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    Unravelling The Neuroprotective Effect of Tinospora Cordifolia in A Parkinsonian Mouse Model Through The Proteomics Approach

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    Hagera Dilnashin

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    ACS Chemical. Neuroscience pubsacrorgiehemnewe Unraveling the Neuroprotective Effect of Tinospora cordifolia in a Parkinsonian Mouse Model through the Proteomics Approach Hareram Birla, Chetan Keswani, Saumitra Sen Singh, Walia Zahra, Hagera Dilnashin, ‘Aaina Singh Rathore, Richa Singh, Monika Rajput, Priyanka Keshri, and Surya Pratap Singh* ce This hetps1/doiorg/10.1021/acschemnewro.1c00881 GO Read Online ACCESS lull Metrics & More | @ Article Recommendations — | © Supporting Information nduced dopaminergic (DAergic) neutonal apc enna cts region is the primary catse of Parkinson's poccieeece nines, dlseate (PD). Following the discovery of t-dopa, multiple drugs 4 y— have been developed to improve the lifestyle of PD patients; . however, none have been suitable for clini! use due to their ? sett rltiple side effects. Tinospora cordifolia has been used in mre ditional medicines to treat neurodegenerative diseases. Pre- viously, we reported the neuroprotective role of Te via inhibition of NE-xB-associated proinilammatory cytokines against MPTP. intoxicated Parkinsonian mice. In the present study, we investigated the neuroprotective molecular mechanism of Te in a rotenone (ROT)-intoxieated mouse model, using 2 proteomics approach. Mice were pretreated with Te extract by oral administration, followed by ROT intoxication. Behavioral tests were performed to check motor functions of mice, Protein was isolated, and label-feee quantification (LEQ) was catied out to identify dliferentially expressed protein (DEP) in control vs PD and PD vs treatment groups. Results were validated by gRT-PCR with the ‘expression of target genes correlating withthe proteomics data. In this study, we report 800 DEPs in control vs PD and 133 in PD vs treatment groups. In size tools demonstrate significant enrichment of biochemical and molecular pathways with DEPs, which are known to be important for PD progression including mitochondrial gene expression, PD pathways, TGE-P signaling, and Alcheimer’s disease. This study provides novel insights into the PD progression as well as new therapeutic targets. More importantly, ome it demonstrates that Tc can exert therapeutic effects by regulating multiple pathways, resulting in neuroprotection. KEYWORDS: Parkinson's disease, neuroprotection, Twospora cordifolia, proteomics, label: free quantification INTRODUCTION ‘The global burden of neurological diseases doubled between the years 1990 and 2015. Among these, Parkinson's disease (PD) groweh was the fastest in prevalence and death.” PD isa ancurological disorder affecting balance, movement, and muscle control with progressive and continuous death of dopaminee gic (DAergic) neurons in the gray matter, followed by a decrease in dopamine (DA) concentration in the striatal region.""* The resulting DA deficiency leads to impaired motor function including tremors, rigidity, bradykinesia, and akinesia® The centerpiece of PD management is to execute symptomatic treatment with L-dopa, but due to its various side cilects like nausea, hallucination, dyskinesiz, lower blood pressure, confusion, and loss of appetite, its use is limited. Tn recent years, environmental factors (vie. exposure to pesticides) have been studied to contribute to PD onset and progression. Rotenone (ROT), a pesticide, which is also an Inhibitor of mitochondrial complex, when administrated for a long period of time, recapitulated PD's features like reactive oxygen species (ROS) generation and degeneration of the nigrostriatal DAergic system." As PD advances the death of ¢ ACS Publications 2% Arwen cimal sony DAergic neurons surges and these neurons Tose the ability to regenerate Because of the limitation of avalable therapeuties used for the tcatment of PD shown only to provide symptomatic relief With serious side effects that affect the quality of life, the development of effective therapeutic agents having the ability to protect the neurons and to diminish side effects is much needed. Scientists have been working on natural altematives, which provide ficient neuroprotection and prevent the subsequent impending basic pathologial conditions." Several redicinal plants, including Mucuna pruriens, Bacaps mornieri Withania somnyera, Tinospora cordifolia (Te), and their active constituents, exhibit neuroprotective activity against neuro degeneration in PD.2-"* Received: July 19, 2021 Accepted: October 21, 2021 ACS Chemical Neuroscience pubsacsorg/ehemneure Behavioral test A r : “Ti on ret ne) Figure 1. Neurobehavioral test of ROT-intosicated mice treated with Te. (A) Time in the cataleptic test. (B) T-tum time in the pole test. (C) ‘Time onthe rotarod tet. (D) Stride forepaw length in Oorrint ts, Vales ate represented inthe form of mean + SEM ( 0.01, and ***p < 0001 Tp e005, pe Studies have reported that Te possesses neuroprotective activity and its coadministration with dopa reduces intra neuronal oxidative stress.” Kosaraju et al, in a @OHDA model of PD, reported that Te exhibits significant neuro- protective effcets as well as improved levels of dopamine and reduees iron toxicity in the rat brain.” Birla et al demonstrated the neuroprotective activity of Te via tyrosine hydroxylase restoration (2 key enzyme for DA synthesis) in MPTP-intoxicated mice. Te also exhibits anti-indammatory activity in PD mice, owing to its ability to inhibit NF-xB and sssociated proinflammatory cytokines including TNF-a, I-12, and IL-1.” Additionally, Sharma et al. reported that the Dutanolic extract of Te reversed glutamateinduced neuro- degeneration in the hippocampal region of the rat brain."° ‘By employing proteomics, we can identify novel molecular pathways involved in the progression of PD. Identiying these pathways can serve to aid in eatly disease diagnosis and prognosis and to monitor the stage of disease development as ‘well as recognition of advanced drug targets." Therefore this study was executed to address the gap between PD pathogenesis and treatment strategies. Our current study focused on identification of new molecular targets in PD and the mechanism of action through which Te reverses the disease progression. In the present study, we used label-free quantification (LFQ) based on LC-MS/MS due to its high sensitivity, reproducibility, and reliability to study the protein capression patterns in the nigrostriatal region of the midbrain of mice.” Using this method, quantification of the difer cntially expressed proteins (DEPs) of the control vs Parkinsonian group and the Parkinsonian vs Te group was performed. Our results revealed that Te treatment improves the behavioral abnormalities in PD mice, and in the label-ree proteome analysis, we report multiple proteins in both groups that were diferenially