Journal Pre-proof

miR-124 and Parkinson’s disease: a biomarker with therapeutic potential

Efthalia Angelopoulou, Yam Nath Paudel, Christina Piperi

PII: S1043-6618(19)31540-3
DOI: https://doi.org/10.1016/j.phrs.2019.104515
Reference: YPHRS 104515

To appear in: Pharmacological Research

Received Date: 29 July 2019

Revised Date: 20 October 2019
Accepted Date: 28 October 2019

Please cite this article as: Angelopoulou E, Nath Paudel Y, Piperi C, miR-124 and Parkinson’s
disease: a biomarker with therapeutic potential, Pharmacological Research (2019),
doi: https://doi.org/10.1016/j.phrs.2019.104515

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© 2019 Published by Elsevier.

miR-124 and Parkinson’s disease: a biomarker with therapeutic

potential

Efthalia Angelopoulou1, Yam Nath Paudel2 and Christina Piperi1*

1
Department of Biological Chemistry, Medical School, National and Kapodistrian

University of Athens, Athens, Greece

2
Neuropharmacology Research Laboratory, Jeffrey Cheah School of Medicine and

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Health Sciences, Monash University Malaysia, Bandar Sunway, Selangor, Malaysia

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*Corresponding Author

Christina Piperi, PhD,

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Department of Biological Chemistry, Medical School,

National and Kapodistrian University of Athens, 75 M. Asias Street, 11527 Athens,

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Greece.

E-mail: cpiperi@med.uoa.gr; Tel.: +30-210-7462610; Fax: +30-210-7462703

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Graphical Abstract
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Highlights
 miR-124 is abundantly expressed in the brain
 miR-124 participates in neurogenesis, synapse morphology,
neurotransmission
 miR-124 regulates cell survival, autophagy, mitochondrial dysfunction in
PD
 miR-124 regulates oxidative damage and neuroinflammation in PD
 plasma miR-124 levels may serve as a potential diagnostic biomarker in
PD
 miR-124 exhibits a neuroprotective role in PD and may be used
therapeutically

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Word count: 6813 (excluding references)

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Figures: 1

Tables: 1
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Abstract:

Parkinson’s disease (PD) is a multifactorial disorder, attributed to a complex interplay

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between genetic and epigenetic factors. Although the exact etiology of the disease

remains elusive, dysregulation of signaling pathways implicated in cell survival,

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apoptosis, protein aggregation, mitochondrial dysfunction, autophagy, oxidative

damage and neuroinflammation, contributes to its pathogenesis. MicroRNAs (miRs)

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are endogenous short non-coding RNA molecules that negatively regulate gene

expression at a post-transcriptional level. MiR-124 is one of the most abundantly

expressed miRs in the brain that participates in neurogenesis, synapse morphology,

neurotransmission, inflammation, autophagy and mitochondrial function.

Accumulating pre-clinical evidence shows that miR-124 may act through calpain

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1/p25/cyclin-dependent kinases 5 (CDK5), nuclear factor-kappa B (NF-κB), signal

transducer and activator of transcription 3 (STAT3), Bcl-2-interacting mediator of cell

death (Bim), 5' adenosine monophosphate-activated protein kinase (AMPK) and

extracellular signal-regulated kinase (ERK)-mediated pathways to regulate cell

survival, apoptosis, autophagy, mitochondrial dysfunction, oxidative damage and

neuroinflammation in PD. Moreover, clinical evidence indicates that reduced plasma

miR-124 levels may serve as a potential diagnostic biomarker in PD.

This review provides an update of the pathogenic implication of miR-124 activity in

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PD and discusses its targeting potential for the development of future therapeutic

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strategies.

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Keywords: miR-124; Parkinson’s disease; neuroprotection; neuroinflammation;
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biomarker
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Abbreviations:
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PD, Parkinson’s disease; SNpc, Substantia nigra pars compacta; CNS, Central

nervous system; LBs, Lewy bodies; DBS, Deep brain stimulation; CREB, cAMP
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response element-binding protein; REST, RE1-silencing transcription factor; MPTP,

N-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine; AMPK, 5' adenosine

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monophosphate-activated protein kinase; 6-OHDA, 6‐ hydroxydopamine; MEF2D,

Myocyte enhancer factor 2; ROS, Reactive oxygen species; FOXO3, Forkhead box

protein 3; AIF, Apoptosis-inducing factor; mTOR, Mammalian target of rapamycin;

STAT3, Signal transducer and activator of transcription 3;ERK, Extracellular signal-

regulated kinase; CDKs, Cyclin-dependent kinases; MEKK3, Mitogen-activated

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 protein kinase kinase kinase 3; MALAT1, Metastasis-associated lung adenocarcinoma

transcript 1; NEAT2, Nuclear-enriched abundant transcript 2; NSCs, Neural stem

cells

1. Introduction:

Parkinson’s disease (PD) is the second most common neurodegenerative disorder

after Alzheimer’s disease (AD), affecting approximately 1-2% of the global

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population over the age of 60 years (1). Neuropathologically, PD is characterized by

loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and

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intracellular accumulation of Lewy bodies (LBs) and neurites, mainly composed of α-

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synuclein (SNCA), leading to selective nigrostriatal neurodegeneration. The main

symptoms of PD patients involve bradykinesia, rigidity and resting tremor whereas

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non-motor manifestations, such as dementia, depression and dysautonomia are also an

integral part of its clinical phenotype. Although the exact etiology of the disease
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remains elusive, several lines of evidence suggest that dysregulation of signaling

pathways implicated in cell survival, apoptosis, protein aggregation, mitochondrial

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dysfunction, autophagy, oxidative damage and neuroinflammation underlies its

pathogenesis (2). PD is considered as a multifactorial disorder, attributed to a complex

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interplay between genetic and environmental factors (1). Smoking and coffee
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consumption have been consistently associated with a reduced risk for PD, whereas

other factors, such as pesticide exposure correlate with increased risk. Despite

extensive research efforts towards the elucidation of PD pathogenesis, current PD

treatment strategies, including levodopa and deep brain stimulation (DBS), are still

only symptomatic and fail to halt disease progression, being often accompanied by

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serious side-effects (3). Therefore, a deeper understanding of the molecular

mechanisms underlying its pathogenesis and the development of novel effective

therapeutics is highly demanded.

In this regard, recent evidence supports the role of epigenetic alterations as a potential

linkage between genetic and environmental interactions underlying the pathogenesis

of neurodegenerative disorders, including PD. Epigenetic mechanisms alter gene

expression without changing DNA sequence, mainly including DNA methylation,

post-transcriptional histone modifications, such as acetylation, methylation,

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phosphorylation, ubiquitination and sumoylation, as well as microRNAs (miRs) (4).

