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Please cite this article in press as: Kanagaraj N et al. Downregulation of miR-124 in MPTP-treated mouse model of Parkinson’s disease and MPP iodide-treated
MN9D cells modulates the expression of the calpain/cdk5 pathway proteins. Neuroscience (2014), http://dx.doi.org/10.1016/j.neuroscience.2014.04.039
NSC 15370 No. of Pages 13, Model 5G
5 May 2014
Neuroscience xxx (2014) xxx–xxx
DOWNREGULATION OF MIR-124 IN MPTP-TREATED MOUSE MODELOF PARKINSON’S DISEASE AND MPP IODIDE-TREATED MN9DCELLS MODULATES THE EXPRESSION OF THE CALPAIN/CDK5PATHWAY PROTEINS
N. KANAGARAJ, H. BEIPING, S. T. DHEEN ANDS. S. W. TAY *
Department of Anatomy, Yong Loo Lin School of Medicine,
National University of Singapore, Singapore 117597, Singapore
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Abstract—Parkinson’s disease (PD) is a debilitating neuro-
degenerative disorder causing severe motor disabilities
resulting from the loss of dopaminergic neurons in the sub-
stantia nigra pars compacta (SNc) region of the midbrain.
MicroRNAs (miRNAs) are small, non-coding RNAs which
play a major role in several cellular processes in health and
disease by regulating gene expression post-transcription-
ally. Aberrant miRNA expression has been detected in
post-mortem PD samples, in vitro and in vivo PD models.
However, none of the studies have focused on the role of
the brain-abundant miR-124 in PD. In this study, we have
evaluated the expression changes of miR-124 in the SN of
the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-
induced PD mouse model. MiRNA expression analysis by
qPCR revealed a decrease in the expression of brain-
enriched miR-124 in the SN of MPTP-treated mice as com-
pared to controls. Further, in vitro study revealed a decrease
in the expression of miR-124 in MN9D dopaminergic neurons
treated with MPP iodide. The expression of calpains 1 and 2
which is modulated by miR-124 was increased in the SNc of
MPTP-treated mice as observed at different time points after
treatment and in the MN9D dopaminergic neurons treated
with MPP iodide leading to increased expression of the p35
cleavage product, p25 and cyclin -dependent kinase 5
(cdk5). Calpain- p25-mediated increase in cdk5 expression
leading to dopaminergic neuronal death has been demon-
strated in human PD and MPTP-PD models. Increased
expression of calpain 1/cdk5 pathway proteins was observed
in anti-miR-124-transfectedMN9Dcells in our studies. Knock-
down of miR-124 led to increased production of reactive oxy-
gen species (ROS) and hydrogen peroxide (H2O2) both
known to increase oxidative stress. Further, experiments
with miR-124 target protector sequences specific to calpain
http://dx.doi.org/10.1016/j.neuroscience.2014.04.0390306-4522/� 2014 Published by Elsevier Ltd. on behalf of IBRO.
*Corresponding author. Address: Department of Anatomy, Yong LooLin School of Medicine, Level 3, MD 10, 4 Medical Drive, Singapore117597, Singapore. Tel: +65-65163210; fax: +65-67787643.
E-mail address: [email protected] (S. S. W. Tay).Abbreviations: cdk5, cyclin-dependent kinase 5; DAT, dopaminetransporter; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetalbovine serum; H2O2, hydrogen peroxide; miRNA, microRNA; MPTP,1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PBS, phosphate-bufferedsaline; PD, Parkinson’s disease; Prx, peroxiredoxin; RNS, reactivenitrogen species; ROS, reactive oxygen species; SDS, substantia nigra;SN, sodium dodecyl sulfate; SNc, substantia nigra pars compacta; TH,tyrosine hydroxylase.
1
1 revealed an interaction of miR-124 with calpain 1. Overex-
pression of miR-124 after MPP iodide treatment on MN9D
cells was found to attenuate the expression of the calpain
1/p25/cdk5 proteins while improving cell survival. These
results suggest thatmiR-124 acts tomodulate the expression
of calpain/cdk5 pathway proteins in the dopaminergic
neurons. A better understanding of the mechanisms control-
ling the expression of miR-124 will aid in targeting miR-124
for better treatment strategies for PD. � 2014 Published by
Elsevier Ltd. on behalf of IBRO.
Key words: Parkinson’s disease, microRNA, miR-124,
MPTP-induced mouse model of Parkinson’s disease, MN9D
cells.
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INTRODUCTION
Parkinson’s disease (PD) is an incapacitating
neurodegenerative disorder characterized by severe
motor symptoms like tremor, muscle rigidity, paucity of
voluntary movements and postural instability (Lang and
Lozano, 1998). The pathological hallmarks of PD are
the specific loss of dopaminergic neurons in the substan-
tia nigra pars compacta (SNc) and the formation of intra-
cytoplasmic Lewy bodies that are mainly composed of
fibrillar a-synuclein (Braak et al., 2003). Although several
PD genes have been identified to date (Abou-Sleiman
et al., 2006), the pathogenic process of PD is still not fully
understood. The neurotoxin, 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine (MPTP) replicates most of the bio-
chemical and pathological alterations in PD, including
the loss of the dopaminergic neurons of the SNc in sev-
eral mammalian species and has thus been widely used
to generate animal models to investigate the molecular
mechanisms involved in human PD and new treatment
strategies (Przedborski et al., 2001). Current therapies
available for PD provide a symptomatic relief rather than
inhibiting the progression of the disease. Owing to the
inadequacy of these drugs to be a cure for PD and their
associated severe side effects on prolonged usage
(Meissner et al., 2011), consistent research efforts are
focused on the search for better neuroprotective strate-
gies. There has been growing evidence of genetic and
epigenetic factor involvement in PD (Habibi et al., 2011;
Coppede, 2012) and an enhanced understanding of these
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factors in PD development and progression can
contribute immensely to the development of effective
and improved treatment strategies.
