Electronic Poster | Session 1

054 – Dying a Fiery Death: Caspases-3 and -7 Drive Pyroptosis in Human Brain Microglia

Jason Fernandes (1) – Brienne McKenzie (1) – Matthew Doan (2) – William Branton (1) – Christopher Power (3)
University of Alberta, Medical Microbiology and Immunology, Edmonton, Canada (1) – University of Alberta, Neuroscience and Mental Health Institute, Edmonton, Canada (2) – University of Alberta, Department of Medicine, Edmonton, Canada (3)

Multiple sclerosis (MS) is a debilitating autoimmune disease of the CNS characterized by inflammatory demyelination and associated neurodegeneration. One of the key factors that drives the progress of MS is the loss of myelin-producing oligodendrocytes. Along with persistent activation of microglia and astrocytes, the profound reduction in myelin leads to cognitive, visual and motor deficiencies in the 2.5m people afflicted with MS globally. We previously identified pyroptosis as a key driver in neuroinflammation and neurodegeneration in MS and its animal model, experimental autoimmune encephalomyelitis. Pyroptosis (“fiery death”) is a type of highly inflammatory programmed cell death, implicated in a wide spectrum of neurological diseases. During pyroptosis, plasma membrane rupture and cell lysis are driven by inflammasome-mediated activation of the pore-forming executioner protein, gasdermin D (GSDMD). Herein, we report for the first time that GSDMD-dependent pyroptosis engages the apoptotic executioner caspases-3 and -7, using microglia as a model system. Caspase-3/7 activation is considered a prototypic hallmark of “immunologically silent” apoptotic cell death. In contrast to this paradigm, we demonstrate that caspase-3/7 activation occurred rapidly following exposure to pyroptotic stimuli and was prevented by inflammasome inhibition. Suppression of caspase-3/7 prevented GSDMD-mediated pyroptosis. We show that plasma membrane rupture, nuclear disintegration, and key morphological features associated with pyroptosis (e.g. pyroptotic bodies on the cell membrane) were rescued with caspase-3/7 inhibition. Specific substrates of caspase-3/7 were cleaved during pyroptosis in both the cytoplasm (ROCK1) and the nucleus (PARP, DFF45), confirming the proteolytic activity of caspase-3/7. Cleavage of these substrates was markedly reduced upon suppression of caspase-3/7. To verify the in vivo relevance of our findings, we also demonstrate co-expression of activated caspase-3 with GSDMD in cerebral white matter. Collectively these results highlight a putative role for caspase 3/7 in facilitating pyroptosis in human microglia.