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Toxicology
jou rn al hom epage: www.elsev ier .com/ locate / tox ico l
-OHDA-induced apoptosis and mitochondrial dysfunction are mediated by earlyodulation of intracellular signals and interaction of Nrf2 and NF-�B factors
ulio C. Tobón-Velascoa,b,1 , Jorge H. Limón-Pachecob,c , Marisol Orozco-Ibarrac , Marina Macías-Silvad ,enaro Vázquez-Victoriod , Elvis Cuevase , Syed F. Alie , Antonio Cuadradof , José Pedraza-Chaverríb,∗∗ ,bel Santamaríaa,e,∗
Laboratorio de Aminoácidos Excitadores, Instituto Nacional de Neurología y Neurocirugía – S.S.A., Mexico City, MexicoDepartamento de Biología, Facultad de Química, Universidad Nacional Autónoma de México, Mexico City, MexicoLaboratorio de Neurobiología Molecular y Celular INNN-UNAM, Instituto Nacional de Neurología y Neurocirugía – S.S.A., Mexico City, MexicoDepartamento de Biología Celular y Desarrollo, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, MexicoDivision of Neurotoxicology, National Center for Toxicological Research – FDA, Jefferson, AR, USADepartamento de Bioquímica e Instituto de Investigaciones Biomédicas “Alberto Sols” UAM-CSIC, Centro de Investigación en Red Sobre Enfermedades Neurodegenerativas (CIBERNED),pain
r t i c l e i n f o
rticle history:eceived 19 October 2012eceived in revised form 2 December 2012ccepted 17 December 2012vailable online 26 December 2012
6-Hydroxydopamine (6-OHDA) is a neurotoxin that generates an experimental model of Parkinson’sdisease in rodents and is commonly employed to induce a lesion in dopaminergic pathways. The charac-terization of those molecular mechanisms linked to 6-OHDA-induced early toxicity is needed to betterunderstand the cellular events further leading to neurodegeneration. The present work explored how6-OHDA triggers early downstream signaling pathways that activate neurotoxicity in the rat striatum.Mitochondrial function, caspases-dependent apoptosis, kinases signaling (Akt, ERK 1/2, SAP/JNK andp38) and crosstalk between nuclear factor kappa B (NF-�B) and nuclear factor-erythroid-2-related fac-tor 2 (Nrf2) were evaluated at early times post-lesion. We found that 6-OHDA initiates cell damage viamitochondrial complex I inhibition, cytochrome c and apoptosis-inducing factor (AIF) release, as well
poptosisinases signaling
as activation of caspases 9 and 3 to induce apoptosis, kinase signaling modulation and NF-�B-mediatedinflammatory responses, accompanied by inhibition of antioxidant systems regulated by the Nrf2 path-way. Our results suggest that kinases SAP/JNK and p38 up-regulation may play a role in the early stagesof 6-OHDA toxicity to trigger intrinsic pathways for apoptosis and enhanced NF-�B activation. In turn,these cellular events inhibit the activation of cytoprotective mechanisms, thereby leading to a conditionof general damage.
. Introduction
Neurodegenerative events in Parkinson’s disease (PD) involve
oxic mechanisms such as oxidative stress, mitochondrial dysfunc-ion, apoptosis and inflammation (Miller et al., 2009). Oxidativetress has been shown to cause extensive damage to lipids, proteins,
∗ Corresponding author at: Laboratorio de Aminoácidos Excitadores, Institutoacional de Neurología y Neurocirugía, SSA, México City 14269, México.el.: +52 55 5606 3822x2013.∗∗ Corresponding author at: Departamento de Biología, Facultad de Química, Uni-ersidad Nacional Autónoma de México, México City 04510, Mexico.el.: +52 55 5622 3878.
E-mail addresses: [email protected] (J. Pedraza-Chaverrí),[email protected] (A. Santamaría).1 Doctoral Program in Biomedical Sciences, Universidad Nacional Autónoma deéxico (UNAM).
and DNA, resulting in cell death toward a variety of mechanisms,including activation of different apoptotic cell signaling molecules(Tobón-Velasco et al., 2010). Typically, unregulated reactive oxy-gen species (ROS) generation results in calcium dysregulation,excitotoxic cell damage and mitochondrial dysfunction, ultimatelyresulting in activation of apoptotic caspase cascades and promotedinflammatory processes by nuclear factor kappa B (NF-�B) activa-tion (Henchcliffe and Beal, 2008; Naoi et al., 2009; Tsang and Chung,2009; Kanthasamy et al., 2010).
6-Hydroxydopamine (6-OHDA), a hydroxylated dopamine (DA)metabolite (Cohen and Heikkila, 1974), is an oxidative neuro-toxin that causes a parkinsonian pattern of dopaminergic neuronalloss in rodents following its intrastriatal injection (Blum et al.,
2001). Although used as an exogenous neurotoxin in this model,6-OHDA can also be formed from dopamine in vivo, and ele-vated levels have been detected in body fluids of patients with PD(Andrew et al., 1993). Notably, 6-OHDA injury recapitulates several
eatures of degenerating neurons observed in human PD tissues.hese include proteasome inhibition, �-synuclein aggregation, oxi-ation and nitration of proteins, increased protein ubiquitination,leaved caspase 3 expression, glutathione depletion, and cytoplas-ic accumulation of activated signaling proteins (Bové and Perier,
012). A better understanding of 6-OHDA-mediated neurotoxicityould lend important insights into injury and degenerative path-ays shared among different causes of dopaminergic neuronaleath.
The mechanisms by which 6-OHDA elicits its neurotoxic effectsave yet to be fully elucidated, although studies implicate aole for oxidative mediators (Glinka and Youdim, 1995). 6-OHDAetabolism generates a series of ROS at physiologic pH, including
ydrogen peroxide, para-quinone, and superoxide and hydroxyladicals (Cohen and Heikkila, 1974). The role of these oxidativepecies in 6-OHDA toxicity and their intracellular sites of actionemain ill defined. Noteworthy, 6-OHDA-induced free radical for-ation and oxidative stress can be blocked by antioxidants, such
s N-acetyl-L-cysteine or C3 carboxyfullerene (Saito et al., 2007),ll of which prevent downstream toxic sequels (Holtz et al., 2005;im-Han and O’Malley, 2007) and cell death (Lotharius et al., 1999).or instance, 6-OHDA-induced protein oxidation causes endoplas-ic reticulum (ER) stress and upregulation of the unfolded protein
esponse (UPR), which in turn regulates protein folding, degra-ation and translation. In addition to UPR, 6-OHDA also inducesOS-dependent apoptosis in dopaminergic cells (Bové and Perier,012).
