Organophosphate pesticide chlorpyrifos impairs STAT1 signaling to induce dopaminergic neurotoxicity: Implications for mitochondria mediated oxidative stress signaling events

Abstract

The organophosphate (OP) pesticide chlorpyrifos (CPF), used in agricultural settings, induces developmental and neurological impairments. Recent studies using in vitro cell culture models have reported CPF exposure to have a positive association with mitochondria-mediated oxidative stress response and dopaminergic cell death; however, the mechanism by which mitochondrial reactive oxygen species (ROS) contribute to dopaminergic cell death remains unclear. Therefore, we hypothesized that STAT1, a transcription factor, causes apoptotic dopaminergic cell death via mitochondria-mediated oxidative stress mechanisms. Here we show that exposure of dopaminergic neuronal cells such as N27 cells (immortalized murine mesencephalic dopaminergic cells) to CPF resulted in a dose-dependent increase in apoptotic cell death as measured by MTS assay and DNA fragmentation. Similar effects were observed in CPF-treated human dopaminergic neuronal cells (LUHMES cells), with an associated increase in mitochondrial dysfunction. Moreover, CPF (10 μM) induced time-dependent increase in STAT1 activation coincided with the collapse of mitochondrial transmembrane potential, increase in ROS generation, proteolytic cleavage of protein kinase C delta (PKCδ), inhibition of the mitochondrial basal oxygen consumption rate (OCR), with a concomitant reduction in ATP-linked OCR and reserve capacity, increase in Bax/Bcl-2 ratio and enhancement of autophagy. Additionally, by chromatin immunoprecipitation (ChIP), we demonstrated that STAT1 bound to a putative regulatory sequence in the NOX1 and Bax promoter regions in response to CPF in N27 cells. Interestingly, overexpression of non-phosphorylatable STAT1 mutants (STAT1Y701F and STAT1S727A) but not STAT1 WT construct attenuated the cleavage of PKCδ and ultimately cell death in CPF-treated cells. Furthermore, small interfering RNA knockdown demonstrated STAT1 to be a critical regulator of autophagy and mitochondria-mediated proapoptotic cell signaling events after CPF treatment in N27 cells. Finally, oral administration of CPF (5 mg/kg) in postnatal rats (PNDs 27-61) induced motor deficits, and nigrostriatal dopaminergic neurodegeneration with a concomitant induction of STAT1-dependent proapoptotic cell signaling events. Conversely, co-treatment with mitoapocynin (a mitochondrially-targeted antioxidant) and CPF rescued motor deficits, and restored dopaminergic neuronal survival via abrogation of STAT1-dependent proapoptotic cell signaling events. Taken together, our study identifies a novel mechanism by which STAT1 regulates mitochondria-mediated oxidative stress response, PKCδ activation and autophagy. In this context, the phosphorylation of Tyrosine 701 and Serine 727 in STAT1 was found to be essential for PKCδ cleavage. By attenuating mitochondrial-derived ROS, mitoapocynin may have therapeutic applications for reversing CPF-induced dopaminergic neurotoxicity and associated neurobehavioral deficits as well as neurodegenerative diseases.

Keywords: Autophagy; Chlorpyrifos; Mito-apocynin; Mitochondrial bioenergetics; Neurotoxicity; Oxidative stress; STAT1.

Figure 1.. CPF-induces dopaminergic cell death via caspase mediated mechanism in N27 dopaminergic neuronal cells.

