Molecular and Biochemical studies in neuronal cells exposed to corticosterone: An In vitro model mimicking stress
Stress is a state of disrupted homeostasis caused by intrinsic or extrinsic stressors and is counteracted by a range of physiological and behavioural responses which aim to re-establish the threatened homeostasis (adaptive stress response). The body’s principal physiological responses to stressful stimuli are mediated by the sympathoadrenal system and the hypothalamic pituitary adrenal (HPA) axis. Important pathophysiology of chronic stress includes elevated glucocorticoid (GC) levels, increased oxidative stress and mitochondrial dysfunction. Hence corticosterone (CORT) administration is used to develop in-vivo and in-vitro model of stress in rodents and cell lines respectively. In this study, we checked the viability of neurons after treatment of CORT using MTT assay on cultured N2A cell line. Levels of reactive oxygen species (ROS) generated during oxidative stress was recorded by flow cytometry using DCFDA and GSH assay by spectrophotometry. JC1 dye was used to measure change of mitochondrial membrane potential (MMP) in neurons under stress. Signalling mechanisms involved were checked by observing change in protein expression of Bax and Bcl-2 of in-vitro samples. Our results demonstrated that chronic exposure of corticosterone influenced neuronal health by inducing ROS generation and modulating apoptotic and anti-apoptoic proteins levels.
Keywords: homeostasis, stress response, HPA axis, N2A, ROS, MMP.
|ROS||Reactive oxygen species|
|MMP||Mitochondrial Membrane potential|
|DMEM||Dulbecco's Modiﬁed Eagle Medium|
|FBS||Fetal Bovine Serum|
|PBS||Phosphate Buffer Saline|
|DTNB||5-5’-dithiobis [2-nitrobenzoic acid]|
|HRP||Horse radish peroxidase|
All vital systems of the body preserve a predefined steady state (homeostasis) which is essential for life and well being. Stress is generally defined as any stimulus that disrupts this homeostasis. This is counteracted by range of physiological and behavioral responses which aim to reestablish the threatened homeostasis (adaptive stress response).
Stress and HPA axis
HPA axis mediates stress reponse
The hypothalamic pituitary adrenal (HPA) axis is responsible for the neuroendocrine adaptation component of the stress response. This response is characterized by hypothalamic release of corticotropin-releasing factor (CRF)  . CRF is also known as corticotropin-releasing hormone (CRH). When CRF binds to CRF receptors on the anterior pituitary gland, adrenocorticotropic hormone (ACTH) is released. ACTH binds to receptors on the adrenal cortex and stimulates adrenal release of glucocorticoids (GCs). In response to stressors, cortisol in humans and corticosterone in rodents, is released for several hours after encountering the stressor. At a certain blood concentration of cortisol when this protection is achieved, the cortisol exerts negative feedback to the hypothalamic release of CRF and the pituitary release of ACTH  . At this point, systemic homeostasis returns.
Acutely, GCs act to inhibit stress‐induced CRF and ACTH secretion through their actions in brain and anterior pituitary (negative feedback). With chronic stress, GC feedback inhibition of ACTH secretion changes markedly  .
The Stress hormone Corticosterone
Corticosterone (CORT) is a glucocorticoid and a major stress hormone produced in the cortex of the adrenal gland and plays a regulatory role in stress-induced HPA axis activity in rodents. Chronically elevated CORT levels activate the chronic stress-response network, and as a result, impact various processes involved in coping with stress. Previous studies showed that chronic CORT administration in rodents inhibits stress-induced serum CORT elevation  and causes increases in serum insulin and leptin  , adipose-derived hormones that regulate food intake and body weight . In addition, chronic CORT administration induces depression in rodents  .
Pathophysiology of Stress
Recently, oxidative stress has been suggested as one important down-stream consequence of chronically elevated GC levels. Oxidative stress is a complex, multi-faceted state that arises in organisms as a consequence of the imbalance between the production of pro-oxidant molecules and antioxidant defences. Reactive oxygen species (ROS) are one main type of pro-oxidants, and partly arise as by-products of cellular respiration in mitochondria. Although ROS have important roles in cell signalling, they act as a double-edged sword damaging macromolecules, cell components and structures when produced in excess. In the past decade several studies have highlighted direct links between chronic stress exposure and/or chronically elevated GC levels and oxidative stress  .