We have recently shown that some evoked antioxidant stimuliay modulate intracellular responses, hence offering protection
gainst toxicity in 6-OHDA treated animals (Tobón-Velasco et al.,012). Moreover, activation of the NF-�B signaling pathway isnown to contribute to 6-OHDA toxicity (Tarabin and Schwaninger,004). Noteworthy, apoptosis signaling mediated by mitochon-rial pathway promotes delayed retrograde cell death in substantiaigra following an intrastriatal injection of 6-OHDA (Hanrott et al.,008). In consideration to the temporal and spatial toxic fea-ures of this model, we hypothesize that 6-OHDA readily elicits aomplementary wave of mitochondrial ROS by mitochondrial com-lex inhibition, caspases activation and inflammatory responsesy NF-�B activation, all of them corresponding to early (minutes-ours) toxic events evoked by 6-OHDA. The activation of thesearly events might lead to toxic processes that can inhibit theompensatory/resistance cell responses, such as the antioxidantesponse coordinated by the Nuclear factor-erythroid-2-relatedactor 2 (Nrf2). Previous studies indicate that there are, indeed,irect and indirect links between the activation of NF-�B and Nrf2Tusi et al., 2010; Lee et al., 2012), and the exploration of this inter-ction can provide important clues on the early cell responses toxidative and inflammatory damages induced by 6-OHDA.
Impaired mitochondrial function in the brain has been showno be experimentally induced by exogenous neurotoxins such as-OHDA. This toxin is able to inhibit complex I in isolated brainitochondria (Glinka and Youdim, 1995). Although this evidence
learly implicates mitochondrial dysfunction directly induced by 6-HDA, the mechanisms underlying in vivo toxicity still require fulllucidation. In order to better understand the mechanistic nature ofhose acute effects of 6-OHDA on mitochondrial function in vivo, asell as the in vivo signaling associated to its early pattern of toxicity
n the brain, we used the intrastriatal lesion produced by 6-OHDA inhe rat model to evaluate some parameters of mitochondrial res-iration and the activity of complexes I and V in isolated striatalitochondria. These parameters were also investigated in regard to
heir possible relation with modulation of early signaling pathwayshich are known to be linked to mitochondrial damage, the asso-
iation between modulation of kinases, the toxin-induced oxida-ive stress and the crosstalk between inflammation-dependent
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activation of NF-�B, as well as the primary antioxidant responseinduced by activation of factor Nrf2. This multiple approach wascarried out with the purpose of offering an integrative pattern ofearly toxicity exerted by 6-OHDA in the rat striatum that mightexplain the late toxicity that has been observed and reported forthis model after several days of its infusion.
2. Materials and methods
2.1. Materials
Most chemicals, including 6-OHDA, ascorbic acid, digitonin, bovine serumalbumin (BSA) carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), 2,6-dichlorophenolindophenol (DCPIP), antimycin A, safranin O, hexokinase, ethyleneglycol tetraacetic acid (EGTA), Ethylenediaminetetraacetic acid (EDTA), adenosinediphosphate (ADP), rotenone, oligomycin A, succinic acid, phosphotungstic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium fluoride (NaF),sodium orthovanadate (Na3VO4), nitroblue tetrazolium (NBT), flavine adenine dinu-cleotide (FAD), nicotinamide adenine dinucleotide phosphate, oxidized (NADP+)and reduced form (NADPH), and phosphomolybdic acid, were obtained from SigmaChemical Company (St. Louis, MO, USA). All other reagents were analytical gradeand commercially available.
2.2. Surgery and 6-OHDA injection
Male Wistar bred-in-house rats (280–320 g) were used throughout the exper-iments. All procedures with animals were carried out according to the NationalInstitutes of Health Guide for the Care and Use of Laboratory Animals and the localguidelines on the ethical use of animals from the Mexico’s Health Ministry. Ani-mals (twenty per group) were anesthetized with sodium pentobarbital (40 mg/kg,i.p.), placed on a stereotaxic frame (Stoelting Co., Wood Dale, IL, USA) with incisorbar fixed at 3.0 mm below the interaural line, and bilaterally injected with vehi-cle (0.01% of ascorbic acid in isotonic saline solution) or 6-OHDA (2 �L [10 �g/�L]dissolved in 0.01% ascorbic acid) into the right striatum, using the following coordi-nates: +1.0 mm anterior to bregma, ±3.2 mm lateral to bregma, and −4.5 mm ventralto the dura (Paxinos and Watson, 1986). Rats were injected into the striatum usinga Hamilton microsyringe. The injection rate was 0.4 �L/min, the needle was left inplace for 5 more min, and then slowly withdrawn in 1 min. Rats were euthanizedafter 30 min, 1, 2 and 4 h post-lesion for biochemical tests and Western blot analyses.Animal brains were obtained by decapitation at the indicated times and the striatawere dissected out, removing the cortex and white matter with the aid of brushes.The striatum was removed either for immediate use or collected and stored at −80 ◦Cfor further analysis.
2.3. Immunoblotting
Striatal tissues of animals exposed to different experimental conditions weredissected, washed once with cold PBS, and lysed on ice with lysis buffer (1% Non-idet P-40, 10% glycerol, 137 mM NaCl, 20 mM Tris–HCl (pH 7.5), 1 �g/mL leupeptin,1 mM phenylmethylsulfonyl fluoride, 20 mM NaF, 1 mM sodium pyrophosphate,and 1 mM Na3VO4; all reagents were obtained from Sigma–Aldrich Co. (St. Louis,MO, USA). Cell lysates were pre-cleared by centrifugation, and protein concentra-tion was quantified by Lowry’s method (Lowry et al., 1951). Then, protein extractswere resolved by sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) using 60 �g (cytoplasmic, nuclear or mitochondrial fraction) of protein perlane, and transferred into Immobilon-P membranes (PVDF, Millipore, MA, USA).Blots were analyzed with the corresponding primary antibodies (dilutions 1:1000or 1:1500): anti-Nrf2, anti-NF-�B (p65), anti-lamin B1, anti-�-tubulin, anti-�-actin(from Santa Cruz Biotechnology), anti-histone-2 and anti-kinases (p-Akt, Akt, p-ERK1/2, ERK 1/2, p-SAPK/JNK, SAPK/JNK, p-p38 and p38 from Cell Signaling Biotechnol-ogy Inc., MA, USA). Antibodies against the antioxidant enzymes NADPH quinoneoxidoreductase 1 (NQO-1), heme oxygenase-1 (HO-1), Cu/Zn-dependent superox-ide dismutase (Cu/Zn-SOD), glutathione peroxidase (GPx) and glutathione reductase(GR), and the inflammatory markers tumor necrotic factor-alpha (TNF-�), induciblenitric oxidase synthase (iNOS) and cyclooxygenase-2 (COX-2), were all from SantaCruz Biotechnology (Santa Cruz, CA, USA). Antibodies against cytochrome c andapoptotic inducing factor (AIF) were from Abcam® Inc. (Cambridge, MA, USA).Peroxidase-conjugated secondary antibodies (1:10,000 or 1:15,000) were used todetect proteins of interest by an enhanced chemiluminescence kit (ECL-Pierce, Mil-lipore). Secondary antibodies were goat anti-rabbit HRP (62-6120, Zymed) and goatanti-mouse HRP (sc-2005, Santa Cruz Biotechnology, Santa Cruz, CA, USA).