expressed, From these results, we selected 21 proteins whose expression was significantly modulated in the PD treatment group vs the untreated group and performed gene ontology (GO) analysis of functional pathway entichment, along with the protein~ protein interaction (PPI) network and gene—gene interaction analyses, to investigate the cortelation with previously eported protein and gene prominent pathways iniluenced by Te for neuroprotective activity. These results may provide insights into farther developing promising novel therapeutic ap proaches for management of PD, II RESULTS AND DISCUSSION Neurobehavioral Parameters. Discovery of dopa was a rmajor step forward in the treatment of PD, but its major dhawhack was the generation of various side effets. In addition, many combinational drugs were reported to reduce the side effects, but the success rate was very low." Besides, researchers also focused on other molecular targets as well as alternative treatment options to improve the lifestyle of PD patients, PD can be attributed to mitochondrial dysfunctions, impairment of protein degradation, oxidative stress, and death, of DAergic neurons in SN, but the contiauous progression of molecular events Teading to cell death remains unclear” Prior to designing any preventive intervention, in-depth knowledge of the molecular mechanisms underlying neurodegeneration in PD is required. ROT is a wellknown pesticide acting selectively on mitochondrial complex, causing its inhibition like MPP=. A rowing body of evidence suggests that the insecticide model offers more advantages over diferent experimental models as it eifectively mimics the behavioral and neucopathological symptoms of the illness through the selective degeneration of DAergic neurons.” PD has various motor (tremor, bradykinesia, muscle stifiness falls and dizziness, freezing, gait imbalance)” and nonmotor (pain, low blood pressute, fatigue, restless legs, swallowing and saliva control, sleep, skin and sweating, bowel and bladder problems) symptoms.” Various studies have reported that nonmator symptoms generally develop at the initial stage of PD progression, which makes it dificult to Ailferentiate PD from other diseases. Zabra etal. reported the characteristic motor impairment in ROT-intoxicated Parkinso- nian mice.” To check the asymmetric motor coordination in ROT-intoxicated PD mice, multiple behavioral assessments were performed such as rotarod, pole, catalepsy, and footprint” (Figure 1). Incistinguishable ‘co-ordinates and deterioration of motor skills were also observed in our result due to ROT toxicity, whereas the behavioral tests demon- strated that Te improves the neurobehavioral shortfall as compared with previous studies.” Alter ROT injection, sifiness was observed in mice by the catalepsy test. The latency in the ROT group tended to increase over time (p < 0.001), showing significant diference when compared with the control group, but the Te-treated st0up showed a shorter latency time (p < 0.001) vs the ROT- Intoxicated group (Figure la). In the pole test, ROT. ACS Chemical Neuroscience cont Ror pubsacsorg/ehemneure Ror+te Figore 2. Immnnathuorescence expression of TH in SNpe. TH postive dopaminergic netrom expression was redoced in ROT-intaicated mice (p 13001), while Te treatment (p « 001) in ROT ntoatsted mice significantly enhanced the expression of TH. intoxicated mice showed a prolonged T-tum in comparison to control mice (p < 001). The T-deseend duration in Te pretreated mice was significantly reduced relative to ROT mice ( < 001) (Figure 1b). The rotared test was performed to check the balance, grip, and movement adjustment of the mice Compared to the contra, the ROT:intoxicated group had a significantly reduced retention time (p < 02001) on a rotarod, and it was observed that mice lost motor coordination and grip strength. Compared to ROT-intoxicated mice, Te treatment signiicantly increased the retention time on the rotarod (p < 0.01) (Figure 1¢). Footprint analysis showed diferences in the average stride length between the control group, the ROT group, and the Te-treated group. The stride diference in the ROT-intoxicated group significantly decreased compared to the control group (p < 0.01), whereas the gait disturbance was signiicantly improved on Te treatment (p < 0.05) (Figure 1d). 1TH is an essential enzyme in the DA synthesis pathway. ‘Changes in TH expression patterns ate directly related to the loss of DAergic neurons expressing in SNpe.”” Our findings showed that ROT intoxication induced a significant amount of DA (p < 0.001) neurons in SNpe, compared with the control group (Figure 2). The treatment of Te in the ROT-intoxicated group displayed substantial protection (p < 0.01) of DA ‘eurons in comparison with the ROT-intoxicated group. Our result shows similarity with previous studies. This indicates that Te exhibits neuroprotective effets on DA. neurons in ROT intoxicated mice Various medicinal herbs have been used for therapeutic fanctions since ancient times in Indi, China, and other Asian countries. Medicinal plants are used to treat various neuro: degenerative diseases, and they show very promising results with minimal side effets, Herbs lke B. monnier, W. sonmifera, 1M. prurins, and Te have been tested for their therapeutic potential against neurodegenerative diseases via redox balancing and immunomodulation activities." In our previous study, we reported that Te has a neuroprotective role va suppressing oxidaive stress as well as neuroinilammation.” ‘Comparative Protein Profiling between Control vs PD and PD vs the Treatment Group. ‘The protein prosling was analyzed through PGLC software (Masslynux 4.1 Water) and compared between control vs Parkinsonian and Parkinsonian vs Te mice groups. The current analysis showed the unique (3244 and 233) and common (1498) proteins discovered between control vs PD and PD vs Te (Figure 3) ‘On the basis of fold change, the significance of differences in the protein expression was also categorized. Proteins with a fold change value of $0.5 were considered downregulated, and Figure 3. Venn diagram. It represents the unigue and common proteins dscovered between contol vv PD and PD vs Te, proteins with a value 22.0-fod were considered upregulated (tables 14, 1B, 24, and 28, Figure 4). "The labelfee LC-MS/ MS results demonstrated that the control vs PD. group contained 800 DEPs, of which 67 were downregulated (Table 1A) and 101 were upregulated (able 1B). In the PD ve TC group, 133 DEPs were obtained, of which 19 (Table 2A) were downregulated and 16 were upregulated (Table 23). Besides this, some proteins that were significantly expressed in the PD group were normalized by Te ({old change inthe range of 0.5~ 2.0) (Figure 4). From this analysis, we identified 21 proteins whose expression was significantly modulated in the ROT. intoxicated Parkinsonian group and was ellcienty restored with the treatment of ‘Te. Some prominent upregulated proteins included the short transient receptor potential channel, glutamate