by Jae Choi, Ph.D.; Janaki Narahari, Ph.D.; Douglas Hughes, Ph.D.; Megan Dobbs, B.S.; Georgyi Los, Ph.D.; Brian Webb, Ph.D. - 04/30/13
Parkinson disease (PD) is a neurodegenerative disorder characterized by the selective loss of dopaminergic neurons and the presence of Lewy bodies in the substantia nigra pars compacta (SNpc) (Lees 2007). However, the molecular events and mechanisms involved remain unknown. Common herbicides and insecticides have been implicated in the pathogenesis of Parkinson’s disease (Tanner 2011). Paraquat is a widely-used agricultural herbicide that, in vivo, has been reported to induce dopaminergic cell loss through oxidative stress (McCormack 2002, Cristovao 2009). Systemic administration of paraquat results in upregulation of α-synuclein expression and the formation of aggregate (McCormack et al., 2002). Rotenone is an insecticide that also inhibits complex I in mitochondria (Greenamyre 2010). Rotenone has been used in rat models to induce a Parkinson-like phenotype characterized by degeneration of the dopaminergic neurons and motor impairment (Sherer et al., 2000). MPP+ is an active metabolite of MPTP, a byproduct of heroin synthesis. Because MPP+ supposedly induces oxidative stress and cell death (Fallon 1997), it serves as a familiar control of neurotoxic effects.
Oxidative stress activates the transcription of a variety of antioxidant genes through cis-acting sequences of antioxidant response element (ARE) (Brigelius-Flohe 2011). Research suggests that oxidative stress also activates NF-κB-dependent transcription of pro-survival genes (Nguyen 2009). Although several studies have identified paraquat, rotenone, and MPP+ as a high risk factor for Parkinson’s disease, the molecular mechanism how these neurotoxic chemicals exert their effect on the dopaminergic neurons is largely unknown. For this reason, we are interested to know if and how these chemicals induce genes in an ARE- or NF-κB-dependent manner. To test these possibilities, we developed a sensitive triple luciferase reporter assay system with Thermo Scientific Pierce Luciferase Assay Vectors and Kits (Gaussia, Cypridina, and Red Firefly luciferases). We used this system with neuroblastoma cells (IMR-32) to simultaneously measure whether or not the parkinsonian neurotoxins paraquat, rotenone and MPP+ can activate ARE- and NF-κB-dependent transcription normalized by the CMV-driven transcription. In so doing, we demonstrate the practical application of this triple luciferase assay to simultaneously monitor two pathways while normalizing to a third reporter of baseline activity.
Before constructing reporter vectors and measuring treatment responses on ARE and NF-κB, we checked for off-target effects of the test chemicals on luciferase activities in vitro by measuring secreted Gaussia and Cypridina luciferase in different concentrations of various neurotoxic chemicals (Figure 1). Luciferase activities of these two enzymes were relatively unaffected by up to 500μM concentrations of various chemicals, including paraquat, rotenone, TCDD (2,3,7,8-tetracholorodibenzo-p-dioxin), tBHQ (tert-butylhydroquinone) and 3-MC (3-methylcholanthrene). However, rifampicin (activator of pregnane X receptor, PXR) inhibits Gaussia activity in high concentration. In addition, it is important to determine ranges of chemical concentration that do not affect cell viability because a lethal concentration of chemicals shut down cellular transcription and translation that makes any measurement of transcriptional activation impossible (Figure 2). Taken together these tests indicate that there are ranges of treatment-concentrations with these compounds whereby changes in luciferase activities in our experiments can be attributed to their biological effects on the expression of the genes to which the respective luciferases are linked as reporters.
Figure 2. IMR-32 cell viability curve with different concentration of pesticides and other test compounds treated for 10 hours. We determined a range of compound concentration that does not affect cell viability. A lethal concentration of chemicals would necessarily interfere with accurate interpretation of transcriptional reporter assay. Yellow-shaded regions represent the ranges used in subsequent experiments (Figures 3 to 5). Plotted values are means ± SD of three replicates.