2.4. Preparation of cytosolic, nuclear and mitochondrial cell fractions
Striatal tissues were washed once with cold phosphate-buffered saline (PBS) andlysed on ice with cold buffer 1 (250 mM sucrose, 20 mM HEPES (pH 7.0), 0.15 mMEDTA, 0.015 mM EGTA, 10 mM KCl, 1 mM phenylmethylsulfonyl fluoride, 20 mMNaF, 1 mM sodium pyrophosphate, 1 mM Na3VO4 and 1 �g/mL leupeptin plus 1%Nonidet P-40). Then, homogenates were centrifuged at 800 × g for 10 min; the
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xanthine, 0.006% bovine serum albumin and 49 mM sodium carbonate, all of whichare final concentrations. Striatal tissue samples were homogenized in 1 mL of phos-phate buffer 50 mM (pH 7.0)/Triton X-100 (1%). Two hundred microliters of striatal
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ecovered supernatants contained the cytosolic fraction. Nuclear pellets wereashed in cold buffer 2 (10 mM HEPES pH 8.0, 0.1 mM EDTA, 1 mM phenylmethyl-
ulfonyl fluoride, 20 mM NaF, 1 mM sodium pyrophosphate, 0.1 M NaCl, 1 �g/mLeupeptin and 1 mM Na3VO4 plus 25% glycerol). After centrifugation at 500 × g for
min, nuclei were resuspended in buffer 3 (RIPA: 50 mM Tris-HCl pH 7.6, 150 mMaCl, 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride,
�g/mL leupeptin, 20 mM NaF, 1 mM sodium pyrophosphate, and 1 mM Na3VO4).ytosolic fractions (supernatants of the first centrifugation) were centrifuged at400 × g for 15 min; the recovered supernatants contained the cytosolic fraction andhe bottom corresponded to the mitochondrial fraction. The mitochondrial pelletas resuspended in buffer 4 (215 mmol/L mannitol, 71 mmol/L sucrose, 10 mmol/L
uccinate, and 10 mmol/L HEPES, pH 7.4) and kept on ice for further analysis. Proteinsrom cytosolic, mitochondrial and nuclear fractions were resolved by SDS-PAGE andmmunoblotted with the indicated antibodies.
.5. Co-immunoprecipitation (Co-IP) assays
Whole protein extracts were obtained from striatal tissues, which were previ-usly washed once with cold PBS and harvested by centrifugation at 1100 rpm for0 min. Cell pellets were resuspended in 0.45 mL of ice-cold RIPA buffer. Nrf2 andF-�B (p65) proteins were immunoprecipitated from the cytoplasmic or nuclearell fractions using 4 �L of anti-Nrf2 or NF-�B antibody per sample. After incuba-ion for 4 h at 4 ◦C in a rotating wheel, gamma-bind Sepharose protein G was addedAmersham Biosciences, Piscataway, NJ, USA) and reincubated for 2 h at 4 ◦C. Theomplexes were harvested by centrifugation and washed 3 times with RIPA buffer.amples were boiled, resolved by SDS-PAGE and immunoblotted with anti-Nrf2,nti-Keap1 or anti-Maf protein (MafK) antibodies for Nrf2 immunoprecipitation, orith anti-p65 and anti-IkB antibodies for NF-�B immunoprecipitation to detect theroteins of interest by an enhanced chemiluminescence kit. Mouse IgG TrueBloteBiosciences, San Diego, CA, USA) was used as a peroxidase-conjugated secondaryntibody (1:10,000 dilutions).
.6. Phase II antioxidant enzymes activities
.6.1. Heme oxygenase (HO) activityTotal HO activity was estimated in striatal homogenates. Striatal tissues were
esuspended in 1 mL of 100 mM phosphate buffer (pH 7.4) with 2 mM MgCl2, soni-ated on ice for 30 s, incubated for 5 min on ice and centrifuged at 10,000 × g for
0 min at 4 ◦C. The supernatants (200 �L) were incubated with a reaction mix-ure consisting of mouse liver cytosol as source of bilirubin reductase (25 �L),emin (20 mM), glucose-6-phosphate (150 mM), glucose-6-phosphate dehydroge-ase (0.2 U/mL) and nicotinamide adenine dinucleotide phosphate (8 mM). Theeaction was conducted at 37 ◦C in the dark for 1 h, and terminated by the addition of
ig. 1. Effect of 6-OHDA on mitochondrial respiration 2 h after 6-OHDA administration. (Aomplex V activity. (D) Transmembrane potential of mitochondria isolated from Sham ased to evaluate differences among groups. * statistically significant differences, with p <
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0.5 mL chloroform. Formed bilirubin was extracted by centrifugation at maximumspeed for 45 s in a bench top centrifuge, and its content was calculated by the differ-ence in absorbance between 464 and 530 nm (ε = 40 mM−1 cm−1). Enzyme activitywas expressed as pmol bilirubin/mg protein.
2.6.2. NADP(H)-quinone oxidoreductase-1 (NQO-1) activityNQO-1 activity was measured in striatal homogenates: striatal lysates (20 �L)
were mixed with 200 �L of the following solution: 0.025 M Tris–HCl pH 7.4,bovine serum albumin (0.7 mg/mL), 0.01% Tween-20, 5 �M FAD, 1 mM glucose-6-phosphate, 250 �M menadione, 300 U of glucose-6-phosphate dehydrogenase,0.3 mg/mL of MTT and 30 �M NADP+. Mixtures were incubated for 5 min at roomtemperature and the reaction was stopped by the addition of 50 �L of 0.3 mMdicumarol. Optical density was measured at 610 nm and the activity was calculatedusing the molar absorption coefficient of MTT of 11,300 M−1 cm−1. Results wereexpressed as nmoles formazan/min/mg protein.