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The expression of SNCA gene, one of the most significant genetic risk factors for PD,

can be modulated by DNA methylation, and peripheral blood leukocytes from PD

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patients were found to exhibit reduced methylation levels in CpG-2 sites of the SNCA
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gene promoter, in comparison to controls (5). Thus, DNA methylation levels could be

used as a potential PD biomarker. Furthermore, paraquat, a neurotoxin associated with

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PD development, was shown to induce hyperacetylation of histone H3 in N27

dopaminergic cells and promote apoptosis, suggesting that dysregulation of H3

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acetylation may underlie pesticide-induced neuronal loss in PD (6).

MiRs are highly conserved, endogenous, short non-coding RNA molecules, consisting
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of about 22 nucleotides, capable to regulate gene expression at a post-transcriptional

level (7). They bind specifically to the 3´-untranslated region (UTR) of multiple
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targeted mRNAs, leading either to inhibition of mRNA translation or induction of

mRNA degradation (8). Thus, miRs are increasingly recognized as crucial regulators

of several cellular processes, including cell proliferation, apoptosis, differentiation,

inflammation, metabolism, mitochondrial function and autophagy (9). Accumulating

evidence shows that aberrant expression of various miRNAs plays a pivotal role in

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cancer, heart disorders as well as neurodegenerative diseases, including PD (10, 11).

More specifically, miR-153 and miR-7 downregulate the SNCA gene expression,

leading to decreased levels of intracellular SNCA (12). Furthermore, miR-133b,

which is specifically expressed in midbrain dopaminergic neurons and is crucial for

their maintenance in the brain, has been found deficient in the midbrain tissues of PD

patients (13). In addition, miR-34b and miR-34c levels are reduced in the SH-SY5Y

dopaminergic cell lines and at the affected brain tissues of PD patients (14). Notably,

these alterations are accompanied by mitochondrial dysfunction, oxidative damage,

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decreased cell viability and reduced expression of Parkin and DJ1, which are mainly

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associated with familial PD cases but also implicated in idiopathic forms (14).

Moreover, miR-181a overexpression reduced neuronal apoptosis and autophagy by

inhibiting JNK pathway (15).

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Interestingly, miR-124 is the most abundantly expressed miR in the brain displaying a

significantly higher expression (about 100 times) in brain parenchyma as compared to

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other mammalian tissues (16).

Up to date, three miR-124 subtypes have been identified, named miR-124-1, -2 and -
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3, with specific chromosome locations (9). MiR-124 can regulate hundreds and

possibly thousands of genes, including those encoding Notch ligand Jagged1, the
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transcription factor Sox9, the SCP1 subunit of RE1-silencing transcription factor

(REST) and cAMP response element-binding protein (CREB) (17). In neurons, miR-
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124 down-regulates the expression of numerous non-neuronal genes, associated with

the acquisition and preservation of their neuronal identity during the embryonic

development of the central nervous system (CNS) as well as with neurogenesis in

adults (10, 18). MiR-124 participates in synapse morphology, neurotransmission,

inflammation, autophagy and mitochondrial function (19). Considerable amount of

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 evidence has revealed that the abnormal expression and neuroprotective role of miR-

124 is associated with various neurological disorders, including stroke (20),

experimental autoimmune encephalomyelitis (EAE), the experimental model of

multiple sclerosis (21) and neurodegenerative diseases, such as AD (22).

Given the abundance of miR-124 in the brain and the fact that the biological processes

affected by miR-124 are impaired in PD, it has been suggested that it could play a

substantial role in its pathogenesis. Notably, the study of the MIRECORDS database

has shown that 40 out of 202 of validated targets of miR-124 are dysregulated in PD

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(23). Furthermore, 8 out of 27 validated targets of miR-124a in HEK-293S cells are

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also dysregulated in PD dopaminergic neurons (24), highlighting the potential pivotal

role of miR-124 in PD development.

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In this review, we will discuss recent pre-clinical and clinical data of the emerging
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role of miR-124 in PD pathophysiology, aiming to shed more light towards the

understanding of the molecular mechanisms involved in PD and the development of

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novel treatment strategies.

2. The neuroprotective role of miR-124 in PD: Insights from in vivo and in vitro
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studies
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Recent findings from in vitro and in vivo studies demonstrate the crucial

neuroprotective effects of miR-124 against dopaminergic cell death, autophagy

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dysregulation, neuroinflammation and oxidative damage in PD models (10, 25-30). In

this context, the expression of miR-124 has been shown to be downregulated in 1-

methyl-4-phenylpyridinium (MPP) iodine-treated MN9D dopaminergic neurons (10),

MPP+-treated SH-SY5Y cells (26-29), 6-hydroxydopamine (6-OHDA)-treated PC12

and SH-SY5Y cells (25), as well as in N-methyl-4-phenyl-1, 2, 3, 6-

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 tetrahydropyridine (MPTP)-treated mice (10, 28, 29). Importantly, miR-124

downregulation was observed during early stages of neurodegeneration in most of

these studies, implying that miR-124 reduction may just not reflect dopaminergic cell

death (10, 29, 30), but rather contribute to the initial biological processes of

neurodegeneration in PD.

2.1.The effects of miR-124 on calpain 1/p25/ cyclin-dependent kinase 5 (CDK5)

pathway in PD

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Calpains are calcium-dependent proteases abundantly expressed in the CNS. There

are two main calpain isoforms; calpain 1 (calpain μ) and calpain 2 (calpain m),

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corresponding to micromolar and millimolar concentrations of Ca2+ required for their

activation (31). A previous study has indicated that the persistent activation of
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calpains mediates dopaminergic neuronal death in MPTP-induced PD mouse models
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and midbrain tissues from PD patients (32). Upon their activation, calpains can

proteolytically cleave p35 and generate the more stable form of the cofactor, p25.
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This in turn activates cdk5 and phosphorylates several downstream factors, including

myocyte enhancer factor 2 (MEF2D) and peroxiredoxin2 (Prx2), thereby inducing

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MPTP-mediated dopaminergic neuronal death (33, 34). Moreover, dysregulated

CDK5 can deactivate Prx2 which acts as a scavenger protein of reactive oxygen
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species (ROS) and H2O2, resulting in oxidative-stress mediated mitochondrial damage

and subsequent neuronal death (10). Since dysregulated mitochondria produce more
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ROS, a vicious cycle is therefore generated. Interestingly, it has been demonstrated

that calpains can be regulated by miR-124 (35). However, the effects of miR-124-

calpains interaction in PD were until recently largely unknown.

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 In this context, overexpression of miR-124 in MN9D dopaminergic neurons treated

with MPP iodide has been shown to improve cell survival and reduce oxidative stress

by downregulating the calpain/ CDK5 pathway (10). In particular, miR-124 can

interact with and downregulate both calpain 1 and 2 (35), although it can significantly

reduce only calpain 1 levels in MPP iodine-treated MN9D dopaminergic neurons

(10). These results can be further explained by the fact that calpain 1 possesses two

conserved binding sites for miR-124, whereas in calpain 2 there is only one, leading

to a probably higher impact of miR-124 on calpain 1 than calpain 2 inhibition (10).

