Downregulation of miR-124 in MPTP-treated mouse model of Parkinson’s disease and MPP...

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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.

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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

http://dx.doi.org/10.1016/j.neuroscience.2014.04.039

mailto:[email protected]



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5 May 2014

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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5 May 2014

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



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




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5 May 2014

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).



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




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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).



5 May 2014

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.



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).




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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).



5 May 2014

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).
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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




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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).



5 May 2014

mRNA expression of cdk5 was also significantly

increased in the anti-miR-124-transfected group as



compared to the negative control transfected group (Fig

6C) as detected by RT-PCR.




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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).



5 May 2014

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).



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;




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5 May 2014

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).



(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,




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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).



5 May 2014

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).



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




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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).



5 May 2014

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.



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




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5 May 2014

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)




Downregulation of miR-124 in MPTP-treated mouse model of Parkinson’s disease and MPP...

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