MicroRNA Expression Profiling Screen miR-3557/324-targeted ...

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6-12-2019

MicroRNA Expression Profiling Screen miR-3557324-targeted MicroRNA Expression Profiling Screen miR-3557324-targeted

CaMKmTOR in the Rat Striatum of Parkinsons Disease in CaMKmTOR in the Rat Striatum of Parkinsons Disease in

Regular Aerobic Exercise Regular Aerobic Exercise

Wenfeng Liu Hunan Normal University wfliuhunnueducn

Li Li Georgia Southern University liligeorgiasouthernedu

Shaopeng Liu Hunan Normal University

Zhiyuan Wang Hunan Normal University

Heyu Kuang Hunan Normal University

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Recommended Citation Recommended Citation Liu Wenfeng Li Li Shaopeng Liu Zhiyuan Wang Heyu Kuang Yan Xia Changfa Tang Dazhong Yin 2019 MicroRNA Expression Profiling Screen miR-3557324-targeted CaMKmTOR in the Rat Striatum of Parkinsons Disease in Regular Aerobic Exercise BioMed Research International 2019 httpsdigitalcommonsgeorgiasoutherneduhealth-kinesiology-facpubs181

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Authors Authors Wenfeng Liu Li Li Shaopeng Liu Zhiyuan Wang Heyu Kuang Yan Xia Changfa Tang and Dazhong Yin

This article is available at Digital CommonsGeorgia Southern httpsdigitalcommonsgeorgiasoutherneduhealth-kinesiology-facpubs181

Research ArticleMicroRNA Expression Profiling Screen miR-3557324-TargetedCaMKmTOR in the Rat Striatum of Parkinsonrsquos Disease inRegular Aerobic Exercise

Wenfeng Liu 123 Li Li 4 Shaopeng Liu1 ZhiyuanWang1 Heyu Kuang1 Yan Xia1

Changfa Tang 1 and Dazhong Yin 35

1Hunan Provincial Key Laboratory of Physical Fitness and Sports Rehabilitation Hunan Normal UniversityChangsha Hunan 410012 China

2Department of Experimental and Clinical Pharmacology University of Minnesota Minneapolis MN 55455 USA3The Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education Hunan Normal UniversityChangsha Hunan 410081 China

4School of Health amp Kinesiology Georgia Southern University Statesboro GA 30460 USA5Qingyuan Peoplersquos Hospital The Sixth Affiliated Hospital Guangzhou Medical University Guangzhou Guangdong 511500 China

Correspondence should be addressed to Wenfeng Liu wfliuhunnueducn Changfa Tang 1104755704qqcomand Dazhong Yin dazhongyingzhmueducn

Received 12 November 2018 Revised 13 May 2019 Accepted 22 May 2019 Published 12 June 2019

Academic Editor Gessica Sala

Copyright copy 2019 Wenfeng Liu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

This study aimed to screen the target miRNAs and to investigate the differential miR-3557324-targeted signal mechanisms in theratsrsquo model of Parkinsonrsquos disease (PD) with regular aerobic exercise Rats were divided into sedentary control PD group (SED-PD n = 18) and aerobic exercise PD group (EX-PD n = 22) After 8 weeks of regular aerobic exercise a 6-hydroxydopamine-(6-OHDA-) induced PD lesion model was constructed Preregular aerobic exercises enhanced the injury resistance of rats with6-OHDA-induced PD The rotational behavior after injection of apomorphine hydrochloride was alleviated Under the scanningelectron microscopy we found the neurons axons and villi of the striatum were clearly and tightly arranged and neurons andaxons significantly becoming larger Tyrosine hydroxylase (TH) was increased significantly and 120572-synuclein protein expressionwas reduced in the EX-PD group compared to the SED-PD group Screening from miRNA microarray chip we further foundupregulation of miR-3557 and downregulation of miR-324 were closely related to the calcium-modulating signaling pathwayremitting the progress of Parkinsonrsquos disease on aerobic exercise Compared to the SED-PD group Ca2+calmodulin dependentprotein kinase II (CaMK2120572) was upregulated but CaMKV and voltage-dependent anion-selective channel protein 1 (Vdac1)were significantly downregulated in the EX-PD group Additionally phosphatidylinositol-3-kinase (PI3K)mammalian target ofrapamycin (mTOR) expression were activated and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) expression was upregulatedin the EX-PD group Conclusions the adaptive mechanism of regular aerobic exercise delaying neurodegenerative diseases andlesions was that miR-3557324 was activated to regulate one of its targets CaMKs signaling pathways CaMKs coordinated withmTOR pathway-related gene expression improved UCH-L1 level to favor for delaying neurodegeneration or improving thepathogenesis of PD lesions

1 Introduction

microRNAs (miRNAs or miR) are associated with variousdiseases such as neurodegenerative diseases [Alzheimerrsquosdisease (AD) Parkinsonrsquos disease (PD) and Schizophrenia]

[1] cardiovascular diseases (myocardial infarction myocar-dial fibrosis and atherosclerosis) [2] and metabolic dis-eases (diabetes and obesity) [3] In the present studyon the miRNAs about 70 miRNAs were expressed inthe brain tissue of mammals such as specific miR-124a

HindawiBioMed Research InternationalVolume 2019 Article ID 7654798 12 pageshttpsdoiorg10115520197654798

2 BioMed Research International

miRNA-128 and miRNA-101 miRNAs were rich in braintissues (such as miR-125b) and important in the regulationof gene expression in different physiological processes andsignaling pathways of the nervous system such as the devel-opment and progression of the nervous system neural stemcell differentiation and apoptosis [4] Liu et al [5] reportedthat Drosophila melanogaster began to express miR-34 inits brain in the adulthood and that if miR-34 was missingprotein misfolding increased neurodegenerative diseases ofthe brain occurred and the life expectancy was shortened

In addition miRNAs may also participate in the occur-rence of neurodegenerative diseases through some specificmolecular mechanisms Nelson et al [6] found that miR-25 downregulated the expression of synaptic proteins in thebrain of patients with AD and synergized with activatedreactive oxygen species (ROS) Wang et al [7] reportedthat miR-34a could induce neuronal apoptosis by inhibitingB-cell lymphoma 2 (Bcl-2) in neuronal development andsenescence and that miR-34a was expressed in patients withAD Lemaitre et al [8] showed that the expression of miR-433 decreased and the expression of fibroblast growth factor20 (FGF20) increased in patients with PD while FGF20promoted the expression of 120572-synuclein which is the mostcritical protein in PD pathogenesis Packer et al [9] foundthat the expression of miR-9 decreased in patients withPD patients which might be involved in the pathogen-esis of PD by modulating the target gene RE1-SilencingTranscription factor (REST)coREST miR-133 regulated theexpression of transcription factorN-myc downregulated gene1 (NGRG1) associated with PD miR-7 miR-153 and miR-433 regulated the expression of 120572-synuclein in PD [10]Dostie et al [11] showed that miR-175 was associated withearly clinical manifestations of PD miR-132 and miR-212were involved in the occurrence of various abnormalitiessuch as synaptic plasticity and connectivity in schizophrenia[12]

Yet no known study has examined how the miRNAs(examined via miRNAs profiling) might differ between PDanimals who regularly exercise interventions vs PD ani-mals who are habitually sedentary A study of this type isneeded to discern whether miRNAs can be employed asa biomarker of neuronal functional decline and whetherregular exercise may more greatly attenuate neurodegener-ation The purpose of the exercise-adaptive stress-reversingexercise was to seek more direct evidence of regulatingmRNA in related pathways thereby identifying the biolog-ical functions of genes that regulated the key interpretablemotor-adapted mechanisms of action We found the adap-tive mechanism of regular aerobic exercise delaying thedevelopment of neurodegenerative diseases and lesions wasto initiate miR-3557 and miR-324 to regulate one of itstarget Ca2+calmodulin dependent protein kinase (CaMKs)signaling pathways and to coordinate with regulating closelyrelated phosphatidylinositol-3-kinase (PI3K) protein kinaseB(Akt) mammalian target of rapamycin (mTOR) path-way protein expression hence delaying the progression ofneurodegeneration or alleviating the development of PDlesions

