Loading...

Summer Research Fellowship Programme of India's Science Academies

Engendering DJ-1 expressing stable transfected astrocytoma cell line

P Anil Sasikumar Menon

Amrita School of Biotechnology, Amritapuri, Clappana P. O. Kollam 690525 Kerala, India

Dr. Jayasri Das Sarma

Neuroscience Laboratory, Indian Institute of Science Education and Research Kolkata, Mohanpur, Nadia District 741246, West Bengal, India

Abstract

DBT is a mouse astrocytoma cell line (a cellular model to study astrocytes) that has been found to get infected with MHV-A59 virus. MHV-A59 is a neurotropic strain of the mouse hepatitis virus that has been found to infect neurons, astrocytes, microglia and oligodendrocytes​. This virus has been found to induce oxidative stress in the nervous system upon infection, which can be considered as one of the underlying mechanism for cellular death caused due to virus infection. One of the cellular defence response proteins against oxidative stress is DJ-1, which functions via multiple pathways to prevent oxidative stress-induced cellular apoptosis. Therefore, the lookout of our study is whether DJ-1 is playing any particular role in MHV-A59 infection in DBT cells. To address this question, we generated DJ-1 stably transfected DBT cell line and studied the effect of MHV-A59 infection in these cells. DJ-1 was previously cloned into pDsRed plasmid (contains G418 antibiotic selection marker). Antibiotic kill curve analysis was performed to determine the minimum G418 concentration required killing maximum non-transfected cells in 48 hours, and 1.5 mg/ml G418 concentration was standardised to be the optimum for selection for DBT cells. DBT cells were then electroporated with DJ-1 containing pDsRed plasmid and selected with G418 containing media. The stable transfection was confirmed by PCR analysis for a DJ-1-RFP fusion gene present in the transcriptome of the transfected cells. DBSDJ (DBT stably transfected with DJ-1) cells were infected with MHV-A59, and no significant loss in cell viability was observed in the transfected cell line. This data was contrary to the non-transfected DBT cells which showed a marked decline in cell viability upon infection. Additionally, oxidative stress generation was not observed in DBSDJ cell line upon infection, but observed in DBT cells. This study will help in understanding the role of DJ-1 in MHV-A59 infection in DBT cells.

Keywords: DBT, DJ-1, MHV-A59, oxidative stress, cell viability

Abbreviations

Abbreviations
DBT Delayed Brain Tumour
MHV-A59 Mouse Hepatitis Virus A59
DJ-1 Protein deglycase-1
XBP1 X-box binding protein 1
Nrf2 Nuclear factor erythroid 2-related factor 2
ASK1 Apoptosis signal-regulating kinase 1
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
DCFDA Dichlorofluorescin diacetate
DBSDJ DBT stably transfected with DJ-1
PCR Polymerase Chain Reaction

INTRODUCTION

MHV-A59 virus has been found to infect astrocytes, which are instrumental in controlling the oxidative balance in the nervous system. Therefore, it is important to investigate whether oxidative stress is involved in the process of viral infection in astrocytes. To address this issue, we used a cell culture model of astrocytes, DBT mouse astrocytoma cell line, which is infected with MHV-A59 virus. Virus infection was found to cause oxidative stress generation and a loss in cell viability in DBT cells.

The next question that arises is whether DJ-1, a cellular response protein against oxidative stress generation, is playing any particular role in the viral infection process. Previous data from our laboratory showed that DJ-1 is downregulated upon viral infection, which can be assessed to contribute to the observed decrease in cell viability due to infection. To address this question, we have generated a stably transfected DJ-1 overexpressing DBT cell line, and assessed any changes in cell viability and oxidative stress generation due to viral infection. We have observed that DJ-1 overexpression in DBT cell line largely ameliorates the decline in cell viability and oxidative stress generation that was previously observed in untransfected DBT cell line. Therefore, it can be concluded that DJ-1 is playing an important role in the cellular defence response against oxidative stress induced cellular death by MHV-A59 infection in DBT cells. Another future question to be investigated is the process of cell death that is followed due to MHV-A59 in DBT cells, and whether DJ-1 overexpression in the cells can ameliorate such processes as well.