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Mechanistically, the supplementation of miR-124 mimics in MPP iodide-treated

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MN9D cells significantly reduced the expression of calpain 1, p35, p25 and CDK5 in

comparison to controls, leading to increased cell viability as well as reduced ROS

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production and H2O2 levels (10). These results demonstrate that miR-124 may inhibit

dopaminergic neuronal death and oxidative damage at least partially by

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downregulating the calpain 1/p25/CDK5 pathway.
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2.2.The role of miR-124 in Bcl-2-interacting mediator of cell death (Bim)-mediated

pathways in PD
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Bim is one of the BH3-only proteins, a subgroup of the proapoptotic bcl-2 family,

which share the short BH3 region (36). Notably, Bim promotes apoptosis in several
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neuronal cell lines, including primary cerebrocortical neurons exposed to β-amyloid

peptide and sympathetic neurons after withdrawal of nerve growth factor (NGF) (37).
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Mechanistically, JNK phosphorylates forkhead box protein 3 (FOXO3) at serine 574.

FOXO3 promotes Bim transcription, inducing the formation of the Bcl-2-associated X

protein (Bax)/Bcl-2 homologous antagonist/killer (Bak)-mediated mitochondrial outer

membrane pore and the following release of proapoptotic factors, cytochrome c and

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apoptosis-inducing factor (AIF) that promote apoptosis (38). Furthermore, in addition

to its role in apoptosis, Bim can also act as an autophagy inhibitor under normal

conditions via its direct interaction with Beclin1 (39). Apoptosis and autophagy

represent two distinct processes of cellular death with their underlying molecular

mechanisms being interconnected. In MPTP-induced mouse models, mitochondrial

Bax translocation induces mitochondrial outer membrane permeabilization (MOMP)

leading to apoptosis (36). On the other hand, lysosomal Bax translocation causes

lysosomal membrane permeabilization (LMP), further impairing autophagy (40).

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Therefore, Bax seems to be a key mediator of both apoptosis and autophagy in PD. In

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particular, JNK-dependent transcriptional upregulation of Bim enhances

mitochondrial-dependent neuronal apoptosis in the SNpc of MPTP mouse models,

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through the translocation of Bax into the mitochondrial outer membrane, the release

of mitochondrial cytochrome c into the cytosol and the subsequent activation of the
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proapoptotic caspase cascade (36).
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Importantly, there is evidence that miRs can repress Bim in other human diseases. For

instance, miR-363 directly inhibits Bim, leading to the promotion of stem cell survival
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in the case of glioblastoma (41) and miR-24 can inhibit apoptosis of cardiomyocytes

through its direct interaction with Bim (42). Consequently, detection of miRs
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targeting Bim in the case of PD has recently gained increased attention.

It is well-established that autophagy plays a key role in PD pathogenesis, since loss of

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basal autophagy and impaired autophagy in neurons could lead to neurodegeneration

(43). A recent systems biology-based integrative computational analysis indicated that

miR-124 could modulate the expression of 52 target genes involved in the autophagy-

lysosomal pathway (35), suggesting its high implication in autophagy and lysosomal

function. Interestingly, it has been shown that miR-124 is an upstream inhibitor of

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 Bim in MPTP-treated mice and MPP+-intoxicated SH-SY5Y cells, resulting in the

attenuation of apoptosis and enhancement of basal autophagy (29). More specifically,

exogenous addition of miR-124 agomir could inhibit the MPTP-induced upregulation

of Bim and the mitochondrial translocation of Bax, resulting in the inhibition of

apoptosis (29). Moreover, miR-124 could suppress the translocation of Bax to the

lysosome by downregulating Bim, leading to the restoration of MPTP-induced

impaired autophagy. Although Bim inhibits autophagy under normal conditions

through its interaction with Beclin1, under stress conditions, such as starvation or

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MPTP treatment, Bim and Beclin1 interaction was found disrupted, leading to

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amelioration of the inhibitory effects of Bim on autophagy. Mechanistically, miR-124

was shown to directly bind to the 3´-UTR regions of the mRNA of Bim, thereby
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inhibiting its expression. As a result, exogenous supplementation of miR-124 rescued

the dopaminergic neuronal cell apoptosis, maintained striatal dopamine levels and
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inhibited impaired autophagy in the SNpc of these MPTP-induced PD mouse models
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(29). Therefore, miR-124 could also alleviate MPTP-induced dopaminergic neuronal

apoptosis and impair autophagy by targeting Bim, resulting in the inhibition of Bax

mitochondrial and lysosomal translocation.

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2.3.MiR-124 is implicated in 5' adenosine monophosphate-activated protein kinase

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(AMPK)-mediated pathways in PD

AMPK is a serine/threonine kinase that gets activated by increased cellular stress or

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low energy levels, in order to restore energy balance. In most studies, AMPK

activation seems to play a neuroprotective role in PD, since it has been associated

with enhanced macroautophagy, increased mitochondrial quality control and cell

survival, as well as anti-inflammatory effects. AMPK enhances autophagy directly by

activating unc-51 like autophagy activating kinase (ULK1), and indirectly through its

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inhibitory effects on mTOR, which suppresses ULK1 (38). ULK1 initiates the

macroautophagic cascade through Beclin-1 phosphorylation (38). AMPK also induces

mitochondrial biogenesis by activating peroxisome proliferator-activated receptor

gamma coactivator 1-alpha , PGC-1α which is downregulated in PD (44). However,

there is also substantial evidence that AMPK activation could lead to opposite

outcomes, since in severe stress conditions it might induce apoptosis in dopaminergic

cells upon treatment with PD-related neurotoxins. Although the reasons for these

discrepancies have not been yet understood, it has been proposed that the detrimental

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effects of AMPK over-activation could be explained by the excessive

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macroautophagy and defective protein synthesis mediated by the inhibition of AMPK-

induced mammalian target of rapamycin (mTOR), which could limit cellular growth

(36).
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Importantly, an in vitro study demonstrated that miR-124 markedly inhibited

dopaminergic neuronal apoptosis and inhibited autophagy in two MPTP-treated

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neuroblastoma SH-SY5Y and SK-N-SH cell lines by modulating the AMPK/mTOR

pathway (27). More specifically, miR-124 suppression was significantly associated

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with increased p-AMPK levels and decreased p-mTOR levels in these cells.

