CNS Inflammasome Activation and Pyroptosis in Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis

• Author / Creator

  McKenzie, Brienne Alexandra Furfaro

• Multiple sclerosis (MS) is a progressive and incurable demyelinating disease of the central nervous system (CNS). The neuropathogenesis of MS and its prototypic animal model, experimental autoimmune encephalomyelitis (EAE), is defined by persistent neuroinflammation, involving the activation of microglia (the resident macrophages of the CNS), as well as infiltrating T lymphocytes, B lymphocytes, and monocyte-derived macrophages. Inflammasomes are multi-protein cytosolic complexes that mediate the maturation and release of proinflammatory cytokines (IL-1β and IL-18); they participate in the pathogenesis of EAE, notably in the activation and recruitment of CNS-infiltrating immune cells. Inflammasomes also initiate a form of lytic programmed cell death termed pyroptosis (“fiery death”), driven by the pore-forming executioner protein, gasdermin D (GSDMD). Neither the presence of GSDMD within CNS-resident cells nor the contribution of pyroptosis to MS or EAE has been investigated to date.

  In this thesis, inflammasome activation and GSDMD-mediated pyroptosis were demonstrated in both myeloid cells (microglia/macrophages) and in myelin-forming oligodendrocytes (ODCs) in the CNS during MS and EAE. Inflammasome activation and pyroptosis occurred in human microglia in vitro after exposure to MS-relevant inflammatory stimuli. GSDMD inhibition by siRNA and caspase-1 inhibition by VX-765 suppressed pyroptosis in human microglia. VX-765 treatment of EAE animals reduced expression of inflammasome- and pyroptosis-associated proteins in the CNS, prevented axonal injury, and improved neurobehavioral performance. Thus, this thesis provided evidence for GSDMD-mediated pyroptosis in multiple glial cell types as a previously unrecognized mechanism contributing to inflammatory demyelination.

  Recent evidence has suggested that subcellular changes, including nuclear condensation, pyroptotic body formation, and mitochondrial dysfunction, precede GSDMD-mediated cell lysis during pyroptosis. These observations are reminiscent of changes observed during apoptosis, which prompted the investigation of apoptotic executioner caspase-3/7 as potential drivers of pyroptosis. This thesis provided the first evidence that human microglia undergoing pyroptosis activate caspase-3/7 downstream of inflammasome activation, which is prevented by caspase-1 inhibition. Specific substrates of caspase-3/7 were cleaved during pyroptosis in the nucleus (DFF45, PARP) and the cytoplasm (ROCK1). The siRNA-mediated suppression of caspase-3/7 interrupted pyroptosis, inhibited nuclear disintegration, reduced pyroptotic body formation, prevented plasma membrane rupture, and diminished proteolytic cleavage of caspase-3/7 substrates. Using human microglia as a model system, these findings reveal a previously undiscovered role for caspase-3/7 as executioners of GSDMD-mediated pyroptosis.

  This thesis offers a novel perspective on inflammatory demyelination in the CNS by identifying a previously unrecognized molecular pathway that perpetuates cell death and neuroinflammation in MS and EAE. In addition, a clinically relevant therapeutic strategy for MS that targets this pathway is proposed. Lastly, the first demonstration of a functional role for caspase-3/7 in driving GSDMD-mediated pyroptosis is provided, which challenges the use of cleaved caspase-3/7 and their substrates as universal biomarkers for apoptosis. These findings represent a substantial conceptual advance in our understanding of the molecular mechanisms underlying pyroptosis.

• Subjects / Keywords

  • multiple scerlosis
  • inflammasome
  • pyroptosis
  • EAE

• Graduation date

  Fall 2019

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-gfwp-5d69

• License

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