receptor inotropic early endosome antigen 1, serine/threonine-protein phosphatase 2A, transcription factor A, aryl hydrocarbon receptor nuclear translocatos, ceakaryotie translation initiation factor SB, prosaposin receptor GPR37, RAC-a serine/threonine-protein kinase, and neutro phil eytosol factor 4 (Table 2A). However, ubiquitin carboxyl terminal hydrolase, DNA. (cjtosine-s)-methyltransferae 1 phosphatidylinositol 3,4,S-trisphosphate S-phosphatase 2, ribosome-releasing factor 2, phospholipase D1, receptor-type tyrosineprotein phosphatase, centromere protein 1, mothers against decapentaplegic homologue 7, Iysine-specfic demethy lase 3A, Telymphoma invasion, and metastasis-inducing protein 1 were the prominently downregulated proteins (table 28). With the application of proteomic profiling analysis, the quantitative and qualitative analyses of thousands of proteins from the tangled mixtures along with the demonstrations of their PTMs could be efficiently performed. Based on our findings, we have identiied various new molecules and pathways as an alternate treatment approach. Here, we used comparative proteome profiling through LFQ/LC-MS/MS to ACS Chemical Neuroscience pubractorglchemneuro Table IA. List of Pownregulated Proteins in Control vs PD Groups seceon Aesctition ove teore ld change pres PH0S80——_teasrption fctor A, mitochondeal Tia 1377 aos70N673 0.00 {Q6026 ALS? Cterina ike protein Alaa sass uae 0 M64 nese protein KIFZ? xa? 710s OLMMIT70O8 QpDU2 —tanmembrae protein 1A Tmentle 847 OSSeROS QBVEAE—aldeketo reductase fay 1 member C19 Asiels Ant orem 0 QsTDI6 protein RUBCNE ke abesl 694 oTerae8s om 90407 clheyelecontlprten SOC Tmemdte 49 OTDEMIO 0 PoeSL allan (1) cain lta 2609 O1MSIBSIS (Q8KIP0 _preemRNA 3’ end procening protein WDRSS wees Rn oes 0 QBCXIE——_ATP-ining csette ubamly B member 8, mitochondria Abebs BAT om09098 QpD84—_premmRNA Send procenng tor HPL putt 6128 ones oo D7PDD4 —tegaarycye and paelthbibitoryeeeptor Go Mpgsb 1559 24staso91 0.05 Q3VORY ia and again acid protin 65 capes $64 0248598970 Q3UZI8 ie elongation complex subane 2 ket 294 o2pIrsOs 008 QBUEQE capping protein Arp2/, and myosin Kner poten 3 Camis 10097 o2sen0sgss om Q6vGSS protein Dape Cedeshe 68 2eTIS28 PLS439 uridine Smonephoepatespthaze Umpe A968 — oxupE007 0 AgIPAS hand ealuninding domalo-consning protein § Beaks m2 o2masn167 S389 serine/threonine protein phosphate 24 stator Pps ai? oanesan2e 001 UAT eydin tz cela oir o2nssoere7 oo (965740 vngs apt protein homologve Wapl 9396 o2N6s0e797 om QBUSHS splicing factor supprestor of white-aprict homelague Sip mas o2esseanis 0 ESPY4S ata ansport protein 140 horelogue musa soa o2skismes 0 (QUBIs transport and Gols arganzaten protein I homologe Miss mss osm oo QUE ile dmnai-contuinng putin 61 eles 6276 0510365985005 QSVCES pecs homology donusin-contsning family H member 3 Pidbhs 4121. ogI9si9e6 oo 40282 WD epea-contining poten 98 Wass aes oazoanst on QPEL —_DPBL- and CUA snoented Ber 6 Deas 2 onsue7 oo PODMA erie protein Fels Gar on29sse9s6 0 QWOVWT ——_AT-hooke conning wanerption factor Ae nn0at 0329558956 001 e9CQ7S—_nephroan Nea 273 onses9ss11 0 QICTRY—_ubigulnke moder actiating ensyme 6 uns mon o3asissss om QQDITE ——_EP-hand demain containing protein, het 1S oasro7 oo S761 dabspeccty prota Kase TK Ta Sst o360se987 00 QZ Fancantaeocated nceae 1 Fant sar? onerer9Hn oo 055123 omen? Mapio 7449 ose7s79441 D353 sorting nen 29 some yo oyrssniom 0 QAVCI2 arcane Byte Ure i794 0a790n3027 008 PSS436 fate reeptor tonotopic Genta sas ogame 0s QOISNS Fe seceptorle protein § Fels 362 o3keran02k 00) Q6IS13 tarpon fear AP26 Thpd 245 96 03945SI70H 0.6 (089032 SHS and PX domainconinng protein 2A stapes 6367030583708 O02 osssds adele eyase pe ‘Adept 2994 oases om QSURWS youn 4 ys 3699 o4n6se9ses 0a {Q692KS——_MORC famly CWaype ane Sage protein 24, Mocta 3688 0406569669002 QBIYRS ——mutyhranerveike protein 13 Mews 7080406569669 0.08 P1784 VDI} recombation-actating protein 2 Rage nm oatermeis 002 QstFFO——_Grcharcenaed protein KIAAISAI Kath 457 oalgTRIA oo MST AFS/EMRD family member 1 an sase oais9sis#7 on Pe7484 ——_leskoct oimanoglbulin ike receptor subfanly B member 3 tals Tay oarriemr 009 QUHIZS ——logecinatedprofsin Kite Kinase kite 13 Maks 9618 oaz7HLa2 004 quer Bin Ein 4169 oasiTIOsss 006 (Q03391 ate seeptrionstopis, NMDA 2 Grad 7379 oaseo4s394 0s AQRS+——_UHREL-ning protein Le Unilbpll — 7399 oaH04si6ss 008 {QSPGG2 GEM interaing protein ans o4atassoss 001 QBCHBS tabula poygatnyase TTLLS Hi oaaisss06s 001 {Q67PRS into of Bran tyrosine Kinase suas oasieu7as OL QIBLRS PHL and SECT domso-contiing protein 4 6996 oasssasas 007 QUBRCS tia and age aztocited protein 91 Sa96 0463013077006 QPQUQS — hort transient seceptor potential chanel 4 432 o4so13077 02 URIBE sit homologe 2 proven 9896 0as7666ISL O18 > esucerane evacuees ACS Chemical Neuroscience Table LA. continued pubsacsorg/ehemneure seceon Asctition rene ore fold ange pre 61739 negra 6 igs S604 0467666891001 {QBJIMS——_-DNA excion cep protein ERCC-6 ike 2 Heck 2609 O477ELIIIL Po7s6s BS ugutinyrotet ligase REBPS Robs aso? aTTLLSILL Q64S21 glycerol phosphate dshpdrognae,mitochondsl pat Mss oatis06581 01 1035595 _protes patched omelugue 2 Pea 4 nanos es COLO import aban 8 Kp? 1569 ateTsa2 Q9ZIN protein une 3 homologue B Uneish 7446 ggigesn0s oo QWVDR? dato of tokinea prten 6 Decks S798 oasisaoe oa QpIW)s Gr upsceam clement Siding protein Fabpt uis29pasieeeaoe 024 Q6VIWE dynein egultry complex sbunt 7 Dre? 4509 0496545310 QHOTHE pecan homology damn contig amy H member 1 Piatt ase D8ZICS_—__AN ype nine dnger protein Zea BIT o4s6stsal one (Q623s2 spe surace protein Spt? Spal? cA7—oasesRS31 0s FBI7S9 RAC serine/threonine peta Kase Au S76 0s0is7607 ons antes ey endosomeansien 1 Eat Mas 05015760701 Q7ENZS serine/threonine eon phenpatve 2A 6S LDeeeulatery sobunt Aa vofoem —Pppaela 1064 OSH68SIS21 1S 153762 a bydrocarbonreepor nuclear anlcstor Ar 656 osmnsrs 06 QOSD#4 eukaryote wanlation ination tr $B Bist mom asim 0 favestigate DED in the nigrostelatal zegion of mice brain. Out cof total $00 DEPs, 101 were upregulated and 76 were downregulated in ROT:intoxicated mice compared to the control; however, out of total 133 DEPs in Tectreated mice, 16 were upregulated and 19 were dovenregulated as compared to ROT. Some proteins that ae significantly expressed inthe PD group were reversed by Te teeatment. On the basis of fapression and coreelation with PD marker proteins, we selected 21 proteins (Vables 2A and 28). Further in size analysis suggested that some of the DEPs (Smad7, ‘fam, Grin2a, Gpr37, and Tiam7) play a key role in the pathogenesis of PD via regulating diffrent molecular pathways (Table 3) and could be a new therapeutic avenue for neuroprotection, Smad? is an inactive record factor in pathogenesis of PD, which is constitutively upregulated in teggered microglia.” It leads DAergic neurons toward apoptosis by means of p53 and TGE-p cascades. Interestingly, Pal et al indicated that activators of either NFB (IL-1, TNE-a) of STAT-1 (IEN- 1) signaling pathorays enhance the expression of Smad7.””"” In fur study, we also found an upregulated expression of Smad? in PD mice as established by Ubetham et al, whereas its caprestion was downregulated ater Te treatment. Upregul tion of Smad? in teggered microglial cell poses a threat to the neuroprotective TGF AI signaling.