PGC-1alfa controls mitochondrial biogenesis and dynamics in lead-induced neurotoxicity

          INTRODUCTION Peroxisome proliferator-activated receptor-γ coactivator- 1α (PGC-1α) is a transcription factor controlling many aspects of oxidative metabolism, including mitochondrial biogenesis, adaptation, respiration, adaptive thermogenesis, gluconeogenesis and oxidative phosphorylation [1, 2]. Signalling for mitochondrial biogenesis (MB) is activated by PGC-1α and involves the expressionof several transcription factors, resulting in the upregulation of proteins encoded by both nuclear and mitochondrial genomes. Oxidative stress from the generation of reactive oxygen species (ROS) has often Research Paper been cited as toxicants, and is believed to originate from mitochondria. Many neurodegenerative conditions have been shown to originate from compromised number, morphology and function of mitochondria. PGC1α has previously been reported to play a protective role against neurodegenerative conditions such as Alzheimer’s disease and is known to respond to ROS by the induction of many ROS-detoxifying enzymes, including superoxide dismutase, Gpx1and γ-glutamylcysteine, regulating the biosynthesis of glutathione. Moreover, Ca2+ and ROS have been shown to regulate mitochondrial biogenesis by activating PGC1α, leading to an increase in mitochondrial mass [3, 4]. PGC‐1αcontrolsmitochondrialbiogenesisanddynamicsinleadinducedneurotoxicity  AleksandraDabrowska1,JoseLuisVenero2,RyotaIwasawa1,Mohammed‐khairHankir3, SunniyatRahman1,AlanBoobis1,andNabilHajji1  1ImperialCollegeLondon,CentreforPharmacologyandTherapeutics,DepartmentofMedicine,London,United Kingdom; 2DepartamentodeBioquímicayBiologíaMolecular.FacultaddeFarmacia,UniversidaddeSevilla,C/Prof.García González,Sevilla,Spain; 3IntegratedResearchandTreatmentCentreforAdiposityDiseases,DepartmentofMedicine,UniversityofLeipzig, Leipzig,Germany  Keywords:neurotoxicity,Lead,mitochondrialbiogenesisanddynamics,PGC‐1α,Drp1,BAP3,calcium Received:05/18/14;Accepted:08/03/15;Published:09/04/15 Correspondenceto:NabilHajji,PhD;E‐mail:[email protected]  Copyright:Dabrowskaetal.Thisisanopen‐accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,which permitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited  Abstract: Duetoitsroleinregulationofmitochondrialfunction,PGC1αisemergingasanimportantplayerinageingand neurodegenerativedisorders.PGC1αexertsitsneuroprotectiveeffectsbypromotingmitochondrialbiogenesis(MB)and functioning.However,thepreciseregulatoryroleofPGC1α inthecontrolofmitochondrialdynamics(MD)and neurotoxicityisstillunknown.HereweelucidatetheroleofPGC1αinvitroandinvivointheregulatorycontextofMBand MDinresponsetolead(II)acetateasarelevantmodelofneurotoxicity.Weshowthatthereisanadaptiveresponse(AR) tolead,orchestratedbytheBAP31‐calciumsignallingsystemoperatingbetweentheERandmitochondria.Wefindthat thishormeticresponseiscontrolledbyacell‐toleratedincreaseofPGC1α expression,whichinturninducesabalanced expressionoffusion/fissiongenesbybindingtotheirpromotersandimplyingitsdirectroleinregulationofMD.However, dysregulationofPGC1αexpressionthrougheitherstabledownregulationoroverexpression,renderscellsmoresusceptible toleadinsultleadingtomitochondrialfragmentationandcelldeath.OurdataprovidenovelevidencethatPGC1α expressionisakeyregulatorofMDandthemaintenanceoftoleratedPGC1αexpressionmayofferapromisingstrategyfor neuroprotectivetherapies.  