2.6.3. Glutathione reductase (GR) and glutathione peroxidase (GPx) activityGR activity was assayed using GSSG as substrate, measuring the disappearance
of NADPH at 340 nm. One unit of GR was defined as the amount of enzyme that oxi-dizes 1 mmol of NADPH/min. The reaction mixture consisted of 100 mM potassiumphosphate, pH 7.6, 0.5 mM EDTA, 1.25 mM oxidized glutathione and 0.1 mM NADPH.Supernatants (0.05 mL) were added to 0.95 mL of mixture and optical density wasrecorded at 340 nm for 3 min. The activity was calculated from the slope of theselines as micromoles of NADPH oxidized per minute. Data were finally expressed asunits per milligram of protein. GPx activity was measured at 340 nm using GR andNADPH in a coupled reaction. One unit of GPx was defined as the amount of enzymethat oxidizes 1 �mol of NADPH/min. Data were expressed as U/mg protein.
2.6.4. Superoxide dismutase (SOD) activityTotal SOD activity was assessed by a competitive inhibition assay, using a xan-
thine/xanthine oxidase system to reduce NBT, which served as the indicator reagent.Briefly, the mixture reaction contained 0.122 mM EDTA, 30.6 �M NBT, 0.122 mM
homogenates were added to 2.45 mL of the mixture described above, and then, 50 �Lof xanthine oxidase (final concentration 2.8 U/L) were added and incubated at 27 ◦Cfor 30 min. Reactions were stopped with 1 mL of 0.8 mM cupric chloride, and opticaldensity was recorded at 560 nm. The amount of striatal protein that inhibited 50%of maximal NBT reduction was defined as one unit of SOD activity.
) The RCR and state 3 and 4. (B) Mitochondrial complex I activity. (C) Mitochondrialnd 6-OHDA rats. Data are expressed as mean ± S.E.M. (n = 6). Student’s t-test was0.05 vs. Sham group.
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.7. Isolation of mitochondrial fraction
Mitochondria were isolated as described by Mirandola et al. (2010) with minor
odifications. The striata were dissected and extensively washed with ice-cold iso-
ation buffer containing 225 mM mannitol, 75 mM sucrose, 1 mM EGTA, 0.05% bovineerum albumin and 10 mM HEPES pH 7.2. Later on, tissue was homogenized andentrifuged at 2000 × g for 5 min; the resulting supernatant was recentrifuged for
ig. 2. 6-OHDA-induced release of cytochrome c and apoptotic inducing factor (AIF),
ytochrome c and AIF levels in cytoplasmic and mitochondrial fractions. (B) Densitometrractions is shown in the right panel. (C) Caspases 9 and 3 and cleaved caspase 3 levels measf caspase 9 (upper panel) and caspase 3 (Pro) and cleaved (Cle) caspase 3 (lower paneifferences among time-groups. * statistically significant differences, with *p < 0.05 and **
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15 min at 12,000 × g. The pellet was resuspended in 2 mL isolation buffer containing3 �l of 10% digitonin and recentrifuged for 15 min at 12,000 × g. The supernatantwas discarded and the final dark pellet gently washed and resuspended in isolation
buffer devoid of EGTA, at an approximate protein concentration of 3–4 mg/mL. Theuse of striatal tissue was assessed by precise dissection, as described in Section 2.2.Mitochondria obtained from Sham animals showed a respiratory control ratio (RCR)over 3.0, using succinate as substrate.
and apoptosis-mediated caspases pathway activation at the indicated times. (A)ic analysis of cytochrome c (upper graphic) and AIF (lower graphic) in cytoplasmicured in cytoplasmic cell fractions. (D) Densitometric quantifications of immunoblotsl). Data are expressed as mean ± S.E.M. (n = 3). Student’s t-test was used to assessp < 0.01 vs. Sham (control).
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Fig. 3. 6-OHDA inhibited Akt and ERK1/2, and activated SAPK/JNK and p38 kinases inthe rat striatum at the indicated times. (A) Expression of phospho-kinases and totalkinases (p-Akt, Akt, p-ERK1/2, ERK1/2, p-SAPK/JNK, SAPK/JNK, p-p38 and p38) incytoplasmic cell fractions. (B) Densitometric quantifications of kinase immunoblots.Df*
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ata are expressed as mean ± S.E.M. (n = 3). Student’s t-test was used to assess dif-erences among time-groups. * statistically significant differences, with *p < 0.05 and*p < 0.01 vs. Sham (control).
.8. Markers of mitochondrial function
.8.1. Oxygen uptake measurementOxygen consumption was measured using a Clark-type electrode (Strathkelvin)
n 0.15 mL of standard reaction medium, in a sealed glass cuvette equipped with aagnetic stirrer. Oxygen uptake measurements were conducted at 37 ◦C in standard
eaction medium containing 70 mM sucrose, 220 mM mannitol, 5 mM MgCl2, 10 mMH2PO4, 25 mM HEPES (pH = 7.2), 1 mM EGTA and 0.2% BSA. Succinate (10 mM)as used as respiratory substrate and complex I activity was inhibited with 5 �M
otenone. State 3 was measured after the addition of 200 �M ADP and the sub-equent state 4 after the complete ADP phosphorylation. RCR was calculated byividing state 3 over state 4 respiratory values.
.8.2. Mitochondrial transmembrane potentialMitochondrial potential was estimated through fluorescence changes of safranin
(Cano-Ramírez et al., 2012). In functional mitochondria, safranin O is “stacking” inhe inner mitochondrial membrane, but addition of an uncoupling agent promotes
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an increase in fluorescence because safranin O is released when transmembrane pro-tential is lost. A solution of 10 �M safranin O plus 14 mM succinate was mixed with25 �g of mitochondrial protein in standard reaction medium, and fluorescence wasrecorded on a spectrofluorometer operating at excitation and emission wavelengthsof 495 and 586 nm, respectively. Later on, 20 �M FCCP was added and the changein fluorescence units recorded. Results were expressed as change in �fluorescenceunits/mg protein.