Therefore, miR-124 may act neuroprotectively by downregulating AMPK and

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upregulating mTOR in these models. In comparison to the abovementioned data

indicating that miR-124 enhanced autophagy (29), miR-124 suppression was found to
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increase the expression of autophagy-associated proteins, including Beclin1, implying

that it may also inhibit autophagy by downregulating AMPK pathway. Consequently,

it is possible that under specific, yet unknown conditions, miR-124 might exhibit

diverse roles in autophagy in PD through different pathways. Although the exact

targets of miR-124 implicated in AMPK pathway regulation are still unknown, it is

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 worth noting that continuous AMPK activation has been associated with enhanced

apoptosis in a Bim-dependent manner (45). Therefore, targeting Bim could also

partially underlie the effects of miR-124 on AMPK pathway. Nevertheless, given the

diverse roles of AMPK activation in PD preclinical studies and the seemingly

contradictory results of the abovementioned studies regarding the role of miR-124 on

autophagy, the effects of miR-124 on AMPK activation and autophagy need to be

further investigated in other types of PD animal models, under different degrees of

stress conditions, as well as in a time-specific manner, in order to clarify its role in

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early versus late stages of PD progression.

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2.4.The effects of miR-124 on Signal transducer and activator of transcription 3

(STAT3)-mediated pathways in PD
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STAT3 is a transcription factor that plays a key role in cancer and inflammation-
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induced injury (46). STAT3 regulates the expression of a wide range of genes

implicated in cell survival, apoptosis, differentiation, proliferation and inflammation

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(47). STAT3 is phosphorylated by Janus kinases (JAK) upon their activation by

growth factor receptors or cytokines, such as interleukin-6 (IL-6), tumor necrosis

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factor (TNF) and epidermal growth factor (EGF). In PD, it has been shown that

manganese exposure can activate JAK2-STAT3 signaling pathway in microglia,

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resulting in the elevated expression of pro-inflammatory cytokines, such as TNF-α

and IL-1β and increased apoptosis of PC12 neuronal cells (48). Apart from its
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regulatory role in neuroinflammation, STAT3 activation is also known to stimulate

caspase-3, a critical activator of the caspase cascade, thereby inducing apoptosis (49).

Interestingly, miRNAs have been shown to directly inhibit STAT3 in other diseases.

For example, miR-17 binds to STAT3 mRNA in a breast cancer cell line (50).

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 Importantly, miR-124 can also directly inhibit STAT3 resulting in the reduction of

immune-suppressive properties of glioma cancer stem cells (gCSC) and the

suppression of glioma growth in vivo (51).

In this context, overexpression of miR-124-3p, a member of the miR-124 family that

displays similar biological activity to other miR-124 subtypes and is found to be

downregulated in neurodegenerative disorders such as AD (52), has been indicated to

attenuate MPP+-induced neurotoxicity in SH-SY5Y cells through direct binding to

STAT3 (26). More specifically, miR-124-3p overexpression increased cell survival,

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reduced apoptosis, exerted anti-inflammatory effects and inhibited oxidative damage

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in MPP+-treated cells. The underlying mechanisms included reduction of caspase-3

activity, reduction of TNF-α and IL-1β levels, low lactate dehydrogenase (LDH)
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activity, enhancement of superoxide dismutase (SOD) activity and subsequent lower
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levels of ROS (26). Importantly, the restoration of STAT3 activity attenuated the

neuroprotective effects of miR-124-3p. Therefore, miR-124 may directly inhibit

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STAT3, resulting in anti-apoptotic, anti-inflammatory and anti-oxidant effects in PD

that need to be further investigated.

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2.5. MiR-124 is implicated in extracellular signal-regulated kinase (ERK)-mediated

pathways in PD
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ERK 1/2 signaling is a critical mitogen-activated protein kinase (MAPK) pathway,

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whose activation regulates key cellular processes, including differentiation,

proliferation, apoptosis and response to oxidative stress (53). Activation of ERK 1/2

pathway is essential for G1/S phase cell cycle progression. ERK1/2 phosphorylates c-

Fos, thereby promoting its interaction with c-Jun and the activation of transcription

factor activator protein 1 (AP-1). AP-1 activation is crucial for the permission of G1/S

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 transition by cyclin D1 that interacts with cyclin-dependent kinases (CDKs). ERK

activation is associated with dopaminergic neuronal apoptosis, mitochondrial

dysfunction and oxidative damage in PD models in several studies (54, 55).

Interestingly, miR-124 also regulates ERK pathway in PD, affecting apoptosis and

oxidative damage. In addition to its binding to calpain 1, Bim and STAT3, miR-124

was shown to exert its neuroprotective effects through its direct interaction with

annexin A5 (ANXA5). ANXA5 is a large protein that can bind to membrane-bound

phosphatidylserine (PS) in a Ca2+-dependent manner, thereby contributing to the

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externalization of PS on the cellular membrane, which is particularly observed during

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early apoptosis (56, 57). In this context, miR-124-3p enhances cell survival and

inhibits apoptosis of 6-OHDA-treated PC12 and SH-SY5Y cells via ANXA5

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interaction (25). More specifically, miR-124-3p was found to directly bind and
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downregulate ANXA5 in this study, leading to the inhibition of ERK phosphorylation

and the 6-OHDA-induced caspase-3 activation (25). MiR-124-3p overexpression was

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also associated with reduced ROS production independently to ANXA5, suggesting

that other proteins may be implicated in the neuroprotective effects of miR-124

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against 6-OHDA-induced oxidative damage. Notably, ERK 1/2 can phosphorylate

STAT3 (58). Therefore, a crosstalk between these two signaling pathways occurs in
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PD and miR-124 seems to play a critical role in this molecular interplay.

2.6. The role of miR-124 in nuclear factor-kappa B (NF-κB) pathway in PD

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Neuroinflammation mediated by microglia is increasingly recognized as a core

pathophysiologic mechanism underlying PD pathogenesis and progression. The

activation of microglia, the macrophage cell types of the CNS, plays a key role in

neuroinflammation and consequent neurodegeneration (59). Microglia can have two

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distinct functional phenotypes; neuroprotective M2 phenotype that monitors the

surrounding microenvironment and proinflammatory M1 phenotype that generates

several pro-inflammatory cytokines, including TNF-α, IL-1β and ROS, thereby

leading to chronic detrimental neuroinflammation and neuronal apoptosis (48).

Regarding the underlying mechanisms, several crucial pathways underlie microglia

activation, including JAK/STAT axis that was described previously, as well as NF-κB

and AP-1 (60). NF-κB is a transcription factor that modulates both immune and cell

survival pathways in various disorders, including PD. Importantly, selective NF-κB

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inhibition can protect against MPTP-induced dopaminergic neurotoxicity (61).