www.impactaging.com AGING, September 2015, Vol 7 N 9   www.impactaging.com    629AGING,September2015,Vol.7No.9 Growing evidence suggests that the delicate equilibrium between mitochondrial fission and fusion is vital for many mitochondrial functions including metabolism, energy production, Ca2+ signalling, ROS production and apoptosis. In many types of neurodegenerative diseases, a significant reduction in the levels of fusion proteins optic atrophy-1 (Opa-1), mitofusin-1 (Mfn-1) and mitofusin-2 (Mfn-2) as well as the fission protein dynamin-related protein-1 (Drp-1) is reported, as well as an increase in the level of the fission related protein-1 (Fis-1) [5]. A number of signalling processes relating to mitochondrial, nuclear and inter-organelle communication, have been shown to be sensitive to ROS. Upon oxidative stress, the mitochondria and the endoplasmic reticulum critically contribute to apoptosis induction via a signalling pathway that establishes a feedback loop by releasing Ca2+ from the ER which in turn activates the mitochondria for apoptosis [6]. The majority of mechanisms proposed for neurotoxicant-induced cellular damage converge on mitochondrial dysfunction and energy deprivation [7].The integrity of this organelle is vital in the context of neurodegeneration due to the high-energy demands of dopaminergic neurons as a result of their unusually long axons [8]. This generates a need for the stringent regulation of mitochondrial biogenesis, function and dynamics in such cells. Disruption of any of these processes can contribute to neurodegenerative disorders [9] such as Parkinson’s [10], Alzheimer’s [11] and Huntington’s disease [12]. However, no study has fully elucidated a link between mitochondrial biogenesis and dynamics in neurotoxicity, highlighting the need for a thorough investigation into PGC1α’s role in those processes in the context of neurological disorders. Chronic exposure to the environmental neurotoxicant lead is believed to be associated with neurodegeneration [13]. While it has been proposed that there is an association between chronic lead exposure and risk of Parkinson’s disease (PD) [14, 15], the exact mechanism is not fully understood. Exposure to lead induces oxidative stress in the form of ROS in a variety of different tissues [16, 17] and also induces Ca2+ release from the ER in hippocampal neurons [18]. However, evidence is lacking with regards to the mechanism of neuronal damage and whether its induction in chronically exposed individuals is due to an increased production of ROS or disruption of calcium homeostasis or both. In this study we investigated the role of PGC1α as a regulator of both mitochondrial biogenesis and dynamics in vitro and in vivo in the context of leadinduced neurotoxicity. Strikingly, the role of PGC1α was found to be equivocal. While a modest increase of PGC1α expression provides a neuroprotective role, dysregulation of expression either by knockdown or overproduction is non-tolerated, leading to neurotoxicity. Our data showed a significant increase of expression of a mitochondrial fission protein, DRP1, in both,neuronsin the rat substantia nigra (SN) and adopaminergic (DA) neuronal N27 cell line. SN cells showed an adaptive response to lead exposure by increasing the transcript levels of PGC1α, its target genes, regulating mitochondrial biogenesis, and mitochondrial fusion- and fission-related genes. ChIP and QPCR analysis revealed that PGC1α directly regulates DRP1expression by binding to its promoter. Our data provides strong evidence that PGC1α is directly involved in the cellular response to lead by regulating mitochondrial dynamics. RESULTS Exposure to lead induces mitochondrial dysfunction, energy depletion and subsequent apoptosis of dopaminergic neurons N27 cells, derived from rat mesencephalon, were used to evaluate the effect of lead on mitochondrial function. Cells were treated with a range of lead (II) acetate concentrations over a period of 48 hours. Cells stained with MitoSOX and analyzed by