2 Materials and Methods

21 Experimental Animals Ethics Statement andExercise Protocol

211 Experimental Animals and Ethics Statement Specific-pathogen-free 13-month-old male SpraguendashDawley rats (n =40 54950plusmn1613 g) were supplied by the Animal Center ofEast Biotechnology Services Company (Changsha HunanChina License number Xiang SCXK 2009-0012) Four orfive rats were housed in one standard cage (three or fourratscage) with free access to food and water All animalswere kept in an air-conditioned room maintained at aconstant temperature of 20∘Cndash25∘C with a relative humidityof 45ndash55They were subjected to a cycle of 12-h light and12-h darkness They were acclimated to laboratory conditionsfor 1 week prior to the start of the experiment A total of 40rats were randomly assigned to 2 groups sedentary controlPD group (SED-PD n = 18) and aerobic exercise PD group(EX-PD n = 22) After 8 weeks of aerobic exercise in EX-PD the SED-PD and EX-PD groups were administered aunilateral injection of 6-OHDA and the no-lesion side (N)and the lesion side (L) were formed respectively

All animal procedures were approved by the local ethicscommittee (the Institutional Review Board of HunanNormalUniversity) and the Guidelines for Care and Use of Labo-ratory Animals (Washington DC 2011) Disposal of animalswas done in accordance with The Guidance on the Care ofLaboratory Animalsrdquo (The provisions were issued in 2006by the Ministry of Science and Technology of the PeoplersquosRepublic of China)

212 Exercise Protocol Recently da Costa and Tuon et al[13 14] corroborated that aerobic treadmill exercise beforeunilateral intrastriatal 6-OHDA injection rescued motordeficits measured by the apomorphine-induced rotationalasymmetry in rats We performed regular aerobic exercisebefore unilateral intrastriatal 6-OHDA injection in accor-dance with newfangled researches [13 15ndash17] and exerciseload standards reference to Bedford et al [18] All animals inthe SED-PDandEX-PDgroupswere first subjected to a 5-dayadaptation period on a rat treadmill (slope gradient 0 ZH-PT-1 Treadmill Li Tai Bio-Equipment Co Ltd HangzhouZhejiang China) Adapted training was carried out at a speedof 10 mmin and a gradient of 0∘ for a gradually increasingduration of time 10 min on the 1st day 20 min on the 2ndday and 25 min on the 3rd-5th day During this period theywere placed on a belt facing away from the electrified grid(06-mA intensity) once per day

For the actual experiment SED-PD was in habitualsedentary behavior but the EX-PD group underwent dailytraining by running at 15 mmin equivalent to approx50ndash60 maximum oxygen consumption (VO2max) [1819] on a slope of 0∘ for a duration of 15 min During thefirst week at the start of the training the exercise durationwas increased gradually from 15 min to 20 min in thesecond week 25 min in the third week 30 min in the fourthweek and 35 min in the fifth and sixth weeks The EX-PD

BioMed Research International 3

group underwent daily training by running at 20 mminand maintained speed for a week (equivalent to approx65ndash70VO2max) [18] on a slope of 0∘ for a duration of 40min in the seventh to the eighth weeks The acceleration ofthe treadmill was set such that the final speed of 22 mminwas achieved in about 3 min after the start of the trainingSound stimulation and a small wooden stick were used tostimulate the animalsrsquo tails when necessary to ensure thatthe animals completed the exercise regime Also electricalstimulation (06 mA intensity) was used to keep the rats ata one-third distance on the treadmill runway The rats wererequired to perform the training 5 days a week for a total of 8weeks

22 Model of PD Induced by 6-OHDA in Rats All surgicalprocedures were completed and performed under anesthesiainjected intraperitoneally with 10 chloral hydrate at 3mLkg body weight All efforts were made to minimize theratsrsquo suffering and distress The EX-PD group fasted for24 h after eight weeks of regular aerobic exercise and therats in SED-PD were carried out in the same way aftereight weeks of habitual sedentary behavior After anesthesiathe brains of the rats were fixed on the brain stereotaxicinstrument The skin was cut and treated with sterilizationto expose the anterior skull and the intersection betweenthe anterior ridge and the midline was determined to bezero in coordinates Referring to the George Paxinos andCharles Watsonrsquos original (Zhuge Qiyin) rat brain stereotaxicmap the right striatum dual coordinates were determinedthe first target 10 mm anterior to the anterior ridge 30 mmto the right of the midline 70 mm subdurally the secondtarget 02 mm posterior to the anterior ridge 26 mm tothe right of the midline and 60 mm subdurally Triangularneedleswere labeled and thewellswere drilled Eachwell waspipetted with 50 120583L of 6-hydroxydopamine (6-OHDA total40120583L 40 120583g120583L) to inject the aforementioned two targets6-OHDA was dissolved in 09 sodium chloride and 02mgmL ascorbic acid was dissolved in 09 sodium chlorideThe injection rate was 050 120583Lmin Each point was injectedfor 10 min After the injection the needle was retained for5 min and then withdrawn at a speed of 10 mmmin Afterthe injection the wound was sealed with bone wax the headskin was sutured and the local skin was routinely disinfectedAt the same time each rat was injected with clindamycinintraperitoneally twice a week to prevent infection and thecage was normally reared Behavioral rotation test tyrosinehydroxylase (TH) and 120572-synuclein was detected to test thePD model induced by 6-OHDA in rats

23 Behavioral Identification on Rat PD Model In the firstpostoperative week the rats were placed in a plastic circulartube with a diameter of about 28 cm and a height of 25 cmor more Before each rat was placed alcohol was disinfectedand deodorized and then 050mgkg body weight of rats wasgiven using apomorphine hydrochlorideThe intraperitonealinjection was used to induce the rotation to the contralateralside and the number of rotations of the rat to the contralateralside was recorded Each measurement lasted 40 min The

number of rotations in the first-10min second-10min andthird-10min were recorded once per week more than sevenrotations per minute was the success model [20]

24 Tissue Specimen Collection and Analysis The rats wereanesthetized with chloral hydrate (400 mgkg intraperi-toneally) and decapitated in two weeks after 6-OHDA injec-tion and lesion maturation [21 22] The striatum (StereotaxicAtlas of the Rat Brain George Paxinos 2005) was dissectedfrom all rats Tissue samples were stored at minus80∘C until readyto be used for miRCURY LNA miRNA Array and real-timefluorescence quantitative PCR All surgical procedures wereperformed under anesthesia induced using chloral hydrateAll efforts were made to minimize suffering and distress inanimalsThe striatum tissue was quickly separatedThe tissueblock was cut into cubes of 3 times 3 mm2 with a razor bladeplaced in a precooled 25 glutaraldehyde solution (ReagentGrade Ameresco BioOhio USA) in a centrifuge tube andkept in a refrigerator at 4∘C for electron microscopy

At least three rats from each group were used for the sam-ple preparation After the ascending aorta was infused withphysiological saline it was perfused with 4 paraformalde-hyde (PFA)01M phosphate buffer saline (PBS pH 74 4∘C400ndash500 mL) until the animalrsquos liver hardened and the tailwas stiff which indicated the completion of the perfusionThe brains were isolated and kept in a 4 PFA 01M PBSovernight at 4∘C (no more than 48 h) The following daythey were transferred to 30 sucrose and dehydrated to alow level They were routinely dehydrated cleared waxedand paraffin embedded The first slice was sectioned from 10mm anterior to the anterior ridge in the right half brain forimmunohistochemical staining

25 Scanning Electron Microscopy (SEM) The rat brain stria-tum tissues were rapidly dissected mounted on toothpicksat resting length and placed in 25 glutaraldehyde in PBSfor overnight fixation at 4∘C Postfixation was performed inaqueous 1 osmium tetroxide for 60 min Specimens weredehydrated with ethanol sequentially 30 50 70 8090 and 100 ethanol (twice) Ethanol was removed anda 11 mixture of isoamyl acetate and ethanol was addedand they were mixed by shaking 10-20 min and the mix-ture was discarded and then slowly infiltrated with epoxyresins (Embed 812Araldite Electron Microscopy SciencesHatfield PA USA) embedded and then heated overnightat 60∘C For SEM the specimens were critical point driedmounted on aluminum stubs sputtered with a gold coat andexamined under a JEOL 6335F field emission gun scanningelectron microscope

26 Immunohistochemical Staining of TH and 120572-Synuclein5120583m slices were sectioned by 820 Rotary Tissue Slicer (AOCompany American) Supersense ImmunohistochemistryDetection System Kit (NoBS13278 Bioworld TechnologyInc St Louis Park USA) was used for the localizationcharacterization and quantification of TH and 120572-synucleinin the striatum The primary antibody anti-TH (NOBS40101200 Bioworld Technology) and anti-120572-synuclein (sim20 kD


NOBS34291200 Bioworld Technology) were added to eachsample 50 120583lslice and kept in the refrigerator at 4∘Covernight The secondary antibody 50120583lslice of Goat anti-Rabbit IgG (HampL)-HRP (12000 NOBS13278 BioworldTechnology) was added dropwise to each sample Then theroutine steps were diaminobenzidine staining hematoxylinand eosin restaining dehydration with gradient alcohol andclearing of tissue sections with xylene

