Glass et al. 2010; Jucker 2010; Nainu et al. 2019; Philip et al. 2002). The misfolding, aggregation, and accumulation of proteins in the brain are hallmark events of NDDs at the cellular level. Although distinct NDDs have different protein aggregates, the protein misfolding process is remarkably similar (Bennett 2005; Ross and Poirier 2004; Soto 2003; Soto and Pritzkow 2018). The accumulated misfolded proteins activate resident immune cells, such as microglia and astrocytes, which induce sustained inflammatory responses. Concomitantly, immune cells release a variety of other neurotoxic factors (Brown and Neher 2010; Glass et al. 2010; Sofroniew 2015), which further contribute to the progression of NDDs (Liang et al. 2017). Previous studies have demonstrated that mitochondrial dysfunction and cumulative oxidative stress are common features of several NDDs, such as AD, PD, ALS, and Friedreich’s ataxia (Federico et al. 2012; Gandhi and Abramov 2012; Kim et al. 2015). Oxidative stress may impair the DNA repair system that accelerates the aging process and development of NDDs (Kim et al. 2015). Furthermore, being associated with inflammatory and immune responses, BBB disruption leads to neuronal injury, synaptic dysfunction, and loss of neuronal connectivity (Sweeney et al. 2018; Zlokovic 2008). miR-124 has been reported to play a neuroprotective role in animal models of NDDs, suggesting that some therapeutic approaches involving this particular miRNA may be effective for treating these diseases (Du et al. 2017; Kong et al. 2015; Zhou et al. 2019).

AD

Approximately 47 million people worldwide have AD, and the prevalence of AD is 15% in those aged 68 years or older in the United States (Arvanitakis et al. 2019). miR-124 has been reported to be downregulated in patients with AD (An et al. 2017a; Burgos et al. 2014; Zhang et al. 2017a). miR-124-1 was found to be hypermethylated in AD brains, which would explain its suppression (Villela et al. 2016). Intracellular aggregates of insoluble tau proteins are one of the characteristics of AD, and one study demonstrated that tau increased the silencing activity of miR-124 through DEAD-box RNA helicase 6 activity (Chauderlier et al. 2018). In addition, downregulation of miR-124 resulted in elevated CAPN1, and CAPN1 induced cleavage of p35 to p25 as well as the formation of the p25/cyclin-dependent kinase 5 (CDK5) complex, which resulted in tau hyperphosphorylation and cellular apoptosis (Zhou et al. 2019). This led to the accumulation of extracellular amyloid plaques, the major cause of AD in vitro. Overexpression of miR-124 suppressed BACE1, an enzyme that plays an indispensable role in the generation of the β-amyloid (Aβ) peptide (An et al. 2017a; Du et al. 2017; Zhao et al. 2019). Abnormal accumulation of Aβ peptide participates in the formation of amyloid plaques (Du et al. 2017). Furthermore, it has been reported that PTBP1 is a target of miR-124 (Mokabber et al. 2019). Through PTBP1, miR-124 was shown to regulate alternative splicing of the amyloid precursor protein (APP) mRNA, and abnormal neuron-specific APP mRNA splicing affects Aβ peptide production (Smith et al. 2011). This suggests that miR-124 could be used in a fine-tuning manner to remedy abnormal neuronal splicing of APP to reduce Aβ peptide production. However, there exists controversial data regarding the alterations of miR-124 in the brains of patients with AD. Wang et al. separately examined different brain regions from AD patients and discovered that miR-124 was increased in the hippocampus and temporal cortex, the first two brain regions damaged in AD (Wang et al. 2018c). In their recent study, abnormally upregulated miR-124 was detected in P301S mice (a well-known model of tau pathology), leading to decreased expression of protein phosphatase 1, accompanied by tau hyperphosphorylation at multiple sites; these findings implied that miR-124 had an impact on the regulation of tau pathology (Hou et al. 2020). Taken together, these findings suggest that disorders involving miR-124 play an important role in various AD pathological processes, and that miR-124 holds therapeutic promise.