The next preliminary steps in our study were (a) to construct reporter plasmids of ARE and NF-κB and (b) to validate their expression in the assay system using known activators. We cloned the ARE gene into pMCS-Gaussia Luc (Part No. 16146) and the NF-κB gene into pMCS-Cypridina Luc (Part No. 16149), including the TATA-minimal promoter. To test functionality, we transfected each reporter plasmid into IMR-32 (neuroblastoma) cells, which we then treated with different concentrations of canonical activators and assayed using respective Pierce Luciferase Flash Assay Kits. L-sulforaphane (an antioxidant) and tumor necrosis factor alpha (TNFα) activated ARE and NF-κB, respectively, in a dose-dependent manner (Figure 3), indicating that the ARE and NF-κB reporter constructs were functional.
Figure 3. Two different response elements are functional after treatment with canonical activator. (A) ARE activation by the antioxidant, L-sulforaphane. (B) NF-κB activation by TNFα. Plotted values are means ± SD of three replicates.
Next, we developed and tested our triple luciferase assay system by examining its ability to measure the extent to which either tBHQ or TNFα can activate both ARE and NF-κB. The assay involves co-transfecting three plasmids (ARE-Gaussia, NF-κB -Cypridina, and CMV-Red Firefly) into neuroblastoma cells. At 24 hours post-transfection, the cells are treated with different concentrations of test compound for a given time (5 hours in this case). The activity of Gaussia (a secreted luciferase) is measured in an aliquot of media to assess ARE activation. After cell lysis, Cypridina and Red Firefly activities are simultaneously measured in lysate using the Pierce Cypridina-Firefly Dual Assay Kit. All relative light unit (RFU) values are normalized to the Red Firefly readings.
The antioxidant tBHQ strongly activates ARE and only weakly activates NF-κB response element as well. (Figure 4A). TNFα strongly activates both NF-κB and ARE (Figure 4B), indicating that there may be a significant cross-talk between TNFα and the ARE pathway upon TNFα activation in neuroblastoma cells.
Figure 4. Triple luciferase assay reveals that both ARE and NF-κB are activated by tBHQ or TNFα. (A) tBHQ strongly activates ARE and weakly activates of NF-κB, (B) TNFα strongly activates both ARE and NF-κB pathways. (Gaussia and Cypridina values are normalized to signal from the co-transfected, constitutively expressed, CMV-Red Firefly Luc reporter.) Plotted values are means ± SD of three replicates.
Ultimately, we wished to determine whether paraquat, rotenone, or MPP+ can activate antioxidant response element (ARE) and NF-κB pathways. Thus, we applied our triple luciferase assay system to measure effects of these compounds on expression of co-transfected ARE and NF-κB reporter plasmids in human IMR-32 cells. At 24 hours post-transfection, the human IMR-32 (neuroblastoma) cells were treated for 5 hours with paraquat, rotenone or MPP+ at the various concentrations. Activation of both pathways occurred in response to paraquat (Figure 5A) but activation was negligible or nonexistent in response to rotenone (5B) and MPP+ (5C).
Paraquat and rotenone have been reported to be tightly linked to the generation of reactive oxygen species (ROS) formation (Castello 2007; Cristovao 2009; Drechsel and Patel 2009). This suggests that oxidative stress activates the transcription of a variety of antioxidant genes through cis-acting sequences known as antioxidant response elements (ARE). Similarly, oxidative stress has been largely demonstrated to activate NF-κB-dependent transcription of pro-survival genes (Brigelius-Flohe and Flohe 2011; Nguyen 2009). We simultaneously measured the activation of both ARE- and NF-κB-driven reporters in response to parkinsonian neurotoxins. Our results show that paraquat, but not rotenone or MPP+, triggered transcriptional activity driven by ARE and NF-κB reporters. These results demonstrate that oxidative stress induced by paraquat, but not by MPP+ or rotenone, is directly linked to ARE-driven transcriptional activation.
Luciferase reporter-based pathway analysis in neuronal cells has been a challenge due to the slow growth and low metabolism of these cells. Here, we demonstrate the efficacy of a triple luciferase assay system to measure transcriptional up-regulation of antioxidant response element (ARE) and NF-κB pathways in response to known activators, antioxidants and pesticide chemicals in neuronal cells.