MicroRNAs (miRNAs) are small (�20–24 nucleotides
long), non-coding RNAs derived from �70 nucleotide
hairpin pre-miRNA’s, constituting about 1–2% of the
genes in worms, flies and mammals (Bartel, 2009; Ebert
and Sharp, 2012). They suppress mRNA expression via
translation inhibition, degradation or deadenylation of
mRNA (Jackson and Standart, 2007; Pillai et al., 2007).
Their role in many fundamental biological processes like
embryonic development, cell proliferation and differentia-
tion is well known (Ambros, 2004; Wienholds et al.,
2005; Wurdinger et al., 2008; Gangaraju and Lin, 2009).
Further, the deregulated expression of specific miRNAs
has been implicated in cancers, heart disorders and neu-
rodegenerative diseases (Volinia et al., 2006; Lukiw,
2007; Johnson et al., 2008; Ha, 2011; Skalsky and
Cullen, 2011). They have an immensely large regulatory
potential owing to the fact that each miRNA can target
multiple mRNAs (Sassen et al., 2008). MiRNAs are prom-
ising therapeutic targets and tools in several major dis-
eases owing to their small size and ease of delivery
(Harraz et al., 2011). With increasing evidence of abnor-
mal miRNA expression in the pathogenesis of PD (Kim
et al., 2007; Minones-Moyano et al., 2011; Mouradian,
2012), alterations in specific miRNAs will provide impor-
tant insights into molecular mechanisms of the disease,
and could help in generating novel targets for therapeutic
intervention (Junn and Mouradian, 2012).
In this context, there have not been many published
data in the literature showing the changes in the
expression of miRNA in the MPTP-induced PD animal
models. Hence, our study was focused on identifying
specific miRNAs that are significantly altered and their
role in inducing gene expression changes that occur in
the MPTP-induced PD mouse model. A qPCR miRNA
expression analysis of the substantia nigra (SN) of
MPTP-induced PD mice revealed significant changes in
the expression levels of miRNA-124.
MiR-124 is a brain-enriched miRNA (He and Hannon,
2004), shown to play a role in neuronal differentiation dur-
ing the development of the central nervous system and in
adult neurogenesis (Lim et al., 2005; Makeyev et al., 2007;
Yu et al., 2008; Cheng et al., 2009). Plasma concentration
of miR-124 has been suggested to be a promising candi-
date biomarker for cerebral infarction (Weng et al.,
2011). Downregulation of miRNA-124 has been implicated
in the progression of medulloblastoma and glioblastoma
(Pierson et al., 2008; Skalsky and Cullen, 2011). The
autophagy-lysosomal pathway has been shown to be sig-
nificantly altered in several neurodegenerative diseases
including PD (Pan et al., 2008; Wong and Cuervo,
2010). MiR-124 has been predicted to target 52 genes of
the autophagy-lysosomal pathway by a systems biology-
based computational analysis (Jegga et al., 2011), impli-
cating a role for miR-124 in the function of the pathway.
Furthermore, a negotiating role between the nervous and
immune systems has also been proposed for miR-124
(Soreq and Wolf, 2011). With such varied roles predicted
for miR-124 and owing to its abundance in the brain,
Please cite this article in press as: Kanagaraj N et al. Downregulation of miR-124 in
MN9D cells modulates the expression of the calpain/cdk5 pathway proteins. Neuro
evaluating its role in PD will provide important insights into
how it regulates the pathogenic processes in PD. The cal-
pains are calcium-activated non-lysosomal proteases,
demonstrated to be involved in the dopaminergic neuron
loss in PD acting through the cyclin-dependent kinase 5
(cdk5) pathway (Crocker et al., 2003; Vosler et al., 2008)
and are known to be modulated by miRNA-124 (Jegga
et al., 2011); yet, the involvement of miRNA-124 in the pro-
cesses leading to their activation in MPTP-induced PD
models has not been established. Hence, our study was
aimed at investigating the role of miRNA-124 in modulat-
ing the expression of the calpain/cdk5 pathway in MPTP-
induced mouse model of PD and MPP iodide-treated
MN9D dopaminergic neurons.
In the present study, we found that miR-124
expression was decreased in the SN of the MPTP-
induced PD mouse model and that the loss of miR-124
in the dopaminergic neurons contributes, in part, to the
increase in expression of the calpain/cdk5 pathway
proteins by interacting with the calpain 1 mRNA. It was
also found that overexpression of miR-124 after MPP+
insult diminishes the expression of calpain 1/p25/cdk5
proteins while improving cell viability compared to the
negative control transfected MPP+-treated group.
EXPERIMENTAL PROCEDURES
Animals and treatment
Eight- to ten-week-old male C57BL/6J mice (weighing 20–
25 g) were used for the MPTP treatment. All animal
experiments were conducted according to protocols
approved by the Institutional Animal Care and Use
Committee, National University of Singapore. Mice were
given four injections of MPTP–HCl (Sigma) at 2-h
intervals (total dosage—72 mg/kg of body weight equally
distributed over four injections) (Jackson-Lewis and
Przedborski, 2007). Control animals were injected an
equal volume of 0.9% sterile saline. Animals were sacri-
ficed 1, 3, 5, 7, 10 days after the last MPTP injection. The
SN was dissected bilaterally and total RNA was isolated
using themiRNeasymini kit (Qiagen) for RT-PCRanalysis.