Neuronal oxidative stress is induced by GCs through enhanced mitochondrial respiration and oxidative phosphorylation. This was demonstrated clearly in a study by Du et al., showing that acute incubation of cortical neurons with corticosterone modulates mitochondrial oxidation, membrane potential, and calcium-holding capacity in a dose and time-dependent manner.
An important pathophysiology of stress is mitochondrial dysfunction since mitochondria are the site of aerobic respiration and a major source of ROS. GCs are known to affect mitochondrial gene expression, mitochondrial biogenesis, and mitochondrial fission/fusion dynamics, influencing both ATP and ROS production. However, the way GCs affect mitochondrial function and consequently oxidative stress and apoptosis could differ depending on GC dose and duration of exposure.
By investigating the effect of CORT induced stress on neuronal cells, and by exploring the underlying mechanism, we pave for a more targeted neuroprotective therapy during condition of chronic stress.
Objectives of the Research
1. To investigate cell viability of neurons at different concentrations of corticosterone in time-dependent manner
2. To investigate the effect the different concentrations of CORT on mitochondrial functionality and oxidative stress
3. To investigate the signalling mechanism involved in CORT induced cellular stress
Stress and Depression
It is estimated, by the World Health Organization, that depression will be the most important cause of disability in the world by the year 2020  . Twin studies suggest that about 25–30% of the variance for depression is genetic and that environmental factors account for about 75% of the variance  . The most important environmental factor is stress  . The connection between stress and depression was initially drawn from observations of over activity of the hypothalamic–pituitary–adrenal (HPA) axis, elevated cortisol levels and disrupted cortisol rhythmicity in depressed patients .
Animal Models for Stress and Depression
Based on such clinical findings, animal models were developed to further study the molecular effects of stress and the underlying neurobiological mechanisms of depression. For pharmacologists, these animal models of depression can also be used to screen for antidepressants or preventive drugs which target the HPA axis or cortisol receptors. Of the animal models that currently exist, those involving repeated exposure to stress hold promise for modeling depression, as they simulate the presumed etiology of the disorder. Animal models of repeated stress have utilized a wide range of stimuli to invoke HPA axis activation, ranging from chronic mild stress exposure to repeated restraint stress  . There is a possibility of lack of control over individual differences in HPA axis activation and subsequent corticosterone levels in terms of experimenter-applied stress models. Stressful stimuli can differ in their physical qualities, and in terms of their psychological qualities  . This may result in differing corticosterone levels between different animals exposed to the same stressor, which in turn could lead to increased experimental variability  .
One way to avoid these problems is by using exogenous corticosterone administration as a means to study the effects of elevated corticosterone levels, which would occur as a consequence of stress exposure.
CORT is the main glucocorticoid in many species and is involved in regulation of energy  , immune responses  and stress response  . CORT has multiple effects on memory. The main effects are seen through the impact of stress on emotional memories as well as long term memory (LTM). With emotional memories, CORT is largely associated with fear memory recognition. Studies have shown that when fear memories are reactivated or consolidated, levels of corticosterone increased and depending on the time at which the administration of corticosterone took place as compared to when the fear conditioning took place; CORT can either facilitate or interrupt conditioned fear  .
CORT-treated rat models exhibit decreased telomerase activity and down-regulated expression of telomere-binding factor 2, correlated with enhanced oxidative damage. This was associated with inhibition of sirtuin 3 leading to reduced activities of superoxide dismutase 2 and glutathione reductase  .
Stress signalling is a cell signalling pathway that promotes a response to cell stress. Depending on the severity of the insult, stress signalling can mitigate the potential damage or induce apoptosis.
Cells respond to stress in a variety of ways like alteration of cell survival pathways as well as modulating programmed cell death. The cell’s initial response to a stressful stimulus is geared towards helping the cell to defend against and recover from the insult. However, if the stimulus is unresolved, then cells activate death signaling pathways. Various types of cellular stress stimuli have been shown to trigger apoptosis, including chemotherapeutic agents, irradiation, oxidative stress, and ER stress. Caspases, a family of cysteine proteases, act as common death effector molecules in various forms of apoptosis  . The mitochondrial pathway to caspase activation is initiated by the release of cytochrome c from the mitochondrial intermembrane space, which results in caspase-3 activation  .
Stress influences biological and physiological processes across the organism, including those inside the cell nucleus where genes are transcriptionally and epigenetically regulated.