°"" TGE- signaling fsvore cell survival by inactivating Bad (prospoptotic protein of the [Bel-2 family), thereby enhancing its phosphorylation through activation ofthe ERK/MAP kinase pathway. TGF pI signaling also suppresses the secondary neuronal damage caused by neuoinflammation.”~* Thus, TGF: /Smad signaling plays a signiticant role in neuronal protection. Ous results also suggest that TTe potentially suppresses the death of DAergic neurone via downregulation of Smad7."” ‘Tfam (Mitochondrial transcription factor A) is a 25 kDa DNA-binding protein contained in mtDNA. Tiam protects mtDNA ffem ROS, and mtDNA protects Tiam from Lon protease degradation." Loss of Tlam expression was found to be linked to degeneration of the DAergic neurons,” whereas ite enhanced expression improves mitochondrial siiment phenotypes via accelerating the mtDNA copy variety and delays neurodegeneration."" Consequently, Tim i good. indicator of defects im mitochondrial integrity. In this stady, we observed that ROT intoxication upregulates Tam expression, ‘whereas its expression was downregulated by ‘Te pretreatment. Let al, also demonstrated a downregulated Tfam expression in an ROT-intoxiated culture of SHLSYSY."" Thus, i is suggested that by downregulating the Tfam expression, Te improved the mitochondrial function, which is a major pathogenic hallmark of PD. ‘Gpr37 isa substrate of the E3 ligase Parkin and henceforth is alko called the Parkin-rlated endothelin-like receptor (Pael: RJ. Missense type of mutation in PARKIN causes aggregation of Gpr37 in the brain of PD patients.” Besides, overerpresson of Gpr37 causes ER stress and neuronal death." On the contrary, itis proposed that native Gpe37 shows a neuroprotective property by binding prosapti¢e and prosaposin." Interestingly, Lundivs et al. reported that ‘overexpression of Gp137 was robustly protected against ROT, MPP+, or 6-OHDAinduced cytotoxicity in N2a cells Furthermore, it is suggested that Gpr37 regulates oligodendrocyte separation and myelination by means of ERK signaling.“ However, Te treatment to PD mice suppresses the expression of Gpr87 in an ROT-intoxicated mice model. Her, we present that Gpr37 plays a broader role in neuroprotective and glioprotecve activities of Te. ‘Tiam1 (Tlymphoma invasion and metastasis 1) is a Race speciic GEF (Dbl family member), potentially involved in neurodegenerative diseases (AD and PD). It is a Ras effector solecue, stimulated by the Ca"-dependent activation of the NMDA receptor (NMDAR).” Tiamal controls the activation of Rho GrPase and plays an important role in PD by regulating oxidative stress and neuroinflammation,”* ‘Tia! controls neurite extension and DAergic neuron diferentia tion.” After exposing hippocampal neurons to amyloid-f peptide, Tiaml is activated and mobilized to the membrane, th may affect the pathology of AD.” In the present study, cxpression of Tiaml is downregulated by Te extract treatment against ROT intoxication, Interestingly, Smith et al. also reported that an elevated expzesion of Tiaml contributes to neuronal damage.* In contrast, Cajinek etal, showed Tiamt as a positive regulator of DAergic neuron diferentiation.”° ‘Together, these studies indicate that ROT-induced neurotoxic ciflocts ae linked with an upregulated expression of Tiaml and ACS Chemical Neuroscience ‘Table 1B. List of Upregulated Proteins in Control vs PD Groups yusi7 cpppvs 93289 sre, sitio wer sire ontess curves ppers ures qusxzi 90520 RAVE yuan oasis uci sinc anas 9054s 755997 ans ono? 92103 piv nowy ascot pwrrs qossi02 scree aanscs ayz80 ossiss Pros arr BoEyK0 ayzsis pune pnts 098393 qs0see 99258 ovess anise qsrsps apnats cyszsi auayet 90767 srry auch cums srpno ‘ppnys anKias cuKsas ayrass cqpnon0 apRsxs nyzxs ‘quovRe Aesriton tine Ager poten 48 prsbable ATP-dependent RNA hace DEX Rho GTPaseacvtng poten 23, yin specie demetiyze 3A cGMP-dependent proten iave 2 staphylococcal ese domain comaning protein 1 ral homeloguesubamly C member 1 “phan pe receptor 6 transient ceeptor potent ction channel subfamily M member 1 ‘led cll domain containing protein 97 BCI domsin fanly member 9 protela dsude-somease TMXS ‘ved amphipathic ke protein Sinda nae eat repeat contuaiog protein fay member 7 [WD repeat containing preten 75 sheer aplnt ecapentplgic homelogue 7 edientor af etobiness protein § canceraevected gone 1 protein berologue ritogen-actiated protein knase Kate kinase 2 Phosphatase 545-tinphorphate Sghowpatae 2 ‘sgn ubfamly A member $ cele prien 14 celagen @1(V) dan ety dependent netroproecorhomesbex poten ‘TELODintracting protein 1 homologue nlidrogrestance anced protein 9 aheson proen-coupled receptor B2 cadherin 22 extovoic earbonypepidv ike protein § inerfronindscble GTPase § ‘ne finger MYND demain cotasing protein 11 Phospholipase DI DNA diced RNA polymers {rabunit RAL homeobox poten cue 2 collagen @2(V1) chin DZ domai-cntaiing protein UWS th RNP sociated prot rue prota § Spemspeic antigen 2 hemalague teasorming srt alr receplor ze 3 ricrombule-avciated serine/threonine protein Knae 2 ‘petdopodion-ecrched spp Kinare 1 cellogen (XK) hain fsncni sveia gop I pcin bomelgse eotosomal poten of 138 kDa ‘eticenslar mater 1 pots Teucnesih repeat tansmembrane neuronal rote 3 ATP ae fly AAA domsin contig poten S Imphocyte antigen 75 Ras GTPaveacthatng protein 4 [acta] monovnygenze MICALS ribosomereleasing fr 2; miochondl ‘Pleekatsn homology ike domain family B member 1 sortag nee-20 yoann ike protein setibindng LIM protein 1 ‘rsa anspert protein Sec tune Sager Ran bindag domair- containing pete entncopetide repeat contig protein 2, miscbendit pling factor 48 Protea FAMI6OB2 pubsacsorg/ehemneure ene zits Diss Ashgap2s Keéms Pag snd Dnaet Eph ‘pat cede held Tm Mish? wars Sind? Decks Cage Mapa Ingplt Goigs Neplt lsat Adsp Tu Abcel2 dg. ca aghs We yaad ist Pots cua, Calén2 Pos Nols ‘Tybes Masse Peat Ccall9at epias Lint ands Lys Ras Mick ‘cin Paabt sao Ab Secitb zeanba Pres Rimi? Famis sas ss9 ns asa 4995 sa 638 waa 7836 sass ssa 496 613, as 9677 783 936 Iss Bt st os st 3a 4u71 S291 236 nss7 301 38 4997 eas sas 4st 4594 sare sas 4826 1827 9375 nant ns 137 m9 fold change ronsoiais 2054433269 2075080687 2ovsos06e7 2095845534 2117000017 2117000017 payer, 22702 259786213 pamassers 2205596474 azevs07e92 22a7sa7992 227907992 aaessis7m, asssie7an 2316366916 21636016 aaasessn0s 2sa964e508 aisesssas 2sso1s0728 aacaieor28 2386910865 24ss602053 2559961412 2ssooni412 2sss7ase2s rarveusas 2ss7944588 armisarit 2944679677 BoMaseass asissiais a0sansenis sasissints 09s6scans sssisa8y4 s2uiseas a2e7081385, a4sse198 ssasannast ansast 3469296095 araanisrs a7s108ss aasransexa 896193358 pres oss ose oss os 096 oss ost 076 os 096 oss ov os ose ost om ose oss 06 ose oss ACS Chemical Neuroscience