The morphometric analysis was performed using theImage-Pro Plus (IPP) Version 60 software (Media Cyber-netics Inc Rockville USA) Five images were taken usingCompix Simple PCI (Compix Inc Imaging Systems Penn-sylvania USA) Five fields were randomly selected undereachmicroscope (times200) for analysisThe immunohistochem-ically positive anti-TH and anti-120572-synuclein were stainedyellow or brown by 33-diaminobenzidine tetrahydrochloride(DAB) (Zhongshan Biotechnology Co Ltd Beijing China)Their integrated optical density (IOD) was evaluated usingthe area of interest method in Image-Pro Plus Version 60(IPP 60)

27 Determination of miRNA Genomics

271 miRCURY LNA miRNA Array The seventh generationof miRCURY LNAmiRNA array (v180) (Exiqon Denmark)contained 3100 capture probes covering all human mouseand ratmiRNAs annotated inmiRBase 180 and all viralmiR-NAs related to these species [23] This array also containedcapture probes for 25 miRPlus human miRNAs These wereproprietary miRNAs not found in miRBase

272 RNA Extraction About 60 mg of rat striatum wasused extracting total RNA with TRIzol reagent (Invitro-gen Carlsbad California USA) and miRNeasy mini kit(Qiagen Germantown MD USA) according to the man-ufacturerrsquos protocol which efficiently recovered all RNAspecies including miRNAs The RNA quality and quantitywere measured using a NanoDrop spectrophotometer (ND-1000 NanoDrop Technologies Wilmington DE USA)and RNA integrity was determined using gel electrophore-sis

273 miRNA Array After RNA isolation from the samplesthemiRCURYHy3Hy5 Power labeling kit (ExiqonVedbaekDenmark) was used according to the manufacturerrsquos guide-line for miRNA labeling One microgram of each sample was3rsquo-end-labeled with a Hy3 fluorescent label using T4 RNAligase After the labeling procedure the Hy3-labeled sam-ples were hybridized on the miRCURY LNA Array (v180)(Exiqon Exiqon AS Skelstedet 16 2950Vedbaek Denmark)according to the array manual The 12-Bay HybridizationSystem (Nimblegen Systems Inc WI USA) was used Thenthe slides were scanned using the Axon GenePix 4000Bmicroarray scanner (Axon Instruments CA USA)

274 miRNA Array Scanning and Data Analysis Scannedimages were then imported into the GenePix Pro 60 software

(Axon Instruments Inc Union City CA USA) for grid align-ment and data extraction Replicated miRNAswere averagedand miRNAs with intensities ge30 in all samples were chosenfor calculating the normalization factor Expressed data werenormalized using the median normalization After nor-malization differentially expressed miRNAs were identifiedusing fold change filtering Finally hierarchical clusteringwasperformed using the Institute for Genomic Research (TIGR)Multiple Experiment Viewer (MEV) software (v46) to showdistinguishable miRNA expression profiling among samplesThe statistical analysiswas performed using theBioconductorDESeq package (httpwwwbioconductororg) to detectdifferential expression levels of miRNAs [24]

275 Bioinformatics Analysis of miRNAs Target Predictionand Functional Analysis All miRNA target data were ana-lyzed using the Ingenuity Pathway Analysis tool and thetarget prediction database in Target Scan Three aspects ofmolecular function biological process and cell compositionwere obtained through the Gene Ontology analysis

The miRDB (httpmirdborgmiRDB) database wasused to predict the target of the differentially expressedknown miRNAs with a predicted score ge60 Functionalannotation of each target gene was performed using theDAVID (the database for annotation visualization and inte-grated discovery) (httpdavidabccncifcrfgov) bioinfor-matics resource (version 67) including an integrated biolog-ical knowledge base and analysis tools Biological significancewas systematically extracted from large geneprotein familiesThe information about metabolism genetic informationprocessing environmental information processing cellularprocesses and human diseases can be obtained from thedatabase of the Kyoto Encyclopedia of Genes and Genomes(KEGG) website

28 Real-Time Fluorescence Quantitative Polymerase ChainReaction of miRNAs and mRNAs Real-time fluorescencequantitative (q) polymerase chain reaction (qPCR) was per-formed as previously described [25] At least three sampleswere randomly assigned to each group and qPCR was per-formed on each of the samples (Table 1) The data regardingthe amplification curves and melting curves of quantitativereal-time PCR and cycle threshold (Ct) values of each genemRNA were recorded The mRNA expression of the targetgene was quantitatively determined using the method of 2-delta Ct as the internal reference

29 Statistical Analysis Data were presented as mean plusmn SEMStatistical analysis was performed using predictive analyticssoftware statistics 160 (SPSS Inc Chicago IL USA) Theexperimental groups were compared using ANOVA withrepeated measures on the behavior test A Tukey post-hoctest was used to follow up with significant ANOVA resultsCohnrsquos D was used to examine the effect size The statisticalsignificance of the effects of the experimental treatment wasdetermined by comparing the areas under the curve (P lt 005Studentrsquos t-test)


Table 1 Real-time fluorescence quantitative PCR primers for target miRNAs and mRNAs

Gene Sequence (51015840mdash31015840)

miRNA reference primers GeneCopoeia Inc USA CatalogRmiRQP9003

rno-miR-3557-3p GeneCopoeia Inc USA CatalogRmiRQP1817


120573-actin F GAGATTACTGCTCTGGCTCCTAR GGACTCATCGTACTCCTGCTTG

Camk 2a GeneCopoeia Inc USA CatalogRQP049239

Camk V GeneCopoeia Inc USA CatalogRQP050747

Vdac1 GeneCopoeia Inc USA CatalogRQP051105

Itpr1(PI3R) GeneCopoeia Inc USA CatalogRQP045271

Akt1 GeneCopoeia Inc USA CatalogRQP051482

mTOR GeneCopoeia Inc USA CatalogRQP050125

UCH-L1 GeneCopoeia Inc USA CatalogRQP049756

Table 2 Rotation experiment in the PD model (unit number of turns)

Groups Injection of apomorphine in the first postoperative week Injection of apomorphine in the second postoperative weekThe first-10min The second-10min The third-10min The first-10min The second-10min The third-10min

SED-PD 800plusmn200 729plusmn130 729plusmn129 775plusmn149 850plusmn105 800plusmn132EX-PD 563plusmn155 620plusmn119 625plusmn158 7plusmn140 76plusmn108 68plusmn116SED-PD is the sedentary control PD group EX-PD is the aerobic exercise PD group

3 Results

31 Behavioral Test on Rat PD Model The successful estab-lishment of the 6-OHDA-induced PD model was confirmedby the rotation test using an intraperitoneal (IP) injection ofapomorphine hydrochloride [13 14] Four rats in SED-PDand six rats in EX-PD were excluded after the first rotationtest (less 4 rotations) Table 2 shows that the number ofrotations in the EX-PD group was significantly reduced butnot statistically significant compared to the SED-PD groupin the first and the second week after surgery (119875 gt 005)

32 Scanning Electron Microscopy of Rat Striatum in the PDModel As shown in Figure 1 the striatum showed fewerneuro-villi and the arrangement was sparse and cluttered inthe no-lesion side of SED-PD at 5000-foldmagnification Theimage at 10000-foldmagnification revealed the neuronal villigrown in a scattered pattern with no entangled plexus andmore nodular synaptic junctions on or between the neuronalvilli The image at 3000-fold magnification revealed atrophyof the neuronal villi in the lesion side of SED-PD A largenumber of neurites and axons were seen which were unclearand resembled a scum-like structure

Figure 1 shows a larger number of nerve villi in thestriatum arranged neatly in bundles Many nodules of synap-tic junctions were observed on or between the neuronalvilli at 5000-fold magnification in the no-lesion side of EX-PD Atrophy of the neuronal villi and a large number ofneurites and axonswere observed in the striatum at 3000-foldmagnification in the lesion side of EX-PD The neurites andaxons were entangled in bundles and neatly arranged Manyneuronal villi were observed to be entangled with the neuritesand axons The image at 10000-fold magnification revealedthat the neurites and axons were thick and still more nodularsynaptic connections were found above or between neuronalvilli Our results showed the neurons axons and neuronalvilli of the striatum were clearly and tightly arranged andneurons and axons were significantly larger in the EX-PDgroup compared to the SED-PD group

33 TH and 120572-Synuclein Expression in the Rat Striatum120572-Synuclein protein aggregation was the major structuralcomponent of the intracellular protein inclusions termedLewy bodies that define the hallmark of PD [26] AndTH expression decreased in the striatum is another markerinvolved in the pathology of PD [27] Immunohistochemical