Recent work has established that, in brain and other organs, the glucocorticoid receptor (GR) is translocated from the cytosol to the mitochondria and that stress and corticosteroids have a direct influence on mtDNA transcription and mitochondrial physiology. They showed degenerated mitochondrial functions are represented by decreased adenosine triphosphate production, decreased nicotinamide adenine dinucleotide content, and decreased activity of nicotinamide phosphoribosyltransferase  .
It has been found that GRs formed a complex with the anti-apoptotic protein Bcl-2 in response to CORT treatment and translocate with Bcl-2 into mitochondria after acute treatment with low or high doses of CORT in primary cortical neurons. However, Bcl-2 levels in the mitochondria of the prefrontal cortex were signiﬁcantly decreased, along with GR levels, after long-term treatment with high-dose CORT in vivo  . These ﬁndings have the potential to contribute to a more complete understanding of the mechanisms by which GCs and chronic stress regulate cellular plasticity and resilience and to inform the future development of improved therapeutics.
Materials and Reagents
- Neuro-2a cell line is mouse neuroblastoma cell line, obtained from the National Centre for Cell Sciences, Pune, India, and maintained at a tissue culture facility of CSIR-CDRI, Lucknow.
- DMEM (ThermoFisher Scientific)
- FBS (HiMedia)
- Trypsin (ThermoFisher Scientific)
- Corticosterone (Sigma Aldrich)
- DMSO (SDFCL, India)
- MTT salt (Sigma Aldrich, USA)
Estimation of ROS levels by flow cytometry
- DCF-DA (Sigma Aldrich, USA)
- Ellman's reagent (DTNB) (SRL Chemicals, India)
Estimation of mitochondrial membrane potential
- JC 1 dye (5,5,6,6’-tetrachloro-1,1’,3,3’ tetraethylbenzimi-dazoylcarbocyanine iodide) (Sigma Aldrich, USA)
|Acrylamide/Bis-acrylamide (30%/0.8% w/v)||4 ml|
|1.5M Tris (pH=8.8)||2.6 ml|
|10% (w/v) SDS||0.1 ml|
|10% (w/v) ammonium persulphate (AP)||100 μl|
|Acrylamide/Bis-acrylamide (30%/0.8% w/v)||0.67 ml|
|1.5M Tris (pH=6.8)||0.5 ml|
|10% (w/v) SDS||40 μl|
|10% (w/v) ammonium persulphate (AP)||30 μl|
- RIPA lysis buffer containing protease inhibitor cocktail (Sigma-Aldrich, St. Louis, USA)
- BCA protein assay kit (Thermo Pierce, Rockford, USA)
- PVDF membranes (Millipore, Billerica, MA, USA)
- BSA (SRL chemicals, India)
- Rabbit anti Bax (1:1000, Thermo scientific) antibody
- Rabbit anti Bcl-2 (1:2000, Millipore, Temecula, CA, USA) antibody
- Mouse anti-β-actin (1:3000, Sigma-Aldrich, St. Louis, USA) antibody
- HRP-conjugated secondary antibodies (Sigma-Aldrich, St. Louis, USA)
- Chemiluminescence substrate kit (Millipore, Billerica, MA, USA)
Neuro-2a cell line was maintained in DMEM/F-12 containing 10% FBS at 37 ºC in a humidified atmosphere of 5% CO2/95% air.
MTT assay for estimation of cell viability
MTT assay was used for the estimation of cell viability according to the reference with some modification  . N2A cells were seeded at the density of 1 ×104 cells/well in 96-well plates. Corticosterone (CORT) was prepared as a fresh stock solution by dissolving it in pure DMSO. Neuro-2a cells were incubated with CORT 25 µM, 50µM and 100µM with allowed concentration of DMSO (0.5% per treatment well). After the treatment period, MTT salt (Thiazolyl Blue Tetrazolium Bromide) (10 µl/well containing 100 µl of cell suspension; 5 mg/ml in PBS) was added. The assay determines the ability of the mitochondria to convert the yellow 3-(4,5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide tetrazolium salt into a purple MTT formazan crystals, by the mitochondrial dehydrogenase activity of live cells, which is measured spectrophotometrically at 595 nm. Results of this assay determine the concentration and time for further experiments.