Table 1B. continued seceson Aeseripion Pisco row histone chromosomal pte HMG-18 auozt0 sirtactind 059226 tarimembrine and cold domain prota 1 BDH 605 rhoromal protein U7 ike F st4ss receptortype fyrosine poten phosphatase ta QBCZIIE eae cail domain containing protein 77 Piss aegative elongation fer B QNBYR? serine hon protein nase LATS 03527 Sssegin wd mctllopoteimtedomsiorentiningpotin 7 QUKIKA centromere protein QS2QK0 condensin? complex bunt DS QURSIE BS abiguinprotein gave MIB2 Qozi90 macrophage tinting protein cesptor QSPRFO HEAT sepetconainingprotela SA 04592 ropoteinconvertace subiin/hesn Ope S QBUWME pine specie demetilae TA RAARAS integrin cot QvOTKO uncharacterized prten KIAALIO? QUBTTS ——Sigetve organ expansion fet homelogse QwOTQ? ——abiguiin ashorytrminlhydelve C¥LD owe al homatogve subfamily B member QUKIN? ——_lechtrn homelogy ke domain family B member 2 QBDBME personal Ianctionalensyne QSCFEH SCY protein QUBW2S ——_Newacerpleasfrase 25, NB aur sua auvces ——pendin-t QYBHSS cn and agelacited protein 69 p97 RING finger protein 17 QpDSAL ——aneesventinal yosieth ase nyomesins QBEPRS —_VPSIO domain containing receptor SonCS2 sess centromere protein} pene eth pol factor 4 puis elaryte teasaton iiaon toe 3 subunit A api.cs apse Q9D4KT——_celledcildomain-cntining protein 105 QUBMAS prota NPAT Q9Q¥s2———_prosponsrecepor GPRST as0si0 "Tiymphoma irason ané mettre indacng protein 1 BOPP ES ubigutinprotein pe TRIM urs. SAM and SH domaincontaining protein 3 pubsacsorg/ehemneure ene seore fad change pace Hovgot 386 1405519997 ' co a7 sagsessas? Toced 10s4 4099956798 pit sas sure Pos na aime eter? eaa9 dames Nae mm dames atl 67 aaeisT6s Adan? us asmmsion 98 engi sos 4617669 OL Nespas st 4sisi76s oss aa vss 461817669 os Mee 3856 s7usessse Hesse 1738 sosmnsn Pests sss Ssassosoye) Kan? 173 saisssi0is ss miss Ssasseiees ape Kasey S636 rr) Diest nat ayn oye ps8 raarai ods Daas 10876 rae ro 48.05 Tousnsis ass Ehbagh nn smones 1 Soi moss Lansi7745 ' M99 L24sssou7 1 404 13.06812357 ' soa s.ase9esae 1 vast wsinaia7t 297920497282 ' sss eo8est ' ma aarisoe717 1 Capes, 3893 ssma7i085 1 caletos uo ator ose pt sor saseg72 ose Gps wise srasr46r99 03s Tamt 707816499371 0 “Tina mss aa7m2st6 1 Sus 4198 «ap0si0809 ' ‘Te reverts the ROT-induced neurotoxicity via downregulating the Tiamt expression, Aka serine/threonine kinase (also known as protein kinase B (PKB)], is necessary for neuronal survival as it phosphorylates its substrates, including GSK3, NF-xB, BAD, and forkhead proteins.” Downregulation of Akt signaling is seen in PD." Numerous studies suggest that neurotoxins that induce Parkinsonian symptoms including 6-OHIDA and MPP+ decrease the expression of pAkt.” Durgadoss et al demonstrated Akt signaling impairment in the MPTP induced mouse model, showing pAkt levels and loss of Akt kinase activity (Thr308 and Ser473)." Similarly, we have also reported a downregulated expression of Aktl in ROT-indueed Parkinsonian mice. Previous studies had suggested the crucial role ofthe mTOR pathway in autophagy and apoptosis leading to neuronal death but later it was found that the inhibiting AKC phosphorylation instead of mTOR activation induced neuronal degeneration.” Raghu etal reported the activation of Akt by G1-4A (a polysaccharide from Te).*" In an attempt to unravel the antiapoptotic action of Te against ROT intoxication, our study revealed an increased expression of AKt, also supported by Salama et al” "The present study showed that Te signiicantly reversed the ROT-induced downregulation of Grin2A expression, thus exhibiting neuroprotective effects against ROT induced neuto- toxicity. Moreover, downregulation of Grin2A (NR2A, a slutamate ionotropic NR-type subunit 2A) expression resulted Ca mediated neurotoxicity. NRe (N-methy-naspartate or NMDA receptors) are crucial factors in the pathogenesis of neurological disorders. NR were found to be downregulated in the patient's brain with neurodegenerative diseases.” NRs entail significant association between GRINI and GRIN2 (A~ 1D) subunits to permit Ca influx into the cell.” GRIN2A regulates excitatory neutotransmission in the brain and thus plausibly influences the course of PD. Kong et al confirmed that Grin? is significantly downregulated in PD fies against c= ACS Chemical Neuroscience pubsacsorg/ehemneure ‘Table 2A. List of Downregulated Proteins in PD vs Treatment Groups a Can Sd QNKHO ATP binding exe soba A member 8B abeath 42205918 797369 neutrophil eto! eter 4 Net wom ames 0 LQSPFAT neurons ron pheiporystedphosphoionte-kase adapter Nyt 491 assess 0 PIMIGL DNA (oosine $) methylase 1 Doml 499, aaLaas QERIQD ——ganni9 sms awol 261846 aot QURSCS sine Enger MYND domsin containing protein 11 Znyndl! 2004 9a7SAT! QBKIKE centromere protein empl «22850404208 QNB2Q7 anaphase promoting complex ssbuit 2 Anya «205860756 Q5920S —AUGGAP wih Rho-GAP domain ANK ropet abd PH domain-contlnng protein” Anp?—=«SB—OMSIG PUITSO RAC serine/threonine protein kinase aed 2 asim ats QSPCMI —Iysinespeiiedemetyase 3A Kimi 441 ag (035253 mothers psn decapentplegie homologse 7 Sms? 3984 aSssH8 Q8PS49——_phowhatdlinentel 34Sesphospate S-pesphate 2 Tnpell 1582056555. (097280 phospolpae DI ist 454s asoesst a HIG bosom reangftor 2, mitachondel Gin nas oss ant (980610 T-ymphoms invasion and metatade inducing protea ‘Tam «SLL a.8s0809 007 QHOTQ? ——abiqutincarhor! eins hyealare CYLD old 16s a6sro47 =o QpQYH? proses ecepter GPRS? Gyar moat genes a7 OSHS ceptortype yrodne protein phoophatae ets Pp 77 asses at ‘Table 2B. List of Upregulated Proteins in PD vs Treatment Groups cei Aesciton ene are fold change ples P3I7S0 RAC serine/threonine protein Kase Au fm Leos P4030 eanaegion fear A, mitochondl Tam sams Lagos QBUHS ——ducoriterctng poten 2 homologue B Dib 33820187532 QPDATS——abrson/NoCat checkpoint repute Zaye? 7352 A.SOTSB 0.96 QNOTH Uber syrome ypetG prtea homelogue we ao ask 753762 al ydroctbonreepor nace anlcstor I6st assis ost Q76NZA stn thconne protein phosphate 2A 6S ADs ceplatry eabunit A iar mais 2334599 a9 QNBLS6 cary endovome antigen I Beal 3406 2095656086 QEPRQ? Rar GTP ate acting protein + Rasat satan Q979Ks askin Cukiot == 2 ae 208 plein AB Plnss = as 39660 P7067) nace plypepte-stociated complex subunit muted fom Nea sid 7690609 QSSXTS nce anemia group J protein homologee 3 aun sa7isy7 ar Oss eakarytie waptation ntition eter 53 Bib aos 1124St6 8 WPQUOS shore tansent receptor potential channel 4 Tipet ob teas PIsa36——_glutunate receptor xaotopig NMDA 2A Gene ’ rsmom 1 synuclein neurotoxicity.” Interestingly, in the present study, ‘we have documented a downregulated expression of Grin2A in ROT-induced Parkinsonian mice, whereas Te upregulates the Grin2A expression against ROT intoxication. Similarly, Simon et al. and Hamza etal also reported Grin2A association with the risk of PD."°”” Downregulation of Grin2A showed a lack of binding to Grint resulting in redueed NMDAR complex formation. By considering all of the aforementioned studies, the potential of targeting Grin2A for the therapeutics of PD pathogenesis is confirmed, Te plays an important role in attenuation of NMDARvdependent Ca!