SED-PD (N) SED-PD (N)

SED-PD (L) SED-PD (L) EX-PD (L) EX-PD (L)

EX-PD (N)EX-PD (N)

Figure 1 Scanning electron microscopy images of the striatum of the rat model of PD SED-PD the sedentary control PD group EX-PD theaerobic exercise PD group no-lesion side (N) and lesion side (L)

staining of TH and 120572-synuclein protein was abundantlyexpressed in the cytoplasm of the striatum and sparinglyexpressed in the stroma as a brownish color with a smallnumber of nuclei also tan and normal nuclei stained blue THand 120572-synuclein positively stained area were large and theprotein stained dark in color in the striatum in SED-PD andEX-PD Compared with the no-lesion side TH expressionwas decreased significantly in the lesion side in both SED-PD and EX-PD groups (119875 lt 001) but compared to theSED-PD group TH expression was significantly increasedin EX-PD preexecuted with regular aerobic exercise (119875 lt001) (Figure 2) Compared to the no-lesion side 120572-synucleinexpression in the lesion side significantly increased in thestriatum in SED-PD and EX-PD (119875 lt 001) Intriguinglycompared to the SED-PD group 120572-synuclein was signifi-cantly decreased in EX-PD (119875 lt 001) (Figure 2)

34 Effect of Differentially Expressed miRNAs in the Striatumof the Rat Model of PD Based on the preimplementationof regular aerobic exercise and the impact of miRNAson PD-related expression profiles this study standardizedand screened the unreliable data and screened 11 miRNAsupregulated by regular aerobic exercise to two-fold or higher(multifold) in the striatum of the rat model of PD miR-1-3p miR-34c-3p miR-31a-3p miR-499-3p miR-200b-5pmiR-465-3pmiR-190a-3pmiR-146a-3pmiR-2985miR-377-3p and miR-3557-3p (Table 3) Further 10 miRNAs weredownregulated by twofold or more miR-136-5p miR-138-5pmiR-324-5p let-7e-3pmiR-770-5pmiR-134-5pmiR-935miR-653-3p miR-331-5p and miR-539-3p (Table 3)

The bioinformatics analysis was performed on the afore-mentioned 21 miRNAs target prediction and functionalanalysis The subcellular localization the role of target genesin known diseases and pathway screening of the target genepairs were investigated according to the expression levelThe reliable miRNA-mRNA interaction network was selectedfrom the target database by predicting transcription factors

and molecular activity miR-3557 and miR-324 were worthfurther exploration in this experiment

35 Differential Screening of miR-3557 and miR-324

351 Gene Ontology (GO) and KEGG Biological PathwayAnalysis According to the GO bioinformatics analysis miR-3557 and miR-324 had molecular functions The analysisshowed that 218 targeted genes were involved in energymetabolism 42 in metabolic regulation 44 in developmentalprocesses 75 in biological regulation and 64 in stimulusresponse Gene-related biological functions were regulationof sensory and pain perception integration of regulatory sig-naling pathways phosphatidylinositol metabolic processespositive regulation of biological processes and positive reg-ulation of transmembrane transporter activity

miR-3557-3p is closely related to CaMKV nuclear het-erogeneous riboproteins and miR-324 is closely related toVdac1 The KEGG biological pathway analysis predictspossible enrichment of biological pathways and classifies theresults with statistical significance These statistically sig-nificant biological pathways include the calcium signal-ing pathway (rno04020) neuroactive ligand-receptor inter-action (rno04080) DNA replication (rno03030) extracel-lular matrix- (ECM-) receptor interaction (rno04512)amphetamine addiction (rno05031) glycosylphosphatidyl-inositol- (GPI-) anchor biosynthesis (rno00563) B cellreceptor signaling pathway (rno04662) endocytosis(rno04144) drug metabolism-cytochrome P450 (rno00982)and nucleotide excision repair (rno03420) These biologicalsignaling pathways may have a statistically significantrelationship with PD

352 Validation of miR-3557 and miR-324 The analysis oftarget genes showed that miR-3557was upregulated andmiR-324was downregulated Real-time quantitative PCRwas usedto verify the consistency of the results of miRNA expression


SED-PD EX-PD SED-PD EX-PD

No-lesion Lesion

TH-synuclein

lowastlowast

lowastlowast

(A) (B)

SED-PD (L) times200

EX-PD (L) times200

SED-PD (N) times200

EX-PD (N) times200

SED-PD (N) times200 SED-PD (L) times200

EX-PD (L) times200EX-PD (N) times200

0

20000

40000

60000

80000

100000

120000

IOD

val

ue

lowastlowast

lowastlowast

Figure 2 TH and 120572-synuclein expression in the striatum of the PD rat model (A) Microscopic image of TH (B) Microscopic image of 120572-synuclein SED-PD is the sedentary control PD group EX-PD is the aerobic exercise PD group lowastlowastΡ lt 001 comparing between SED-PD andEX-PD Ρ lt 001 comparing between no-lesion side (N) and lesion side (L) n=4-6 no-lesion side (N) and lesion side (L) TH tyrosinehydroxylase

andmicroarrayThe expression of miR-3557was significantlyupregulated in the EX-PD group compared to the SED-PD group (119875 lt 001) (Figure 3) Further the expressionof miR-3557 was significantly downregulated in Lesion sidecompared to no-lesion side in EX-PD (119875 lt 005)

Figure 3 also shows that the expression of miR-324 wassignificantly downregulated in the lesion side of EX-PDgroup compared to the SED-PD group (119875 lt 001) but theexpression of miR-324 was slightly downregulated in thelesion side compared to the no-lesion side in SED-PD Theexpression of miR-324was significantly downregulated in thelesion side compared to the no-lesion side in EX-PD (119875 lt005)

36 Effect of CaMKsmTOR Signaling Pathway in the Striatumof the RatModel of PD Figure 4 shows thatCamk2120572 CamkVandVdac1mRNA expression was significantly upregulated inthe lesion side compared to the no-lesion side in SED-PD and

EX-PD (119875 lt 001) CamkV and Vdac1mRNA expression wasdownregulated in the lesion side of EX-PD compared withthe SED-PD (119875 lt 001) butCamk2120572was slightly upregulatedIntriguingly Camk2120572 and CamkV mRNA expression wasalso downregulated in the no-lesion side of EX-PD comparedwith the SED-PD and Vdac1 significantly downregulated(119875 lt 001)

The real-time quantitative PCR analysis showed thatPI3R Akt and mTOR mRNA expression (Figure 4) wassignificantly upregulated in the lesion side compared to theno-lesion side in SED-PD (119875 lt 001) and EX-PD group exceptAkt1 (119875 lt 001) PI3R Akt and mTOR mRNA expressionwas downregulated in the no-lesion side of EX-PD comparedto SED-PD However PI3R and mTOR mRNA expressionwas slightly upregulated in the lesion side of EX-PD groupcompared to the SED-PD group but the expression of Akt1was significantly downregulated (119875 lt 001) In summaryregular aerobic exercise activated the effect of PI3R andmTOR mRNA expression


Table 3 Differential miRNAs in the striatum of the rat model of PD on regular aerobic exercise

ID Gene log2 fold change P-valueMore than two-fold upregulated miRNAs14294 rno-miR-1-3p 65784 P lt00142767 rno-miR-34c-3p 36394 P lt00546320 rno-miR-31a-3p 43727 P lt001145692 rno-miR-499-3p 108036 P lt001145974 rno-miR-200b-5p 73004 P lt001148207 rno-miR-465-3p 22084 P lt005148330 rno-miR-190a-3p 30857 P lt005148365 rno-miR-146a-3p 34221 P lt005148439 rno-miR-2985 39897 P lt005148549 rno-miR-377-3p 31776 P lt005148581 rno-miR-3557-3p 212246 P lt001More than two-fold downregulated miRNAs10943 rno-miR-136-5p 05312 P lt00113140 rno-miR-138-5p 05830 P lt00142477 rno-miR-324-5p 04613 P lt00542743 rno-let-7e-3p 04045 P lt00142817 rno-miR-770-5p 04294 P lt00142942 rno-miR-134-5p 05081 P lt005145742 rno-miR-935 04532 P lt005148174 rno-miR-653-3p 01252 P lt001148385 rno-miR-331-5p 03686 P lt005148507 rno-miR-539-3p 03345 P lt005

