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Necroptotic–Apoptotic Regulation in an Endothelin-1 Model of Cerebral Ischemia

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Abstract

The primary forms of cell death seen in ischemic stroke are of two major types: a necrotic/necroptotic form, and an apoptotic form that is frequently seen in penumbral regions of injury. Typically apoptotic versus necroptotic programmed cell death is described as competitive in nature, where necroptosis is often described as playing a backup role to apoptosis. In the present study, we examined the relationship between these two forms of cell death in a murine endothelin-1 model of ischemia–reperfusion injury in wildtype and caspase-3 null mice with and without addition of the pharmacologic RIPK1 phosphorylation inhibitor necrostatin-1. Analyses of ischemic brain injury were performed via both cellular and volumetric assessments, electron microscopy, TUNEL staining, activated caspase-3 and caspase-7 staining, as well as CD11b and F4/80 staining. Inhibition of caspase-3 or RIPK1 phosphorylation demonstrates significant neural protective effects which are non-additive and exhibit significant overlap in protected regions. Interestingly, morphologic analysis of the cortex demonstrates reduced apoptosis following RIPK1 inhibition. Consistent with this, RIPK1 inhibition reduces the levels of both caspase-3 and caspase-7 activation. Additionally, this protection appears independent of secondary inflammatory mediators. Together, these observations demonstrate that the necroptotic protein RIPK1 modifies caspase-3/-7 activity, ultimately resulting in decreased neuronal apoptosis. These findings thus modify the traditional exclusionary view of apoptotic/necroptotic signaling, revealing a new form of interaction between these dominant forms of cell death.

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Acknowledgements

We would like to thank Ali Darbandi and William Martin at the Nanoscale Biomedical Imaging Facility at SickKids hospital, Lindsey Fiddes at the University of Toronto for electron microscopy assistance and Lora Stepanian for quantitation assistance.

Funding

This study has been funded by grants awarded to J.T.H. from Natural Sciences and Engineering Research Council of Canada (NSERC) (RGPIN 298553-12) and Heart and Stroke Society of Canada (72043506). C.D.S. additionally received scholarship funding from Natural Sciences and Engineering Research Council of Canada (NSERC) and Queen Elizabeth II Graduate Scholarships in Science & Technology—Merck Company of Canada.

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Authors and Affiliations

  1. Department of Pharmaceutical Sciences, University of Toronto, 144 College St. Rm 962, Toronto, ON, M5S 3M2, Canada

    Chesarahmia Dojo Soeandy, Christopher Rodgers, Shudi Huang, Andrea C. Elia & Jeffrey T. Henderson

  2. Princess Margaret Cancer Center, University Health Network, 610 University Avenue Rm 7-323, Toronto, ON, M5G 2C1, Canada

    Andrew J. Elia

  3. Department of Medical Biophysics, University of Toronto, 101 College Street Rm 15-701, Toronto, ON, M5G 1L7, Canada

    Andrew J. Elia

  4. Department of Pharmaceutical Sciences, University of Toronto, 144 College St. Rm 1010, Toronto, ON, M5S 3M2, Canada

    Yanshan Cao

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  1. Chesarahmia Dojo Soeandy
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Contributions

CDS designed and performed the majority of experiments, analyzed and interpreted the data, and wrote the manuscript; AJE aided with CD11b and F4/80 staining; YC performed animal care; CR, SH, and ACE aided quantitation and analysis; JTH directed the overall study, designed experiments, interpreted the data, and wrote the manuscript.

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Correspondence to Jeffrey T. Henderson.

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All animal experimental procedures were performed in accordance with standards outlined by the Local Animal Care Committee and the University of Toronto, following the guidelines set by Ontario's Animal for Research Act and the federal Canadian Council on Animal Care.

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Dojo Soeandy, C., Elia, A.J., Cao, Y. et al. Necroptotic–Apoptotic Regulation in an Endothelin-1 Model of Cerebral Ischemia. Cell Mol Neurobiol 41, 1727–1742 (2021). https://doi.org/10.1007/s10571-020-00942-y

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