CPF reduces cell viability in a dose-dependent manner in N27 and differentiated LUHMES dopaminergic neurons. N27 cells treated with increasing concentrations of CPF (100nM-300nM) for 24h and cell death was evaluated using both (A) MTS assay and (B) DNA fragmentation assay. (A) The effects of CPF on dopaminergic neuronal cell viability evaluated using the MTS assay. N27 dopaminergic neuronal cells were exposed to increasing concentrations of CPF and IC50 was determined at the end of 24h using the MTS assay. Results are expressed as percentage of vehicle treated cells. (B) Quantification of CPF-induced apoptosis of N27 cells using DNA fragmentation analysis. The relative concentration of nucleosomes in cells treated with or without CPF was determined by Elisa based cell death detection ELISA^plus assay. Results are representative of at least 3 independent experiments and expressed as Mean ± SEM, N=6. *p<0.05, **p<0.01 and ***p<0.001 indicates level of significant difference between CPF versus Control group. (C) Differentiated LUHMES cells were treated with increasing concentrations of CPF (10nΜ−100μΜ) for 24h and cell viability was determined using the DNA fragmentation assay. The relative concentration of nucleosomes in cells treated with or without CPF was determined by Elisa based cell death detection ELISA^plus assay. Results are representative of at least 3 independent experiments and expressed as Mean ± SEM, N=4. *p<0.05, **p<0.01 and ***p<0.001 indicates level of significant difference between CPF versus Control group. (D) CPF treatment reduces neuritic length in differentiated LUHMES human dopaminergic neurons. ICC analysis of differentiated LUHMES cells exposed to 300nM CPF for 24. Scale bar: 100μm. The bar graph represents the neurite length, measured using imageJ software. Results are representative of at least 3 independent experiments and expressed as Mean ± SEM, (N=6).***p<0.001 indicate significant difference between Control versus CPF treated group. (E) CPF-induces a time dependent increase in caspase 3 activity. N27 cells were treated with or without CPF (10μΜ) for various time periods (6–24h). Cell lysates were prepared and incubated with Ac-DEVD-AMC, a caspase 3 specific substrate, at 37°C for lh. Caspase 3 activity was determined spectrophotometrically using Ac-DEVD-AMC substrate and outcomes are expressed as of FU/mg protein. Data calculated as percentage of control and are representative of at least 3 independent experiments. Data expressed as Mean ± SEM. Each time point was performed in triplicates (N=3). *p<0.05, **p<0.01 and ***p<0.001 vs control. (F) CPF induce time-dependent increase in PARP cleavage in N27 cells. Post treatment with CPF, cell lysates were prepared and membranes were probed with anti-PARP antibody, which recognize both native (116kDa) and cleaved (89kDa) PARP fragment. Cleaved PARP band density was normalized to β-actin. The bar graph represents the mean ± SEM of atleast 4 independent experiments. *p<0.05 and **p<0.01 vs Control group. (G) Effect of pan caspase and caspase-3 inhibitor on CPF induced neuronal cell death. N27 cells were pretreated with Z-VAD-FMK (pan caspase inhibitor; 100μΜ) or Z-DEVD-FMK (Caspase-3 inhibitor; 50μΜ) for lh followed by incubation with CPF 10μΜ for another 24h. Post treatment cytotoxicity was assessed via DNA fragmentation analysis and results were expressed as percentage of control. Data expressed as mean ± SEM, N=6. *p<0.05, **p<0.01 and *** p<0.001 indicates significant change from Control group, while ^###p<0.001 indicate significant difference between CPF and CPF+ caspase inhibitor (s) treated group.

Fig. 2.. Oxidative stress response is a critical determinant of CPF-induced cell death.

Effect of CPF on ROS production. (A) N27 cells were treated with or without CPF (10μΜ) for the indicated time period. Post treatment cells were incubated with ROS sensitive H2DCFDA dye and DCF fluorescence was determined 60min later. Change in the fluorescence reading, was determined via spectrophotometer. Each data point was converted to percentage of control. Data expressed as mean ± SEM and representative of at least 3 independent experiments performed in six replicates. (B) Time dependent increase in mitochondria derived superoxide levels, as assessed via MitoSOX™ Red dye. Each data point was converted to percentage of control. Data expressed as mean ± SEM and representative of at least 2 independent experiments performed in six replicates per group. (C) CPF-induces time-dependent reduction in GSH levels in N27 cells. N27 dopaminergic cells were treated with 10μΜ CPF for the indicated time period. Post treatment, CPF-induced changes in glutathione levels were measured spectrophotometrically using monochlorobimane based fluorometric assay. Data expressed as mean ± SEM and representative of at least 2 independent experiments with N=6 per group. (D, E) N-acetyl cysteine (NAC) attenuated CPF-induced ROS generation and cytotoxicity in N27 cells. N27 cells were pretreated with NAC (5mM) for 12h followed by treatment with and without CPF (10μΜ) for another 24h and the levels of CPF-induced ROS generation (D) and cell viability (E) was assessed spectrophotometrically by redox sensitive CM-H2DCFDA dye and DNA fragmentation analysis, respectively. Data expressed as mean ± SEM (N=6) and representative of at least 2 independent experiments performed in six replicates per group per group. *p<0.05, **p<0.01 and ***p<0.001 vs Control group; ^##p<0.01 and^###p<0.001 indicates significant differences between CPF and CPF + NAC treated cells.