Several miRs have been demonstrated to regulate NF-κB pathway including miR-26b

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which inhibits NF-κB signaling in hepatocellular carcinoma cells through targeting of
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TAK adaptor 3 (TAB3) that downregulates NF-κB pathway (62). Interestingly,

mitogen-activated protein kinase kinase kinase 3 (MEKK3) can trigger NF-κB

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activation (63). Notably, miR-124 acts as a crucial mediator of inflammatory
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responses in the CNS (21). Under normal conditions, miR-124 is highly expressed in

microglia, but not in peripheral macrophages. In microglia, miR-124 exerts anti-

inflammatory effects by downregulating TNF-α and reducing ROS generation (64).

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However, the exact underlying mechanisms have not been clarified yet.
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Interestingly, miR-124 overexpression was shown to attenuate lipopolysaccharide

(LPS)-induced pro-inflammatory response by microglia and the production of pro-

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inflammatory cytokines by targeting MEKK3/ NF-κB pathway (30). In particular,

miR-124 overexpression in BV2 cells was shown to reduce the levels of the pro-

inflammatory cytokines iNOS, IL-6, and TNF-α whereas it increased the expression

of anti-inflammatory cytokines, TGF-β1 and IL-10. Importantly, miR-124

overexpression in BV2 cells inhibited apoptosis of SH-SY5Y cells in the microglial

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 culture supernatant (MCS) transfer model. Exogenous supplementation of miR-124

inhibited microglia activation and apoptosis in SNpc of MPTP-induced PD mouse

models in this study (30). Mechanistically, it was shown that miR-124 exerted these

effects by targeting MEKK3. Therefore, miR-124 seems to inhibit neuroinflammation

in PD by targeting MEKK3/ NF-κB axis in microglia, resulting in the suppression of

apoptosis of neuronal cells.

Of note, miR-124 has been detected in exosomes released from neuronal cells,

affecting neighboring cells, such as microglia (64). This implies that neuronal miRs

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may also be implicated in inflammatory responses by acting on nearby glial cells.

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Therefore, inflammatory regulation by miRs is very complex and this spatiotemporal

fluctuation should be carefully evaluated (64).

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Another important issue that should be considered is the dopaminergic/cholinergic
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imbalance observed in PD that could affect the outcome of the NF-κB signaling

inhibition (65). In the CNS, acetylcholine exerts anti-inflammatory effects through

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binding to α7 nicotinic acetylcholine receptors (nAChRs) on macrophages/microglia

by inhibiting NF-κB pathway and suppressing the secretion of pro-inflammatory

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cytokines (66). Activation of the cholinergic anti-inflammatory pathway has been

shown to inhibit microglia activation and protect dopaminergic neurons in a MPTP-

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induced mice model of PD, and the use of α7 nAChR agonists could suppress

neuroinflammation in this study (67). Since nigrostriatal damage also significantly

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affects nicotinic receptor-mediated dopaminergic signaling (65), inhibition of NF-κB

signaling by miR-124-targeted strategies could also enhance cholinergic anti-

inflammatory effects.

2.7.Other potential targets of miR-124 in neurodegenerative disorders

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Several preclinical studies have investigated the role of miR-124 in other

neurodegenerative disorders, including Alzheimer’s disease (AD), Amyotrophic

Lateral Sclerosis (ALS) and Huntington’s disease (HD), shedding more light on the

molecular mechanisms underlying its effects on neurodegeneration (68). A study of

PC12 cell line and primary hippocampal neurons demonstrated that miR-124 could

target the β-site APP cleaving enzyme 1 (BACE1), which cleaves amyloid precursor

protein (APP), playing a key role in β-amyloid production and AD pathogenesis (69).

MiR-124 was also overexpressed in the hippocampus of Tg2576 mouse model of AD

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and at specific brain regions of AD patients (70). Mechanistically, tyrosine-protein

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phosphatase non-receptor type 1 (PTPN1) was found to be a direct target of miR-124

in this study, and regulating miR-124/PTPN1 axis was able to restore synaptic failure
-p
and memory impairment in these AD models. In another experiment, miR-124-3p was

significantly downregulated in N2a/APP695swe cells while miR-124-3p mimics were

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shown to attenuate hyperphosphorylation of tau protein-induced neuronal apoptosis
lP

via the Caveolin-1/phosphoinositide 3-kinase (PI3K)/phospho-Akt (Akt-Ser473)/Akt

and phospho-glycogen synthase kinase-3 beta (GSK-3β-Ser9)/GSK-3β pathways (52).

Additionally, miR-124 was found decreased in Drosophila AD models and the Notch
na

ligand Delta was detected as a direct target of miR-124 (71). Moreover, miR-124 has

been shown to regulate neuronal splicing regulation of APP, by targeting

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Polypyrimidine tract-binding protein 1 (PTBP1) (72). Furthermore, miR-124

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regulated mitochondrial localization and activity in mouse primary motor neurons by

targeting the intermediate filament Vimentin (Vim), a key regulator of mitochondrial

motility and function (73). In addition, altered miR-124 expression was associated

with astrocyte differentiation by targeting Sox2 and Sox9 in G93A-SOD1 transgenic

mouse models of ALS (74).

18
 MiR-124 has also been found down-regulated in the brain of HD patients with SRY-

related HMG box transcription factor 9 (SOX9), PTB1 and PGC1 being some of its

targets (75, 76). Collectively, these additional mechanisms may also apply in the case

of PD, and investigating these potential miR-124 targets in PD could represent a

promising future research field.

3. Biomarker potential of circulating cell-free miR-124 in PD

Given the fact that there is still no reliable test for early PD diagnosis, molecular

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biomarkers are currently investigated as potentially useful clinical tools towards this

direction. Taken into consideration that miRs display tissue-specificity and high

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stability in extracellular body fluids and their possible detection at low concentrations

by quantitative PCR (77), it has been suggested that their plasma or cerebrospinal
-p
fluid levels could serve as non-invasive diagnostic biomarkers for a wide range of
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human diseases. MiRs can be released from cells within exosomes, transfer their

content and act as intercellular signaling factors to target cells within the CNS (78).
lP

Identification of specific patterns of altered miRNA expression during the course of

neurodegenerative disorders could represent disease signature and may be used along
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with other diagnostic tools in order to increase diagnostic accuracy (64). Interestingly,

plasma concentration of miR-124 has been shown to represent a promising biomarker

ur

for cerebral infarction (79). Given its important role in PD pathogenesis, it has been

reasonably suggested that it could serve as a potential biomarker for PD diagnosis and
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progression. Α recent study of 60 PD patients and 60 control subjects indicated that

plasma levels of miR-124 were significantly reduced in PD patients in comparison to

controls (80). However, no statistical association was obtained between miR-124

levels and the severity of motor symptoms, as assessed by UPDRS and Hoehn and

Yahr scale, possibly due to low sample number.