PD

Worldwide, the incidence estimates of PD range from 5 to 35 new cases per 100,000 individuals yearly (Twelves et al. 2003). The prevalence increases with age, and it is about 2–3% in those aged 65 years or older (Lappin et al. 2018). One study estimated that the average disease duration until death is between 6.9 and 14.3 years (Jiao et al. 2018). PD is characterized by the selective loss of midbrain dopaminergic (DA) neurons. It has been demonstrated that miR-124 levels are significantly lower in the plasma of patients with PD, PD mouse models induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and DA neuronal cell lines treated with 6-hydroxydopamine or methyl phenyl pyridinium (Dong et al. 2018; Kanagaraj et al. 2014; Li et al. 2017; Rosas-Hernandez et al. 2018; Yao et al. 2019).

In the development of PD, upregulation of miR-124 may regulate several pathogenetic events involved in anti-inflammatory effects, decreasing oxidative stress, and anti-apoptotic effects, thus reducing the loss of DA neurons. The roles of miR-124 in inhibiting neuroinflammation, decreasing ROS production and neuronal apoptosis, and improving cell viability are related to the regulation of JAK/STAT3, MEKK3/NF-κB, AnnexinA5 (ANXA5)/ERK, and calpain/CDK5 signals, respectively (Dong et al. 2018; Geng et al. 2017; Kanagaraj et al. 2014; Yao et al. 2018). Moreover, miR-124 targets Bim, a vital protein regulating the apoptosis and autophagy of DA neurons in the pathogenesis of PD, to reduce Bax translocation into the mitochondria and lysosomes. This relationship was implicated in the alleviation of apoptosis and promotion of autophagy in DA neurons (Wang et al. 2016a). P38 and p62 are upregulated in MPTP-induced PD model; therefore, by targeting p38 and p62, overexpression of miR-124 could suppress the secretion of proinflammatory mediators and promote autophagy in the inflammatory response of PD (Yao et al. 2019). Overexpression of death-associated protein kinase 1 (DAPK1) resulted in DA injury and locomotor disabilities in mice with PD; however, miR-124 rescued impairment in PD by targeting DAPK1 (Lu et al. 2020; Su et al. 2019). These results indicate that miR-124 could be a potential therapeutic target for regulating the pathological process in PD.

ALS

In Europe, the incidence of ALS is about 2.6–3.0 cases per 100,000 people, with a prevalence of 5.4 per 100,000 people (Cicero et al. 2017; van Es et al. 2017). ALS is a fatal neurodegenerative disease characterized by progressive motoneuron loss, and most patients with ALS die of respiratory complications (Niedermeyer et al. 2019). miR-124 may be a possible indicator of disease stage/progression in ALS. A recent study using the SOD1 G93A model of ALS showed that the spinal motor neuron-derived exosomal miR-124 and its extracellular localization are increased even at the pre-symptomatic stage; this event occurs before a large number of spinal motor neurons undergo degeneration, indicating that the extracellular change in miR-124 is likely an early pathological event (Yelick et al. 2020). Expression of miR-124 is increased in the brain of ALS model mice in the late stages of the disease, and is significantly upregulated in the hippocampus, subventricular zone, brainstem motor nuclei, and primary motor cortex of 18-week-old ALS mice compared to controls (Marcuzzo et al. 2015). Similarly, miR-124 was increased in differentiating ependymal stem cell progenitors in vitro, particularly in stressed neurons, as well as linked to neurodegeneration in 18-week-old ALS mice (Marcuzzo et al. 2015, 2014). Increased miR-124 levels were also found in mSOD1 NSC-34 cells (an in vitro ALS model) and their derived exosomes (Pinto et al. 2017). The activation of neuroinflammation and neurotoxic effects has been shown to contribute to motoneuron degeneration (Brites and Vaz 2014; Cunha et al. 2018). Overexpression of miR-124 seems to directly or indirectly reduce the differentiation of astrocytes and increase neuronal differentiation (Krichevsky et al. 2006; Marcuzzo et al. 2014). However, alterations in miRNA localization can lead to different effects and outcomes. The ability of neurite development may be lost in the early stages of ALS and thereby contribute to the progressive nature of axonal degeneration. However, miR-124 has been shown to play an important role in promoting neurogenesis, which has been reviewed in another section of this article (Ischemic stroke). These findings suggest a complex mechanism underlying the relationship between miR-124 and ALS.