flow cytometry revealed a 25% increase in mitochondrial superoxide at 5 µM and 100 µM concentrations and almost a 100% increase at 500 µM (Fig. 1A). DiOC6(3), a selective mitochondrial dye, and propidium iodide (PI) staining revealed that lead causes mitochondrial membrane depolarization and neuronal cell death, which was much more marked at 500 µM than at 5 or 100 µM (Fig. 1B, C). PARP cleavage analysis (Fig. 1D) confirmed this, with the effect being far more pronounced at the highest lead concentration. At 5µM and 100µM, effects on mitochondrial dysfunction and cell death were similar (Fig. 1A-D). Apoptotic cell death was confirmed by an increased activation of caspases3/7 and caspase 8 in N27 cells exposed to 100µM and 500µM lead (Supplementary Figure1-S-1, A-a and b). Morphological characteristics of apoptotic cells, including condensed nuclei, were also evident in cells treated with 500µM lead (Supplementary Figure1-S-1, B and C). Morphological changes in the cells were apparent only at higher concentrations than changes in molecular markers of apoptosis, most likely reflecting the temporal engagement of the respective hallmarks of apoptosis.   www.impactaging.com    630AGING,September2015,Vol.7No.9 Mitochondrial function was assessed by Seahorse XF24 analysis allowing for the measurement of a number of parameters including mitochondrial basal respiration, ATP production, oxygen consumption rate (OCR), maximum respiratory capacity and mitochondrial spare respiratory capacity. While a reduction in basal respiration was observed in cells treated with 100 and 500 µM lead (Fig. 1E), this was not statistically significant. There was a concentration-dependent decrease in ATP production (Fig. 1F), with the highest lead concentration causing a statistically significant (P<0.01) decrease of more than 50%. Proton leak was not significantly affected by lead treatment (Fig. 1G). Maximal respiration decreased in a concentrationdependent manner (Fig. 1H). Interestingly, mitochondrial reserve capacity levels were very low across all of the lead-treated cells (P<0.01) including 5 µM, a concentration which did not have a significant impact on other aspects of mitochondrial function investigated with the Seahorse XF24 compared with control (Fig. 1I). Figure1.ExposuretoleadinducesmitochondrialdysfunctionandsubsequentapoptosisofN27dopaminergic neurons.Cellswereincubatedfor48‐hourswithleadacetate,attheconcentrationsshown.(A)Mitochondrialsuperoxide levelsweremeasuredusingMitoSOXstainingfollowedbyFACSanalysis.(B)Theeffectofleadtreatmentonmitochondrial membranepotential(MMP)losswasdetectedbyDiOC6(3).(C)Analysisofcelldeathmeasuredbypropidiumiodide(PI).(D) WesternblotanalysisofPARPcleavage(PARPtoPARPc)followingleadexposure.Profilesofdifferentparametersof mitochondrialfunction(E‐I)weredeterminedusingSeahorseXF24Analyser.*P<0.05,**P<0.01,***P<0.001,n=3;mean±SE.   www.impactaging.com    631AGING,September2015,Vol.7No.9 Figure2.PGC1αprotectsN27dopaminergicneuronalcellsfromPb2+‐inducedneurotoxicity.Cellswereincubated withleadacetateattheconcentrationsshown.(A,B)qRT‐PCRandwesternblottingmeasurementofmRNAandprotein expressionlevels,respectivelyofPgc1αinN27cellstreatedwithleadfor48‐hours.(C)mRNAlevelsofPgc1αtargetgenes (Tfam,Nrf1)inN27cellstreatedwithleadfor48‐hours.(D)N27Pgc1αmRNAlevelsafter3‐hoursleadtreatment.(E)Pgc1α mRNAlevelsand(F)colonyformationinN27cellswithstabledownregulationofPGC1α.(G)Mitochondrialsuperoxidelevels weremeasuredinshPGC1αcellsusingMitoSOXstainingand(H)theeffectofleadtreatmentonmitochondrialmembrane potentiallossinshPGC1αwasdetectedbyDiOC6(3)inN27cellstreatedwithleadfor48‐hours.(I,K)Confocalimaginganalysis ofmitochondrialmorphologyinwildtypePGC1αandinoverexpressedPGC1αN27cells(↑Pgc1α)wasinvestigatedusingMito TrackerandDAPIstainingrecordedbyfluorescencemicroscopy.