Cell culture and MPP iodide treatment
MN9D cells (obtained from Drs. Alfred Heller and Lisa
Won, University of Chicago) were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) with 10% fetal bovine
serum (FBS) (Choi et al., 1991). Cells were differentiated
using 1.5 mM sodium butyrate in 10% FBS+ DMEM for
5 days before being subjected to further experiments. Dif-
ferentiated cells were treated with 100 and 200 lM MPP
iodide (Sigma Aldrich) for 24 h to mimic the MPTP-
induced in vivo model based on Chee et al. (2005). Cells
were plated on poly-L-lysine-coated cover slips and differ-
entiated for immunofluorescence studies and in situhybridization.
Cresyl Violet (Nissl) staining
Brain sections of thickness 20 lm cut using the cryostat
were immersed sequentially in 100%, 75% and 50%
MPTP-treated mouse model of Parkinson’s disease and MPP iodide-treated
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ethanol. Sections were then washed in water and
immersed in Cresyl Violet stain for 20 min. Slides were
then passed through 50%, 75%, 100% ethanol and
finally washed twice in histoclear. Slides were then
mounted and left to dry. Dopaminergic neurons were
visualized and captured using a light microscope
attached with a camera.
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Stereological analysis
Tissue sections of thickness 20 lm were cut through the
SNc region in the frozen brain tissue. Every fourth section
was used for stereological counting. Sections were
incubated with rabbit anti-mouse tyrosine hydroxylase
(TH) (Sigma) and the signal was developed using the
Vectastain ABC kit (Vector, CA, USA) according to the
manufacturer’s instructions. TH-positive neurons were
then counted using the optical fractionator method
(Chan et al., 1997).
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Immunofluorescence and laser capturemicrodissection
Brains removed from the control and MPTP-induced mice
sacrificed 7 days after the last MPTP injection were fixed
by immersing in 4% PF for 2 h and subsequently
transferred to 30% RNAse-free sucrose solution for 3 h.
Brain samples were then embedded in the cryo-
embedding medium and snap frozen in liquid nitrogen.
Samples were stored at �80 �C until further processing.
Coronal sections of the brain (thickness 20 lm) were
cut using Leica cryostat and processed for
immunofluorescence staining. Sections were washed in
phosphate-buffered saline (PBS) and incubated with
rabbit anti-mouse TH (Sigma) overnight following which
they were incubated with goat anti-rabbit Cy3 and
counterstained with the nuclear stain DAPI. The MN9D
cells were fixed with 4% PF for 20 min and then washed
with PBS. Staining was performed using anti-NeuN and
anti-TH.
For laser capture microdissection, 10-lm-thick
coronal sections of the brain samples (day 5) were
obtained using a cryostat. Sections were stained using
the Cresyl Violet LCM staining kit (Ambion) following the
manufacturer’s instructions. The SN region was then
isolated by laser capture microdissection (Espina et al.,
2006). Samples were stored in qiazol (Qiagen) and then
analyzed for miRNA expression by qPCR array (Exiqon).
The miRNA Ready-to-Use PCR panels were used for
sensitive high-throughput expression profiling from
minimal amounts of RNA obtained from LCM samples.
The PCR array uses a combination of a Universal cDNA
synthesis reaction and Ready-to-Use PCR panels to
profile the expression of 742 mouse miRNAs.
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In situ hybridization
MN9D cells seeded onto poly-L-lysine-coated cover slips
in 24-well plates were differentiated for 5 days using
sodium butyrate and then subjected to in situhybridization using miR-124 fluorescence-labeled in situ
Please cite this article in press as: Kanagaraj N et al. Downregulation of miR-124 in
MN9D cells modulates the expression of the calpain/cdk5 pathway proteins. Neuro
probes and the hybridization kit (Exiqon) as per the
manufacturer’s instructions.
Knockdown studies
Differentiated MN9D cells were transfected with anti-
miR-124 oligos and negative control oligos (Exiqon) at
30 lM concentration using lipofectamine transfection
reagent (Invitrogen). Protein samples were isolated 48 h
after transfection using the mammalian protein
extraction reagent MPER (Pierce/Thermo Fisher
Scientific) for Western blotting analysis. Cell viability
after anti-miR-124 transfection was also assessed at
48 h after transfection.
Overexpression studies
To observe if the overexpression of miR-124 after MPP
iodide treatment can reduce the expression of calpains 1
and 2, p35/25 and cdk5 induced by MPP+, differentiated
MN9D cells were treated with MPP iodide for 24 h after
which they were transfected with negative control and
miR-124 mimics. Proteins were isolated from both sets of
samples for Western blotting analysis. Viability of the
cells was measured after 48 h of transfection.
Measurement of extracellular reactive oxygenspecies (ROS) and hydrogen peroxide (H2O2)
The conditioned medium from negative control and anti-
miR-124-transfected MN9D cells was used to measure
the total ROS/reactive nitrogen species (RNS) levels
released by these cells. The concentration of ROS/RNS
and H2O2 liberated was measured using Oxiselect in vitroROS/RNS assay kit (Cell Biolabs) according to the
manufacturer’s instructions. The relative fluorescence of
the samples and the standards was read at 480 nm
excitation/530 nm emissions using SpectraMaxM5
microplate reader (Molecular Devices). The concentration
of the total ROS/RNS and H2O2 released was calculated
using the 20,70-dichlorodihydrofluorescein standard curve
and H2O2 standard curve.
