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Impaired Autophagy Flux is Associated with Proinflammatory Microglia Activation Following Japanese Encephalitis Virus Infection

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Abstract

Role of autophagy in Japanese encephalitis viral (JEV) infection is not well known. In the present study, we reported the role of autophagy flux in microglia activation, neurobehavioral function and neuronal death using a mouse model of JEV. Markers for autophagy (LC3-II/I, SQSTM1/P62, phos-Akt, phos-AMPK), and neuronal death (cleaved caspase 12, H2Ax, polyubiquitin) were investigated by western blot at 1, 3 and 7 days post inoculation. Cathepsin D was measured in cerebral cotex of JEV infected mice spectrophotometrically. Microglia activation and pro-inflammatory cytokines (IL1β, TNF-α, IFNγ, IL6) were measured by immunohistochemistry, western blot and qPCR analysis. In order to determine the neuroinflammatory changes and autophagy mediated neuronal cell death, BV2-microglia and N2a-neuronal cells were used. Autophagy activation marker LC3-II/I and its substrate SQSTM1/P62 were significantly increased while cathepsin D activity was decreased on day 7 post inoculation in cerebral cortex. Microglia in cortex were activated and showed higher expression of proinflammatory mRNA of IL1β, TNF-α, IFNγ and IL6, with increased DNA damage (H2AX) and neuronal cell death pathways in hippocampus and neurobehavioral dysfunction. Similar observations on JEV infection mediated autophagy flux inhibition and neuronal cell death was found in N2a neuronal cell. Collectively, our study provides evidence on the role of autophagy regulation, microglial activation and neurodegeneration following JEV infection.

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Acknowledgements

Authors acknowledge Ms. Swati Singh, Mr. Firoz Ahmed for technical assistant in animal experiments.

Funding

This study was supported by the Ramalingaswami re-entry fellowship (BT/RLF/Re-entry/13/2014) from Department of Biotechnology, Ministry of Science and Technology, Govt. of India to A.K. and ICMR (59/05/2019/ONLINE/BMS/TRM) and SERB (SRG/2019/000398) to RAS.

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Authors and Affiliations

  1. Department of Molecular Medicine and Biotechnology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareily Road, Lucknow, 226014, Uttar Pradesh, India

    Alok Kumar, Gajendra Singh & Swasti Tiwari

  2. Department of Neurology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareily Road, Lucknow, 226014, Uttar Pradesh, India

    J. Kalita & U. K. Misra

  3. Department of Endocrinology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareily Road, Lucknow, 226014, Uttar Pradesh, India

    Rohit A. Sinha & Anjum B

  4. Department of Microbiology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareily Road, Lucknow, 226014, Uttar Pradesh, India

    Mukti Shukla & T. N. Dhole

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  1. Alok Kumar
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Contributions

AK: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Visualization. AK and UK: Writing—original draft, RAS, GS, AB, MS: Methodology, Validation, JK, RAS, ST, TND: Resources, Supervision. All authors reviewed the manuscript.

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Correspondence to Alok Kumar or U. K. Misra.

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Kumar, A., Kalita, J., Sinha, R.A. et al. Impaired Autophagy Flux is Associated with Proinflammatory Microglia Activation Following Japanese Encephalitis Virus Infection. Neurochem Res 45, 2184–2195 (2020). https://doi.org/10.1007/s11064-020-03080-5

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