LITERATURE REVIEW

Astrocytes are specialized glial cells that perform many essential complex functions in the central nervous system. Major astrocyte functions are trophic support, providing guidance for neuronal migration during development, maintenance of the neural microenvironment and modulation of immune reactions by serving as antigen-presenting cells Constantinescu CS and Tani M and Ransohoff RM and Wysocka M and Hilliard B and Fujioka T and Murphy S and Tighe PJ and Das Sarma J and Trinchieri G and Rostami A, 2005 McBean GJ, 2018Sofroniew MV and Vinters HV, 2010​ . One of the cellular models to study the astrocytic cells is DBT cells Nagakubo D and Taira T and Kitaura H and Ikeda M and Tamai K and Iguchi-Ariga SM and Ariga H, 1997 . DBT cells has been found to be infected with Mouse Hepatitis Virus A59 (MHV-A59), a neurotropic strain of mouse hepatitis virus, resulting in loss in cell viability.

MHV-A59 has been found to infect neurons, microglia, astrocytes and oligodendrocytes. Infection by MHV-A59 has been found to induce oxidative stress in the nervous system Khan RS and Dine K and Das Sarma J and Shindler KS, 2014 . Oxidative stress is defined as the imbalance between oxidants and antioxidants in favour of the oxidants, potentially leading to cellular apoptosis Kannan K and Jain SK, 2000 . Recent studies have demonstrated that oxidative stress has profound implication in several progressive diseases, namely Parkinson’s disease, Alzheimer’s disease, rheumatoid arthritis, diabetes, inflammatory bowel disease etc.

DJ-1 is a reactive oxygen species sensor protein which gets activated upon oxidative stress induction in cells ​​Nagakubo D and Taira T and Kitaura H and Ikeda M and Tamai K and Iguchi-Ariga SM and Ariga H, 1997​​. DJ-1 is found to be ubiquitously expressed in several cells of the body. DJ-1 expression has been found to be induced via activation of ER stress response protein XBP1 under stress condition. DJ-1 gets activated by oxygen radicals generated during stress conditions. Upon activation, DJ-1 has unique role in both the cytoplasm as well as in the nucleus. In the cytoplasm, it activates Nrf2, a master regulator of several anti-oxidant genes. DJ-1, upon activation, also translocates from the cytoplasm to the nucleus, where it prevents ASK1-mediated cellular apoptosis​​. Thus DJ-1 has multi-faceted role in evoking the cellular defence response against oxidative stress ​Ariga H and Takahashi-Niki K and Kato I and Maita H and Niki T and Iguchi-Ariga SM, 2013​ .

Since MHV-A59 infection in DBT cells results in loss of cell viability Das Sarma J and Kenyon LC and Hingley ST and Shindler KS, 2009 , it is important to look into the role of DJ-1 in MHV-A59 infection of DBT cells. To address the question, we have stably transfected DJ-1 gene in DBT cell line. The stably transfected DBT cell line was infected with MHV-A59 and any changes in cell viability or oxidative stress generation was analysed. Results from our study suggested that the loss of cell viability in DBT cells upon MHV-A59 infection is greatly ameliorated upon DJ-1 transfection. Similar downregulation in oxidative stress generation was also observed upon MHV-A59 infection in DBT cells upon DJ-1 transfection. Such results provide an indication of the role of DJ-1 in conferring cellular protection against MHV-A59 infection in DBT cells.

METHODOLOGY

Cell Culture Conditions

DBT cells were cultured in DMEM media containing 10% Foetal Bovine Serum (FBS) and 1% Penicillin-Streptamycin anitibiotic combination. All the subsequent experiments are performed at 80-90% confluency.

Kill Curve Analysis using MTT Assay

Antibiotic kill curve is a dose response experiment to determine the minimum concentration of an antibiotic required to kill maximum cells in a specific period of time. This is a crucial step before using a selection antibiotic to kill untransfected cells and generate stable cell lines.

DBT cells were cultured in 96 well plate till 90% confluency and were treated with G418 antibiotic in DMEM media (Containing 10% FBS and 1% Pen-Strep antibiotic) at concentrations- 0.25, 0.5, 0.75, 1, 2 mg/mL for 48 Hours. MTT Assay was performed and absorbance was measured at 570 nm (details of the assay are described later).