Figure 3.. CPF-induced mitochondrial depolarization is accompanied by mitochondria mediated proapoptotic cell signaling events.

(A) Effect of CPF on mitochondrial depolarization. N27 cells were treated with and without 10μΜ CPF for the indicated time periods. CPF-induced mitochondrial depolarization cells were quantified fluorometrically using JC-1 dye. Representative data from three independent experiments are shown and the numerical values represents Mean± S.E.M. (N= 6). (B) Effect of CPF on Bax/Bcl-2 ratio. N27 cells were treated with or without 10μΜ CPF for the indicated time periods. Cell lysates were prepared and immunoblotted for Bcl-2 and Bax and tubulin was used as the loading control. All blots shown are representative of atleast 3 independent experiments. Panel on the right hand side shows the densitometric scanning analysis for Bcl2 and Bax was performed, and the Bax/Bcl2 ratio was determined. The results represent mean ± S.E.M. from at least 3 independent experiments. *p<0.05, **p<0.01, ***p<0.001 indicates significant difference between CPF treated and control group. (C) CPF-induced time dependent increase in PKCδ cleavage. N27 cells were exposed to the indicated time period and cell lysates were immunoblotted for PKCδ using an antibody, which recognize both native and cleaved bands. β-actin was used as the loading control. All blots shown are representative of at least 4 independent experiments. Right panel, quantification of relative band intensity of cleaved PKCδ and normalized to β-actin. (D) PKCδ genetic knockdown attenuates CPF-induced dopaminergic cell death. N27 were transfected with PKCδ specific siRNA or scrambled siRNA for 48h and CPF (10μΜ) was added for another 24h. At the end of the incubation period, the extent of apoptotic cell death was determined spectrophotometrically using ELISA-based cell death assay kit. The results are expressed as Mean ± S.E.M. performed in replicates of six and performed at least three independent experiments. Data are expressed as percentage of control. *p<0.05, **p<0.01 and ***p<0.001 indicates level of significant difference from Control group, whereas ^{# # #}p<0,001 indicates significant differences between CPF-treated N27 were transfected with PKCδ siRNA or scrambled siRNA. (E) Over expression of caspase-3 cleavage resistant mutant of PKCδ attenuates CPF-induced cell death. N27 cells stably expressing the Lac Z vector or caspase-3 cleavage resistant PKCδ (CRM; PKCδ^D327A) were treated with and without CPF. The extent of DNA fragmentation measured spectrophotometrically as described above. Results shown are Mean ± S.E.M. from at least three independent experiments and expressed as % of control. ***p<0,001 represent significant difference between CPF treated LacZ empty vector transfected cells vs CPF-treated PKCδ-CRM-V5 mutant group.

Figure 4.. CPF-induces STAT1 activation in N27 dopaminergic neuronal cells.

Time-dependent increase in phosphorylation of STAT1 at both Tyr⁷⁰¹ and Ser⁷²⁷ and its nuclear import. (A) N27 cells were treated with CPF (10μΜ) for the indicated time period and the cell lysates were immunoblotted for phospho-Tyr701 (pY701), phospho-Ser727 (pS727) and STAT1. Bottom panel represents quantification of the relative band intensity normalized to total STAT1 levels. Results are normalized to control values and are shown as Mean ± S.E.M. from atleast three independent experiments. *p< 0.05, **p< 0.01 and ***p<0.001 vs Control group. (B) CPF induced intracellular translocation of phosphorylated STATE Representative immunofluorescent images obtained from LUHMES cells that were treated with or without CPF (300nM) for 12h. Phosphorylated STAT1 levels as visualized using an antibody against STAT1 (p-Ser727) (red fluorescence) and subsequently counterstained with Hoechst (blue). Experiments were performed in triplicates and representative of at least 3 independent experiments. Scale bar in C=100μM. (C) STAT1 activation determined by luminescence assay in N27 cells stimulated with or without CPF. To measure STAT1 activation, N27 cells were transfected with the STAT1-responsive GAS reporter/ luciferase construct. The cells were subsequently treated with and without CPF(50μM) for 4 hours and the cells were harvested at the end of the incubation period for the determination of STAT1 activation using luminescence based dual luciferase assay. The firefly luciferase results were normalized using the internal Renilla luciferase activity. The assays were performed in duplicate, N=8. The results were expressed as relative luciferase unit, Mean ± SD. **p<0.01 represent significant difference between control and CPF treated group.