Measurement of chronic corticosterone esposure induced ROS generation by flow cytometry in Neuro-2a cell line
Intracellular ROS (H2O2 and/or hydroxyl radical) in Neuro-2a cells was determined through a flow cytometric assay using 2,7-dichlorofluorescin diacetate (DCF-DA). The cell samples were incubated with 25μM, 50μM, 100μM CORT for 48 hours. DCF-DA, a non-fluorescent cell permeant compound, is cleaved by endogenous esterases and the de-esterified product becomes fluorescent upon oxidation by ROS. Cells were incubated with DCF-DA at a final working concentration of 0.1µM for 20 min at 37 ºC, washed twice in PBS, and their fluorescence intensity was assessed by acquiring 10,000 events using FACS Calibur and analyzed by Cell Quest program. The data of treated group was compared to control in terms of mean fluorescence intensity (MFI) 
Estimation of reduced Glutathione level
The GSH content in the supernatant of cell lysate was measured after the end point of corticosterone treatment by following the Ellman method (1959). Briefly, 100 µl of supernatant of cells lysate was mixed with 100 µl of 0.1 M sodium phosphate buffer (pH 8.0) and 50 µl of 6 mM DTNB. The reaction mixture was then incubated at room temperature for 10 min and optical density (O.D) of coloured product was measured at 412 nm, using ELISA plate reader (BIOTEK, USA). GSH concentration was calculated by using standard curve prepared with reduced glutathione and expressed as µg/mg protein .
Estimation of mitochondrial membrane potential (ᴪM, MMP) in corticosterone treated Neuronal cells
N2A Cells (1×105 cells/well) were seeded on cell culture treated 12 well plates and maintained at 37°C with 5% CO2 exposure. The culture medium is changed every other day until the cells reach 60-70% confluency and treatment of CORT was given for 48 hours followed by changing the media. At the end time point of corticosterone treatment, the media is removed from culture wells by aspiration and then suffiecient trypsin or trypsin/EDTA solution is dispensed into culture wells(s) to cover the monolayer of cells completely. Then it is placed in 37°C incubator 2 min and double amount of warm cell culture medium with 10% FBS was added into each well to stop the action of trypsin/EDTA solution. The material from each well was collected into sterile FACS tubes and centrifuged for 5 min at 25°C at 2500 rpm. Then media was removed from the tube by aspiration and the pellet was washed twice in 1X PBS. Cell sample in each FACS tube were stained with fluorescent probe JC-1 at 2.5 μg/ml and incubated for 30 minutes at 37°C. Total 10,000 events were acquired and analysed by FACS Calibur.
Western blot analysis
After 48 hours of CORT treatment, cells were extracted in ice-cold RIPA lysis buffer containing protease inhibitor cocktail, and total protein concentration in the samples was determined by the BCA protein assay kit. Protein samples of 40 μg were subjected to SDS-PAGE gel (12%) than further transferred onto PVDF membranes. 5% BSA was used for blocking PVDF membrane at room temperature for 2 h further. PVDF membranes were incubated overnight at 4 °C with primary antibodies rabbit anti Bax (1:1000), rabbit anti Bcl-2 (1:2000) and mouse anti-β-actin (1:3000, Sigma-Aldrich, St. Louis, USA). After washing with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at room temperature. Immunoreactive bands were detected with an enhanced chemiluminescence substrate kit, and the density of protein band was quantified by my Image analysis software (Thermo Scientific, Rockford, USA) .
RESULTS AND DISCUSSION
No significant change in the viability of the neuronal cells is observed after 12 and 24 hours of CORT treatment. After 48 hours, cell viability of N2a cells incubated with 100μM CORT dropped significantly compared to control.
Levels of ROS Generated
By flow cytometry
After diffusion in to the cell, DCFDA is deacetylated by cellular esterases to a non-fluorescent compound, which is later oxidized by ROS into DCF which is a highly fluorescent compound. Using flow cytometry, we saw that ROS levels increased with increase in concentration of CORT and there is significant increase in N2a cells incubated with 100μM CORT.
By GSH assay
Reduced glutathione is a prominent antioxidant and low levels of GSH is correlated with increased oxidative stress and ROS generation in N2a cells RENE KIZEK, 2012 . There is significant reduction in GSH levels with increasing concentrations of CORT.