Electroporation, Production and Confirmation of Stable Transfected cell Line

DBT cells were electroporated with pDsRed plasmid cloned with DJ-1 gene. G418 (at an optimal concentration) containing media was added 48 hours after electroporation.

Total RNA was isolated from the cells by Trizol method and cDNA was prepared by High-capacity cDNA reverse transcription kit. PCR amplification was performed with forward primer complementary to a portion of the DJ-1 gene and reverse primer complementary to a portion of the RFP gene and agarose gel electrophoresis was performed.

Virus Infection of Cells

Two viruses were used for the experiment- MHV-A59, and a recombinant strain of MHV-A59 virus which expresses EGFP called RSA59 virus (Recombinant strain of MHV-A59). For both the virus, the cells were infected at MOI= 2. The virus was added to the cells in DMEM media containing 2% FBS and incubated for 75 minutes. The virus containing media is discarded and fresh cell culture media was added. The virus infection was then continued for variable length of time depending on the subsequent experiments.

MTT Cell Viability Assay

MTT cell viability assay was performed in 96-well plate for both DBT and DBSDJ (DBT cells stably tranfected with DJ-1 gene) cell lines upon infection with RSA59 virus at increasing time points. The media was discarded, the cells were washed with 1X PBS and fresh media was added. 20µl of MTT reagent (5mg/ml in 1XPBS) was added to each well and incubated at 37˚C, 5%CO2 for 6 hours. The entire solution was discarded carefully without disturbing the purple-colored formazan crystals formed on the surface of the well.

100µl of DMSO was added to each well and Absorbance was measured at 595 nm using Hidex Chameleon Plate reader. Statistical analysis was done using One-way Anova and Fisher’s LSD Multiple Comparison Test. Only the significant comparisons were represented in diagrams as significance bars depicting the level of significance. The statistical significance parameter was set at p<0.05.

DCFDA Assay

Both DBT and DBSDJ cells were grown in 6 well plates till 85-90% confluency. MHV-A59 infection was done at MOI= 2 for increasing time points. 2% DMEM was used for mock infection of the cells as separate sets. The cells were then washed with 1X PBS (ice cold) and scrapped in 1X PBS containing 0.1mM EDTA solution. The cells were pelleted down at 2000 rpm for 5 minutes and resuspended in DCFDA solution (20 µM DCFDA in 1XPBS) and collected into flow cytometer tubes. The tubes were kept at 37˚C, 5% CO2 for 20 minutes in dark. The flow cytometer readings of the green fluorescence were then taken and quantified at 485Abs/535Ext nm wavelength in BDFACS Calibur Flow cytometer (BD Company, Franklin Lakes, NJ, USA). Statistical analysis was done using One-way Anova and Fisher’s LSD Multiple Comparison Test. Only the significant comparisons were represented in diagrams as significance bars depicting the level of significance. The statistical significance parameter was set at p<0.05.

RESULTS AND DISCUSSION

DBT cells were treated with G418 antibiotic at increasing concentration and MTT assay was performed to analyse changes in cell viability due to the treatment. Statistically significant decrease in cell viability was observed at 0.5, 0.75, 1 and 2 mg/mL G418 concentration. However, maximum decrease in cell viability was observed at 1 and 2 mg/ml concentration, implying cellular toxicity at these concentrations. Therefore, 1.5 mg/ml G418 concentration is considered for antibiotic selection for DBT cells for further experiments. The decrease in cell viability at different concentrations of G418 is tabulated in table 1 and graphically represented in Fig. 1 (DF= 41, N= 6).

: Decrease in cell viability of DBT cell line upon increasing concentration of G418 treatment.
G418 Concentration Decrease in Cell Viability
   
0.1 mg/ml -2.406% ± 1.484%
0.25 mg/ml -7.699% ± 1.331%
0.5 mg/ml -22.95% ± 1.627%, ****p<0.0001
0.75 mg/ml -30.95% ± 0.9373%, ****p<0.0001
1 mg/ml -37.38% ± 2.222%, ****p<0.0001
2 mg/ml -88.35% ± 4.869%, ****p<0.0001

The ‘-’ sign indicates decrease in cell viability.

fig1.jpg
    : Kill Curve Analysis to optimize G418 concentration for stable transfection of DBT cells. DBT cells were treated with 0.1, 0.25, 0.5, 0.75, 1 and 2 mg/ml concentration of G418 antibiotic for 48 hours. Statistically significant decrease in cell viability was observed at 0.5, 0.75, 1 and 2 mg/ml G418 concentration.