Figure 5.. STAT1 mediates CPF-induced cytotoxicity in N27 cells.

(A) N27 cells were transfected with scrambled or STAT1 siRNA, 48h post transfection, STAT1 knockdown efficiency was evaluated by immunoblotting for STATE Right panel represents densitometric scanning analysis for STATE Results are normalized to control values and are shown as Mean ± S.E.M. from at least three independent experiments. *p<0.05, and ***p<0.001 vs Control group (Scrambled siRNA transfected cells exposed to vehicle). (B, C) STAT1 KD confers resistance against CPF-induced cell death. Scrambled and STAT1 siRNA transfected N27 cells were treated with CPF (10μΜ) for another 24h and cell viability was determined using the DNA fragmentation assay (B) or MTS assay (C). Results are expressed as percentage of control and each value represent Mean ± SEM from atleast 3 independent experiments. *p<0.05; ***p<0.001 vs scrambled siRNA transfected vehicle treated N27 cells, while ^##p<0.01 and ^###p<0.001 vs scrambled siRNA transfected CPF-treated N27 cells. (D) STAT1 KD markedly reduces CPF induced cell shrinkage in N27 cells. Phase contrast light microscopy images reveal that STAT1 KD attenuated CPF-induced reduction in cell density and cell death associated morphological changes. Representative image (20x) from 3 independent experiments. (E) Effect of STAT1 KD on CPF-induced caspase-3 activity. N27 cells were transfected with scrambled or STAT1 siRNA as detailed in methods and subsequently were treated with CPF (10μΜ) for 18h. CPF-induced caspase 3 activity was measured fluorometrically by detecting free cleaved product from the caspase 3 specific peptide substrate, DEVD-AFC. Results are expressed as FU/mg protein. Results are expressed as percentage of control and each value represent Mean ± SEM (N=6). ***p<0.001 vs scrambled transfected siRNA transfected vehicle treated N27 cells, ^#p<0.05 vs scrambled siRNA transfected CPF-treated N27 cells. (F) STAT1 regulates CPF-induced PARP cleavage. N27 cells were transfected with scrambled or STAT1 siRNA, followed by treatment with CPF (10μΜ) for 24h, cell lysates were prepared and immunoblotted for PARP with antibody recognizing parental and cleaved PARP (89kDa). (Right panel) Densitometric scanning analysis revealing attenuation of CPF-induced PARP cleavage in STAT1 KD cells. Data shown are mean ± S.E.M, and represented as % control. Immunoblot representative of atleast three independent experiments. **p<0.01 vs scrambled siRNA transfected cells exposed to vehicle, ^#p<0.05 vs scrambled siRNA transfected cells exposed to CPF.

Figure 6.. STAT1 regulates NOX-l-mediated oxidative stress and GSH levels in CPF- treated cells.