2.8.3. Complex I (NADH-ubiquinone oxidoreductase) activityThis was evaluated through a spectrophotometric assay, as previously described
(Long et al., 2009). Briefly, the reaction medium was prepared with 3 mg/mL BSA,60 �M decylubiquinone, 2 �M antimycin A, 2 mM KCN, 80 �M NADH and 160 �MDCPIP in standard reaction medium. The reaction was initiated by adding 30 �gof previously sonicated mitochondrial protein and the DCPIP absorbance decreasewas followed at 600 nm. The rotenone-insensitive activity was determined as thedifference in activity in the absence and in the presence of rotenone (4 �M). Resultswere expressed as nmol/min/mg protein.
2.8.4. Complex V activityThe rate of ATP synthesis was measured using an enzyme-linked assay following
the reduction of NADP+ at 340 nm (Cano-Ramírez et al., 2012). Succinate (14 mM)was used as respiratory substrate and the enzyme-linked assay was: 4 U/mL hex-okinase, 2 U/mL glucose-6-phosphate dehydrogenase, 20 mM glucose and 1.4 mMNADP+ in standard reaction medium. The reaction was started with 200 �M ADP.Specific activity of complex V was expressed as nmol/min/mg protein.
2.9. Statistical analysis
Results were expressed as mean ± one S.E.M. All data were statistically analyzedusing one-way analysis of variance (ANOVA) for repeated measures, followed bypost hoc Tukey’s test. Student’s t-test was also used to make specific comparisonsbetween Sham and 6-OHDA groups. All analytical procedures were performed usingthe scientific statistic software GraphPad Prism 5 (GraphPad Scientific, San Diego,CA, USA). Differences of p < 0.05 were considered as statistically significant.
3. Results
3.1. 6-OHDA-induced mitochondrial dysfunction and inhibitionof respiratory complex I activity
Since the highest release of cytochrome c induced by short-term administration of 6-OHDA was observed at 2 h, we studiedthe effects of this neurotoxin on the striatal mitochondrial functionat the same time-point. In Sham rats, the mean active respirationrate or state 3 was 110 ± 6.6 and the mean of resting respirationrate, or state 4, was 31 ± 4.9. Respiratory control ratio (RCR) in stri-atal isolated mitochondria indicated by state 3/state 4 resulted ina mean value of 3.9 ± 0.4. Intrastriatal administration of 6-OHDAfor 2 h caused a significant reduction in the RCR rate and the oxy-gen consumption at state 3, without any significant change instate 4 (Fig. 1A). The mitochondrial complex I of 6-OHDA-treatedrats showed a significant reduction in its activity (around 35%)when compared with mitochondrial complex I activity of Shamrats (Fig. 1B). In contrast, 6-OHDA did not induce any change inthe mitochondrial membrane potential, or complex V activity, atthe time-point evaluated (Fig. 1C and D).
3.2. 6-OHDA-induced apoptotic pathways by cytochrome c andAIF release, and caspases activation
To further investigate the early toxic effect of 6-OHDA on striatalcells, we examined markers of intrinsic-apoptotic pathway at dif-ferent times (0.5, 1.0, 2.0 and 4 h). 6-OHDA-induced mitochondrialdysfunction is initiated by oxidative stress and inhibition of mito-chondrial complex I, and it has been shown to induce the releaseof cytochrome c and AIF, with the consequent activation of procas-pase 9, the formation of apoptosome, and the activation of caspase3.
First, at short times after its infusion, 6-OHDA induced anincrease in cytochrome c release of 115, 95, 201 and 215% at 0.5,1, 2 and 4 h, and this effect was accompanied by an increase inthe AIF in the cytosolic compartment of 35, 41, 73 and 62% at 0.5,
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, 2 and 4 h (Fig. 2A and B). Consistent with the concept that theaspases pathway participates in the 6-OHDA-induced cell death,e further evaluated the pattern of expression of caspases 9 and
in striatal tissue. The intrastriatal infusion of 6-OHDA to ratsaused a significant increase in capases 9/3 expression/activation.pecifically, 6-OHDA induced an increase of caspase 9 by 31, 91 and2 at 1, 2 and 4 h post-lesion (Fig. 2C and D), when compared withham group. Caspase 3 (cleaved casp3) was increased by the toxiny 39 and 45% at 2 and 4 h post-lesion (Fig. 2C and D).
.3. 6-OHDA modulated some kinases signaling pathways in thetriatum: Enhanced stress-activated pathway (JNK and p38) andnhibited typical survivor pathway (Akt and ERK 1/2)
Among several brain regions, we analyzed the striatum, a regionarticularly relevant in the toxic paradigm evoked by 6-OHDA.ere, we studied the early effect (0.5, 1.0, 2.0 and 4 h) of 6-OHDAn the canonical signaling pathways Akt and ERK1/2 (activated byome neurotrophins and growth factors) as well as SAP/JNK and38MAPK (activated by stress factors). Phosphorylation of signalinginases, indicative of their activation, was analyzed by westernlot with phospho specific antibodies that recognized the activeorms of Akt, ERK1/2, SAP/JNK and p38 (Fig. 3A). Densitometricata are presented as relative intensity obtained at 0.5, 1, 2 and
h after the striatal infusion of 6-OHDA (Fig. 3B). Intra-striatal
njection of 6-OHDA led to an increase of 52% in phospho-Aktnly at 1 h post-lesion, but later there was a decrease at 2 and 4 host-lesion. Although phospho-ERK was slightly decreased, no sig-ificant changes were observed when compared with Sham group.
ig. 4. 6-OHDA induced inflammatory responses mediated by NF-�B activation in the ratut in cytoplasmic cell fractions with anti-NF-�B antibody. p65 was analyzed by immunoell fractions with anti-NF-�B antibody. (C) IkB-alpha was analyzed by immunoblottingevels measured in cytoplasmic cell fractions; densitometric analysis is shown in the lowssess differences among groups. * statistically significant differences with *p < 0.05 and *
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The response of MAPKs to 6-OHDA-induced oxidative stress wascharacterized by an increase in phospho-SAP/JNK of 92, 123 and101% at 1, 2 and 4 h post-lesion, and p38 37, 35 and 39% at 1, 2and 4 h post-lesion (Fig. 3A and B). To our knowledge, this is oneof the few studies where signaling pathways have been analyzedin the rat striatum in response to a given neurotoxin. Our findingsclearly indicate that 6-OHDA inhibits the canonical survival path-way represented by the Ser/Thr kinase and promotes de signalingactivation of stress at early times.