Calpain 1 target protector studies
Miscript target protectors specific to miR-124 binding site
on calpain 1 and negative controls (Qiagen, Germany)
were transfected into MN9D cells at a final
concentration of 500 nM to study the interaction of miR-
124 with calpain 1 mRNA. Protein samples were
isolated 48 h after transfection for analysis of calpain 1
expression by Western blotting.
Real-time RT-PCR
Total RNA isolated was converted to cDNA for gene
expression analysis using superscript (Invitrogen) and
for miRNA expression analysis using the Universal
cDNA conversion kit (Exiqon). Primers for TH, dopamine
transporter (DAT), cdk5 were purchased from 1st Base,
Singapore. Beta actin was used as the normalization
control for these genes. Primer for miR-124 was
purchased from Exiqon. U6 primers were used as
MPTP-treated mouse model of Parkinson’s disease and MPP iodide-treated
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normalization control for miR-124 expression. RT-PCR
was performed in an ABi 7900 Real time-PCR system
(Applied Biosystems).
Western blotting
Mammalian protein extraction reagent (Pierce/Thermo
Fisher Scientific) was used to extract proteins from cell
cultures. All procedures were performed at 4 �C.Homogenates were centrifuged at 14,000 rpm for 20 min
and the supernatant collected. Protein concentration
was measured using a Bio-Rad colorimetric protein
assay kit following the manufacturer’s instructions (Bio
Rad). Samples of supernatants containing 40 lg of
proteins were heated to 95 �C for 5 min and were
separated by sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS–PAGE) in 10% SDS gels. Protein
bands were electroblotted onto 0.45-lm polyvinylidene
difluoride membranes (Bio-Rad) and were blocked with
5% (w/v) nonfat-dried milk overnight at 4 �C.Membranes were incubated with dilutions of anti-calpain
1 (1:1000), anti-calpain 2 (1:1000), anti-p35 (1:500),
anti-p25 (1:500), anti-cdk5 (1:500). They were then
incubated with horseradish peroxidase-conjugated
secondary antibodies (BioRad). An enhanced
chemiluminescence kit (Pierce/Thermo Fisher Scientific)
was used following the manufacturer’s instructions to
visualize the protein bands. The bands were quantified
using the BioRad Quantity one image quantification
software (BioRad, USA). Each band density was
normalized against the respective beta actin band
intensity and the values were then normalized against
the control band intensity to obtain the change in protein
expression in treatment groups in fold change as
compared to control group.
Statistical analysis
Data were presented as mean ± SEM. Statistical
significance of differences between control and MPTP
treatment groups and control, MPP+-treated cells and
miR-124 knockdown cells was calculated using a one-
way analysis of variance (ANOVA) (Dunnett’s test).
Statistical significance between the groups was
represented as ⁄p< 0.05 and ⁄⁄p< 0.01.
RESULTS
MPTP lesion
We analyzed the loss of neurons by Cresyl Violet staining
and TH immunostaining on day 7 after MPTP treatment.
There was a reduction in the number of nissl-stained
neurons in the SN as seen from the Cresyl-Violet
staining and a reduction in the number and intensity of
TH-immunostained neurons (Fig 1A). A decrease in TH
expression (Fig 1B) and DAT expression (Fig 1C) was
observed in the MPTP-induced SN as compared to
controls at different time points (days 1, 3, 5, 7, 10) after
MPTP treatment by RT-PCR. A significant decrease in
the number of TH-positive neurons was observed on
day 7 after MPTP treatment as shown by stereological
counting (1D).
Please cite this article in press as: Kanagaraj N et al. Downregulation of miR-124 in
MN9D cells modulates the expression of the calpain/cdk5 pathway proteins. Neuro
MiR-124 expression was downregulated in theMPTP-lesioned mice
RT-PCR techniques were used to study the expression
changes of miR-124 in the SNc of MPTP-treated mice.
The expression of miR-124 in the SN of mice treated
with MPTP, isolated by laser capture microdissection,
was found to be decreased on day 5 post-treatment as
compared to saline-injected control mice from a miRNA
qPCR array (Exiqon, Denmark) (data not shown).
Expression changes in miR-124 at different time points
after MPTP treatment were then analyzed by RT-PCR.
When compared to control mice, MPTP-treated mice
showed a significant decrease in the expression of miR-
124 after MPTP treatment (Fig 2A) as observed by RT-
PCR at different time points (days 1, 3, 5, 7, 10). The
decrease was evident even on day 1 after the last
MPTP injection.
MN9D cells as in vitro PD model
MN9D cells were used to model PD in vitro to study the
impact of miR-124 expression on dopaminergic neurons
without the influence of the other cell types present in
the SNc and surrounding brain regions. Differentiated
MN9D cells in culture developed processes (Fig 2B) and
were confirmed to be dopaminergic neurons by
immunostaining with NeuN (Fig 2C) and TH (Fig 2D). In
situ hybridization analysis showed the expression of
miR-124 in the MN9D dopaminergic neurons (Fig 2E).
In response to treatment with MPP iodide, the
expression of miRNA-124 was downregulated in MN9D
cells as detected by RT-PCR (Fig 2F).