SED-PD EX-PD

No-lesionLesion

SED-PD EX-PDNo-lesionLesion

lowastlowastlowastlowast

lowastlowast

0

02

04

06

08

1

12

14

16

18

miR

-355

7 re

lativ

e exp

ress

ion

0

02

04

06

08

1

12

miR

NA

324

relat

ive e

xpre

ssio

n

Figure 3MiR-3557 and miR-324 expression by real-time quantitative PCR assay lowastlowast119875 lt 001 comparing between SED-PD and EX-PD 119875 lt005 comparing between no-lesion side (N) and lesion side (L) SED-PD is the sedentary control PD group EX-PD is the aerobic exercisePD group

37 Effect of Downstream Molecules in the Rat Model ofPD U-ubiquitin C-terminal hydrolase 1 (UCH-L1) mRNAexpression (Figure 5) was significantly upregulated in thelesion side compared to the no-lesion side in SED-PD andEX-PD (119875 lt 001) However UCH-L1mRNA expression wassignificantly downregulated in the no-lesion side in EX-PDcompared to SED-PD (119875 lt 005) But intriguingly UCH-L1 mRNA expression was upregulated in the lesion side of

the EX-PD group compared to the SED-PD group (119875 lt001)

4 Discussion

Exercising both before and after 6-OHDA could maximizeand improve the possibility of motor function by exercise-induced improvements in PD rats [15 28 29] Increasing



No-lesionLesion

No-lesionLesion

No-lesionLesion

No-lesionLesion

No-lesionLesion

SED-PD EX-PD

lowastlowast

SED-PD EX-PD

lowastlowast

lowastlowast

SED-PD EX-PD SED-PD EX-PD SED-PD EX-PD

lowastlowast

lowastlowast

lowastlowast

002040608

112141618

Cam

k2a r

elat

ive e

xpre

ssio

n

002040608

1121416

Cam

kV re

lativ

e exp

ress

ion

002040608

112141618

PI3R

relat

ive e

xpre

ssio

n

002040608

112141618

Akt

1 re

lativ

e exp

ress

ion

002040608

112141618

2

mTO

R re

lativ

e exp

ress

ion

002040608

112141618

2

Vdac

1 re

lativ

e exp

ress

ion

Figure 4 Expression levels of CaMKsmTOR signal related mRNA by real-time quantitative PCR assay lowastlowast119875 lt 001 comparing between SED-PD and EX-PD 119875 lt 005 119875 lt 001 comparing between no-lesion side (N) and lesion side (L) SED-PD is the sedentary control PD groupEX-PD is the aerobic exercise PD group

SED-PD EX-PD

No-lesionLesion

lowast

lowastlowast

0

05

1

15

2

25

UCH

-L re

lativ

e exp

ress

ion

Figure 5 Expression levels of UCH-L 1 mRNA by real-time quantita-tive PCR assay lowast119875 lt 005 lowastlowast119875 lt 001 comparing between SED-PDand EX-PD 119875 lt 001 comparing between no-lesion side (N) andlesion side (L) SED-PD is the sedentary control PD group EX-PDis the aerobic exercise PD group

aerobic treadmill exercise before unilateral intrastriatal 6-OHDA injection has been shown to rescue motor deficitsmeasured by the apomorphine-induced rotational asymme-try in rats [13 14] In this study regular aerobic exercise before

unilateral intrastriatal 6-OHDA injection improved the PDratrsquos behavior and saved the structure of striatum in PDrat from scanning electron microscopy observation Regularaerobic exercise significantly increased TH expression anddecreased 120572-synuclein expression Our experimental resultsprovided evidence that regular aerobic exercise alleviatedthe neurodegenerative process of PD lesions The specificmechanism of exercise effects is still unclear

MiRNAs act on many targeted genes involved in thedevelopment of neurological diseases or neurodegenerativediseases Hua et al [30] discovered more than 30 specific andhighly expressed miRNAs in rat neural tissue and speculatedthat these miRNAs with specific high expression in ratneural tissue might help in the diagnosis and treatment ofneurological diseases Rogaev et al [31] found that miRNAswere involved in the occurrence of many neuropsychiatricdisorders especially those associated with developmentalabnormalities Yet no known study has examined howthe miRNAs (examined via miRNAs profiling) might differbetween PD animals who regularly exercise interventions andPD animals who are habitually sedentary We further foundthat 11 miRNAs such as miR-3557-3p were upregulated bytwofold or more in the striatum of rats with PD on thepreimplementation of regular aerobic exercise And we alsofound 10 miRNAs such as miR-324-5p were downregulatedby twofold or more Feng et al [32] found upregulation ofrno-miR-3557-5p could be seen as biomarkers of prognosis inclinical therapy of heart failure Diaz et al [33] found that the


overexpression of miR-324-3p promoted the dysregulation ofthe expression of genes involved in cell death and apoptosiscAMP and Ca2+ signaling cell stress and metabolism Insummary the subcellular localization the role of target genesin known diseases and pathway screening of the target genepairs were investigated according to the expression level inthis study Also the most reliable miRNA-mRNA interactionnetwork was screened by predicting transcription factors andmolecular activity The results showed that miR-3557 andmiR-324 deserve further exploration

The bioinformatics analysis showed that miR-3557-3pwas closely related to CaMKV nuclear heterogeneous ribo-proteins and macro-related proteins miR-324 was closelyrelated to Vdac1 The KEGG biological pathway analysispredicted the possible enrichment of biological pathwaysincluding calcium signaling pathways stimulated nervoussystem interactions with receptors and DNA replicationpathways CaMKs is highly expressed in neurons and hasa specific subcellular localization It converts intracellularlyelevated calcium signals into a range of target proteinsincluding ion channels and transcriptional activators andregulates synaptic transmissions related to the downstreamsignaling pathway [34 35] Firstly CaMKs regulate synapticplasticity and neurotransmitter release Zhu et al [36] showedthat CaMKs could activate TH TH is a rate-limiting enzymethat catalyzes tyrosine to form dopa After dopa forma-tion it can be further transformed into neurotransmittersnorepinephrine and DA Phosphorylated TH can increasethe synthesis of catecholamines CaMKs can also directlypromote DA release Secondly CaMKs play a vital role inthe Ca2+CaM signaling system different concentrations andsubcellular regions of Ca2+ activate a range of substrates ofCaMK2 or PP1 (SerThr phosphatase) which appears to becritical in controlling CaMKs-dependent neuronal signaling[37]

CaMKs pathways are involved in many upstream signal-ing pathways such as growth factor signaling PI3KAkt andcalcium signaling pathway [38] mTOR is a serinethreonineprotein kinase that serves as the site of energy-sensitive path-ways for PI3KAkt and serinethreonine kinase II [39]mTORnot only regulates cell proliferation and survival but alsoparticipates in membrane transport and protein degradationespecially in the regulation of protein translation levelsRecently mTOR has been shown to promote autophagyremove abnormal proteins such as amyloid polypeptidein AD and mutated 120572-synuclein in familial PD and thustreat related diseases [40] This provides a new treatmentstrategy for preventing and treating geriatric diseases Ourresults showed that PI3R and mTOR mRNA expression wasslightly upregulated in the lesion side in EX-PD comparedwith the SED-PD group and showed that regular aerobicexercise improved the effect size of PI3R and mTOR mRNAexpression This indicated that preregular aerobic exerciseinduced the activation of mTOR signaling pathway andregulating to remove abnormal proteins mutated 120572-synucleinin the PD model rat striatum Ankovic [41] reported thatmTOR had the potential to treat PD Pan et al [42] found thatthe autophagy inducer rapamycin specifically targeting the

autophagy-gated mTOR could alleviate this phenomenonInhibition of autophagy exacerbated the accumulation of120572-synuclein leading to cell death rapamycin-induced thedegradation of 120572-synuclein and cell survival

The secretion and expression of UCH-L1 could beinvolved in the pathology of Parkinsonrsquos disease Lowe et al[43] reportedmutations in 120572-synuclein and UCH-L1 genes infamilial PD and the presence of these proteins in Lewy bodiesof sporadic PD indicating errors in the production of theseproteins during folding and degradation which could causenerve cell degeneration Mandel et al [44] found the increasein the UCH-L1 level is favorable for DA neurons to clearabnormal proteins delay the accumulation of abnormal pro-teins and inhibit the formation of Lewy bodies Manago etal [45] found that UCH-L1 increased ATP-induced currentswere dependent on cAMP-dependent protein kinase (PKA)and CaMK2 Zhang et al [46] reported that conventionalprotein kinase C (cPKC) could regulate oxidative stress andapoptosis through UCH-L1 inhibiting UCH-L1 expressioncan negatively regulate autophagy through the extracellularsignal-regulated kinase (ERK)-mTORpathwayWe found theexpression of UCH-L1 mRNA expression was upregulatedin the lesion side of EX-PD group compared with SED-PD group It is shown that preregular aerobic exercise alsoactivated the expression of UCH-L1 in the PD lesions Ourresults indicated that regular aerobic exercise alleviated theneurodegenerative process of PD lesions by improving UCH-L1 level whichmight be the downstream of CaMKs ormTORsignal regulation Kanthasamy et al [47] found that ROS andoxidative damage were involved in the disease process of PDUCH-L1 could effectively remove ROS and protect neuronsfrom oxidative damage Therefore UCH-L1 reflects the self-protection mechanism of neurons in experimental animalsWhether the change in the UCH-L1 level can inhibit theoccurrence or development of PD lesions on aerobic exercisemay be worth further exploring