(A, B) Effect of STAT1 knock down on CPF-induced ROS production and NOX-1 protein expression. N27 cells were transfected with either scrambled siRNA or STAT1-specific siRNA as described above, subsequently, N27 cells were exposed to CPF (10μΜ) for 24h, followed by incubation with CM-H2DCFDA for 60min and intracellular ROS levels were measured fluorometrically using Spectramax microplate reader (A). The data represent Mean ± S.E.M. of at least 3 independent experiments. Each treatment group was performed in replicates of six. (B) STAT1 KD attenuates CPF-induced NOX-1 upregulation. Representative immunoblot for NOX-1 from N27 cells transfected with scrambled siRNA or STAT1 siRNA followed by vehicle or CPF (10μΜ, 24h) treatment. Scrambled and STAT 1 siRNA transfected N27 cells were treated with CPF (10μΜ) for 24h. Cell lysates were prepared & immunoblotted for NOX-1. Histogram representing NOX-1 band intensity normalized to β-actin. Data represented as mean ± S.E.M. The blots are representative of at least 3 independent experiments. (C) Chromatin immunoprecipitation analysis of GAS/ISRE element within NOX-1 promoter region. Time-dependent binding of STAT1 transcription factors to the proximal promoter region of NOX-1 in N27 cell line treated with CPF. The N27 cell were treated with CPF 50μΜ for 4h and 6h, post incubation cells were fixed and DNA was extracted and subjected to chromatin immunoprecipitation (CHIP) assay using the STAT1 antibodies and primers specific to the proximal promoter of NOX-1. Representative image of amplified PCR product resolved on 1% agarose gel is shown in figure. Input lanes confirms that equal amount of DNA was used for the initial immunoprecipitation assay. Data is representative of 3 independent experiments. (D) STAT1 KD restores CPF-induced GSH depletion. N27 cells were transfected with either scrambled siRNA or STAT1-specific siRNA and exposed to either vehicle or CPF. 24h post CPF treatment, the intracellular GSH levels were determined spectrophotometrically. The data is presented as percentage of control. Data represented as mean ± S.E.M (N=6). ***p<0.001 vs vehicle treated scrambled siRNA transfected cells, ^#p<0.05 and ^{# #}p<0,01 vs scrambled siRNA transfected cells exposed to CPF.

Figure 7.. Effects of STAT1 knock down on CPF-induced mitochondrial bioenergetics deficits in N27 cells.

STAT-1 KD preserves mitochondrial bioenergetic profile following CPF treatment in N27 dopaminergic cells. (A) Schematic representation of the mitochondrial bioenergetics profile in relation to OCR levels, in both scrambled and STAT1 KD N27 cells treated with or without CPF (10μΜ) for 12h. Post treatment, the change in the Oxygen consumption rate (OCR) levels were determined using sea horse XF-24 analyzer. Basal respiration (B), maximal respiration (C), spare respiratory capacity (D) and ATP-linked respiration/ATP levels (E) were calculated by measuring change in OCR levels post sequential addition of oligomycin (1μΜ), FCCP (1μΜ), and Rotenone/Antimycin A (0.5μΜ), respectively, as indicated by arrow in the figure 7A. (F) ATP levels, measured at 24h post CPF (10μΜ) treatment via CellTiter-Glo® Luminescent assay. The data is presented as percentage of control. Data represented as mean ± S.E.M (N=6). *p<0.05 and ***p<0.001 vs Control group, ^#p<0.05 and ^{# #}p<0.01 Vs CPF-treated scrambled siRNA transfected N27 cells. Results are representative of atleast 3 independent experiment performed in replicates of three. Data expressed as mean ± S.E.M. *p<0.05, ***p<0.001 vs vehicle treated scrambled siRNA transfected cells, whereas, ^#p<0.05 and ^##p<0.001 vs CPF treated scrambled siRNA transfected cells.

Figure 8.. Effects of STAT-1 knockdown on CPF-induced modulation of Bax/Bcl2 ratio and mitochondrial release of cytochrome C

(A) STAT1 KD attenuates CPF-induced collapse of mitochondrial membrane potential. N27 cells were transfected with scrambled or STAT1 siRNA as detailed above and subsequently treated with 10μΜ CPF for 24h. Post treatment, CPF- induced mitochondrial depolarization was quantified fluorometrically using JC-1 dye as described in methods section. (B) STAT1 knockdown preserves mitochondrial function by upregulating Bcl-2 and down regulating Bax expression. N27 cells were transfected with scrambled siRNA or STAT1 siRNA and treated with 10μΜ CPF for 24h, cell lysates were prepared and immunoblotted for Bcl2 and Bax. β-actin was used as an internal control. Panel on the right hand side shows the densitometric scanning analysis for Bcl2 and Bax was performed, and the Bax/Bcl2 ratio was determined. The results represent mean ± S.E.M. from atleast 3 independent experiments. *p<0.05, ***p<0.001 vs vehicle treated scrambled siRNA transfected cells; ^##p<0.01 vs CPF treated scrambled siRNA transfected cells. (C) STAT1 binds to Bax promoter post CPF treatment. N27 cells were treated with CPF 50μΜ for 4h and 6h, respectively. Post treatment, cells were harvested and processed for chromatin extraction as described in material method section. The same experimental conditions for CHIP related experiments as detailed above, was implemented here. The immunoblot is representative of atleast 3 independent experiments. (D) Effect of STAT1 KD on CPF-induced cytosolic translocation of cytochrome c. Scrambled siRNA or STAT1 siRNA transfected N27 cells were treated with CPF 10μΜ for another 18h, the cytosolic fraction and mitochondrial fraction were extracted and cytochrome c release was determined using a spectrophotometer. The results are expressed as percentage of control and expressed as Mean ± SEM, N=4. *p<0.05, ***p<0.001 vs vehicle treated scrambled siRNA transfected cells; ^##p<0.01 vs CPF treated scrambled siRNA transfected cells.