miR-124 in Epilepsy

Epilepsy is a common, recurrent, and intractable seizure disorder, affecting over 70 million people worldwide (Thijs et al. 2019). It is bimodally distributed with two peaks: infants less than 1 year old and in people over the age of 50 years (Thijs et al. 2019). The pathogenesis of epilepsy is thought to result from disrupted gene expression, leading to an imbalance between excitatory and inhibitory activity within a neuronal network, a disorder in synaptic structure, cell death, and inflammation. Anti-epileptic drugs are effective in less than half of the patients, and specific anti-epileptic drugs are not available (Walker 2018). Emerging evidence indicates that certain miRNAs play important roles in epileptogenesis (Jimenez-Mateos et al. 2012; Tan et al. 2013; Zheng et al. 2016). No compelling evidence has been found to date relating to the risk of epilepsy to mutations or variations in the miR-124 gene (Manna et al. 2016), although significant upregulation of miR-124 was detected in the seizure-related stages in children with mesial temporal lobe epilepsy (Peng et al. 2013). However, Wang et al. (2016a, b) observed that miR-124 was downregulated in epilepsy models injected intraperitoneally with lithium chloride, as intra-hippocampal supplementation with miR-124 inhibited neuronal firing and excitability as well as susceptibility to epileptic seizures, by targeting CREB1. Moreover, Brennan et al. (2016) demonstrated that miR-124 played dual and opposing roles in epilepsy, attenuating epileptogenesis via the neuron restrictive silencer factor, while promoting epilepsy via inflammation, showing that miR-124 might be aberrantly expressed during epileptogenesis and may be a key regulator and promising therapeutic target for epilepsy. However, in a recent study, a combination of anti-miRNA oligonucleotides (AMOs) against miR-124 and miR-137 administered locally to the dentate gyrus normalized neuroblast proliferation and prevented NSCs loss upon non-convulsive seizures in a subpopulation of patients with epilepsy (Bielefeld et al. 2019). In addition, the effects of AMO co-administration against miR-124 and miR-137 were better than individual AMO administration, which supports the theory that the action mediated by miRNA synergy is necessary. There might be more synergistic effects between miR-124 and other miRNAs, and their co-administration could play more functions in the treatment of CNS diseases. In general, because of the complexity of epilepsy, the few studies published to date are insufficient to draw any conclusion on the comprehensive function and mechanism of miR-124.

miR-124 in MS

MS is a chronic inflammatory autoimmune disease of the CNS. Today, 2–3 million people are affected by MS, and almost three out of four people with MS are women (Compston and Coles 2002; Dobson and Giovannoni 2019). MS is affected by many factors, including environmental and genetic factors; according to epidemiological studies, environmental factors are more important than genetic factors. Low levels of vitamin D, smoking, obesity, exposure to sunlight, and Epstein–Barr virus infection play important roles in MS development. At present, more than 150 genes have been associated with MS; among them, the most significant one is the HLA DRB1*15:01 haplotype (Dobson and Giovannoni 2019; Reich et al. 2018).

Evidence indicates that the pathogenesis of MS is related to a series of pathological events, such as activation of microglia and macrophages, neuronal injury, and glial reaction (Thompson et al. 2018). The main symptoms of MS are neuralgia, cognitive impairment, spinal neuritis, and disability, which affect the normal life of young people (Correale et al. 2017). At present, the major treatments for MS are disease-modifying therapies—the use of drugs, such as dimethyl fumarate, natalizumab, and ocrelizumab; however, all these drugs have side effects, presumably since they do not take into account the patient’s disease phenotype, prognostic factors, acceptable risk preferences, etc. (Tintore et al. 2019).

Activated T cells and B cells are found in the CNS of patients with MS. Activated T cells produce cytokines (IL-17, IFN-γ, etc.) to activate microglia and macrophages. Activation of macrophages (mainly type M1, positive expression of MHC II) can improve the antigen-presenting effect of antigen-presenting cells, promote autoimmunity and inflammatory reactions caused by T and B cells, and cause tissue damage (Baecher-Allan et al. 2018; Essandoh et al. 2016). The activated microglia can release proinflammatory factors, ROS, glutamic acid, etc., which further cause inflammatory reactions, demyelination, and neuronal injury (Faissner et al. 2019). miR-124, which is a marker of both anti-inflammation and immune cell quiescence, was strongly downregulated in progressive MS patients, suggesting the therapeutic opportunities of MS by targeting inflammatory reactions (Amoruso et al. 2020).