3.4. 6-OHDA promoted NF-�B activation and enhancedinflammatory markers
6-OHDA induced a prominent inflammatory response byinducing NF-�B activation. 6-OHDA infusion in the striatumenhanced NF-�B factor and pro-inflammatory proteins, includingTNF-�, iNOS and COX-2 at 4 h post-lesion (Fig. 4A–E). In order toestablish possible correlations between NF-�B and IkB levels in thecytoplasmic compartment, striatal protein lysates from animalsexposed for 4 h to vehicle or 6-OHDA injections were processed toobtain cytoplasmic fractions and immunoprecipitations with anti-NF-�B antibody. Co-IP was assayed to determine the interactionof NF-�B (p65) as control of pull down (Fig. 4A and B) and anti-IkB(Fig. 4C and D) with its repressor. Results of these approachessuggest that 6-OHDA-treatment promotes the dissociation of
NF-�B from the NF-�B/IkB complex, as the levels of protein foundshowed an increase in NF-�B of about 2.1-fold above Sham group,accompanied by a similar level of IkB compared with Sham group.The increased levels of NF-�B induced by 6-OHDA produced
striatum at 4 h post-lesion. (A) Co-immunoprecipitation (Co-IP) assay was carriedblotting. (B) Co-immunoprecipitation (Co-IP) assay was carried out in cytoplasmic. Densitometric analysis is shown in the lower panel. (D) TNF-�, COX-2 and iNOSer panel. (E) Data are expressed as mean ± SEM (n = 3). Student’s t-test was used to*p < 0.01 vs. Sham (control).
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n increase in the protein levels of TNF-� (2.6-fold), COX-21.7-fold) and iNOS (2.0-fold) when compared with Sham groupFig. 4D and E).
.5. 6-OHDA promoted Nrf2 inhibition by enhanced Keap1/Nrf2omplex association
We further analyzed Nrf2 protein levels in striatal fractionsrom animals submitted to vehicle (Sham) or 6-OHDA throughn anti-Nrf2 antibody, as shown in Fig. 5A and B (cytoplasmic
ig. 5. 6-OHDA inhibited Nrf2 factor in the rat striatum. (A) Nrf2 levels measured in cytoprom Nrf2 in cytoplasmic and nuclear cell fractions, respectively. These determinations warried out in cytoplasmic cell fractions with anti-Nrf2 antibody. (D) Nrf2 and Keap1 wehe lower panel. (E) Co-IP assay was carried out in nuclear cell fractions with anti-Nrf2
ensitometric analysis is shown in the lower panel (F). The data presented in panels C–F
-test was used to assess differences among groups. * statistically significant differences w
ogy 304 (2013) 109– 119 115
and nuclear fractions). Noteworthy, rats receiving 6-OHDA thatwere submitted to the same protocol exhibited enhanced levels ofnuclear Nrf2 translocation at 30 min and 1 h post-lesion, probablyas a compensatory response to the toxic insult, gradually returningto basal levels. The 6-OHDA-induced decreased Nrf2 levels incytoplasmic fractions were found around 9 and 18% at 2 and 4 h
after 6-OHDA infusion and by 16 and 29% at 2 and 4 h in nuclearfractions after 6-OHDA infusion, respectively (Fig. 5A and B). Inorder to evidence possible correlations between Nrf2/Keap1 at thecytoplasmic domain, and Nrf2/MafK at the nuclear compartment,
lasmic and nuclear cell fractions. (B) Densitometric quantifications of immunoblotsere performed at 0.5, 1, 2 and 4 h. (C) Co-immunoprecipitation (Co-IP) assay was
re analyzed by immunoblotting (upper panel); densitometric analysis is shown inantibodies, and Nrf2 and MafK were analyzed by immunoblotting (upper panel);were obtained 4 h post-lesion. Data are expressed as mean ± SEM (n = 3). Student’sith *p < 0.05 and **p < 0.01 vs. Sham (control).
Fig. 6. 6-OHDA inhibited the expression of antioxidant enzymes in the rat striatum. (A) Immunoblotting for the following antioxidant enzymes: HO-1, NQO-1, GR, GPx andSOD1, respectively, at 4 h post-lesion. (B) densitometric analyses for antioxidant enzymes, respectively. (C) and (D) antioxidant enzyme activities at 4 h post-lesion. HO (pmolbilirubin/h/mg protein), NQO-1 (nmoles of formazan/min/mg protein), GR (�mol NADPH oxidized/min/mL/mg protein), GPx (�mol NADPH oxidized/min/mL/mg protein)and SOD (U/mg protein). Data are expressed as mean ± SEM (n = 3–6). Student’s t-test was used to assess differences among groups. * statistically significant differences with*p < 0.05 vs. Sham (control).
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triatal protein lysates from animals exposed for 4 h to vehicler 6-OHDA were processed to obtain cytoplasmic and nuclearractions and immunoprecipitation was carried out with anti-rf2 antibody. Cytosolic and nuclear immunoprecipitates were
mmuno-blotted with anti-Nrf2 as control of pull down and eithernti-Keap1 (Fig. 5C and D) or anti-MafK (Fig. 5E and F) antibodies.n the 6-OHDA-treated animals, the amount of Nrf2 that wasmmunoprecipitated in cytoplasmic fractions was around 2.0-foldbout Sham group and an enhanced interaction with Keap1 wasetected (Fig. 5C). On the other hand, 6-OHDA promoted a slightecrease in the interaction with MafK when compared to Shamroup, as determined by the levels of the nuclear heterodimeronstructed by Nrf2 and MafK (Fig. 5E and F). Altogether, theseesults indicate that 6-OHDA increases the Nrf2 arrest by Keap1
rotein and decreases the interaction of Nrf2/MafK heterodimer0.58-fold below the Sham group) in the nucleus, which inurn regulates the induction of antioxidant and detoxificationenes.
3.6. 6-OHDA decreased the antioxidant enzymes protein contentand activity in the rat striatum
The impact of 6-OHDA on the striatal protein content of typicallyNrf2-regulated phase II antioxidant enzymes is depicted in Fig. 6.The intrastriatal injection of 6-OHDA to rats produced a decrease inHO-1, NQO-1, GR and Cu/Zn-SOD protein levels at 4 h post-lesion(Fig. 6A and B). The enzymes expression decreased by 25% (HO-1), 45% (NQO-1), 43% (GR) and 39% (SOD1) (Fig. 6A and B). GPxexhibited increased protein levels only at 4 h post-surgery by 41%when compared with Sham group (Fig. 6A and B).