5 Conclusions

The adaptive mechanism of regular aerobic exercise delayingthe development of neurodegenerative diseases and lesionswas that miR-3557324 was activated to regulate one ofits targets CaMKs signaling pathways CaMKs coordinatedwith mTOR pathway-related gene expression and improvedUCH-L1 level to favor for tyrosine hydroxylase and clearabnormal 120572-synuclein thereby delaying neurodegenerationor improving the pathogenesis of PD lesions This studyprovided important evidence and theoretical guidance formotor rehabilitation

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declared no conflict of interest


Authorsrsquo Contributions

Wenfeng Liu performed the experiments provided the studyconception participated in the statistical analysis and par-ticipated in writing the paper Professor Li Li participated inthe statistical analysis and participated in writing and editingthe paper Shaopeng Liu Zhiyuan Wang Heyu Kuang andYan Xia performed the experiments and participated in thestatistical analysis Professor Changfa Tang and ProfessorDazhong Yin provided the study conception designed theexperiments and participated in revising the manuscript

Acknowledgments

The current study was supported by the National NaturalScience Foundation of China (Grant No 81702236) CSC(GrantNo 201706720028)HunanProvincialNatural ScienceFoundation of China (Grant No 2018JJ3363) the ScientificResearch Fund of Hunan Provincial Education Department(Grant No 12B088) and the Hunan Normal University forDistinguished Young Scholars (Grant No ET1507)

References

[1] L Yin Y Sun J Wu et al ldquoDiscovering novel microRNAsand age-related nonlinear changes in rat brains using deepsequencingrdquoNeurobiology of Aging vol 36 no 2 pp 1037ndash10442015

[2] J Fiedler V Jazbutyte B C Kirchmaier et al ldquoMicroRNA-24regulates vascularity after myocardial infarctionrdquo Circulationvol 124 no 6 pp 720ndash730 2011

[3] R Takanabe K Ono Y Abe et al ldquoUp-regulated expressionof microRNA-143 in association with obesity in adipose tissueof mice fed high-fat dietrdquo Biochemical and Biophysical ResearchCommunications vol 376 no 4 pp 728ndash732 2008

[4] F-B Gao ldquoPosttranscriptional control of neuronal develop-ment by microRNA networksrdquo Trends in Neurosciences vol 31no 1 pp 20ndash26 2008

[5] N Liu M Landreh K Cao et al ldquoThe microRNA miR-34 modulates ageing and neurodegeneration in DrosophilardquoNature vol 482 no 7386 pp 519ndash523 2012

[6] P T Nelson W-X Wang and B W Rajeev ldquoMicroRNAs(miRNAs) in neurodegenerative diseasesrdquo Brain Pathology vol18 no 1 pp 130ndash138 2008

[7] X Wang P Liu H Zhu et al ldquomiR-34a a microRNA up-regulated in a double transgenic mouse model of Alzheimerrsquosdisease inhibits bcl2 translationrdquo Brain Research Bulletin vol80 no 4-5 pp 268ndash273 2009

[8] H Lemaitre V S Mattay F Sambataro et al ldquoGenetic variationin FGF20 modulates hippocampal biologyrdquo The Journal ofNeuroscience vol 30 no 17 pp 5992ndash5997 2010

[9] A N Packer Y Xing S Q Harper L Jones and B L DavidsonldquoThe bifunctional microRNA miR-9miR-9lowast regulates RESTandCoREST and is downregulated inHuntingtonrsquos diseaserdquoTheJournal of Neuroscience vol 28 no 53 pp 14341ndash14346 2008

[10] E Salta and B De Strooper ldquoNon-coding RNAs with essentialroles in neurodegenerative disordersrdquo The Lancet Neurologyvol 11 no 2 pp 189ndash200 2012

[11] J Dostie Z Mourelatos M Yang A Sharma and G DreyfussldquoNumerous microRNPs in neuronal cells containing novelmicroRNAsrdquo RNA vol 9 no 2 pp 180ndash186 2003

[12] S Wang A B Aurora B A Johnson et al ldquoThe endothelial-specific microRNA miR-126 governs vascular integrity andangiogenesisrdquo Developmental Cell vol 15 no 2 pp 261ndash2712008

[13] R O Da Costa C V J Gadelha-Filho A E M Da Costa et alldquoThe treadmill exercise protects against dopaminergic neuronloss and brain oxidative stress in Parkinsonian Ratsrdquo OxidativeMedicine and Cellular Longevity vol 2017 Article ID 213816910 pages 2017

[14] T Tuon S S Valvassori J Lopes-Borges et al ldquoPhysicaltraining exerts neuroprotective effects in the regulation of neu-rochemical factors in an animal model of Parkinsonrsquos diseaserdquoNeuroscience vol 227 pp 305ndash312 2012

[15] E Crowley Y Nolan and A Sullivan ldquoExercise as a therapeuticintervention formotor andnon-motor symptoms inParkinsonrsquosdisease Evidence from rodent modelsrdquo Progress in Neurobiol-ogy vol 172 pp 2ndash22 2019

[16] A S Aguiar M Duzzioni A P Remor et al ldquoModerate-intensity physical exercise protects against experimental6-hydroxydopamine-induced hemiparkinsonism throughNrf2-antioxidant response element pathwayrdquo NeurochemicalResearch vol 41 no 1-2 pp 64ndash72 2016

[17] C C Real A F B Ferreira G P Chaves-Kirsten A S Torrao RS Pires and L R G Britto ldquoBDNF receptor blockade hindersthe beneficial effects of exercise in a rat model of Parkinsonrsquosdiseaserdquo Neuroscience vol 237 pp 118ndash129 2013

[18] T G Bedford C M Tipton N C Wilson R A Oppligerand C V Gisolfi ldquoMaximum oxygen consumption of rats andits changes with various experimental proceduresrdquo Journal ofApplied Physiology vol 47 no 6 pp 1278ndash1283 1979

[19] W Liu G Chen F Li C Tang and D Yin ldquoCalcineurin-NFATsignaling and neurotrophins control transformation of myosinheavy chain isoforms in rat soleusmuscle in response to aerobictreadmill trainingrdquo Journal of Sports Science and Medicine vol13 no 4 pp 934ndash944 2014

[20] J Thomas J Wang H Takubo J Sheng S de Jesus andK S Bankiewicz ldquoA 6-hydroxydopamine-induced selectiveparkinsonian rat model further biochemical and behavioralcharacterizationrdquo Experimental Neurology vol 126 no 2 pp159ndash167 1994

[21] Z Wang K G Myers Y Guo et al ldquoFunctional reorganizationofmotor and limbic circuits after exercise training in a ratmodelof bilateral parkinsonismrdquo PLoS ONE vol 8 no 11 Article IDe80058 2013

[22] M V Mabandla L A Kellaway W M U Daniels and V ARussell ldquoEffect of exercise on dopamine neuron survival inprenatally stressed ratsrdquoMetabolic Brain Disease vol 24 no 4pp 525ndash539 2009

[23] Y Zhou P Gu W Shi et al ldquoMicroRNA-29a induces insulinresistance by targeting PPAR120575 in skeletal muscle cellsrdquo Interna-tional Journal of MolecularMedicine vol 37 no 4 pp 931ndash9382016

[24] S Anders and W Huber ldquoDifferential expression analysis forsequence count datardquoGenome Biology vol 11 no 10 Article IDR106 2010

[25] W Liu L Li H Kuang et al ldquoProteomic profile of carbonylatedproteins screen regulation of apoptosis via CaMK signalingin response to regular aerobic exerciserdquo BioMed ResearchInternational vol 2018 Article ID 2828143 13 pages 2018

[26] A Bellucci L Navarria M Zaltieri et al ldquoInduction of theunfolded protein response by 120572-synuclein in experimental


models of Parkinsonrsquos diseaserdquo Journal of Neurochemistry vol116 no 4 pp 588ndash605 2011

[27] N Tajiri T Yasuhara T Shingo et al ldquoExercise exerts neuro-protective effects on Parkinsonrsquos disease model of ratsrdquo BrainResearch vol 1310 pp 200ndash207 2010