Resveratrol was found to affect the apoptosis of T cells by regulating the miR-124/SK1 pathway in EAE. Resveratrol upregulated miR-124 by targeting SK1, thus affecting the development of T cells (Gandy et al. 2019). T helper (Th) 1 and Th17, two mature Th effector cells, are important in the process of MS, since they can release IFN-α and IL-17A (Baecher-Allan et al. 2018). miR-124 can inhibit the expression of cytokine signal transduction inhibitor 5, and thereby promote the expression of STAT1 and STAT3, which are very important for the differentiation of Th1 and Th17 cells, and subsequently promote the maturation and differentiation of CD4+ cells (Jiang et al. 2014). It has been shown that miR-124 can inhibit the production of IL-6 and TNF-α by targeting STAT3 and TACE, which can further reduce the release of LPS-induced cytokines (Sun et al. 2013; Wang et al. 2017). In addition, the role of miR-124 in M2 polarization might shed light on the therapy of MS symptoms (Mikita et al. 2011; Weng et al. 2019).

In addition, Glu-mediated excitotoxicity is an important process in the pathogenesis of MS, leading to neuronal death by overstimulation of glutaminergic receptors and increased axonal calcium (Ciccarelli et al. 2014). miR-124 interacted with Glutamate Ionotropic Receptor AMPA Type Subunit 2 (GRIA2, receptors mediated excitatory neurotransmission) mRNA and downregulated its translation in the hippocampus (Ho et al. 2014). In contrast, it was found that following hippocampal demyelination in mice, miR-124 levels in the hippocampus increased, and it was associated with the downregulation of GRIA2 content, resulting in a decrease in synaptic plasticity (Dutta et al. 2013). These results suggest that the different expression patterns of miR-124 might be closely related to the progression of MS, and changes in miR-124 levels could address memory decline in patients with MS.

miR-124 in GBM

The grade IV type tumor arising from astrocytes, which is infiltrative and undifferentiated from other normal cells, is called glioblastoma, also known as glioblastoma multiforme (GBM). GBM is the most common malignant gliomas of the CNS, accounting for approximately 60–70% of all malignant gliomas, with the highest incidence rate (3.19 per 100,000 before 2009) (Louis et al. 2007). The incidence of GBM increases dramatically after the age of 54 years, with a peak incidence of 15.24 per 100,000 at the age of 75–84 years. The median overall survival of GBM patients is only about 12–15 months (Ostrom et al. 2015). Thus, GBM are widely noted for their high incidence and low five-year survival rate (no more than 5%) (Dolecek et al. 2012). The role of angiogenesis and the high invasiveness of GBM, which are the main causes of poor prognosis and therapeutic tolerance, are particularly pivotal and involve a variety of molecular mechanisms and signaling pathways. Hypoxia, which acts as the starting signal, prompts the expression of hypoxia-inducible factor 1, resulting in an increase in vascular endothelial growth factor (VEGF) (Jain 2014). In addition, the activation of mitogen signaling pathways (such as PI3K and MAPK) (Jayson et al. 2016), the secretion of extracellular matrix proteins (MMP-2, TWIST1) (Florczyk et al. 2013), and altered cellular metabolism (Beyer et al. 2017) contribute to the progression of GBM. Owing to their multiple pathogeneses, clinical symptoms, like progressive headaches, focal neurologic deficits, and seizures, are complex and intractable (Huse et al. 2011). Although there have been improvements in the clinical therapy of glioblastomas, such as surgical excision combined with DNA methylation agent-mediated chemotherapy and radiotherapy, the prognosis and overall survival of patients have not changed significantly (Gittleman et al. 2018; Xue et al. 2020). Therefore, research on targeted molecular therapy is warranted.