Calpain 1 and calpain 2 expression in MPTP-treatedmouse SNc
Calpains 1 and 2 are predicted to be targets of miR-124
by targetscan (release 6.2) (Fig 3A). The expression of
calpains 1 and 2 was found to be significantly increased
in the SNc of MPTP-treated samples in a time-
dependent manner on days 3, 5, 7, 10 as compared to
control mice. Immunoreactive bands for calpain 1 and
calpain 2 both appeared at 80 kDa (Fig 3B) and were
significantly increased in expression as shown in Fig
3C, D respectively. However, the mRNA expression of
calpains 1 and 2 did not show any significant changes
upon MPTP treatment (data not shown).
Expression of calpain 1 and calpain 2 in MPPiodide-treated and miR-124 knockdown MN9D cells
In order to study the effect of the loss of miR-124 on
calpains 1 and 2 in the dopaminergic neurons, the
expression of calpains 1 and 2 in MPP iodide-treated and
miR-124 inhibitor-transfected MN9D cells was analyzed.
Protein expression of calpain 1 showed a significant
difference in the MPP iodide-treated group and anti-miR-
124-transfected group as compared to controls.
Immunoreactive bands for calpain 1 appeared at 80 kDa
(Fig 4A) and was significantly increased in the MPP
iodide-treated group (200 lM) and the anti-miR-124-
transfected group (Fig 4B). The two groups showed
MPTP-treated mouse model of Parkinson’s disease and MPP iodide-treated
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Fig. 1. (A) Nissl staining of control and MPTP-treated SNc on day 7 after treatment (Upper panel), TH immunostaining of control and MPTP-treated
SNc on day 7 after treatment. (Lower panel) (B) TH expression on days 1, 3, 5, 7, 10 after MPTP treatment. (C) DAT expression on days 1, 3, 5, 7,
10 after MPTP treatment. (D) Number of TH-positive neurons in the SNc on day 7 after MPTP treatment. Each bar represents mean ± SEM.⁄Represents statistical significance p< 0.05 and ⁄⁄represents p< 0.01 (one-way ANOVA, n= 5).
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similar levels of increase in protein expression. The
100 lM MPP iodide-treated group also showed an
increase though not significant as compared to the
control group.
Immunoreactive bands for calpain 2 appeared at
80 kDa (Fig 4A) and showed a significant increase in
the MPP iodide-treated (200 lM) though not in the anti-
miR-124-transfected group (Fig 4C).
To identify the impact of reduced miR-124 expression
and increased calpain 1 and 2 expression on the
downstream proteins in the calpain/cdk5 pathway,
expression of p35, p25 and cdk5 was analyzed in the
MPP iodide-treated and miR-124-inhibitor-transfected
cells.
Please cite this article in press as: Kanagaraj N et al. Downregulation of miR-124 in
MN9D cells modulates the expression of the calpain/cdk5 pathway proteins. Neuro
Expression of p35 and p25 increased on MPP iodidetreatment and miR-124 knockdown
Western blot analysis showed a significant increase in
p25 protein expression in both the MPP iodide-treated
groups and the anti-miR-124-transfected group. The
expression of p25 (immunoreactive band at 25 kDa- Fig
5A) increased with the dose of drug used and was
significantly increased in the 200 lM MPP iodide-treated
group as compared to the control group (Fig 5C). The
anti-miR-124-transfected group also showed a
significant increase in p25 expression as compared to
the control group with the levels being similar to the
200 lM MPP iodide-treated group (Fig 5C).
MPTP-treated mouse model of Parkinson’s disease and MPP iodide-treated
science (2014), http://dx.doi.org/10.1016/j.neuroscience.2014.04.039
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Fig. 2. (A) miR-124 expression in SNc on days 1, 3, 5, 7, 10 after MPTP treatment. (B) BF image of differentiated MN9D cells. (C) Confocal images
of MN9D cells stained with neuronal marker NeuN. (D) Confocal images of MN9D cells stained with dopaminergic marker TH, (E) in situhybridization images of MN9D cells showing miR-124 expression and negative control using the scrambled probe (F) miR-124 expression in MN9D
cells after 24 h of MPP+ exposure at 100 and 200 lM. Each bar represents mean ± SEM. ⁄⁄Represents statistical significance p< 0.01 (one-way
ANOVA, n= 5).
6 N. Kanagaraj et al. / Neuroscience xxx (2014) xxx–xxx
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Immunoreactive bands for p35 at 35 kDa (Fig 5A) was
found to be slightly increased in both MPP iodide-treated
groups and the anti-miR-124-transfected group as
compared to the control group, though not statistically
significant (Fig 5B).
413Please cite this article in press as: Kanagaraj N et al. Downregulation of miR-124 in
MN9D cells modulates the expression of the calpain/cdk5 pathway proteins. Neuro
Increased cdk5 expression
Cdk5 immunoreactive band appeared at 30 kDa (Fig 6A)
and was significantly increased in the 200 lM MPP
iodide-treated group and the anti-miR-124-transfected
group as compared to the control group (Fig 6B). The
MPTP-treated mouse model of Parkinson’s disease and MPP iodide-treated
science (2014), http://dx.doi.org/10.1016/j.neuroscience.2014.04.039
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Fig. 3. (A) Target interaction of miR-124 with 30UTR of calpain 1 and calpain 2 as predicted by the miRNA target prediction algorithm—Targetscan
(version 6.2). Western blot showing the protein expression of calpain 1 and calpain 2 in SNc of MPTP-treated mice with controls at days 3, 5, 7 and
10 after MPTP treatment. (B) Immunoreactive bands of calpain 1 (80 kDa), calpain 2 (80 kDa) and beta actin (42 kDa). (C) Bar graph showing the
fold changes in calpain 1 expression in each group as compared to controls. (D) Bar graph showing the fold changes in calpain 2 expression in each
group as compared to control. Each bar represents mean ± SEM. ⁄Represents statistical significance p< 0.05 and ⁄⁄represents p< 0.01 (one-
way ANOVA, n= 3).