(J)qRT‐PCRanalysisofPgc1αinN27cellsexpressing exogenousPgc1αversus↑Pgc1αcells,treatedwithleadfor48‐hours.*P<0.05,**P<0.01,n=3;mean±SE.   www.impactaging.com    632AGING,September2015,Vol.7No.9 PGC1α protects DA neurons from neurotoxic insults from lead Real-time QPCR and protein expression analyses were performed to investigate the expression of Pgc1α and its downstream targets, which regulate mitochondrial biogenesis, in lead-treated N27 cells. Interestingly, only the 100 µM treatment with lead for 48 h caused a significant increase of Pgc1α expression, at both the RNA and protein levels (Fig. 2A, 2B). Downstream targets of PGC1α, Tfam and Nrf1 showed a similar pattern of expression, with changes apparent only at 100 µM lead (Fig. 2C). In order to determine whether the highest lead concentration affects Pgc1α expression at an early exposure time point, Pgc1α mRNA levels were investigated after a 3-hour lead exposure. Surprisingly, the expression of Pgc1α mRNA levels was markedly upregulated 3-hour post-treatment, with a ten-fold increase observed at 500 µM (Fig. 2D). Since no significant difference was observed between cells treated with 5 µM and 100 µM in terms of disruption of mitochondrial function, ROS generation or cell death(Fig. 1A-D), but there was a significant difference in Pgc1α mRNA levels between the two groups, we sought to investigate whether Pgc1α plays a protective role against lead insult at 100 µM. To interrogate this hypothesis, we produced a stable knockdown of Pgc1α using a lentiviral shRNA vector. Successful knock-down was confirmed by Pgc1α mRNA levels(Fig. 2E) and clear downregulation of PGC-1α protein levels, as shown by immunocytochemistry (Supplementary Figure3-S3, A). sh-PGC1α transduction did not affect colony formation (Fig. 2E and F). FACS analysis of cells stained independently with MitoSOX and DiOC6(3) showed that reduction of Pgc1α expression renders N27 cells more susceptible to lead-induced mitochondrial stress (Fig. 2G, H), with morphological characteristics of apoptosis apparent in cells treated with 100µM or 500µM lead (Supplementary Figure1-S1, B). The influence of lead treatment on mitochondrial organisation was investigated by immunofluorescence (Fig. 2I). Exposure to 100 µM lead caused mitochondrial filament elongation, while mitochondria in cells exposed to 500 µM Pb2+ were very fragmented and localized to the nuclear perimeter (Fig. 2I). An equivocal role of PGC1α in neuroprotection and toxicity To determine whether up-regulation of PGC1α was responsible for the changes in mitochondrial morphology and organization and subsequent protection of DA neurons from lead insult, we examined the effects of PGC1α overexpression on the structure of mitochondria (Fig. 2J and K) and (Fig. 3A and B). Intriguingly, overexpression of PGC1α actually increased susceptibility of N27 cells to lead toxicity at both 100 and 500 µM, as manifested by increased fragmentation of mitochondria, measured as a decrease in mitochondrial surface area (Fig. 3A and B).Whereas a two-fold increase in PGC1α expression protected cells against lead-induced neurotoxicity (endogenous levels of the protein doubled in response to 100 µM lead), higher expression levels (>50 fold) of the protein had the opposite effect and exacerbated the toxicity of lead (Fig. 2J and K). Furthermore, cells overexpressing PGC1α treated with 100µM and 500µM leaded showed condensed nuclei, an apoptotic hallmark (Supplementary Figure1-S1, C). Since exposure to lead induced changes in mitochondrial organization, the expression profiles of genes encoding proteins regulating mitochondrial dynamics were