19
Interestingly, miR-124 has been found upregulated in a chronic stress model of

depression (81), possibly by targeting CAMP responsive element binding (CREB)

protein that enhances 5-HT-dependent long-term facilitation (82). In this context, the

abovementioned study also investigated the correlation between circulating miR-124

levels and the co-occurrence of depression in PD patients, without however

significant association being observed (80). These results could be attributed to the

opposite trends of miR-124 levels in depression and PD, as well as to the distinct

pathophysiologic background of PD-associated depression, compared to general

of
depression (80).

ro
However, it is important to mention that so far, the number of studies investigating the

levels of various circulating miRs from PD patients is relatively small, and all
-p
published data show a particularly limited overlap between the detected miRs targets.
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In this regard, only miR-221-3p, miR-214-3p and miR-29c-3p have been validated by

several studies and there is further study confirming the biomarker potential of miR-
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124 (83). Thus, future research is definitely needed towards this direction, as well as

standardization of the applied methods, validation of cohorts from multiple centers,

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larger patient samples and ideally post-mortal diagnosis of PD, in order to obtain

clinically meaningful results.

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Moreover, we should also mention the results of clinical studies investigating

potential associations between miR-124 and other neurodegenerative disorders at a

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clinical level. In particular, a case-control association study demonstrated no

significant association between the single nucleotide polymorphism (SNP) rs531564

of miR-124, which can increase the amount of mature miR-124, and AD risk in a

Mongolian population (84). However, the sample size of this study was relatively

small. Regarding ALS, miR-124 was found to be one of the 11 differentially

20
 expressed miRNAs in the cerebrospinal fluid of sporadic ALS patients, as evaluated

by small RNA sequencing (85). In accordance, whole transcriptome sequencing from

spinal cord ventral horns of post-mortem sporadic ALS patients and healthy controls

revealed that mir-124-3p was detected as one of the 18 differentially expressed miRs

(86). In this regard, whole transcriptome sequencing of PD post-mortem tissues and

respective controls could provide an accurate and complete expression profiling,

enhancing the elucidation of the role of miR-124 in PD patients and its biomarker

potential.

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Therefore, miR-124 may potentially serve as diagnostic biomarker in PD, although

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further studies with larger sample sizes incorporating patients with other neurological

disorders and particularly synucleinopathies, such as multiple system atrophy (MSA)

-p
are definitely needed in order to obtain clinically meaningful results. Moreover, since
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PD motor symptoms develop only when the majority of dopaminergic neurons are

lost in SNpc, the detection of preclinical biomarkers of PD are of paramount

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importance. Given the critical implication of miR-124 in PD pathogenesis even at

initial stages as described above, we can speculate that it could also serve as a
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preclinical biomarker at prosymptomatic stages especially for populations at risk.

4. Therapeutic potential of miR-124 in PD

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Currently, PD treatment remains only symptomatic, since there are still no available
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effective strategies to halt the progression of the disease. Therefore, detection of novel

effective therapeutic targets particularly involved in the early stages of the disease is

urgently needed.

MiRs are increasingly considered as promising molecular targets in the treatment of

several neurodegenerative diseases, including PD (87). MiR mimics or antagonists

21
which enhance or inhibit the activity of the targeted miR respectively, can be used for

this purpose (88). MiR mimics are synthetic double-stranded small RNA molecules

which match the sequence of the mRNA target, thus restoring the deregulated miR

activity. However, the development of safe and efficient methods for their delivery

into the cells is challenging due to their short half-life and relatively low stability (89).

For this reason, nucleic acid chemical modifications that improve their stability and

effective delivery, including locked nucleic acids (LNA), 2′-O-methyl modification

(2′-O-Me) and phosphorothioate backbones have been developed. In the case of PD,

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suitable carriers acting as miRs transporters that can cross blood brain barrier (BBB)

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are needed (90). Viral vectors can effectively deliver miRs intracellularly, although

immunogenicity and the risk of oncogenic transformation raise safety issues and limit
-p
their clinical use (90). Non-viral vectors, such as lipid-based carriers that encapsulate

miR and can easily cross BBB are also under investigation. Interestingly, polymeric
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nanoparticles (NPs) have been successfully used for the delivery of miRs into the
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cells in vivo (91). Therefore, given the critical neuroprotective role of miR-124 in PD,

it has been recently suggested that increasing its intracellular levels could represent a

novel promising therapeutic strategy.

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In this context, a novel polymeric NP formulation, made of poly lactic acid-co-

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glycolic acid (PLGA) and perfluoro-1,5-crown ether (PFCE) and coated with

protamine sulphate to form a complex with miR-124, has been recently developed
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(92). Interestingly, miR-124-loaded NPs (miR-124 NPs) could enhance brain repair

mechanisms, by triggering the differentiation and migration of neural stem cells

(NSCs) in PD brain. Adult neurogenesis mainly occurs in the subventricular zone

(SVZ), which is located between the striatum and the lateral ventricles. Under normal

conditions, neuroblasts generated in SVZ can migrate towards the olfactory bulb (OB)

22
and differentiate into mature neurons. However, this process is deregulated in several

neurological disorders, including PD. Hence, NSCs are increasingly recognized as a

potential therapeutic strategy against neurodegenerative disorders and PD, due to their

ability to self-renew, proliferate and replace dead dopaminergic neurons. Boosting the

regenerative potential of endogenous NSCs by efficient delivery of specific pro-

neurogenic factors to them has gained increased attention. In this context, miR-124

NPs promoted neuronal differentiation and axonogenesis in vitro by downregulating

Sox9 and Jagged1, two known targets of miR-124. Furthermore, the

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intracerebroventricular administration of miR-124 NPs into the lateral ventricles of 6-

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OHDA-induced PD mouse models increased the migration of neuroblasts towards the

granule cell layer (GCL) of the OB and the migration of SVZ-derived new neurons
-p
into the striatum. Importantly, these effects were accompanied by amelioration of

motor deficits as evaluated by the apomorphine-induced rotation test. Hence, miR-124

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NPs represent a novel promising therapeutic strategy against PD, since their use can
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promote endogenous brain repair mechanisms during neurodegeneration.