*-mediated signaling via downregulating Grin2A. Epigenetic dysregulation has emerged as_an_ important component in PD pathogenesis.” Kdm3A is a JejC Jumonji) domain-containing lysine (K)-specific demethylase 3A, Kdm3A-mediated demethylation of PGC-1a (peroxisome prolierator-activated receptor y coactivator I-a) causes Inhibition of subsequent mitochondrial biogenesis and oxidative metabolism via reduction in PGC-la and Neil/2 binding and Nefl/2dependent ‘Tiam expression.” Damtl [DNA (cytosine-s)-methyltransferase 1] is an_enzyme catalyzing DNA methylation via methylating CpGs on hhemimethylated DNA. Hypermethylation of the PGC-1a promoter resulls in the reduction of PGC-la protein expression. Downregulated PGC-1a exacerbates age-depend- ent neuroinflammation by enhancing the release of 1L6."” Gene Ontology and Functional Pathway Enrichment Analysis. To comprehend the potential functions of DEPs in PD, these DEPs were examined. GO analysis (hep: /iww. gencontology.org) of most significant DEPs based on bio- logical process (BP), molecular function (ME), and celular component (CC) has been shown in Figure 5. This analysis revealed that the dopamine metabolic process, catechol- containing compound metabolic process, catecholamine retabolie process, adherens junction assembly, mitochondrial gene expression, phenol-containing compound metabolic process, oligodendrocyte diferentiation, adherens junction ‘organization, positive regulation of protein modification by small protein conjugation or removal, and response to toxie substances were found to be signicantly enriched in BP. ACS Chemical Neuroscience ae EE pubsacsorg/ehemneure Figure 4. Construction of heat map. The heat map represents the expression level of the resulting proteins. Here, the resheoloed regions indieate {he high level of prozein expression, the orange regions represent the mid-value ofthe protein expression and the yellow regions indicate the low capretion of the protelns, Samples are clustered on the bass of relative expresion, accession number, and pale ‘Table 3. Enriched Wikipathway" gene st eset te wraes Hie eect on hepa production 7 wens ‘mitochondrial gone eesion » wrinss thet wou for dg alton 3 wrssis Parkinsons dear pathway » wrus “TGF signing pathway 2 rss hang trois a wr207s ‘Aimer dea 1s wrass ESC plonpotencypathwaye us wens? odorant GPCRS 189 wens fherokine signing patwny 190 epee lo pave FOR 0.007% ist 0781 1 anos asars 020459 ' ones ai osama n01ass oasis ones ' sess waa vossan3 1 056382 15098 sosisas ' ons 78081 oni 1 2063 sare son 1 “Here, gene ets indicate the pathway ID in vikpathway, Description represents the name ofthe pathways in which resulting DEDs were found Sine indicates the total number of proteins in each pathway. False discovery rate (FDR): for dark blue color FDR < 005 and fr lightlue color FDR > 0.05. Enrichment ratio the rato ofthe observed count over the expected count by chance. Furthermore, the NMDA-selectve glutamate receptor com: plex, neuron spine, cation channel complex, extrinsic component of the plasma membrane, cell-cell junction, cell junction, cell surfce, transmembrane transporter comple, ion channel complex, and extrinsic component of the membrane ‘were significantly enriched in CC. Moreover, the TGF-f receptor, eytoplasmic mediator activity, and ligand-gated ion channel activity play key roles in maintaining the presynaptic ‘membrane potential; Ca*-transmembrane transporter activity, P-catenin binding, heat shock protein binding, NMDA lutamate receptor activity, S-catenin binding, and Ca" channel activity were significantly enriched in MF. A p-value -<0005 was considered significant for enriched GO analysis. To explore the enriched pathways associated with PD Webgestalt (burps Awwnewebgestalt.ong) sofware was used. In this study, ‘we analyzed various significant pathways through Wikipathway bttps/ /wwew.wikipathovaysong/index php, WikiPathways), a8 has been shown in Table 3. Enriched pathway analysis demonstrated that DEPs were highly involved in the PD pathway (Gpr37), Alzheimer's disease pathway, hypothetical network for drug addiction (Grin2a), mitochondrial gene capression (Tam), lung fibrosis, TGF-f signaling pathway, ie effect on hepcidin production, ESC pluipotency (Smad”), and chemokine signaling pathway (Tiam!). {In Silico Analysis through the PPI Network. PPIs play a crucial role in understanding the molecular function of DEPs, responsible for PD onset. Ar the outcome suggests, hub genes like AKTI, PARK2, MTOR, TFAM, PINK, TH, SNCA, PIDI, PARKT, LRRK2, and App were found with the highest degrees of connection in the network (Figure 6A). Ina contacted network, DEPs connected to each other generally have analogous functions and ean be considered as functional genes. A gene—gene interaction network wat constructed to Jnestigate th interaction of significant proteins inthe network (Figure 6B. Further Confirmation of the Neuroprotective Role of Te by Regulating Various Signaling Pathways. Using RT-PCR. This result confirmed the therapeutic tole of Te in rodulating pathogenesis of PD in the mouse model through modulating mulple genes. gRT-PCR was performed (Figsre 1) with GAPDH as-an intemal mRNA control. The result revealed upregulation of SMAD? (1.97-fold), KDMBA (1.97- fold), DNMTI (2.93-ld), and TIAMI (290-018) in ROT- induced PD mice in comparison to healthy contol, while ‘TRAM (033-fld), AKT! (037-fld), GPRS? (0.46 fold) and ACS Chemical Neuroscience ubsacsorg/chemneuro en eee my ‘cement ene cn Figure 5. Gene ontology ensichment analysis on DEPs. A Web>bated tool Webgestalt was used to perform the GO analysis, which Includes three rain modiles, Le, biological processes, molecular fanction, and cellular component. Grin2a (0.23-f0ld) were seen to be downregulated. Following ‘Te treatment, SMAD7 (0.17-f0ld) expression was seen to be significantly reduced, along with KDM3A (0.27-fold), DNMTI (0.32-fold), and TIAMI (0.62-fld), and an incre- ‘ment was found in the levels of TEAM (1.10-fold), ARTI. (1.394old), GPR37 (1.5S-fold), and GRIN2A (1.73-fold), as shown in Figure § displaying the fact that Te plays a significant potential role in PD pathogenesis via regulating different signaling pathways. From the result of RT-PCR, itis clestly evident that Smad7, Gpr37, Kdm3a, Damt, and Tiaml are upregulated in ROT-injected mice, whereas significant restoration was observed in the Te-treated group. Alternatively, the mRNA levels of Téam, AKtI, and Grin2a were reduced in the ROT group, whereas they were restored in the Te-treated group. These results demonstrate the neuroprotective activity of Te in ROT-injected mice via regulating diferent signaling pathways at the mRNA level ‘Overall, epigenetic methylation of PGC-1a positively regulates initiation and progression of PD. Downregulation of PGC-1a via inhibition of Dnmsts-mediated hypermethyla- tion, or Kdm3A-mediated demethylation, plays a central rle in PD pathogenesis by improving mitochondial dysfunction