Recent studies and evidence have shown that different miRNA expression profiles play distinct roles in the progression of GBM (Fowler et al. 2011). Quantitative RT-PCR was used to investigate the expression profiles of 192 miRNAs in primary brain tumor and non-tumor brain tissues; the results showed that miR-124 was downregulated in GBM (Silber et al. 2008; Yang et al. 2013). In addition, the expression of miR-124 decreased progressively in glioma tissues from WHO grades II to IV (Deng et al. 2019). Another study that examined 119 clinical GBM patients found that those with lower levels of miR-124 had shorter survival times (Fowler et al. 2011). At present, the mechanisms that suppress miR-124 in GBM remain unclear. The following two mechanisms are recognized. The first mechanism is the overexpression of the transcriptional repressor RE1 silencing transcriptional factor (REST), which represses miR-124 to regulate the oncogenic properties of high-REST GBM stem-like cells (HR-GSCs) in humans. The study showed that REST-miR-124 pathways could regulate self-renewal, apoptosis, and invasion in GSCs (Marisetty et al. 2017). The second mechanism is growth factor signaling. In a mouse NSC culture without growth factors, miR-124 expression is increased, indicating that growth factors may promote GBM formation by repressing miR-124 (Silber et al. 2008).

miR-124 regulates cell growth, differentiation, invasion, and apoptosis by targeting multiple genes or proteins. A previous study showed that miR-124 inhibits the proliferation of C6 glioma cells by targeting Smad4 (Zhang et al. 2017b). Transfection of miR-124 induced the arrest of GBM cells in the G0/G1 phase, thus inhibiting the growth and proliferation of GBM (Chen et al. 2014; Silber et al. 2008; Skalsky and Cullen 2011). One study showed that increasing the expression of miR-124 affects the ability of GBM cells to survive and absorb nutrients and oxygen. Moreover, miR-124 overexpression improved the survival rate of tumor-bearing mice (Mucaj et al. 2015). In addition, overexpression of miR-124 significantly inhibited the invasion of U87-124 and U373-124 cells through a Matrigel membrane in vitro (Xia et al. 2012). At present, several targets, associated with the abovementioned behaviors, have been identified.

Upregulation of miR-124 in glioma cancer stem cells inhibited immunosuppression mediated by the IL-6/JAK/STAT3 signaling pathway, indicating that miR-124 functions through downregulation of STAT3 (Johnson et al. 2018; Wei et al. 2013). Transfection of T cells along with miR-124 administration in patients with GBM affected the expression of cytokines (IL-2, IFN-1, and TNF-α), implying that the therapeutic effect of miR-124, which targets STAT3, relies on the T cell-mediated immune response, which is a potential immunotherapeutic agent (Bo et al. 2013). The miR-124/SOX9 axis also exerts an effect on stem cells differentiating into neurons. Overexpression of miR-124 was shown to silence SOX9 in patient-derived GBM cells, reducing the radiation resistance and tumorigenicity of patients with GBM (Sabelström et al. 2019). miR-124 has been reported to directly target the Ras viral oncogene homolog (R-Ras) and the neuroblastoma Ras viral oncogene homolog (N-Ras) in glioma cells, showing a negative correlation (Shi et al. 2014). Furthermore, Akt, ERK1/2, and VEGF, which are downstream effectors of R-Ras and N-Ras pathways in cell proliferation and survival, are suppressed by miR-124 in GBM cells (Chappell et al. 2011; Shi et al. 2014). A recent study focused on the functions of p62 and demonstrated that the accumulation of p62 could promote glioma progression by regulating autophagy, proliferation, migration, apoptosis, TMZ resistance, and the NF-κB signaling pathway, and its functions could be partially reversed by miR-124 overexpression (Deng et al. 2019). In view of the unique benefits of miR-124 in negatively regulating the proliferation and invasiveness of GBM involving various molecular mechanisms, targeting the delivery of miR-124 to GBM tumor cells may be therapeutically valuable for GBM treatment.

Methods for the Delivery of miR-124-Based Drugs to the CNS

The basis of drug development is a deep understanding of the molecular mechanisms of pathological processes. The development of miRNA-related drugs requires systematic analysis of patient samples to clarify the pathogenesis and biological relationship between miRNAs and diseases through in vivo and in vitro models. Although miR-124 is identified as a promising therapeutic for treating CNS diseases and injuries, the BBB essentially restricts the entry of therapeutic drugs into the brain or spinal cord. The challenges, described below, include the design of an miR-124 delivery system that crosses the BBB and enables tissue/cell-specific targeting. Several methods have been developed for the delivery of miR-124 or other miRNAs to the CNS for the treatment of inflammation, stroke, PD, and GBM.