     Confirmation of Transfection of DJ-1-pDsRed Plasmid by PCR Method

    PCR analysis by DJ-1-RFP primer pair revealed positive band at 180bp location in the DBSDJ cell line. No such bands were observed in DBT cell line. The data is represented in Fig 2.

    Fig 2_1.png
      : DJ-1-RFP fusion gene band obtained after PCR amplification. No DNA band was obtained in the untransfected cell line. Transfected DBT cell line shows presence of band at ~180 bp region. DNA band was obtained at the same position in case of positive control (pDsRed-DJ-1plasmid). All the PCR products are run on a 2% agarose gel and stained with Ethidium bromide (final concentration- 0.5 µg/ml).

      DJ-1 prevents Loss of Cell Viability due to RSA59 Infection in DBSDJ Cells

      DBT cells showed statistically insignificant decrease in cell viability upon RSA59 viral infection at 8 hours. However, the decrease in cell viability was found to be significant at 10 hours, 12 hours, 14 hours, 24 hours, 36 hours and 48 hours of viral infection. (DF= 47, N= 6).

      DBSDJ cells showed statistically insignificant increase in cell viability at 6 hours and 10 hours of RSA59 viral infection. Minute but statistically significant increase in cell viability was observed at 8, 12, 14, and 17 hours of RSA59 viral infection. Statistically insignificant decrease in cell viability was observed at 24 hours post infection. The only statistically significant decrease in cell viability was observed at 48 hours of viral infection. (DF=53, N=6)

      The statistical changes in cell viability for both DBT and DBSDJ cells upon RSA59 viral infection are tabulated in table 2 and represented in Fig. 3.

      : Comparative analysis of loss in cell viability in DBT and DBSDJ cells upon RSA59 infection.
      Viral Infection Time Points DBT cells DBSDJ cells
           
      6 hours - 4.774% ± 1.652%
      8 hours -2.331% ± 10.71% 9.834% ± 3.136%, **p<0.01
      10 hours -31.37% ± 5.363%, ***p<0.001 6.081% ± 3.979%
      12 hours -50.39% ± 3.738%, ****p<0.0001 14.84% ± 2.493%, ***p<0.001
      14 hours -52.36% ± 6.635%, ****p<0.0001 11.61% ± 2.921%, **p<0.01
      17 hours - 9.498% ± 1.744%, *p<0.05
      24 hours -56.78% ± 4.993%, ****p<0.0001 -6.156% ± 2.935%
      36 hours -48.8% ± 6.536%, ****p<0.0001 -
      48 hours -50.57% ± 6.536%, ****p<0.0001 -17.04% ± 1.985%, ****p<0.0001

      The ‘+’ sign indicates increase in cell viability, ‘-’ sign indicates decrease in cell viability.

      Fig 3.tif
        : DJ-1 prevents loss of cell viability due to RSA59 infection in DBSDJ cell line. Statistically significant loss in cell viability was observed upon RSA59 infection in DBT cells line upon increasing time points of viral infection starting from 10 hours. However, the only significant decrease in DBSDJ cell viability was observed at 48 hours of RSA59 viral infection.

        DJ-1 prevents Generation of Oxidative Stress due to RSA59 Infection in DBSDJ Cells

        DCFDA assay showed that there has been a significant generation of reactive oxygen species upon MHV-A59 infection in DBT cells (****p<0.0001, DF= 3, N=3-5). The significant upregulation was obtained at 9 hours and 12 hours of virus infection. DBSDJ showed statistically significant decrease in ROS levels upon MHV-A59 infection at 9 hours and 15 hours of viral infection.

        The detailed regulation of ROS generation is represented in the Fig. 4 and the table 3.