investigated. RT-QPCR analysis revealed that 100 µM lead shifts the mitochondrial dynamic profile towards fusion (Fig. 3C). While Mfn2 mRNA levels were increased, the transcript levels of Drp1, a fission promoting factor, were significantly decreased upon treatment. The Fis1 mRNA expression profile remained unchanged. To investigate the role of PGC1α alone in regulation of mitochondrial dynamics, Drp1 and Mfn1expression levels were investigated in PGC1α-overexpressing cells. Levels of Mfn1 expression mRNA) were significantly increased whilst those of Drp1 were significantly decreased (mRNA and protein) (Fig. 3D and Supplementary Figure 3-S3, B). This suggested that overexpression of PGC1α was accompanied by a shift of the mitochondrial dynamics profile towards fusion (Fig. 3D). ChIP assay was performed to identify regulatory targets of PGC1α. This revealed significantly increased binding of PGC1α to its own promoter and unexpected binding to the Drp1 promoter (Fig. 3E). Moreover, PGC1α bound to Nrf1 and SOD2 promoters. In order to compare the effect of lead on neuronal cells in vitro and in vivo, tissue samples were obtained from substantia nigra of rats given drinking water containing 500 ppm lead acetate for a period of 14 weeks as well as from control animals given water with acetate only. Real time QPCR analysis showed that Pgc1α mRNA levels, as well as levels of its downstream targets (Nrf1, Nrf2, Tfam) were significantly increased in the substantia nigra of animals exposed to lead (Fig. 4A). Expression of both mitofusins was also significantly induced by lead (Fig. 4A). Interestingly, unlike N27   www.impactaging.com    633AGING,September2015,Vol.7No.9 dopaminergic neurons exposed to lead for 48-hours in vitro, levels of theDrp1 transcript were increased together with levels of Mfn1 and Mfn2 transcipts, after lead exposure in vivo, with no decrease in the number of tyrosine hydroxylase-expressing, dopaminergic SN neurons cells (Fig. 4B and C). Since SN tissue used in the experiment was a heterogeneous mixture of different cell types, including neurons, we performed dual fluorescence immunohistochemistry of DRP1 and NeuN (a neuronal specific marker) in the ventral mesencephalon to determine whether the previously observed increase in Drp1 expression was specific to DA neurons (Fig. 4D). Further analysis confirmed that the expression of DRP1 protein was significantly induced by lead in SN pars compact a neurons only (Fig. 4E). As adult rats are less susceptible to lead toxicity than young rats, and a significant increase of Drp1 expression was observed in vivo at 48-hours, we looked at Drp1 expression in vitro after short-term exposure (Fig. 4F). After 3-hours of lead exposure in N27 cells, an elevation of Drp1 mRNA levels was observed (P<0.01). Figure3.Lead‐inducedchangesinmitochondrialmorphologyanddynamics. Immunocytochemistryand confocalimaginganalysisofN27cellsco‐transfectedwithanemptyvectorandmitoYFP(A,a‐c)orPGC1α‐encoding vectorandmitoYFP(A,d‐f).Theextentofthemitochondrialnetworkbothunderbasalconditions(a),Pb2+100µM (b),Pb2+500µM(c)andPGC1α overexpressionalone(d)orcombinedwithPb2+100µM(e)or500µM(f)was measuredbySpectrum.(A,a’‐f’)mitochondrialareasurfacedeterminedusingImageJ‐>threshold‐ >Intermodes,(B,W).(B)MitochondrialsurfaceareaincellswasdeterminedusingImageJsoftware.(C)N27cells treatedwith100µMleadfor48‐hourswereanalysedfortheexpressionofmitochondrialfusion(Mfn2)andfission (Drp1,Fis1)genesbyqRT‐PCR.(D)qRT‐PCRanalysisoftheexpressionofDrp1andMfn1inPGC1αoverexpressing cells.