Figure 9.. Effects of STAT1 knockdown and overexpression of STAT1 on CPF-induced PKCδ cleavage.

(A) STAT1 KD inhibited the proteolytic cleavage of PKCδ in CPF-treated cells. N27 cells were transfected with either scrambled or STAT1 siRNA as detailed above. Post treatment with CPF (10μΜ) for 24h, PKCδ cleavage was quantified using Western blotting analysis. The results are expressed as percentage of control and expressed as Mean ± S.E.M. The blots are representative of at least three independent experiments. ***p<0.001 vs vehicle treated scrambled siRNA transfected cells; ^{# #}p<0.01 vs CPF treated scrambled siRNA transfected cells. (B) Overexpression of STAT1 exacerbated proteolytic cleavage of PKCδ in CPF-treated N27 cells. N27 cells were transfected with either eGFP empty vector, eGFP WT STAT1, eGFPSTATlY701F or eGFP STAT1S727 mutant. Upon treatment completion (12h), cell lysates were prepared and immunoblotted for PKCδ and tubulin. (RHS panel) Blots were quantified using densitometric scanning analysis. Blot shown are mean ratio of cleaved PKC δ to tubulin. Data shown as Mean ± SD of at least three independent experiments. *p<0.05, ***p<0.001, ^###p<0.001 vs CPF treated eGFP empty vector transfected N27 cells.

Figure 10.. Analysis of autophagy and its regulation by STAT1 during CPF-induced dopaminergic cell death.

(A) Effect of CPF on autophagic markers expression in N27 cells. N27 cells were treated with CPF (10μΜ) for the indicated time period. Cell lysates were prepared and immunoblotted for autophagy markers. Equal protein loading was ensured by using β-actin as loading control. Representative blot from 4 independent experiments is presented. Quantification of blots using densitometric scanning analysis (right panel). (B) Immunofluorescence images depicting the extent of autophagic vacuole formation in CPF treated N27 cells. N27 cells were treated with 10μΜ of CPF for 6h and 18h followed by immunostaining for LC3. The cells were counterstained with Hoechst stain for nuclear labelling. CPF-induced significant time dependent increase in LC3 positive punctate structures, while a diffused pattern of distribution was evidenced in vehicle treated controls cells. (C) Effect of pharmacological interference of autophagy during CPF-induced cytotoxicity. N27 cells were pretreated with either Bafilomycin (10nM) or rapamycin (10nM) for lh followed by treatment with or without CPF (10μΜ) for another 24h and the levels of CPF-mediated cytotoxicity was assessed by the DNA fragmentation assay. The data represent mean ± SEM, N=6. ***p<0.001 vs vehicle treated control group; ^#p<0.05 vs CPF treated group. (D) LC3B siRNA knockdown sensitizes cells to CPF-induced cytotoxicity. N27 cells were transfected with scrambled or LC3B siRNA for 48h, subsequently treated with CPF (10μΜ) for another 24h and cell viability was determined using DNA fragmentation analysis. Results are expressed as percentage of control and presented as Mean ± SEM, N=6–8. ***p<0.001 versus scramble control cells exposed to vehicle, ^##p<0.01 vs scrambled siRNA transfected CPF treated group. (E) STAT1 knockdown attenuates CPF-induced upregulation of autophagic markers. N27 cells were transfected with scrambled or STAT1 siRNA as described above and incubated with CPF (10μΜ) for another 24h. LC3B, beclinl and p62 protein expressions were compared between whole cell lysates of scrambled transfected control cells and STAT1 siRNA transfected cells that were treated with or without CPF. β-actin is used as a loading control. The normalized densitometric band intensity analysis was presented as Mean ± S.E.M. Data representative of at least 4 independent experiments**p<0.01 and ***p<0.001 versus scrambled control cells exposed to vehicle, ^#p<0.05 vs scrambled siRNA transfected CPF exposed group.