Notably, the fact that miR-124 is the most abundantly expressed miR in the human
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brain raises some additional practical issues regarding the design of potential

therapeutic strategies. Relatively high doses of targeted oligonucleotides may be

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needed in order to effectively alter miR-124 levels in the CNS, and developing the

most appropriate delivery system for this purpose is quite challenging. Moreover, the
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usage of high doses of miR-124 mimics increases the potentially detrimental side

effects when these agents are also delivered to non-target human tissues that do not

normally express miR-124 at high levels. Therefore, non-viral vectors transferring

miR-124 mimics that can easily penetrate BBB and target brain tissue in a highly-

specific manner are required. In this context, attachment of the brain-targeted rabies

23
 virus glycoprotein (RVG) that specifically binds to nicotinic acetylcholine receptors

on neuronal cells, to intravenously injected exosomes carrying small interfering

RNAs (siRNAs) was reported to result in brain-specific delivery of the

oligonucleotides (93). Given the common structural and pharmacokinetic properties

of miRs and siRNAs (94), this strategy could be potentially applied for the transfer of

miR-124 in a neuronal-specific manner in PD. In this regard, RVG-labeled polymer

polyethylene imine (PEI) nanomaterials have been successfully used to deliver miR-

124a in the brain of mice and reduce the PEI-associated toxicity, although the

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combined use of mannitol was required to disrupt BBB and overcome the limitations

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due to the size of PEI nanocarriers (95). Importantly, exosome-based delivery of miR-

124 into the striatum of R6/2 transgenic HD mice induced a reduction in the
-p
expression of the target gene RE1-Silencing Transcription Factor (REST), without

however any observed clinical improvement (96). Collectively, further investigation

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of the use of RVG or other brain-specific targets in exosomes or other small carriers
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that are able to penetrate BBB would be of great significance for improving the

therapeutic value and limiting the potential side effects of miR-124 or other miRs in

neurodegenerative disorders.
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However, since miR-124 can target multiple genes and is involved in various
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pathways in a cell-type specific manner, its biological effects should be carefully

investigated, before the translation of miR-124-based therapies into clinic.

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Importantly, although thousands of research projects have investigated the potential of

various miRs in human diseases, the most challenging part in this field is choosing the

“right” miR among a large pool of potential candidates (97). Based on the above

evidence, miR-124 may serve as a promising target in PD that deserves further study.

5. Conclusion and future perspectives

24
PD is a multifactorial disorder and its pathophysiology is characterized by a wide

range of complex interconnected molecular interactions. Recently, accumulating

evidence indicates that miR-124 regulates numerous targets implicated in cell

survival, apoptosis, autophagy, mitochondrial dysfunction, oxidative damage and

neuroinflammation in PD through activation of several pathways, including calpain

1/p25/cdk5, Bim, AMPK, STAT3, ERK and NF-κB (Figure 1, Table 1). Therefore,

miR-124 may be implicated in the core pathophysiologic processes underlying PD

pathogenesis. Moreover, it can also serve as a potential diagnostic biomarker and

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therapeutic target in PD by enhancing endogenous brain repair mechanisms.

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Despite the pivotal role of miRs and particularly miR-124 in PD pathogenesis, the

regulatory mechanism of its expression remains still largely unknown. In this context,
-p
a recent study indicated that miR-124 was downregulated in MPP+-treated SH-SY5Y
re
cells and MPTP-induced PD mice while the metastasis-associated lung

adenocarcinoma transcript 1 (MALAT1) was upregulated (28). MALAT1, also

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known as nuclear-enriched abundant transcript 2 (NEAT2), is a spliced long non-

coding RNA (lncRNA), abnormally expressed in neurons that regulates the

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expression of various genes implicated in synapse formation and maintenance (98).

MALAT1 can directly bind to miR-124, thereby inhibiting its expression whereas its
ur

knockdown was demonstrated to increase miR-124 levels and suppress apoptosis in

vitro. Since LncRNAs can act as miRNA sponges or antagomirs that competitively
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modulate the expression and function of their target miRs (99), MALAT1 may serve

as an upstream negative regulator of miR-124 in PD and needs to be investigated

further.

However, apart from miR-124, MALAT1 has several other targets due to its

implication in various physiologic processes, such as alternative splicing, epigenetic

25
regulation of gene expression, apoptosis, synapse formation, neural development,

skeletal myogenesis, vascular growth BBB permeability and neuroinflammation,

mediated through binding to serine/arginine-rich (SR)-related proteins, polycomb

repressive complex 2 (PRC2) and the transcription factor TEAD (100). In PD,

MALAT1/miR-205-5p axis has been shown to modulate MPP+-induced apoptosis of

MN9D cells by directly targeting leucine-rich repeat kinase (LRRK2), a genetic

causative factor of PD (101). Furthermore, MALAT1 has been demonstrated to

associate with SNCA, resulting in its increased stability and expression in SH-SY5Y

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cells (102). Therefore, therapeutic modulation of miR-124 levels may also cause a

ro
domino effect potentially affecting many of these off-target miRs, thus adding another

level of complexity to the evaluation of miR-124-targeted strategies in clinical

settings.
-p
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Another aspect that should be taken into consideration is the reported destruction of

specific miRs upon their hybridization with some of their endogenous or synthetic
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targets, a process known as target RNA-directed miRNA degradation (TDMD) (103).

Although miRs are relatively stable, upon partial pairing with their RNA target,
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extensive complementarity may result during 3΄ trimming (shortening from the 3′

end), tailing (addition of non-templated nucleotides, usually U’s or A’s) and

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destruction of miRs (104), suggesting that miR targets can potentially affect the

stability and activity of miRs (103). Although to the best of our knowledge this
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emerging bidirectional modulation has not been reported for miR-124, it should be

taken into account since it could significantly affect the efficacy of miR-targeted

therapeutic strategies.

Importantly, several other mechanisms regulating the expression of mature miRs have

also been unraveled, including circular RNAs and competing endogenous RNAs

26
(ceRNAs). In this context, miR precursors can modulate post-transcriptionally miR

activity, through competition with their mature counterparts for the binding to the

overlapping miR response elements (MRE) in the 3′UTR of their target genes (105).

Interestingly, the Snail Family Transcriptional Repressor 2 (SNAI2), an oncogenic

transcription factor has been shown to act as a miR precursor of miR-124 (106). In the

case of cancer, the reduced expression of several miRs is due to the impaired

processing of their transcribed precursors (105). Hence, we can speculate that

deregulated miR-124 precursor processing and the consequent imbalance of

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precursor/mature miR-124 levels could also play a role in PD pathogenesis, indicating

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a potential field of future research.

Furthermore, since PD patients may also suffer from other diseases or use
-p
pharmaceutical agents that could potentially affect their brain tissue transcriptional
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profiles, a potentially different mode of action of miR-124-targeted therapies may be

detected. Importantly, AD is a more common neurodegenerative disorder than PD and

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a considerable proportion of PD patients may also suffer from AD (107). Although

miR-124 has also been suggested to exert neuroprotective effects in AD, the final
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outcome of the potentially interconnected effects of its administration is still largely

unknown.
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A recent study demonstrated that nuclear enriched assembly transcript 1 (NEAT1)

levels were increased in the substantia nigra of PD patients as compared to controls,

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and the administration of neuroprotective agents, such as fenofibrate and simvastatin

increased NEAT1 levels in cells under paraquat-induced oxidative damage (108).

These findings suggest that the use of various pharmaceutical agents, including

statins, may alter miR profile in the brain of PD patients, which should also be taken

into account while evaluating the effects of miR-124-targeted therapies.