[28] S J OrsquoDell N B Gross A N Fricks B D Casiano T BNguyen and J F Marshall ldquoRunning wheel exercise enhancesrecovery from nigrostriatal dopamine injury without inducingneuroprotectionrdquo Neuroscience vol 144 no 3 pp 1141ndash11512007

[29] I A Moroz S Pecina T Schallert and J Stewart ldquoSpar-ing of behavior and basal extracellular dopamine after 6-hydroxydopamine lesions of the nigrostriatal pathway in ratsexposed to a prelesion sensitizing regimen of amphetaminerdquoExperimental Neurology vol 189 no 1 pp 78ndash93 2004

[30] Y-J Hua Z-Y Tang K Tu et al ldquoIdentification and targetprediction of miRNAs specifically expressed in rat neuraltissuerdquo BMC Genomics vol 10 Article ID 214 2009

[31] E I Rogaev ldquoSmall RNAs in human brain development anddisordersrdquoBiochemistry (Moscow) vol 70 no 12 pp 1404ndash14072005

[32] H J Feng W Ouyang J H Liu et al ldquoGlobal microRNAprofiles and signaling pathways in the development of car-diac hypertrophyrdquo Brazilian Journal of Medical and BiologicalResearch vol 47 no 5 pp 361ndash368 2014

[33] I Dıaz E Calderon-Sanchez R D Toro et al ldquomiR-125a miR-139 and miR-324 contribute to Urocortin protection againstmyocardial ischemia-reperfusion injuryrdquo Scientific Reports vol7 no 1 Article ID 8898 2017

[34] C E Grueter R J Colbran and M E Anderson ldquoCaMKII anemerging molecular driver for calcium homeostasis arrhyth-mias and cardiac dysfunctionrdquo Journal of Molecular Medicinevol 85 no 1 pp 5ndash14 2007

[35] W Lucchesi K Mizuno and K P Giese ldquoNovel insights intoCaMKII function and regulation during memory formationrdquoBrain Research Bulletin vol 85 no 1-2 pp 2ndash8 2011

[36] G Zhu M Okada S Yoshida S Hirose and S KanekoldquoPharmacological discrimination of protein kinase associ-ated exocytosis mechanisms between dopamine and 34-dihydroxyphenylalanine in rat striatum using in vivo micro-dialysisrdquo Neuroscience Letters vol 363 no 2 pp 120ndash124 2004

[37] N Shioda and K Fukunaga ldquoPhysiological and pathologicalroles of CaMKII-PP1 signaling in the brainrdquo InternationalJournal of Molecular Sciences vol 19 no 1 p 20 2018

[38] A Thorburn ldquoApoptosis and autophagy Regulatory connec-tions between two supposedly different processesrdquo Apoptosisvol 13 no 1 pp 1ndash9 2008

[39] XWan andL J Helman ldquoThe biology behindmTOR inhibitionin sarcomardquoThe Oncologist vol 12 no 8 pp 1007ndash1018 2007

[40] MA Lynch-Day KMao KWangM Zhao andD J KlionskyldquoThe role of autophagy in Parkinsonrsquos diseaserdquo Cold SpringHarbor Perspectives inMedicine vol 2 no 4 Article ID a0093572012

[41] J Jankovic ldquoProgression of parkinson disease are we makingprogress in charting the courserdquo JAMA Neurology vol 62 no3 pp 351-352 2005

[42] T Pan S Kondo W Zhu W Xie J Jankovic and W LeldquoNeuroprotection of rapamycin in lactacystin-induced neu-rodegeneration via autophagy enhancementrdquo Neurobiology ofDisease vol 32 no 1 pp 16ndash25 2008

[43] J Lowe H McDermott M Landon R J Mayer and K DWilkinson ldquoUbiquitin carboxyl-terminal hydrolase (PGP 95)is selectively present in ubiquitinated inclusion bodies charac-teristic of human neurodegenerative diseasesrdquo The Journal ofPathology vol 161 no 2 pp 153ndash160 1990

[44] S Mandel E Grunblatt P Riederer M Gerlach Y LevitesandM BH Youdim ldquoNeuroprotective strategies in Parkinsonrsquosdisease An update on progressrdquo CNS Drugs vol 17 no 10 pp729ndash762 2003

[45] Y Manago Y Kanahori A Shimada et al ldquoPotentiation ofATP-induced currents due to the activation of P2X receptorsby ubiquitin carboxy-terminal hydrolase L1rdquo Journal of Neuro-chemistry vol 92 no 5 pp 1061ndash1072 2005

[46] D Zhang S Han S Wang Y Luo L Zhao and J Li ldquo cPKC120574-mediated down-regulation of UCHL1 alleviates ischaemic neu-ronal injuries by decreasing autophagy via ERK-mTOR path-way rdquo Journal of Cellular andMolecularMedicine vol 21 no 12pp 3641ndash3657 2017

[47] A G Kanthasamy M Kitazawa A Kanthasamy and VAnantharam ldquoDieldrin-induced neurotoxicity relevance toParkinsonrsquos disease pathogenesisrdquo NeuroToxicology vol 26 no4 pp 701ndash719 2005

Hindawiwwwhindawicom Volume 2018

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Neurology Research International


Alzheimerrsquos DiseaseHindawiwwwhindawicom Volume 2018

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Research and TreatmentSchizophrenia

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

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Volume 2018Hindawiwwwhindawicom Volume 2018

Neural PlasticityScienticaHindawiwwwhindawicom Volume 2018


Parkinsonrsquos Disease

Sleep DisordersHindawiwwwhindawicom Volume 2018


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Submit your manuscripts atwwwhindawicom

MicroRNA Expression Profiling Screen miR-3557324-targeted CaMKmTOR in the Rat Striatum of Parkinsons Disease in Regular Aerobic Exercise

Recommended Citation

Authors

tmp1620926819pdfo4bMY

Authors Authors Wenfeng Liu Li Li Shaopeng Liu Zhiyuan Wang Heyu Kuang Yan Xia Changfa Tang and Dazhong Yin

This article is available at Digital CommonsGeorgia Southern httpsdigitalcommonsgeorgiasoutherneduhealth-kinesiology-facpubs181












1 Introduction





















































3 Results








EX-PD (N)EX-PD (N)












No-lesion Lesion

TH-synuclein

lowastlowast

lowastlowast

(A) (B)

SED-PD (L) times200

EX-PD (L) times200

SED-PD (N) times200

EX-PD (N) times200



0

20000

40000

60000

80000

100000

120000

IOD

val

ue

lowastlowast

lowastlowast










SED-PD EX-PD

No-lesionLesion



lowastlowast

0

02

04

06

08

1

12

14

16

18

miR

-355

7 re

lativ

e exp

ress

ion

0

02

04

06

08

1

12

miR

NA

324

relat

ive e

xpre

ssio

n




4 Discussion




No-lesionLesion

No-lesionLesion

No-lesionLesion

No-lesionLesion

No-lesionLesion

SED-PD EX-PD

lowastlowast

SED-PD EX-PD

lowastlowast

lowastlowast


lowastlowast

lowastlowast

lowastlowast

002040608

112141618

Cam

k2a r

elat

ive e

xpre

ssio

n

002040608

1121416

Cam

kV re

lativ

e exp

ress

ion

002040608

112141618

PI3R

relat

ive e

xpre

ssio

n

002040608

112141618

Akt

1 re

lativ

e exp

ress

ion

002040608

112141618

2

mTO

R re

lativ

e exp

ress

ion

002040608

112141618

2

Vdac

1 re

lativ

e exp

ress

ion


SED-PD EX-PD

No-lesionLesion

lowast

lowastlowast

0

05

1

15

2

25

UCH

-L re

lativ

e exp

ress

ion











5 Conclusions


Data Availability







Acknowledgments


References






































































MedicineAdvances in



Psychiatry Journal












Authors













1 Introduction





















































3 Results








EX-PD (N)EX-PD (N)












No-lesion Lesion

TH-synuclein

lowastlowast

lowastlowast

(A) (B)

