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Loss of Microglial Parkin Inhibits Necroptosis and Contributes to Neuroinflammation

  • Pedro Elói Antunes Dionísio
  • Sara Rodrigues Oliveira
  • Joana São José Dias Amaral
  • Cecília Maria Pereira Rodrigues
Article

Abstract

Parkin is an E3 ubiquitin ligase involved in Parkinson’s disease (PD). Necroptosis is a regulated form of cell death that depends on receptor interacting protein 1 (RIP1) and 3 (RIP3). Importantly, parkin has been implicated in ubiquitination events that can alter inflammation and necroptosis. Here, we investigated how parkin influences microglial function. Incubation of BV-2 microglial cells with zVAD.fmk (zVAD) induced high levels of cell death and viability loss, while N9 microglial cells and primary microglia required further stimuli. Importantly, necrostatin-1 (Nec-1), an inhibitor of RIP1 kinase activity, abrogated cell death, thus implicating RIP1-dependent necroptosis in cell death. Cell death was characterized by necrosome assembly, as determined by sequestration of RIP1/RIP3 in insoluble fractions and by MLKL phosphorylation, which were all abolished by Nec-1. Also, necroptosis-inducing conditions led to TNF-α secretion, which may in turn contribute to autocrine necroptosis activation. Interestingly, parkin knockdown protected BV-2 cells from zVAD-induced necroptosis, which may depend on the higher RIP1 ubiquitination levels detected in siRNA-PARK2 transfected cells. This effect was independent of inflammation, since pro-inflammatory stimulation of BV-2 and primary microglia with silenced parkin resulted in stronger pro-inflammatory gene expression, an opposite observation from zVAD-exposed BV-2 cells. LPS-mediated inflammation was exacerbated by NF-κB/JNK over-activation. Finally, no alterations in mitochondrial ROS production were detected in any condition, thereby excluding the role of parkin in mitophagy. In conclusion, here, we reveal that parkin may have unsuspected roles in microglia by modulating ubiquitination. Parkin loss exacerbates inflammation and promotes survival of activated microglia, thus contributing to chronic neuroinflammation.

Keywords

Inflammation Microglia Necroptosis Parkin Ubiquitination 

Notes

Acknowledgements

This work was supported by Fundação para a Ciência e a Tecnologia (FCT) through iMed.ULisboa grant UID/DTP/04138/2013 and individual fellowships SFRH/BPD/100961/2014, PD/BD/128332/2017 and SFRH/BD/102771/2014.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. 1.Research Institute for Medicines (iMed.ULisboa), Faculty of PharmacyUniversidade de LisboaLisbonPortugal

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