The activities of the antioxidant enzymes HO, NQO-1, GR, GPxand SOD (Cu/Zn- and Mn-dependent) were measured in the striatafrom four groups of rats studied 4 h after surgery. The results of
protein expression correlated with the quantified enzyme activity.6-OHDA induced an inhibition in the activities of HO by 13%, NQO-1 by 29%, GR by 9%, and SOD (Cu/Zn-SOD and Mn-SOD) by 24% at4 h post-lesion (Fig. 6C and D). 6-OHDA also induced a significant
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ncrease in the activity of GPx by 31% at 4 h post-lesion (Fig. 6Cnd D). These data are consistent with the observation that Nrf2 isnhibited by 6-OHDA.
. Discussion
Results of this study demonstrate that the commonly usedarkinsonian neurotoxin, 6-OHDA, readily induces early in vivoeurotoxicity in striatal cells via mitochondrial damage and oxida-ive stress. It can be assumed that oxidative byproducts of 6-OHDAan initiate intracellular oxidative stress, implicating hydrogeneroxide and other ROS in first place as key mediators of 6-OHDA-
nduced cell death. In turn, mitochondrial inhibition seems toromote a combination of both necrotic and apoptotic mechanisms,
ncluding the activation of the mitochondrial-caspase 3 apoptoticascade and other intracellular signals.
During respiration, 85% of the molecular oxygen is utilizedy mitochondria to produce ATP for a number of cellular func-ions. In this organelle, ROS are normally produced as byproducts,nd include hydrogen peroxide and superoxide radicals. How-ver, antioxidant systems inside the mitochondria maintain theasal levels of ROS under control (Zhou et al., 2007). Impaireditochondrial function increases the production of superoxide,hich in turn may form hydroxyl radicals, or react with nitric
xide to form peroxynitrite (Brown and Borutaite, 2004). In turn,OS are known to cause cellular damage by reacting with nucleiccids, proteins and lipids, and even the electron transport chaintself could be a target for these oxidants in mitochondria (Cohen,000), leading to further mitochondrial damage and more ROSroduction. Mitochondrial dysfunction has been implicated ineurodegenerative disorders. Particularly, loss of activity of com-lex I at the mitochondrial electron transport chain has beenbserved in idiopathic Parkinson’s disease (Smigrodzki et al.,004; Zhou et al., 2007). Here we employed the hemiparkinso-ian model induced by the intrastriatal injection of 6-OHDA toats, and 2 h later mitochondria were isolated from the striatand subjected to analysis. In this regard, the use of 6-OHDA inn vitro models has served to study its toxic effects on mitochon-rial function. Of note, this is one of the few studies availablexploring in vivo toxic events produced by the toxin at earlytages.
In this study, the RCR observed in the 6-OHDA group indicates direct effect of the toxin on mitochondrial respiration in the stri-tum when compared with mitochondria from Sham animals. Thisata agree with a recent study where mitochondria isolated fromat forebrain exhibited a reduction in the RCR after exposure to 6-HDA at different concentrations (Iglesias-González et al., 2012).oreover, in our work, state 3 was significantly decreased, without
hanges in state 4, which is according with the mentioned study.his indicates that metabolically preserved mitochondria can stille obtained in the toxic model of 6-OHDA.
Also in this report, the intrastriatal administration of 6-OHDAroduced a reduction in the activity of mitochondrial complex I.his finding is in agreement with previous studies using isolateditochondria from total brain (Glinka and Youdim, 1995; Glinka
t al., 1996, 1998) and mitochondria isolated from neuroblastomaH-SY5Y, or the rat forebrain (Iglesias-González et al., 2012). Basedn early evidence collected in vitro, the inhibition of complex I by-OHDA appears to obey to a direct inhibitory effect (Glinka et al.,996, 1997), although the neurotoxin can also act as a generator ofree radicals (Glinka et al., 1996). However, this idea remains to be
ested in the in vivo model. Also, this concept does not exclude thatnder in vivo conditions, oxidative mechanisms exerted by 6-OHDAay lead to damage of other cellular components, as indicated
y the release of cytochrome c, or the possibility that during the
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long-term exposure of mitochondria to 6-OHDA, the inhibition ofthe respiratory chain occurred by ROS generation and oxidativestress.
Interestingly, we were unable to observe changes in the activ-ity of complex V, which is directly related to ATP production. Thisis in agreement with a previous study where 6-OHDA toxicity inSY5Y neuroblastoma cells was not accompanied by a reduction ofATP production, ATP/ADP ratio or NAD+ cellular content (Storchet al., 2000). These results suggest that mitochondria probablyactivate adaptive mechanisms to transiently maintain the energyproduction at initial stages of 6-OHDA toxicity. Finally, we nei-ther observed changes in the mitochondrial membrane potential, asindicated by the evaluation of transmembrane potential and state4, in contrast with previous studies where 6-OHDA showed a ROS-related collapse in mitochondrial membrane potential (Lothariuset al., 1999). Although we do not have an explanation for this find-ing, we hypothesize that this could be related with an undetectedtransient fall in membrane potential due to the experimental con-ditions employed in our study (only one time-point estimated).Further studies characterizing more time-points for this and othermarkers are needed to clarify this issue.
Our findings on 6-OHDA-induced oxidative stress are likely tobe the result of an imbalance between ROS generation, antioxidantlevels and/or depletion of enzymatic and non-enzymatic ROS sca-vengers. This neurotoxin, a hydroxylated metabolite of dopamine,enters the cells via dopamine transporters, and once inside, Fe2+
and H2O2 enhance the formation of 6-OHDA by non-enzymatichydroxylation of dopamine (Linert et al., 1996). We have recentlydemonstrated that this toxin enhances different markers of oxida-tive damage (ROS formation, lipid peroxidation and nitric oxideproduction) in an acute dose-dependent manner, peaking between2 and 4 h post-lesion (Tobón-Velasco et al., 2012). These changescould be closely related with the inhibited expression exertedby 6-OHDA on different antioxidant enzymes in the striatum atfour h post-lesion, hence supporting the concept that oxidativedamage is part of the acute pattern of toxicity exerted by thismolecule.