Exosome Delivery System

Exosomes are 40–100 nm vesicles released as a consequence of multivesicular endosome fusion with the plasma membrane (Raposo and Stoorvogel 2013). The exosome is a well-studied vesicle, which can cross the BBB and carry membrane and cytosolic proteins, lipids, and RNA cargos to mediate brain remodeling after diseases and injuries (Haney et al. 2015; Valadi et al. 2007; Xin et al. 2014). Several miRNA mimetics and inhibitors have been effectively delivered to the brain using exosomes, and could protect against CNS injury (Alvarez-Erviti et al. 2011; Kim et al. 2020; Lakhal and Wood 2011; Xin et al. 2012). Other than promoting microglial M2 polarization, miR-124 is also detected to be abundant in the M2-microglia-derived exosomes, which can be administered intravenously to inhibit neuronal inflammation and neuronal autophagy, enhance neurogenesis, and improve neurological outcome following traumatic brain injury in mice (Ge et al. 2020; Huang et al. 2018; Li et al. 2019c; Yang et al. 2019). Mesenchymal stem cells can migrate to cancer sites, including GBM, and exert anti-tumor and neuroprotective effects (Otero-Ortega et al. 2019; Sharif et al. 2018). In addition, it has been demonstrated that mesenchymal stem cell exosomes loaded with miR-124 significantly inhibit the activity of CDK6, enhance the chemosensitivity of GBM cells to temozolomide, and decrease the migration of GBM cells (Sharif et al. 2018). Exosomal miR-124 from M2-microglia also promoted neurogenesis and protected the brain from ischemic stroke or ischemia–reperfusion injury (Song et al. 2019b). Therefore, identification of the cellular source of exosomes seems important; the native cargos might play a synergistic role with miR-124 to achieve better effects. To achieve neuron-specific targeting, miR-124-loaded exosomes could also be modified. Exosomes modified by RVG-Lamp2b and injected after brain ischemia have been shown to promote cortical neuronal progenitor cell differentiation, cortical neurogenesis, and protect against ischemic brain injury (Yang et al. 2017). The challenges in exosomal miR-124 application might include identifying the best exosome-secreted cells and developing appropriate exosome modification.

Nanoparticles

Delivery systems for miRNA precursors or antagonists can also be approached with nanoparticles, which also have the potential to bypass the BBB and cell-specific delivery after modifications. Nanoparticles encapsulate and protect miRNAs from degradation, enhancing circulation time and targeted accumulation (Blanco et al. 2015). The nanoparticles used for therapeutic miRNA delivery have a size range of 1–500 nm (Boca et al. 2020). In addition to size, the shape, surface chemistry, and charge of nanoparticles affect their capability to specifically target cells and the way cells internalize them (Behzadi et al. 2017). On this basis, the choice of the biomaterial that constitutes the nanoparticles is extremely important, as it dictates the final properties and structure of the nanoparticles. Multiple nanoparticles for miRNA delivery has been extensively investigated, due to their efficient cargo release within the cell cytoplasm and their easy synthesis and functionalization, including liposomes, synthetic polymers, natural polymers, as well as inorganic nanoparticles, such as gold, calcium phosphate, silica, and iron oxides, which has been well-reviewed elsewhere (Lee et al. 2019). miR-124-loaded nanoparticles were shown to reverse tumor-mediated immune suppression and prolong survival in a murine glioma model system by regulating the intracellular signaling pathway (Yaghi et al. 2017). This delivery approach is also applicable to other malignancies, such as non-small cell lung, breast, and ovarian cancers (Lin et al. 2016; Seviour et al. 2016). Intracerebroventricular injection of miR-124-loaded nanoparticles reduced inflammatory cytokine levels and enhanced brain repair in PD (Gan et al. 2019; Saraiva et al. 2016). However, intravenous injections of miR-124-loaded nanoparticles increased NSCs survival and neuronal differentiation in vitro but did not contribute to stroke outcome in vivo (Saraiva et al. 2018). Additional studies might concentrate on achieving the targeted distribution and reducing toxic and off-target effects.