Our assay system easily detected the effects of L-sulforaphane, TNFa, tBHQ and paraquat on ARE and NF-κB-dependent transcriptional activation in human IMR-32 neuroblastoma cells. Most notable among these findings is that paraquat is identified as an activator for ARE-driven reporter assays. What is not clear is whether ARE activation by paraquat eventually contributes neuronal toxicity or protection. Nevertheless, we conclude that the assay system can be used to elucidate molecular mechanisms of paraquat effects or to screen for small-molecule ARE modulators to identify potential positive therapeutic effects on Parkinson Disease and treatment.
Cell culture: IMR-32 (ATCC, CCL-127) and HepG2 (ATCC, HB8065) cells were cultured at 37°C with 5% CO2 in Eagle’s minimum Essential Medium with 10% fetal bovine serum. Cells were passaged every two days and maintained at 50-70% confluence. Log-phase cells were plated in 96-well plates for 16 hours before transfection to ensure that they were healthy.
Transfection: In each well of a 96-well plate, 1.0 x 10^5 log-phase IMR-32 cells were seeded in 0.1mL of in Eagle’s minimum Essential Medium 16 hours before transfection. The three plasmids (50/50/30ng of DNA, respectively) were diluted in 10μL of serum-free media. TurboFect Transfection Reagent (0.3μL; Part No. R0533) was added to the diluted DNA and mixed by pipetting. The mixture was added drop-wise to each well. The cells were incubated at 37°C in a 5% CO2 incubator for 24 hours before any treatment.
Cell viability assay: In each well of a 96-well plate, 1.0 x 10^5 log-phase IMR-32 cells were seeded in Eagle’s minimum Essential Medium 16 hours before compound treatment. The fresh media (100 μL) containing a series of dilution of compounds were added to a well in the plate and continued to incubate for 10 hours at 37°C in a 5% CO2 incubator for 10 hours before cell viability assay. After 10 hours, 10μL of Thermo Scientific alamarBlue Cell Viability Assay Reagent (Part No. 88951) was added to each well, continued to incubate the plate for 3.5 hours, and then the plates were measured at 545nm/590nm (Ex/Em) using the Thermo Scientific Varioskan Flash Multimode Plate Reader.
Luciferase reporter assays: 24 hours after transfection, the media was collected for activity measurement. The cells on the bottom of the plate were lysed with 100μL of Pierce Luciferase Cell Lysis Buffer (Part No. 16189). Either the collected media or lysates was used for luciferase activity measurement. The Gaussia activity was determined using the Pierce Gaussia Luciferase Flash Assay Kit (Part No. 16158). The Cypridina activity was determined using the Pierce Cypridina Luciferase Flash Assay Kit (Part No. 16168). The Gaussia-firefly activity was determined using the Pierce Gaussia-firefly Luciferase Dual Assay Kit (Part No. 16181). The Cypridina-firefly activity was determined using the Pierce Cypridina-firefly Luciferase Dual Assay Kit (Part No. 16181). Bioluminescence signals (RLUs) were detected using a Thermo Scientific Varioskan Flash Luminometer equipped with reagent injectors (Signal integration time = 1 second).
The ARE-Gaussia and NFkB-Cypridina Luc reporter plasmids constructed by cloning each response element plus minimal promoter into Pierce promoterless pMCS-Luciferase Reporter Plasmids (Part No. 16146) (Part No. 16149) (Part No. 16152) were transfected into 1.0 x 10^5 cells for 24 hours using Pierce TurboFect Transfection Reagent (Part No. R0533). At 24 hours post-transfection, cells were treated with chemical activator for 4 to 5 hours to activate the signal transduction pathway. The media was carefully collected for activity measurement. The cells were lysed with Pierce Luciferase Cell Lysis Buffer (Part No. 16189). Activity in media and lysate were determined using the appropriate luciferase flash assay kit (see previous paragraph). Bioluminescence signals (RLUs) were detected using a Varioskan Flash Luminometer equipped with reagent injectors (signal integration time = 1 sec) and filter sets in the ranges of 425 to 525nm for Cypridina Luc and 615nm LP for Red Firefly Luc.
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