        Fig 4.jpg
          : DJ-1 prevents oxidative stress generation by MHV-A59 infection in DBSDJ cell line. MHV-A59 infection results in generation of oxidative stress at 9 and 12 hours post infection in DBT cell line. However, no oxidative stress generation took place upon MHV-A59 infection at increasing time points of viral infection in DBSDJ cells (DBT cells stably transfected with DJ-1 gene).
          : Comparative analysis of ROS levels in DBT and DBSDJ cells upon MHV-A59 infection
          Viral Infection time points (in hours) DBT DBSDJ
          3 -12.71 ± 2.310 -
          6 +0.3615 ± 2.846 -
          9 +16.40 ± 4.737, *p=0.024 -19.98% ± 5.646%, , **p<0.01
          12 +43.06 ± 9.676, ****p<0.0001 -7.861% ± 5.007%
          15 -6.185 ± 3.057 -20.37% ± 2.574%, **p<0.01

          The ‘+’ sign indicated an increase in ROS level; ‘-’ indicated a decrease in ROS level.

          CONCLUSION AND RECOMMENDATIONS

          Since the advent of discovery of DJ-1 and its function by Nagakubo, D. et. al. in 1997, DJ-1 has been always considered as one of the important regulators of the oxidative-stress induced cellular defense against cellular apoptosis. Since the protein is ubiquitously expressed in several tissues, the protein is considered to evoke the anti-apoptotic response against oxidative stress. MHV-A59 infection has been found to evoke oxidative stress in neural cells, which can have potential role in the virus-induced loss of cell viability. Our cell of interest is the astrocytes, which are instrumental in maintaining the homeostasis of the neural environment and is particularly important in the anti-oxidant response generated against oxidative stress condition. We have observed that MHV-A59 infection results in drastic loss in cell viability in DBT cells (cellular model for astrocytes). Moreover, we have also observed that virus infection in the cells also generate oxidative stress. Therefore, we looked into the possible role of the DJ-1 protein in this scenario, by stably transfected DJ-1 gene in the DBT cells, and then performing further analysis with this transfected cell line.

          Firstly, kill curve analysis was performed on DBT cells with increasing concentration of G418 antibiotic and 1.5 mg/ml concentration of G418 antibiotic was confirmed to be optimal for selection of transfected cells v/s non-transfected cells. The DJ-1 gene was initially transformed in pDsRed plasmid and this plasmid containing the DJ-1 insert is then transfected into the DBT cells by electroporation. The confirmation of transfection was done by PCR analysis for a DJ-1 RFP fusion gene in the stably-transfected cell line. The stably transfected DBT cell line is termed as DBSDJ (DBT stably transfected with DJ-1).

          Both the DBT and the DBSDJ cells were then infected with RSA59 virus to look into its effects on cell viability of both the cells. RSA59 virus was found to cause drastic decrease in cell viability in DBT cells as early as 10 hours of infection and the decrease is also observed in subsequent time points. However, no such decrease in cell viability was observed in DBSDJ cells, with a small but statistically significant change was observed as late as 48 hours if viral infection. MHV-A59 was also observed to cause generation of oxidative stress in DBT cells. However, no such oxidative stress generation was observed in DBSDJ cells upon MHV-A59 infection.

          These results suggest that DJ-1 has an important role to play in conferring cellular protection against MHV-A59 infection in DBT cells. Previous data from our laboratory suggested a decrease in the DJ-1 expression level due to RSA59 infection in DBT cells, which can contribute to the loss of cell viability and generation of oxidative stress observed in upon virus infection in the cells. Stable expression of DJ-1 in the cells was found to greatly ameliorate these effects. Therefore, it can be comprehended that stable expression of DJ-1 might act as an additive effect to the cellular defence response against MHV-A59 infection, by the virtue of which loss of cell viability is greatly decreased. One important future lookout from this data is to found out whether the process of apoptosis is at play during MHV-induced loss in cell viability in DBT cells, and whether such effects can be reduced due to stable expression of DJ-1 in the DBT cells.

          REFERENCES

          1. Constantinescu, C.S., et al., Astrocytes as antigen-presenting cells: expression of IL-12/IL-23. J Neurochem, 2005. 95(2): p. 331-40.

          2. McBean, G.J., Astrocyte Antioxidant Systems. Antioxidants (Basel), 2018. 7(9)

          3. Sofroniew, M.V. and H.V. Vinters, Astrocytes: biology and pathology. Acta Neuropathol, 2010. 119(1): p. 7-35.

          4. Nagakubo, D., et al., DJ-1, a novel oncogene which transforms mouse NIH3T3 cells in cooperation with ras. Biochem Biophys Res Commun, 1997. 231(2): p. 509-13.