Fig. 4. Western blot showing the protein expression of calpain 1 and calpain 2 in MPP+-treated and anti-miR-124-transfected MN9D cells with
controls. (A) Immunoreactive bands of calpain 1 (80 kDa), calpain 2 (80 kDa) and beta actin (42 kDa). (B) Bar graph showing the fold changes in
calpain 1 expression in each group as compared to controls. (C) Bar graph showing the fold changes in calpain 2 expression in each group as
compared to control. Each bar represents mean ± SEM. ⁄Represents statistical significance p < 0.05 and ⁄⁄Represents p < 0.01 (one-way
ANOVA, n= 3).
N. Kanagaraj et al. / Neuroscience xxx (2014) xxx–xxx 7
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mRNA expression of cdk5 was also significantly
increased in the anti-miR-124-transfected group as
Please cite this article in press as: Kanagaraj N et al. Downregulation of miR-124 in
MN9D cells modulates the expression of the calpain/cdk5 pathway proteins. Neuro
compared to the negative control transfected group (Fig
6C) as detected by RT-PCR.
MPTP-treated mouse model of Parkinson’s disease and MPP iodide-treated
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Fig. 5. Western blot showing the protein expression of p35 and p25 in MPP+-treated and anti-miR-124-transfected MN9D cells as compared to
controls. (A) Immunoreactive bands of p35 (35 kDa), p25 (25 kDa) and beta actin (42 kDa). (B) Bar graph showing the fold changes in p35
expression in each group as compared to control. (C) Bar graph showing the fold changes in p25 expression in each group as compared to control.
Each bar represents mean ± SEM. ⁄Represents statistical significance p < 0.05 and ⁄⁄represents p < 0.01 (one-way ANOVA, n= 3).
Fig. 6. Western blot showing the protein expression of cdk5 in MPP+-treated and anti-miR-124-transfected MN9D cells with controls. (A)
Immunoreactive bands of cdk5 (30 kDa) and beta actin (42 kDa). (B) Bar graph showing the fold changes in cdk5 expression in each group as
compared to controls. (C) mRNA expression of cdk5 in anti-miR-124-transfected MN9D cells as compared to the negative control. (D) mRNA
expression of cdk4 in control and MPTP-treated mice on days 1, 3, 5, 7, 10 after treatment. Each bar represents mean ± SEM. ⁄Representsstatistical significance p < 0.05 and ⁄⁄Represents p < 0.01 (one-way ANOVA, n= 3 for cells and n= 5 for animals).
8 N. Kanagaraj et al. / Neuroscience xxx (2014) xxx–xxx
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The MPTP treatment induced an increase in cdk5
mRNA expression as compared to control mice in a
time-dependent manner as observed by RT-PCR
analysis of the SN tissue at different time points after
the last MPTP injection (Fig 6D).
Please cite this article in press as: Kanagaraj N et al. Downregulation of miR-124 in
MN9D cells modulates the expression of the calpain/cdk5 pathway proteins. Neuro
ROS/RNS and H2O2 production and MN9D cellviability after knockdown or overexpression ofmiR-124
Oxidative stress is an important event playing a role in
neurodegeneration in human PD and MPTP-models;
MPTP-treated mouse model of Parkinson’s disease and MPP iodide-treated
science (2014), http://dx.doi.org/10.1016/j.neuroscience.2014.04.039
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hence we evaluated ROS and H2O2 production in miR-
124-depleted and miR-124-overexpressing MN9D cells.
A small but significant increase in the production of
ROS (Fig 7A) and H2O2 (Fig 7B) was observed in the
anti-miR-124-transfected cells as compared to the
negative control. However, transfection of miR-124
mimics did not induce any significant changes in the
ROS/RNS and H2O2 levels. There was also a significant
reduction in MN9D cell viability after transfection of miR-
124 inhibitors (Fig 7C). When miR-124 mimics were
transfected into MPP+-treated MN9D cells we
observed that there was lesser cell death as compared
to the cells transfected with negative controls and
MPP+-treated MN9D cells (Fig 7D).
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Studies on overexpression of miR-124 in MN9D cells
In order to analyze if supplementing the MPP iodide-
treated cells with miR-124 could mitigate the expression
of calpains, p35, p25 and cdk5, we transfected miR-124
mimics and negative controls into MN9D cells treated
with MPP iodide (200 lM). Transfection with the
negative control mimics into the MPP+-treated cells
showed similar expression of calpain 1, calpain 2, p35,
p25 and cdk5 as the MPP iodide-treated cells as
demonstrated by Western blotting analysis. However,
transfection with miR-124 mimics was found to mitigate
the expression of calpain 1 (Fig 8A), p35 (Fig 8C), p25
Fig. 7. (A) ROS/RNS production in anti-miR-124-transfected MN9D cells as c
in anti-miR-124-transfected MN9D cells as compared to negative control tran
124 knockdown cells and (D) MPP+-treated MN9D cells transfected with
Statistical significance is represented by ⁄p< 0.05 and ⁄⁄p< 0.01 (Student
n= 3).
Please cite this article in press as: Kanagaraj N et al. Downregulation of miR-124 in
MN9D cells modulates the expression of the calpain/cdk5 pathway proteins. Neuro
(Fig 8D) and cdk5 (Fig 8E). Calpain 2, however, did not
show any change in expression upon miR-124
overexpression (Fig 8B). The representative blots are
shown in Fig 8F.