Adeno-Associated Viruses (AAV)

AAV capsids are a rapidly emerging gene therapy delivery system for the treatment of neurological diseases. AAVs have an unprecedented ability to transfer genes to the CNS and achieve efficient and stable therapeutic levels of miRNAs (Deverman et al. 2018a). AAVs are 25 nm non-enveloped viruses with a single-stranded genome of about 4800 nucleotides that replicate only in the presence of several proteins complemented by adenoviruses, hence the name (Schaffer et al. 2008). Currently, more than 100 natural AAV variants comprising at least 8 serotypes have been identified from vertebrates (Gao et al. 2003). Noteworthy, many of them, such as AAV1, AAV2, AAV5, AAV9, AAVrh.10, and AAV-DJ8, have been evaluated to spread broadly in the CNS and transduce cells with high efficiency (Deverman et al. 2018b). The AAV genome contains two open reading frames (ORF) flanked by inverted terminal repeat elements (ITR), which are the minimal cis-acting elements necessary for viral genome integration, replication, and packaging into particles (Srivastava et al. 1983). The first ORF (rep) encodes four rep proteins that are involved in the replication of the viral genome, whereas the second ORF (cap) encodes three structural proteins (VP1, VP2, and VP3). The VP proteins self-assemble to form the viral capsid, into which the viral genome then loads, and cap, therefore, plays a great role in the viral gene transduction properties (Schaffer et al. 2008). To generate a recombinant AAV for gene delivery, rep and cap are excised from between the ITRs, replaced with the therapeutic transgenes and promoters in their place, and the two viral ORFs are supplied as helper genes in trans to package the transgenes inside the capsid (Kwon and Schaffer 2008). In the CNS, most cells are post-mitotic, and many chronic neurological diseases necessitate long-term transgene expression, which further contributes to the application prospects of AAVs. Transgenes that encode therapeutic genes have been successfully delivered to a variety of tissues and cell types within the CNS with AAVs, as reviewed elsewhere (Deverman et al. 2018b; Ojala et al. 2015). Engineered artificial anti-C9orf72-targeting miRNA AAV was intrathecally injected in an ALS mouse model to reduce the repeat-containing transcripts and showed a significant reduction in the toxicity caused by C9orf72 transcripts after treatment (Martier et al. 2019). Cappella et al. reviewed a series of studies in detail showed that intrathecal AAV-mediated miRNAs delivery could be used as a potential treatment for SOD1-linked ALS, although additional studies and further improvements are required to determine whether the approach is safe and above all efficient (Cappella et al. 2021).

Conclusions and Perspectives

The cellular and molecular changes underlying CNS injuries provide substantial potential biomarkers and therapeutic targets. miRNA research has become a rapidly growing field since their discovery more than two decades ago. Based on their one-to-many molecular regulatory properties, miRNAs contribute to diverse physiological and pathophysiological functions in a global pattern. In this review, we have discussed the significant roles of miR-124 in pathological processes involved in CNS disorders (Table 1). Although some disease-specific alterations of miR-124 in the CNS await further investigation, the biological significance and utility of miR-124 in CNS disease is becoming evident. The challenge lies in the productive exploitation of miRNA-based therapeutics, including designing a miR-124 delivery system that can cross the BBB. It will also be necessary to enable tissue/cell-specific targeting, while conferring higher stability and avoiding potential off-target effects, as well as determining therapeutic windows and modes of treatment (injected intravenously or positionally) according to individual features.

Table 1 Targets of miR-124 and their functions in CNS diseases and injuries
Full size table

Another emerging area of research is the systematic pharmacokinetics of novel miRNA drugs. At present, little is known about the therapeutic combination of miRNAs, and delivery systems have pharmacokinetic characteristics, defined by the target cell, half-life, biological distribution, metabolic pathway, and organ effects. In general, miRNA-related therapies require interdisciplinary studies and are promising in providing more practical insights into current biomedical problems. With miRNAs as novel biological targets, we look forward to future progress of miR-124-based therapies in CNS diseases and injuries in the next decade.