SED-PD (L) times200

EX-PD (L) times200

SED-PD (N) times200

EX-PD (N) times200



0

20000

40000

60000

80000

100000

120000

IOD

val

ue

lowastlowast

lowastlowast










SED-PD EX-PD

No-lesionLesion



lowastlowast

0

02

04

06

08

1

12

14

16

18

miR

-355

7 re

lativ

e exp

ress

ion

0

02

04

06

08

1

12

miR

NA

324

relat

ive e

xpre

ssio

n




4 Discussion




No-lesionLesion

No-lesionLesion

No-lesionLesion

No-lesionLesion

No-lesionLesion

SED-PD EX-PD

lowastlowast

SED-PD EX-PD

lowastlowast

lowastlowast


lowastlowast

lowastlowast

lowastlowast

002040608

112141618

Cam

k2a r

elat

ive e

xpre

ssio

n

002040608

1121416

Cam

kV re

lativ

e exp

ress

ion

002040608

112141618

PI3R

relat

ive e

xpre

ssio

n

002040608

112141618

Akt

1 re

lativ

e exp

ress

ion

002040608

112141618

2

mTO

R re

lativ

e exp

ress

ion

002040608

112141618

2

Vdac

1 re

lativ

e exp

ress

ion


SED-PD EX-PD

No-lesionLesion

lowast

lowastlowast

0

05

1

15

2

25

UCH

-L re

lativ

e exp

ress

ion











5 Conclusions


Data Availability







Acknowledgments


References






































































MedicineAdvances in



Psychiatry Journal












Authors



















































3 Results








EX-PD (N)EX-PD (N)












No-lesion Lesion

TH-synuclein

lowastlowast

lowastlowast

(A) (B)

SED-PD (L) times200

EX-PD (L) times200

SED-PD (N) times200

EX-PD (N) times200



0

20000

40000

60000

80000

100000

120000

IOD

val

ue

lowastlowast

lowastlowast










SED-PD EX-PD

No-lesionLesion



lowastlowast

0

02

04

06

08

1

12

14

16

18

miR

-355

7 re

lativ

e exp

ress

ion

0

02

04

06

08

1

12

miR

NA

324

relat

ive e

xpre

ssio

n




4 Discussion




No-lesionLesion

No-lesionLesion

No-lesionLesion

No-lesionLesion

No-lesionLesion

SED-PD EX-PD

lowastlowast

SED-PD EX-PD

lowastlowast

lowastlowast


lowastlowast

lowastlowast

lowastlowast

002040608

112141618

Cam

k2a r

elat

ive e

xpre

ssio

n

002040608

1121416

Cam

kV re

lativ

e exp

ress

ion

002040608

112141618

PI3R

relat

ive e

xpre

ssio

n

002040608

112141618

Akt

1 re

lativ

e exp

ress

ion

002040608

112141618

2

mTO

R re

lativ

e exp

ress

ion

002040608

112141618

2

Vdac

1 re

lativ

e exp

ress

ion


SED-PD EX-PD

No-lesionLesion

lowast

lowastlowast

0

05

1

15

2

25

UCH

-L re

lativ

e exp

ress

ion











5 Conclusions


Data Availability







Acknowledgments


References






































































MedicineAdvances in



Psychiatry Journal












Authors





EX-PD (N)EX-PD (N)












No-lesion Lesion

TH-synuclein

lowastlowast

lowastlowast

(A) (B)

SED-PD (L) times200

EX-PD (L) times200

SED-PD (N) times200

EX-PD (N) times200



0

20000

40000

60000

80000

100000

120000

IOD

val

ue

lowastlowast

lowastlowast










SED-PD EX-PD

No-lesionLesion



lowastlowast

0

02

04

06

08

1

12

14

16

18

miR

-355

7 re

lativ

e exp

ress

ion

0

02

04

06

08

1

12

miR

NA

324

relat

ive e

xpre

ssio

n




4 Discussion




No-lesionLesion

No-lesionLesion

No-lesionLesion

No-lesionLesion

No-lesionLesion

SED-PD EX-PD

lowastlowast

SED-PD EX-PD

lowastlowast

lowastlowast


lowastlowast

lowastlowast

lowastlowast

002040608

112141618

Cam

k2a r

elat

ive e

xpre

ssio

n

002040608

1121416

Cam

kV re

lativ

e exp

ress

ion

002040608

112141618

PI3R

relat

ive e

xpre

ssio

n

002040608

112141618

Akt

1 re

lativ

e exp

ress

ion

002040608

112141618

2

mTO

R re

lativ

e exp

ress

ion

002040608

112141618

2

Vdac

1 re

lativ

e exp

ress

ion


SED-PD EX-PD

No-lesionLesion

lowast

lowastlowast

0

05

1

15

2

25

UCH

-L re

lativ

e exp

ress

ion











5 Conclusions


Data Availability







Acknowledgments


References






































































MedicineAdvances in



Psychiatry Journal












Authors




No-lesion Lesion

TH-synuclein

lowastlowast

lowastlowast

(A) (B)

SED-PD (L) times200

EX-PD (L) times200

SED-PD (N) times200

EX-PD (N) times200



0

20000

40000

60000

80000

100000

120000

IOD

val

ue

lowastlowast

lowastlowast










SED-PD EX-PD

No-lesionLesion



lowastlowast

0

02

04

06

08

1

12

14

16

18

miR

-355

7 re

lativ

e exp

ress

ion

0

02

04

06

08

1

12

miR

NA

324

relat

ive e

xpre

ssio

n




4 Discussion




No-lesionLesion

No-lesionLesion

No-lesionLesion

No-lesionLesion

No-lesionLesion

SED-PD EX-PD

lowastlowast

SED-PD EX-PD

lowastlowast

lowastlowast


lowastlowast

lowastlowast

lowastlowast

002040608

112141618

Cam

k2a r

elat

ive e

xpre

ssio

n

002040608

1121416

Cam

kV re

lativ

e exp

ress

ion

002040608

112141618

PI3R

relat

ive e

xpre

ssio

n

002040608

112141618

Akt

1 re

lativ

e exp

ress

ion

002040608

112141618

2

mTO

R re

lativ

e exp

ress

ion

002040608

112141618

2

Vdac

1 re

lativ

e exp

ress

ion


SED-PD EX-PD

No-lesionLesion

lowast

lowastlowast

0

05

1

15

2

25

UCH

-L re

lativ

e exp

ress

ion











5 Conclusions


Data Availability







Acknowledgments


References






































































MedicineAdvances in



Psychiatry Journal












Authors





SED-PD EX-PD

No-lesionLesion



lowastlowast

0

02

04

06

08

1

12

14

16

18

miR

-355

7 re

lativ

e exp

ress

ion

0

02

04

06

08

1

12

miR

NA

324

relat

ive e

xpre

ssio

n




4 Discussion




No-lesionLesion

No-lesionLesion

No-lesionLesion

No-lesionLesion

No-lesionLesion

SED-PD EX-PD

lowastlowast

SED-PD EX-PD

lowastlowast

lowastlowast


lowastlowast

lowastlowast

lowastlowast

002040608

112141618

Cam

k2a r

elat

ive e

xpre

ssio

n

002040608

1121416

Cam

kV re

lativ

e exp

ress

ion

002040608

112141618

PI3R

relat

ive e

xpre

ssio

n

002040608

112141618

Akt

1 re

lativ

e exp

ress

ion

002040608

112141618

2

mTO

R re

lativ

e exp

ress

ion

002040608

112141618

2

Vdac

1 re

lativ

e exp

ress

ion


SED-PD EX-PD

No-lesionLesion

lowast

lowastlowast

0

05

1

15

2

25

UCH

-L re

lativ

e exp

ress

ion











5 Conclusions


Data Availability







Acknowledgments


References






































































MedicineAdvances in



Psychiatry Journal












Authors




No-lesionLesion

No-lesionLesion

No-lesionLesion

No-lesionLesion

No-lesionLesion

SED-PD EX-PD

lowastlowast

SED-PD EX-PD

lowastlowast

lowastlowast


lowastlowast

lowastlowast

lowastlowast

002040608

112141618

Cam

k2a r

elat

ive e

xpre

ssio

n

002040608

1121416

Cam

kV re

lativ

e exp

ress

ion

002040608

112141618

PI3R

relat

ive e

xpre

ssio

n

002040608

112141618

Akt

1 re

lativ

e exp

ress

ion

002040608

112141618

2

mTO

R re

lativ

e exp

ress

ion

002040608

112141618

2

Vdac

1 re

lativ

e exp

ress

ion


SED-PD EX-PD

No-lesionLesion

lowast

lowastlowast

0

05

1

15

2

25

UCH

-L re

lativ

e exp

ress

ion











5 Conclusions


Data Availability







Acknowledgments


References






































































MedicineAdvances in



Psychiatry Journal












Authors








5 Conclusions


Data Availability







Acknowledgments


References






































































MedicineAdvances in



Psychiatry Journal












Authors





Acknowledgments


References






































































MedicineAdvances in



Psychiatry Journal












Authors













































MedicineAdvances in



Psychiatry Journal












Authors


MicroRNA Expression Profiling Screen miR-3557/324-targeted ...

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