(E)Chromatinimmunoprecipitation(ChIP)withaPGC1αantibodywasperformedtoinvestigatePGC1αprotein bindingtoitspromoter,Nrf1,Drp1andSOD2genepromoters,*P<0.05,**P<0.01,***P<0.001,n=3;mean±SE.   www.impactaging.com    634AGING,September2015,Vol.7No.9 Figure4.Invivosub‐lethalleadconcentrationindrinkingwaterincreasestheexpressionofgenescontrolling mitochondrialbiogenesisanddynamics.Adultratsreceiveddrinkingwatercontaining500ppmleadacetatefor14weeksand controlanimalsreceivedwaterwithacetateonly.SubstantianigratissuewasisolatedandmRNAwasextractedandprocessedfor RealtimeQPCRanalysis.(A)mRNAlevelsofPgc1αanditstargetgenesNrf1,Nrf2andTfamaswellasMfn1,Mfn2andDrp1were analysedbyqRT‐PCR;(B)Immunohistochemistrywasperformedtodetecttyrosinehydroxylase(TH)positiveneuronsand(C) stereologicalcountingofTH‐positiveneuronsinthesubstantianigraofratsgivendrinkingwatercontaining500ppmleadacetate for14weeks.(D)ImmunohistochemistryofDRP1andNeuNco‐localisationintheventralmesencephalonneuronalcells.(E)Drp1 signalintensitywasanalysedbyconfocallaserscanningmicroscope(ZeissLSM7DUO),usingtheassociatedsoftwarepackage(ZEN 2010)andquantifiedusinganImageAnalysisProgramfromSoftImagingSystem(analySIS®,Germany).(F)MfnIandDrp1mRNA levelsinN27cellsafter3‐hoursofleadexposure.*P<0.05,**P<0.01,***P<0.001,n=6(F,n=3);mean±SE.   www.impactaging.com    635AGING,September2015,Vol.7No.9 Figure5.Leadtreatmentcausesadisruptionofcellularcalciumbalance.Pre‐treatmentofN27cellswith1 mMN‐acetylcysteine(NAC)for1hourfollowedbya48‐hourslead‐exposurewasusedtoinvestigatetheimpacton mitochondrialROS(A)andmitochondrialdepolarisation(MMP)(B)N27cellstreatedwithleadfor48‐hourswere testedforcytoplasmic(C)andERcalciumlevels(D)usingthapsigargin(Tg)andRhod‐2stainingfollowedbyflow cytometryanalysis(D,aandb).Calcineurintranscriptlevels48‐hourspost‐leadexposurewereinvestigatedusing qRT‐PCR(E).Pre‐treatmentofN27cellswith5µMBAPTA‐AMfor1hourfollowedbya48‐hourslead‐exposurewas usedtoinvestigatetheimpactofcalciumonmitochondrialdepolarisation(F)andcelldeath(G)followinglead treatment.(H)WesternblotofBAP31proteinlevelsafter48‐hoursleadtreatment.(I)WesternblotanalysisofBAP31 levelsinshBAP31N27cells.N27cellswithstabledownregulationofBAP31(shBAP31)wereusedtoinvestigatethe involvementofBAP31inlead‐inducedmitochondrialsuperoxidegeneration(J),mitochondrialdepolarisation(MMP) (K)andactivationofcaspases3/7(L)and8(M).*P<0.05,**P<0.01,***P<0.001,n=3;mean±SE.   www.impactaging.com    636AGING,September2015,Vol.7No.9 Lead treatment causes a disruption of cellular calcium balance The possible role of ROS in lead-induced cell death in N27 cells was investigated using N-acetylcysteine (NAC), a well-known ROS scavenger. However, NAC pre-treatment of cells had no effect on lead-induced changes in mitochondrial ROS or membrane potential (Fig.5A and B). Therefore, we explored the possibility that changes in intracellular Ca2+ levels might mediate the effects of lead, since lead is known to mimic this cation inside the cell and compete with it for binding to Ca2+-dependent proteins. Moreover, calcium-regulated proteins have been shown to play a role in control of PGC1α expression [19]. Staining of cells with Fluo4-AM, a cytoplasmic calcium dye, revealed that lead causes an increase of cytosolic Ca2+ with depletion within the ER (Fig. 5C and D). Pretreatment of cells with BAPTA-AM, a cytoplasmic calcium chelator, conferred protection against lead insult (Fig. 5F and G). This finding, together with a lack of response to NAC on cellular susceptibility to lead, implicates a primary role for calcium in the response of dopaminergic neurons to lead. mRNA levels of calcineurin, a modulator of a number of key Ca2+ signalling pathways in neurons, were significantly increased on exposure to higher concentrations of lead, with peak expression occurring at 100 µM (Fig. 5E). Calcineurin mRNA levels at 100 µM and 500 µM mirrored those of Pgc1α and its target genes Nrf1 and