Figure 11.. Mito apocynin (Mito-apo) protects against CPF induced neurotoxicity in N27 cells via amelioration of ROS generation and inhibition of STAT1 phosphorylation.

Dopaminergic N27 neuronal cells were pretreated with either the vehicle or the indicated concentration of mito-apo for lh followed by CPF (10μΜ) for another 24h. The cultures were assayed for cell death, oxidative species generation, STAT1 activation and autophagic markers (A) Mitoapocynin attenuate CPF-induced DNA fragmentation in N27 cells. Estimation of DNA fragmentation in N27 cells treated with CPF in the presence or absence of mitoapocynin. Cell death was assessed via Roche cell death ELISA kit and the data expressed as percentage of the control group. Data represented as mean ± SEM, (N=6). (B) Mitoapocynin attenuates CPF-induced ROS generation. Fluorometric analysis of cellular ROS measured using CM-H2DCFDA in dopaminergic neuronal cells. DCF fluorescence was normalized to the control group. Data presented as Mean ± SEM (N=6). Mitoapocynin attenuate CPF-induced Mitosox generation in N27 cells. (C) Mitochondria-derived superoxide was determined using Mito-Sox Red® fluorogenic dye in N27 cells exposed to CPF in the presence or absence of mitoapocynin. Change in fluorescence intensity was calculated as percentage of control (vehicle treated group) and represented as Mean ± SEM (N=6). (D, E) Mitoapocynin attenuates STAT1 activation and upregulation of autophagic markers. (D) Immunoblotting analysis of lysates prepared from N27 cells treated with CPF in the presence or absence of mitoapocynin. Densitometric scanning analysis revealed that mitoapocynin attenuated STAT1 phosphorylation in CPF treated cells. Membranes were probed with STAT1 and p-STATl (pS727) antibodies and were normalized to β-actin (used as a loading control). Data shown are Mean ± SEM from at least 3 independent experiments. (E) Quantification of bands revealed a reduction in the levels of autophagic markers in cells that received a combination of mitoapocynin and CPF. Data represented as percentage of control group. Each value represents the Mean ± SEM from at least 3 independent experiments. *p<0.001, **p<0.01 and ***p<0.001 indicates significant difference from Control group, while ^#p<0.001, ^##p<0.01 and ^###p<0.001 indicates significant differences between CPF and CPF + mito-apo treated cells.

Figure 12.. Mito-apo attenuated the CPF-mediated locomotor deficit and induction of STAT1 dependent proapoptotic cell signaling events in the rat substantia nigra (SNpc).

Neurobehavioral impairments in CPF (5mg/kg) (35-day repeated dose regimen) treated rats. Both male and female SD rats were subjected to daily oral administrations of CPF (5 mg/kg) or corn oil or CPF/mitoapocynin combination for 35 consecutive days (from PND 27 to 61) as described in methods section. 24h after the last dose, rats were subjected to locomotor analysis using VersaMax open field apparatus. (A-D) Versa plot showing movement track of rats collected over 10 min and various locomotor activity parameters were measured concurrently. Quantification of locomotor activity using the VersaMax Analyzer. Representative (B) horizontal activity; (C) vertical activity and (D) total distance travelled (cm). Mitoapocynin attenuated CPF-induced TH depletion and upregulation of STAT-1 and other proapoptotic markers in the substantia nigra. Mice were sacrificed at the end of behavioral analysis and SN was extracted and lysates were prepared for WB analysis. Representative blots showing the expression of ΤΗ (E), STAT1 and phosphorylated STAT1 (Y701 and S727 residue) (F); Bax and Bcl-2 (G); caspase-3 (H); PKCδ (I) and LC3 (J). Data are represented as means ± SEM of 6–8 rats per group. *p<0.001, **p<0.01 and ***p<0.001 versus the Control group, while ^#p<0.001 and ^{# #}p<0,01 indicates significant differences between CPF and CPF + mito-apo treated group.

Figure 12.. Mito-apo attenuated the CPF-mediated locomotor deficit and induction of STAT1 dependent proapoptotic cell signaling events in the rat substantia nigra (SNpc).