27
Another factor that could affect circulating miR levels in PD patients is deep brain

stimulation (DBS) therapy. In this regard, bilateral subthalamic DBS neurosurgery

was shown to alter the circulating levels of 4 out of 13 long non-coding RNAs that

were differentially expressed in leucocytes of PD patients before DBS therapy in

comparison to healthy controls (109). Moreover, the expression of 11 out of 16 miRs

was also changed upon DBS treatment (110). These alterations were completely

reversed 1 hour following the cessation of the electric stimulation, reflecting the rapid

regulation of miRs expression by environmental factors and the significant dynamics

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characterizing miRs activity (110). In vivo evidence has shown that DBS of

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subthalamic nucleus may significantly alter the translational profile of striatopallidal

neurons in 6-OHDA-lesioned animal models of PD, in regard to synaptogenesis,

-p
apoptosis, dendritic excitability, calcium and cholesterol metabolism as well as the

fusion of vesicles (111). Given the fact that some of these neuronal functions are also
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mediated by miR-124, we can speculate that DBS may also affect its levels. However,
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since miR-124 was not specifically investigated in the abovementioned clinical

studies, further and larger studies are needed for the validation of this specific miR

profiling in PD patients undergoing DBS treatment.

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Notably, as already mentioned, PD is a multifactorial disorder, with environmental

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factors such as smoking and coffee consumption been consistently associated with a

reduced risk, whereas other factors, such as exposure to pesticides increasing its risk.
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Interestingly, it has been indicated that miRs can be differentially expressed in

smokers compared to non-smokers. A recent study showed that non-smokers or

former smokers displayed lower miR-124-3p expression levels in their monocytes

compared to smokers, suggesting that smoking could increase miR-124-3p expression

for a relative short period (112). Based on these findings, we can speculate that

28
smoking-induced differential miR-124 expression could at least partially underlie the

molecular basis of the protective role of smoking against PD which should be further

investigated.

In conclusion, miR-124, being implicated in the core pathophysiologic mechanisms

underlying PD pathogenesis may serve as a potential diagnostic biomarker, with

however, larger studies been highly demanded to carefully elucidate its biological and

clinical outcomes. Despite the practical limitations regarding its effective delivery to

the CNS, the development of novel strategies that are currently under investigation

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are expected to pave the way for the clinical application of miR-124-based therapies

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in PD.

Authors’ contribution
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EA carried out the literature review, conceptualized, and prepared the initial draft. YNP

edited and contributed in the final manuscript. CP provided critical inputs, edited and
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contributed to the final version of the manuscript. All authors read and approved the final

manuscript.
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Funding

This work did not receive any sort of financial assistance from funding agencies in the
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public, commercial, or not-for-profit sectors.

Acknowledgement

YNP would like to acknowledge Monash University Malaysia for awarding with HDR

scholarship.

29
Conflict of Interest

None

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na
ur
Jo

30
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 Table 1. The role of miR-124 in Parkinson’s disease: evidence from in vitro and

in vivo studies

Type Model Target Molecular Outcomes References

of gene mechanisms
Study
In vitro MPP iodine- Calpain 1 -Down- -Increased cell 10
treated MN9D regulation of survival
dopaminergic calpain -Reduced ROS
neurons 1/p25/CDK5 production
pathway
MPP+- Bim -Inhibition of -Inhibition of 29
intoxicated Bax apoptosis
SH-SY5Y mitochondrial -Restoration of

of
cells and lysosomal impaired
translocation autophagy
MPTP-treated Bim or - -Inhibition of 27

ro
SH-SY5Y and other yet Downregulation apoptosis
SK-N-SH cells unknown of AMPK and -Inhibition of
upregulation of autophagy

MPP+-treated
SH-SY5Y
STAT3
mTOR
-Reduction of
caspase-3 and
-p
-Increased cell
survival
26
re
cells LDH activity -Inhibition of
-Reduction of apoptosis
TNF-α and IL- -Anti-
1β levels inflammatory
lP

-Increased SOD effects

activity -Inhibition of
oxidative damage
6-OHDA- AnnexinA5 -Inhibition of -Increased cell 25
na

treated PC12 ERK survival

and SH-SY5Y phosphorylation -Inhibition of
cells and Caspase-3 apoptosis
activation
ur

MCS transfer MEKK3 -MEKK3/NF- -Inhibition of SH- 30

model (BV2 κΒ pathway SY5Y cell
cells, SH- -Reduction of apoptosis
Jo

SY5Y cells) iNOS, IL-6 and -Anti-

TNF-α levels inflammatory
-Increase of effects
TGF-β1 and IL-
10 levels
In vivo MPTP-treated Bim -Inhibition of -Inhibition of 29
mice Bax apoptosis
mitochondrial -Restoration of
and lysosomal impaired
translocation autophagy

47
MPTP- MEKK3 -MEKK3/NF- -Inhibition of 30
induced PD κΒ pathway apoptosis
mouse models -Inhibition of
microglia
activation

PD, Parkinson disease; MPTP, N-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine;

AMPK, 5' adenosine monophosphate-activated protein kinase; 6-OHDA, 6‐

hydroxydopamine; ROS, Reactive oxygen species; mTOR, Mammalian target of

rapamycin; STAT3, Signal transducer and activator of transcription 3; ERK,

of
Extracellular signal-regulated kinase; MEKK3, Mitogen-activated protein kinase

kinase kinase 3; CDK5, cyclin-dependent kinase 5; Bim, Bcl-2-interacting mediator

ro
of cell death; IL-6, interleukin-6; TNF, tumor necrosis factor; SOD, superoxide

-p
dismutase; LDH, lactate dehydrogenase; ERK, extracellular signal-regulated kinase;

NF-κΒ, nuclear factor-kappa Β; MCS, microglial culture supernatant

re
lP
na
ur
Jo

48
Figure legend

Figure 1: Proposed molecular targets and signaling pathways underlying the

effects of miR-124 in Parkinson’s disease

MiR-124 can directly target the calcium-dependent intracellular proteinase calpain 1

and downregulate the Calpain 1/p25/CDK5 signaling pathway to inhibit neuronal

apoptosis. Moreover, it can target Bim resulting in the inhibition of Bax mitochondrial

and lysosomal translocation, downregulate AMPK and upregulate of mTOR pathway

of
to increase neuronal cell survival. MiR-124 can also target STAT3 and reduce

ro
caspase-3 and LDH activity, decrease TNF-α, IL-1β levels and increase SOD activity,

to reduce oxidative damage. AnnexinA5 is another target of miR-124 that mediates its
-p
inhibition on ERK phosphorylation and caspase-3 activation to further inhibit
re
neuroinflammation. Finally, miR-124 can target MEKK3 and induce downregulation

of the MEKK3/NF-κB signaling pathway to attenuate impaired autophagy.

lP
na
ur
Jo

49