Tfam (Fig 1A-C). Caspase-8 cleavage of BAP31 at the ER stimulates Ca2+-dependent mitochondrial fission and subsequently enhances pro-apoptotic signals between the ER and mitochondria [6, 20]. Since the protein level of BAP31 increased after exposure to 100µM and 500µM lead (Fig. 5H), we investigated whether BAP31 downregulation had any neuroprotective effect against lead insult. shRNA delivered by a lentiviral vector was used to knock down Bap31 expression in N27 cells (Fig. 5I).shRNA-Bap31 conferred significant protection against lead toxicity (Fig. 5J-M) implying an important role of BAP31 in the response to lead and in calcium pathway regulation. DISCUSSION In this study, we demonstrate for the first time that PGC1α regulates both mitochondrial biogenesis and dynamics in dopaminergic neurons exposed to neurotoxic lead (II) acetate. Despite a 20-fold concentration difference between 5µM and 100µM, the effects of lead acetate on mitochondrial superoxide generation and membrane polarization were comparable. As both of these concentrations exhibited minimal toxicity, they were classified as tolerated. However, at 500 µM, lead had a much more deleterious effect on mitochondrial function and subsequently induced excessive cell death; therefore this concentration was classified as toxic. Interestingly, while most parameters of mitochondrial function remained unaffected by tolerated concentrations of lead, spare respiratory capacity (SRC) was lost at all concentrations, a previously unreported effect of lead. This finding is consistent with the stochastic mitochondrial failure model proposed by Choi, et al. [21] to explain the importance of mitochondrial SRC in neurodegeneration. SRC is used here to describe the amount of extra ATP that can be produced by oxidative phosphorylation in case of a sudden increase in energy demand. Depletion of the SRC has been related to a range of pathologies affecting high energy requiring cells such as neurons [21, 22]. As N27 cells were able to tolerate concentrations of lead up to a toxic threshold, we suspected the presence of a protective mechanism, to help cells survive lead insult. PGC1α is a well-established master regulator of mitochondrial metabolism [1] and has previously been reported to play a protective role against a variety of neurodegenerative conditions [23, 24]. PGC1α is also known to respond to ROS by inducing a cellular ROS scavenging program [25]. Due to these diverse functions, we investigated whether PGC1α plays a role in the cellular response to lead exposure. Exposure of cells to 100 µM lead for 48-hours indeed resulted in increased expression of PGC1α and modulation of its downstream targets, including genes regulating mitochondrial biogenesis. Stable down-regulation of Pgc1α expression in DA cells increases their vulnerability to lead insult. Thus, we show for the first time that exposure to sub-lethal doses of lead induces low-level stress, which triggers an adaptive response mediated by PGC1α. This supports the hypothesis that PGC1α acts to protect the cells by promoting mitochondrial biogenesis, boosting their function and suppression of ROS [26]. We believe that at low lead levels, slightly increased ROS production, which acts as a retrograde nuclear signal, can improve mitochondrial function with adaptation to the stressor, a phenomenon known as mitohormesis [27]. Short exposure (3-hours) to lead induces a significant increase of Pgc1α transcript levels at 100µM and 500µM. However, whereas at 100µM the increase is sustained at 48-hours, at 500 µM, levels return completely to baseline by this time. Hence, lead modulates Pgc1α expression in a dose-and time-   www.impactaging.com    637AGING,September2015,Vol.7No.9