Neurobehavioral impairments in CPF (5mg/kg) (35-day repeated dose regimen) treated rats. Both male and female SD rats were subjected to daily oral administrations of CPF (5 mg/kg) or corn oil or CPF/mitoapocynin combination for 35 consecutive days (from PND 27 to 61) as described in methods section. 24h after the last dose, rats were subjected to locomotor analysis using VersaMax open field apparatus. (A-D) Versa plot showing movement track of rats collected over 10 min and various locomotor activity parameters were measured concurrently. Quantification of locomotor activity using the VersaMax Analyzer. Representative (B) horizontal activity; (C) vertical activity and (D) total distance travelled (cm). Mitoapocynin attenuated CPF-induced TH depletion and upregulation of STAT-1 and other proapoptotic markers in the substantia nigra. Mice were sacrificed at the end of behavioral analysis and SN was extracted and lysates were prepared for WB analysis. Representative blots showing the expression of ΤΗ (E), STAT1 and phosphorylated STAT1 (Y701 and S727 residue) (F); Bax and Bcl-2 (G); caspase-3 (H); PKCδ (I) and LC3 (J). Data are represented as means ± SEM of 6–8 rats per group. *p<0.001, **p<0.01 and ***p<0.001 versus the Control group, while ^#p<0.001 and ^{# #}p<0,01 indicates significant differences between CPF and CPF + mito-apo treated group.

Figure 13.. Mito apo confers resistance against dopaminergic neurotoxicity in the striata of CPF treated rats.

Rats subjected to the same treatment conditions as described in Fig. 12. 24h after the last CPF dose rats were sacrificed and the striata was harvested for biochemical analysis. (A) Mitoapocynin confers resistance against CPF-induced depletion of DA and its metabolite DOPAC levels. Quantification of striatal dopamine and its metabolite DOPAC levels by HPLC analysis in the striatum of CPF treated rats treated with or without mitoapocynin. DA and dihydrophenyactic acid (DOPAC) levels were determined in extracts prepared from striata of CPF treated rats with or without mitoapocynin using HPLC with coulometric detection. Results are represented as mean ± SEM with 6–8 rats per group. (B) Mitoapocynin attenuate CPF-induced modulation of TH and STAT-1 associated proapoptotic markers in the striata. Western blotting analysis of lysates prepared from striatal tissues. Representative immunoblots depicting expression of TH (B); STAT1, pSTATl Y701 and pSTATl S727 (C); Bax and Bcl-2 (D); caspase-3 (E); PKCδ (F); and LC3 (F). β-actin was used as a loading control. The densitometric scanning analysis indicate the normalized band intensity and are represented as mean ± SEM, N=6 rats per group. *p<0.001, **p<0.01 and ***p<0.001 vs Control group, while ^#p<0.05 and ^##p<0.01 indicates significant differences between CPF and CPF + mito-apo treated group.

Figure 13.. Mito apo confers resistance against dopaminergic neurotoxicity in the striata of CPF treated rats.

Rats subjected to the same treatment conditions as described in Fig. 12. 24h after the last CPF dose rats were sacrificed and the striata was harvested for biochemical analysis. (A) Mitoapocynin confers resistance against CPF-induced depletion of DA and its metabolite DOPAC levels. Quantification of striatal dopamine and its metabolite DOPAC levels by HPLC analysis in the striatum of CPF treated rats treated with or without mitoapocynin. DA and dihydrophenyactic acid (DOPAC) levels were determined in extracts prepared from striata of CPF treated rats with or without mitoapocynin using HPLC with coulometric detection. Results are represented as mean ± SEM with 6–8 rats per group. (B) Mitoapocynin attenuate CPF-induced modulation of TH and STAT-1 associated proapoptotic markers in the striata. Western blotting analysis of lysates prepared from striatal tissues. Representative immunoblots depicting expression of TH (B); STAT1, pSTATl Y701 and pSTATl S727 (C); Bax and Bcl-2 (D); caspase-3 (E); PKCδ (F); and LC3 (F). β-actin was used as a loading control. The densitometric scanning analysis indicate the normalized band intensity and are represented as mean ± SEM, N=6 rats per group. *p<0.001, **p<0.01 and ***p<0.001 vs Control group, while ^#p<0.05 and ^##p<0.01 indicates significant differences between CPF and CPF + mito-apo treated group.