and oxidative and inflammatory stress. In this study, results have shown the downregulation of Dnmts and Kdm3A genes after ‘Te treatment compared to ROT-induced Parkinsonian mice Hence, Te may be a potential therapeutic agent for cpigenctically modified targets in PD pathogenesis (Figure 8). CONCLUSIONS ‘The present study explored the potential neuroprotective property of Te in an ROT-intoxicated PD mouse model Besides, we have also tried to investigate the underlying ‘mechanism of action in restoring the level of various protein molecules including Aktl, Smad?, Gpr37, Grin2A, Tiaml, "Ae Gem erne ACS Chemical Neuroscience e A B gure 6, Protein~protin interaction network construction among DEPs, long witha gene—gene interaction network (A) ‘The PPL network was cotracted by the database named sing. (B) In gene~gene interaction, nodes with fines on there were our renin pts, whereas nodes ‘without lines on them were the proteins fom the database and it was pexformed by Genemana. In both networks, the diferent clared edges, Indicate the dierent interactions among resulting DEP. The width ofthe edge eepresents how sng the interaction is between the resulting DBs and among predicted proteins p< oot “*pcoor : *p<00s igre 7, Fold change in gee expression of alos gone a ROT: expressed at mean = SEM (n= 3) (4p < 001, **4p «0001, tosleated and Covteated mice, in eompatton to healthy contol Values are ). I EXPERIMENTAL PROCEDURE Plant Extract Preparation, ‘Te stems were procured fom Botanical Garden, Insitute of Science, Banaras Hindu University, Varanasi, Indi, in Jansary 2019. Te stems were died for 7=10 days ‘Tfam, Damtl, and Kdm3a through proteomics and qRT-PCR. In conclusion, the overall findings of this study provide a new insight that Te exerts therapeutic effects through the regulation cof various signaling pathways by protecting the DAergic neurons and restoring the mitochondrial function, However, farther studies are needed to explore the bioactive components of Te that play essential roles in regulation ofthe disease. in shade and pulverized with the help of an electee grinder. The deed sample war exracted with ethanol and water solvent st ratio of 70:30 (200g of power into 1000 mL) at 40 °C for 16h in a Soslet apparatus. Subsequently the plat extract was filtered theough 3 045 we fer, and the residue was concentrated under reduced pressure "AS Ge Near IL pubsacsorg/ehemneure — d S). _® SB | — All A Figure 8. Graphical representation ofthe methodology wsed for LEQ analysis of control, PD, and treatment groups, Proteins were iolated and digested with multiple proteases followed by LC-MS/MS, Bach sample was processed in tplicate. ing a ote vaca evaporator. The plant exacts ate expressed in terms of diy weight Ethical Statement. The case and maintenance of the cexperinentl animale were caried out in st acordance withthe final eis procedure and guidelines ofthe Institutional Animal shical Committee ofthe Banaras Hinds University, Varanasi India (Gof no. BHLY/DoZ/IABC/2018-19/032). Al eforts were made to ce suring of mice used in the sud Male Swiss albino mice (25 Sg) were obtained from the animal fact a the Insitute of Medial Science, Banaras Hindu University, YVarana (tna). The mice were housed in clean polypropylene cages swith constant lght—sark eyles (12/12) prior tothe experiment. Ui the dosing was completed, mice were supplied with water and standard det pelts ad Mbitum. ‘The experimental protocol was tablished in acordance with the guidlines ofthe Animal Ethics Committes, Banaras Hinds University, Varanasi Indi, ‘Animal Dosing. Animals were randomly assigned into three roups with eight mie per group: contol (vehicle group), rotenone {G mri body wt/day), and rotenone + Te (200 mg/kg body wt! day) Rotenone was subeataneouly injected for 35 day, whereas Te ‘was orally administered for 7 days before the ROT intoxcaton and concurrent for 21 dayz!”?* Upon completion of the dosing, ‘behavioral analysis was eared out, andthe mice were scriced to tolate the Brains for proteome analy, All experiments were cased ue in tiple, Behavioral Analysis. ‘To determine the effect of ROT Intosteation on the motor activty im the Parkionian mouse rmodel, behavioral parameters including the catalepy test, rotarod text, poe test, and footprint assay were performed Catalepsy Test, Sines inthe muscles of mice canbe estnated by placing the forelimbs of mice onan elevated bar andthe hind libs on the ground. Catalepsy intensity war measured by recording the time when the mice moved thi ind lim fom wooden platform {Gem in eight) to correct thelr posture. The time was recorded in seconds andthe test was discontinued when the tine exceeded 180, Pole Test. For two consecutive days, the mice were trained. The periormance was recorded after the last ROT injection. Mice were Placed heas-up on the top ofa rod (10 mm in diameter, $2 em in eight, with a rough surface), Tura (tne taken to orient downwatd) and T-descend (Uime require forthe mouse to step down the rod Teogth) were recorded in seconds, and the experiment wat Sitconinsed ser 300.” Rotarod Test. For the rtrd test, each mouse was tained ina rotaod before the experiment for thee comectie days, a 4 set, ‘peed of 1 rpm. The time was recorded wnt the morsel fom the ‘Ptrod and eags were recoded up on. For ech mouse, the text war repented ive time, and the mean time war eecodeds The difrence in fal sme observed between the PD group andthe Te- treated group isan indicator af muscle rlzation Footprint Assay. Inthe fotpin te the mice were tained to walk on a whitepaper for thtce consecutive days. The length of the ‘ide wae calated by immersing the mouse’ forefoot in Back nk tnd hindfoot in red ink and then meaeurng the gap between the ‘eps, om the mile toe (st tp) tothe hel (econ step) onthe same side ofthe Body. Footpent aay was conducted tice for ‘ch abil Stade forepaw lengthe wee recorded sn cetinetrs Immunofluorescence Staining of TH in SNpe. Immunohis tochemicl taining of TH. postive DA nedrons in the SNpe pon of the brain wat pesformed wing standard. proceduses"™ In shor, intacardally perfused mouse Brains were caleted and porzed (Gos PEA), th ation, «cryostat (Lea, Wet, Germany) was ed to cal a 26 m thick coronary br alee atthe SNe level Sreons were wased twice wih PDS (001 M pit 74) and thea incubated witha blocking agent (IO NGS-PBST) for 1h The sections were then incubated wih a primary polyclonal antimouse tbody aunt TH (11000) at °C fr 16h Sections were washed Ses wth PBST, andthe secondary antibody (Cy2-onjugated) wa incabated in a'1% BSA Blocking solution for 1'h at RI The Jeans were then washed 3 nes and mounted wih the mounting media (Fluoroshel, Sims Aldrich). Images at 20% magnieation were taken with a Buoresence microzcope (Nikon, Kyoto, ap8). Iinages were analyzed by liege} softwate (NIH) and reported at sean % see values Tissue Collection. After behavioral tests ice were sariiced by cervical dislocation flowed hy despttion tense mira pin. “The brain ofeach mosse was elated and instantly