Calpain 1 expression increased significantly aftermiR-124 target protector transfection
In order to determine if the increase in calpain 1
expression was mediated by direct interaction of miR-
124 with the calpain 1 mRNA, we transfected miR-124
target protectors specific to calpain 1 in the
differentiated MN9D cells. An increase in the expression
of calpain 1 (immunoreactive band at 80 kDa) (Fig 9A)
compared to the negative control transfected cells was
observed by Western blotting analysis (Fig 9B).
DISCUSSION
The role of miRNAs in neurodegenerative diseases could
be multifaceted. They could lead to accumulation of toxic
proteins which is a major hallmark of several
neurodegenerative diseases or contribute to the altered
expression of proteins which promote or inhibit cell
survival (Eacker et al., 2009). Though there have been
a few studies focusing on the role of individual miRNAs
in PD (Harraz et al., 2011), with increasing numbers of
miRNAs being identified there is no dearth to the possibil-
ompared to negative control transfected cells and (B) H2O2 production
sfected cells measured in RFU. (C) Neuronal cell viability in the miR-
miR-124 and control mimics expressed as a percentage of control.
’s t-test for knockdown, n= 3, one-way ANOVA for overexpression,
MPTP-treated mouse model of Parkinson’s disease and MPP iodide-treated
science (2014), http://dx.doi.org/10.1016/j.neuroscience.2014.04.039
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Fig. 8. Western blot showing the protein expression of calpain 1, calpain 2, p35, p25 and cdk5 in the MPP iodide-treated MN9D cells which are
transfected with miR-124 mimics and negative controls. Bar graph showing the fold changes in expression of (A) calpain 1, (B) calpain 2, (C) p35, D)
p25 and (E) cdk5. (F) Immunoreactive bands of calpain 1 (80 kDa), calpain 2 (80 kDa), p35 (35 kDa), p25 (25 kDa), cdk5 (30 kDa) and beta actin
(42 kDa). Statistical significance is represented by ⁄p< 0.05 and ⁄⁄p< 0.01 (one-way ANOVA, n= 3).
10 N. Kanagaraj et al. / Neuroscience xxx (2014) xxx–xxx
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ities of finding many other miRNAs which might contribute
significantly to PD pathogenesis.
The MPTP-induced PD model is considered the gold
standard for studying the molecular mechanisms of
pathogenesis of PD as it induces specific degeneration
of dopaminergic neurons in the SN along with a
decrease in TH and DAT expression similar to human
PD (Dauer and Przedborski, 2003; Jakowec et al., 2004).
Please cite this article in press as: Kanagaraj N et al. Downregulation of miR-124 in
MN9D cells modulates the expression of the calpain/cdk5 pathway proteins. Neuro
Considering the fact that miRNAs are stoichiometric
inhibitors of mRNA translation, the most abundant
miRNAs in a particular tissue or cell type might be the
most pertinent to the biology of the tissue or cell type
(Eacker et al., 2009). In the present study, expression of
miR-124, which is abundantly expressed in the neurons,
was found to be downregulated in the SN of MPTP-
induced PD mouse model and in MN9D dopaminergic
MPTP-treated mouse model of Parkinson’s disease and MPP iodide-treated
science (2014), http://dx.doi.org/10.1016/j.neuroscience.2014.04.039
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Fig. 9. Western blot showing the protein expression of calpain 1 after
transfection of miR-124 target protectors. (A) Bar graph showing the
fold changes in calpain 1 expression as compared to the negative
control. (B) Immunoreactive bands of calpain 1 (80 kDa) and beta
actin (42 kDa). Statistical significance is represented by ⁄p< 0.05
and ⁄⁄p< 0.01 (Student’s t-test, n= 3).
N. Kanagaraj et al. / Neuroscience xxx (2014) xxx–xxx 11
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neurons treated with MPP iodide. Though miR-124 has
been implicated in several brain-related diseases, its role
in PD has not yet been elucidated. Owing to its high
expression in the neurons, the decrease in miR-124
expression could be considered a side-effect of the neuro-
nal death in the MPTP-induced PD model. However,
downregulation of miR-124 expression was observed as
early as 24 h after the last MPTP injection when there is
not much dopaminergic neuronal loss, suggesting a likely
role for miR-124 in the pathogenic process.
Calpains, which are calcium-dependent proteases,
are predicted to be targets of miR-124 (Targetscan,
release 6.2, IPA-Ingenuity systems). The consistent
activation of calpains has been shown to contribute to
dopaminergic neuronal death in MPTP-induced PD
models and in human PD (Crocker et al., 2003). In our
study, we observed an increased expression of calpains
1 and 2 in the SNc of MPTP-treated mice at different time
points after treatment and in the MPP iodide-treated
MN9D cells. Although miR-124 can modulate the expres-
sion of both calpains 1 and 2 as described by Jegga et al.
(2011), reduction in the expression of miR-124 by trans-
fection of miR-124 inhibitors in MN9D cells appears to eli-
cit a significant increase only in calpain 1 expression. This
suggests a role for miR-124 in controlling the expression
levels of calpain 1 in addition to other calpain activating
mechanisms in these cells. While the increase in the
expression of calpain 2 was significant in the MPP
iodide-treated group it was only moderate in the anti-
miR-124-transfected groups. As predicted by the target
prediction algorithm Targetscan (release 6.2), calpain 1
has two conserved binding sites for miR-124 while calpain
2 has only one, which could be the reason for the higher
impact of miR-124 loss on calpain 1 than calpain 2.