MMP of neurons in stress
The membrane-permeant JC-1 dye is widely used in apoptosis studies to monitor mitochondrial health. JC-1 dye exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (~529 nm) to red (~590 nm). Consequently, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio. We observed that mitochondria are healthy at low CORT concentration incubation, but significantly depolarised at 100μM CORT treatment in neuronal cells.
Protein Expression in Cell Apoptotic Pathway
Level of Bax, a pro-apoptotic protein was significantly higher in CORT treated neurons as compared to control group.
B cell lymphoma-2 (Bcl-2), a cell survival related protein, was expressed less in CORT treated neurons compared to the control, untreated cells.
In this study, we demonstrated the effect of CORT on neuronal N2a cells. We found chronic exposure of CORT affects cell viability in a dose dependent manner. This is supported by studies stating the biphasic action of GCs where at low doses, they show neuroprotective effect and high doses induce neurotoxicity in cortical neurons  .
The brain has a high oxygen turnover and is susceptible to damage from ROS  . This is kept in check by the antioxidant defense system. Our study shows that CORT treatment results in increased levels of ROS which is indicator of neurons in oxidative stress. This may be due to an imbalance between the oxidative species and antioxidant system. GSH is a major intracellular thiol compound and it is the main mechanism of antioxidant defense against ROS  . Hence, to validate our results for oxidative stress, we performed GSH assay which shows low levels of GSH at all doses of CORT after 48 hours exposure.
Previous studies have shown that mitochondria is a primary source of ROS generated as a by-product in Electron Transport Chain reaction  . Recent work shows that mitochondria are also a major target for the detrimental effects of ROS   . Based on literature, mitochondrial permeability transition pore is responsible for maintaining the membrane potential. The presence of thiol groups makes it susceptible to oxygenation and subsequent collapse of MMP  . In concurrence with previous studies, our results show that CORT treatment in neurons results in ROS generation and subsequent mitochondrial membrane depolarisation.
Literature suggests that cells under stress activate cell survival and cell death signalling pathways. Cell death signalling frequently converges on mitochondria, a process that is controlled by the activities of pro- and anti-apoptotic proteins. To avoid undesired cell death, this apoptotic pathway is tightly regulated by members of the Bcl-2 protein family, which interact on the outer mitochondrial membrane (OMM), and modulate its permeability to apoptotic factors, controlling their release into the cytosol. Bax is an inactive monomer under non-apoptotic conditions. Following an apoptotic stimulus, Bax is activated and undergoes oligomerisation in the OMM which releases cytochrome c and other mitochondrial proteins into the cytosol  . Furthermore, in our study, oxidative stress induced by CORT is found to be associated with modulation of pro- and anti- apoptotic proteins, Bax and Bcl-2. Bax protein expression was significantly higher after CORT exposure as compared to control group. In contrast, Bcl-2 protein levels were reduced in CORT incubated neurons in comparison with control group.
In conclusion, our study demonstrates a direct pro-oxidant effect of corticosterone (stress hormone) on neuronal cells, which indicates an important implication for oxidative stress as a major pathological mechanism during chronic stress and a consequent target option for anti-stress therapeutic interventions.
The possibility that agents that enhance mitochondrial function may be useful in countering the deleterious effects of excessive glucocorticoid secretion observed in mental health related pathologies like anxiety and depression is an exciting prospect for future investigation.
I would like to express my gratitude to all the people, without whose whole hearted encouragement, support and cooperation, this summer research training work would not have been possible.
First and foremost, I would like to extend my sincere thanks to Indian Academy of Sciences for giving me this opportunity. I am extremely grateful to my guide, Dr Shubha Shukla, for her constant guidance and advice throughout my stay. I also take this opportunity to confer my thanks to Dr Tapas K Kundu, Director, CSIR- Central Drug Research Institute for the facilities I have been provided with.
I express my sincere thanks to all the lab members that I have been associated with. Parul, Akanksha Mishra, Virendra Tiwari, Pratibha Tripathi, Shivangi Gupta, Jitendra Singh and Mrs Sachi Bharti, who have always helped me with my questions and helped set the foundation for any future research work I plan to pursue. Their work and diligence will continue to inspire me and it is my privilege to have learnt from them.
I would also like to thank my family and friends, whose blessings and support helped me undertake this internship.
Finally, I thank my college, National Institute of Science Education and Research (NISER), Bhubaneswar and the Kishore Vaigyanik Protsahan Yojana (KVPY), Department of Science and Technology (DST) for motivating and supporting me.
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