          5. Khan, R.S., et al., SIRT1 activating compounds reduce oxidative stress mediated neuronal loss in viral induced CNS demyelinating disease. Acta Neuropathol Commun, 2014. 2: p. 3.

          6. Kannan, K. and S.K. Jain, Oxidative stress and apoptosis. Pathophysiology, 2000. 7(3): p. 153-163

          7. Ariga, H., et al., Neuroprotective function of DJ-1 in Parkinson's disease. Oxid Med Cell Longev, 2013. 2013: p. 683920.

          8.Das Sarma, J., et al., Mechanisms of primary axonal damage in a viral model of multiple sclerosis. J Neurosci, 2009. 29(33): p. 10272-80.

          ACKNOWLEDGEMENTS

          Knowledge and experience are the two major pillars for successful scientific research. Both these virtues are not restricted to a particular place, and visiting different places enriches both of them, which are instrumental in shaping up a career in research. On this note, I would like to acknowledge the Indian Academy of Sciences, for providing me such a wonderful opportunity to expose myself to the plethora of different approaches that are followed in pursuit of successful research.

          I would also like to thank Dr. Jayasri Das Sarma, for accepting me to learn different techniques in her laboratory. In spite of her busy schedule, she provided utmost care and dedication to our research, and gave her valuable suggestions in shaping up the project.

          Thirdly, I would like to thank Ms. Soma Nag, our lab manager, for teaching me the value of discipline in the laboratory. Her strict manners helped in embedding the virtue in me, which will certainly help me in the long run.

          I would also like to thank Mr. Soumya Kundu, PH. D. student in the laboratory, who was endowed with the responsibility of teaching me the techniques and guiding me in the project. He taught me all the related techniques and described the principle behind them. I am enthralled at his pleasant approach towards each experiment, and the way he analysed each data and come to a conclusion.

          I am also thankful to the other members of the laboratory, Mrs. Mahua Maulik, Mr. Manmeet Singh, Mr. Abhishek Bose, Mrs. Fareeha Saadi, Ms. Debanjana Chakravarty, Ms. Lucky Sarkar, Mr. Sourodip Sengupta, Ms. Vaishali Mulchandani, Mr. Saurav Rohilla, Mr. Abbas, Mr. Akash Das as well as other project students, Mr. Saurav Rout, Ms. Teesta Roy Chowdhury, Ms. Shilauni Dadwal, and Mr. Santos K. Samal, for making my stay at IISER- Kolkata a memorable experience.

          Lastly, I would like to thank my parents, my teachers, and my family member, for making me eligible for such a wonderful and fruitful experience at IISER- Kolkata.

          References

          • Constantinescu CS and Tani M and Ransohoff RM and Wysocka M and Hilliard B and Fujioka T and Murphy S and Tighe PJ and Das Sarma J and Trinchieri G and Rostami A (2005). Astrocytes as antigen-presenting cells: expression of IL-12/IL-23.. 95,

          • McBean GJ (2018). Astrocyte Antioxidant Systems.. 7,

          • Sofroniew MV and Vinters HV (2010). Astrocytes: biology and pathology.. 119,

          • Nagakubo D and Taira T and Kitaura H and Ikeda M and Tamai K and Iguchi-Ariga SM and Ariga H (1997). DJ-1, a novel oncogene which transforms mouse NIH3T3 cells in cooperation with ras.. 231,

          • Khan RS and Dine K and Das Sarma J and Shindler KS (2014). SIRT1 activating compounds reduce oxidative stress mediated neuronal loss in viral induced CNS demyelinating disease.. 2,

          • Kannan K and Jain SK (2000). Oxidative stress and apoptosis.. 7,

          • Ariga H and Takahashi-Niki K and Kato I and Maita H and Niki T and Iguchi-Ariga SM (2013). Neuroprotective function of DJ-1 in Parkinson's disease.. 2013,

          • Das Sarma J and Kenyon LC and Hingley ST and Shindler KS (2009). Mechanisms of primary axonal damage in a viral model of multiple sclerosis.. 29,

          More
          Written, reviewed, revised, proofed and published with