Please cite this article in press as: Kanagaraj N et al. Downregulation of miR-124 in
MN9D cells modulates the expression of the calpain/cdk5 pathway proteins. Neuro
In order to analyze if the increase in calpain 1
expression is due to the direct interaction of miR-124
with the 30-UTR of calpain 1, we used commercially
available target protector sequences which can interfere
with the interaction of miR-124 and the calpain 1 mRNA
sequence. Transfection of the target protectors induced
an increase in the expression of calpain 1 as shown by
Western blotting analysis thereby demonstrating an
interaction between miR-124 and calpain 1 mRNA,
which has not been established previously.
Calpains increase the expression of stable p25
formed by the cleavage of p35, both of which are known
to increase cdk5 expression and activity (Lee et al.,
2000). The calpains contribute to dopaminergic neuronal
death by increasing the expression and activity of cdk5
(Levy et al., 2009). Cdk5 phosphorylates various down-
stream proteins like myocyte enhancer factor 2D
(MEF2D) (Smith et al., 2006), peroxiredoxin2 (Prx2) (Qu
et al., 2007) which mediate the dopaminergic neuronal
death.
In our studies, MPP iodide treatment and anti-miR-124
transfection both induced a significant increase in the
expression of p25. The expression of p25 in anti-miR-
124-transfected cells increased as in MPP iodide-treated
cells suggesting a possible role for miR-124 in the process.
The increased expression of p25 is known to increase
cdk5 expression and activity (Patrick et al., 1999), and
therefore we analyzed the protein expression of cdk5 in
the MPP iodide-treated and anti-miR-124-transfected
MN9D cells. It is clearly evident that the loss of miR-124
can induce an increase in cdk5 expression in the anti-
miR-124-transfected group as compared to the control
group. In the SN of MPTP-induced mice, we found a
time-dependent increase in cdk5 mRNA expression, as
assessed at different time points starting from 24 h after
the last MPTP injection up to 10 days after MPTP
injections, as compared to control mice. Increase in
cdk5 activity is known to increase ROS production (Qu
et al., 2007; Sun et al., 2008) which can lead to mitochon-
drial damage and cell death. The transfection of miR-124
inhibitors in MN9D cells induced a significant increase in
total ROS/RNS production as compared to controls imply-
ing that the reduction of miR-124 can increase oxidative
stress in cells. Further, an increase in H2O2 levels was
also observed on knockdown of miR-124 in the MN9D
cells. Deregulated cdk5 can inactivate Prxs, which
scavenge H2O2 and ROS, thereby leading to oxidative
stress-mediated mitochondrial dysfunction and neuronal
death. Since damaged mitochondria elevate the
expression of ROS, a vicious cycle of cellular damage is
activated. From our observations, miR-124 appears to
contribute to the increased oxidative stress. However,
further studies are warranted to analyze if the reduction
in miR-124 can induce changes in the expression of mito-
chondrial component proteins leading to the dysfunction
of mitochondria in the MPTP model.
In order to better understand the function of miR-124
in the MPP iodide-induced death of MN9D cells, we
transfected miR-124 mimics into MPP iodide-treated
MN9D cells. We observed a significant decrease in the
expression of calpain 1, p35, p25 and cdk5 in the
MPTP-treated mouse model of Parkinson’s disease and MPP iodide-treated
science (2014), http://dx.doi.org/10.1016/j.neuroscience.2014.04.039
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miR-124-transfected cells as compared to the negative
control transfected cells indicating that supplementing
miR-124 could mitigate the expression of these proteins
after MPP+ treatment. In addition, the viability of the
cells overexpressing miR-124 was higher than that of
negative controls suggesting that miR-124 could help in
the survival of the MN9D cells after MPP iodide
treatment. However, the levels of ROS/RNS and H2O2
did not show much change in both groups. There is a
high basal expression of miR-124 expression in the
MN9D cells and overexpression of miR-124 in the
normal MN9D cells might not be of relevance since PD
is a disease where there is a significant loss of neuronal
viability by the time it is diagnosed. Hence, we adopted
the approach of overexpressing miR-124 in the MPP
iodide-treated cells to mimic the diseased state.
While our study demonstrates a role for miR-124 in
the calpain/cdk5 pathway mediated dopaminergic
neuronal death, additional studies are warranted to
identify the molecular events leading to its reduction in
the MPTP-treated animals and MPP iodide-treated cells.
An in-depth analysis of the mechanisms involved in the
control of miR-124 can provide the strategy to
manipulate it for therapeutic purposes.
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CONCLUSION
The present study has shown that the expression of miR-
124 is reduced in the SN of MPTP-induced-PD mice and
in MPP iodide-treated MN9D cells. The results suggest
that miR-124 is a contributor to the increase in the
expression of cdk5 mediated by calpain 1/p25 in the
MPTP-mouse model of PD and in MN9D cells treated
with MPP iodide. Supplementing miR-124 in the MPP
iodide-treated MN9D cells improved cell viability and
attenuated the MPP-induced increase in the expression
of the calpain 1/cdk5 pathway proteins. A better
understanding of the mechanisms controlling miR-124
expression will help in using miR-124 as a therapeutic
target for effective treatment of PD.
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Acknowledgments—This study was supported by a grant MOE
2009-T2-1-061 from the Ministry of Education (MOE), Singapore.
The authors declare that they have no conflict of interest.
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(Accepted 16 April 2014)(Available online xxxx)
MPTP-treated mouse model of Parkinson’s disease and MPP iodide-treated
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