frozen. Late, he brain war divected under ice-cold conditions to late the rigrostratal tue fom the midbrain and war stored at —80 °C ol fuer experiment were performed. ‘Sample Preparation for LFQ Analysis. Protein extraction was done following the previous method of Gupta etal with sght ‘modifications The ban tue war thawed and homogenized using ACS Chemical Neuroscience pubsacsorg/ehemneure ski mie ‘cra - li ante y ar Figure 9 Pictorial representation ofthe pathways found tobe involved in PD progression and modulated by Te treatment in the Patkinsonian mice model the ysis bute (RIPA and proteae cocktail inkbitor) an incubated for 90 min on ie. The homogenate was centrged a 12000 rpm at 4 °C for 20 min, and the supernatant war calleted in a fresh Eppendorf tabe. The proteins were quand by the Bradiordauay and expresied in terms of mg/mL /gUsue”* Quantitative assessment ofthe protein samples was performed by SDS-PAGE. For digestion, 100g ofthe protein sample was taken from each roup. The samples were disted with $0 mM NHLCO, protein followed by treatment with 100 miM dithiothreitol (DTT) at 85 °C for 1b Then, 260 mM iodoucetamide (IDA) was aed at RT inthe dark and incubated for 48 min. The samples were then digested with “Trypsin (Trypsin god Promega) and incubated overnight at RT. The reving sample was vacuum dred and dioved in $0 al of 01% formic acid (FA) in water, followed by centfgation at 10000 rps, and che supernatant was collected into 4 separate tube. Injection volume of 10 pl was used on the BEH C18 UPLC column for separation of peptides. The mobile phae contained 0.1% FA in water {Quter A) and 0.196 FA in acetone (98:2 v/ for 1~30 ml, 50:S0 ‘iv for 3040 min, 20:80 v/v for 40-50 min, and then 98:2 vv for {he next 30 min), and the flow rte was set at 03 ml/min. The sfotamate level was enamined at a wavelength of 472 nim, and the concenttaton was determined using a standard curve. Thee runs pet sample were cared out for LFQ The separated peptides on the column were directed to Waters Synapt G2. Q-TOF instrument for (MS and MS/MS analyes. The raw data was procesed by MassLynx 41 WATERS. The individual peptide MS/MS spectra were matched to the database sequence on PLGS software (Proteiniynx Global Server Sofware scores cach protein based on the significance of the protein beng denied, bated om the MS pattem and MS/MS pattern ‘matching foreach peptide as well asthe coverage) and WATERS for protein identification (Figure 8). The instrument wed for acquiring Mass Spee Data was connected with Waters Synapt G2 (Q.TOF), ‘The iutrument parameters ured for Wdentication were a follows peptide mass tolerance at the MSI level: 0 ppm and fragment mass tolerance a the MS? level: 100 ppm, During processing ofthe sample cysteine sites were mofed to carbamiomethylated eysteine and the methionine ates, belag prone to oxidation, were considered a a ‘arable modification tothe mass””** I BIOINFORMATICS ANALYSIS Gene Ontology and Pathway Enrichment Analysis. ‘The biological significance of DEGs was investigated using WebGestat as itis a free online Web-based gene set analysis toolkit for fonetional enrichment analysis in different biological levels. In GO and pathway enrichment analyses, we obtained sore valuable knowledge about cellular, molecular, and biological processes. A p-value <0.05 was considered a significant enrichment.” Protein-Protein Interaction Network Analysis. Search tool forthe retrieval of interacting genes database (STRING) is a database and Web resource for forecasting PPI networks. "To investigate the relationship between DEGs, the STRING database was used. Our active interaction source of data was text mining, experiments, database, coexpression, gene fasion, and co-occurrence. The minimum required score is set at 0.7 (higher confidence). By analyzing the predicted interaction networks, we can propose new directions for future ‘experimental esearch and come up with cross-species forecasts for systematic interconnecting mapping.” Investigation through KEGG Mapper Tool and Genemania. The KEGG mapper tool is used for eellular or biological interpretation of large-scale datasets like genome or meta genome sequence.” It comprises three diferent databases, Brite, Pathway, and Module, which further contain caperimental information from the already published literature and represented in the form of these three liferent pathways Bioinformatics tool such as Genemania was used for imaging and visualizing the different interacting networks between different genes.”> The interaction between genes may be physical, correlational, colocalized, and genetic interactions. It also provides tinge information about the interaction with ‘unknown genes from already published literature. ‘Quantitative Real-Time Reverse Transcriptase Poly- merase Chain Reaction (qRT-PCR). For gene expression analysis, total RNA was isolated from the SNpe region of different groups of mice according to the manufacturer's ACS Chemical Neuroscience protocol (Invitrogen, Carlsbad, CA). After isolation, RNA {quantifcation and purity were estimated on A260/A280 using Nanodrop 1000. cDNA was prepared by the reverse tan scription process, and for ths, 2 4g of total RNA (kept equal for each amplieation), 20U M-MLV reverse transcriptase (Fermantas, Germany), 20 mM NTPs (New England Biolabs), 1x RT butfer, 20U RNasin (Fermentas, Germany), 100 ng of random hexamers (Fermentas, Germany), and 0.1 M DTT with DEPC-treated water were used Gene expression profile analysis was performed on the ABI7S00 Fast system, ‘The PCR reaction was carted out at stated in previously reported studies witha few modifications." ‘The real-time mix includes $ yL of SYBER green master mix (Applied Biosystem), 0S Leach of forward primers and reverse primers (domtl, Akt1, Tiam1, Yam, Gpr37, smad7, Grin2s, Kdm3a), 1 ul of eDNA, 1 iL of RNase inhibitor, and 2 pL of nuclease-fce water. The PCR reaction was set up according to the following protocal: initial incubation at $0 °C for 2 min, denaturation at 95 °C for 10 min, and 40 eyeles a 95 °C for 15 60 °C for 1 min, and 72 °C for 40 s. The abundance or decline of mRNA was normalized to the geometric average of the endogenous control GAPDH for AC, The fold change (AC) was ealeulated by the 2° 84° method and reported as an arbiteary unit Statistical Analysis, Statistical analysis of behavioral data ‘was done by one-way analysis of variance (ANOVA) using the Student—Newman—Keuls test by GraphPad Prism 60. For gene expression, we applied the unpaired Students fest to ‘unequal variances to find the p-value fr each gene. The results are expressed as mean + SEM. pwalues <0,05 were considered statistically significant ASSOCIATED CONTENT @ supporting information The Supporting Information is avaiable fre of charge at butps/pubsaesorg/doi/10.1021 /aescherneuro.1<0048 Te dose-dependent response on body weight (Supple mentary figure 1A); Te causes reduction of the DPPH radical (Supplementary gure 1B); and dose

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