It has been reported that alterations in mitochondrial mem-brane permeability result in the release of cytochrome c intothe cytosol (Fiskum et al., 2003). Here we found that striatal tis-sue exposed to 6-OHDA showed cell death in a time-dependentmanner. Also, 6-OHDA induced altered mitochondrial activity andfunction, and these alterations were followed by an increase incytosolic cytochrome c and AIF levels. Once cytochrome c isreleased from mitochondria, it forms a complex with apoptosisprotease-activating factor-1 (Apaf-1) to further activate caspase-9, which in turn activates caspase-3 (Yuyama et al., 2003; Zhouand Tang, 2002), thereby establishing a causal role of mitochon-drial dysfunction on oxidative cell toxicity in this toxic paradigmat early times. Our results are also in agreement with previousstudies in which oxidative stress, alterations in mitochondrial func-tion, cytochrome c release, and caspase-9 and caspase-3 activationoccur during 6-OHDA-induced cell death in PC12 pheochromo-cytoma cells (Elkon et al., 2004), SH-SY5Y human neuroblastomacells (Jordan et al., 2004), MN9D murine dopaminergic cells (Choiet al., 1999) and cerebellar granule cells (Dodel et al., 1999). Still,the question remains on whether mitochondrial dysfunction is theearly chronological origin of dopaminergic toxicity in the toxicmodel evoked by 6-OHDA. Although more detailed studies areneeded to clarify this issue, here we provide initial evidence sug-gesting that mitochondrial dysfunction is relevant for triggeringtoxic cascades at short times after the toxic insult has been ini-
tiated in the rat striatum. Noteworthy, since we did not collectstrict evidence on a causal relationship between mitochondrial dys-function, apoptosis, cytochrome c release and signaling pathways,we are unable to declare that one is chronologically responsible of
Fig. 7. Schematic representation of the early mechanisms involved in the toxic effects of 6-OHDA in the rat striatum. The focal infusion of 6-OHDA promotes ROS formationand damage to lipids, proteins, enzymes, and DNA. The mitochondrial complex I inhibition by 6-OHDA promotes mitochondrial damage, which is associated with anincrease of cytochrome c and apoptotic inducing factor (AIF) release, and its further association with caspase 9 for the formation of the apoptosome; this event, most likelyoccurring in neurons, leads to the activation of caspase-mediated apoptosis, involving caspase 3, which in turn induces DNA damage and promotes cell death. Furthermore,6 the aci and r
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-OHDA-mediated inflammatory processes (likely occurring in glial cells) promotes responsible for the arrest of the compensatory cell response by inhibition of Nrf2
ach other. For instance, given the time points evaluated here, oneould not say that mitochondrial dysfunction precedes cytochrome
release. From this point of view, it is difficult to define whetherytochrome c release would precedes all other effects. Moreover,hile it is clear that the damage generated in mitochondria is
esponsible for apoptotic activation, it remains elusive whether its directly responsible for activation and interaction of other path-
ays. In this regard we could also speculate that different scenariosan occur simultaneously: mitochondrial inhibition, augmentedytochrome c release, caspase activation, ROS formation, and JNKnd p38 activation mostly occurring in DA neurons, all matchingith Nrf2 inhibition and NF-�B activation most probably in glial
ells. If this is the case, the other pathways explored here will notecessarily depend on 6-OHDA-induced apoptotic activation. In theeantime, our data agree with those observed in neurons of cortex,
ippocampus, sympathetic neurons and cerebellar granule neuronsy other authors (Deshmukh et al., 2000; Krohn et al., 1999; Stefanist al., 1999; Wigdal et al., 2002), where the cytochrome c releaseccurs without immediate mitochondrial membrane potential loss.n addition, the initiation of the intrinsic pathway in this model doesot exclude at all that other caspases associated with the extrinsicathway were also activated. Suggestive data for this possibilityan be obtained in our study when considering that TNF-� expres-ion was augmented by 6-OHDA. Then, if receptors for TNF-� arectivated, the changes for an activation of the extrinsic pathway areresent.
On the other hand, our study also revealed an early-triggeredignaling component evoked by 6-OHDA and characterized byinase modulation and NF-�B-mediated inflammatory responses,ccompanied by inhibition of Nrf2. Our results support the concepthat upregulation of kinases SAP/JNK and p38 play a role in thearly stages of 6-OHDA toxicity by triggering intrinsic pathways
or apoptosis and enhanced NF-�B activation. Hence, this earlyignaling component seems to be relevant to explain dopaminer-ic damage as it can be the result of a coordinated toxic strategyonsistent of simultaneously stimulating NF-�B while silencing
tivation of NF-�B and further release of proinflammatory cytokines, which in turnelated antioxidant responses.
Nrf2. This crosstalk is particularly relevant in our study and oth-ers as it has been shown that, for instance, attenuating NF-�Band activating Nrf2 constitutes a protective strategy to preservePC12 cells against apoptosis (Tusi et al., 2010). Therefore, the sim-ple cost of modifying the intricate relationship between NF-�Band Nrf2 is altering the balance between ROS and inflammatorycytokines formation and the depletion of antioxidant enzymessuch as hemeoxygenase-1 (Andreadi et al., 2006), which in turnmay contribute to explain the complex pattern of toxicity elicitedby 6-OHDA since the early stages. In contrast, stabilization ofNrf2 by electrophiles such as tert-butylhydroquinone is known toprevent PC12 from LPS-induced apoptosis while inhibits NF-�B andother proteins with deleterious actions, including COX-2, TNF-�and caspase 3 (Khodagholi and Tusi, 2011). In addition, it has beendemonstrated that, under normal conditions, NF-�B regulates sur-vival responses in PC12 cells, but when cells are stimulated with6-HODA, this factor mediates pro-apoptotic actions (Tarabin andSchwaninger, 2004). Whether these pathways and those directlyrelated with kinases modulation are contributing to early the 6-OHDA-induced damage is an issue that will catch our attentionin the next years. In the meantime, here we have demonstratedthat these altered signaling pathways contribute to the toxic patterelicited by 6-OHDA. Finally, based on all findings collected in thisstudy, we hypothesize the early mechanisms of toxicity exerted by6-OHDA taking place in the rat striatum in Fig. 7.
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgments
This work was supported in part by PAPIIT/UNAM (IN201910)and CONACyT (129838 J.P.-C.) and (168356 MO-I). J.C. Tobón-Velasco is scholarship holder from CONACyT-Mexico (239757,46497), and gratefully acknowledges Santander Bank for the
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ellowship and scholarship student exchange. J.H. Limón-Pachecos grant-fellowship from CONACYT 129838- J.P.-C. Authors wouldike to thank Dr. Isabel Lastres-Becker (IIB-UAM and CIBERNED)nd Dr. Monica Torres-Ramos (INNN-MVS/SSA) for critical readingf the manuscript and stimulating discussions. We also thankrs. Ismael Torres and Enrique Pinzón for their support in sup-lying experimental animals, and M.Sc. Omar N. Medina-CamposFQ-UNAM) for technical support.
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