Abstract
Microglia respond to cytotoxic protein aggregates associated with the progression of neurodegenerative disease. Pathological protein aggregates activate the microglial NLRP3 inflammasome resulting in proinflammatory signaling, secretion, and potentially pyroptotic cell death. We characterized mixed sex primary mouse microglia exposed to microbial stressors and alpha synuclein preformed fibrils (αsyn PFFs) to identify cellular mechanisms related to Parkinson’s disease. Microglia package and release the endosome fate regulator Coronin1A (Coro1A) in EVs in an Nlrp3-dependent manner in widely used experimental activation conditions. We were surprised to find that Coro1A packaging and release was not Nlrp3-dependent in αsyn PFF exposure conditions. Coro1A-/- microglia exposed to αsyn PFFs trafficked more αsyn to lysosomal compartment increasing lysosomal membrane permeabilization. This corresponds to a decrease in αsyn released in EVs suggesting that Coro1A functions to shunt pathological proteins to a secretory pathway to attenuate lysosomal stress. αsyn PFF driven lysosomal stress resulting from Coro1a loss was associated with enhanced cytotoxicity. Intrinsic apoptosis signaling was unaffected, but we observed elevated cytosolic cathepsin B and the presence of a cathepsin associated 55kD PARP cleavage product. Post-mortem analysis of the PD mesencephalon supported a role for Coro1a in microglia, revealing elevated levels of Coro1A protein in human PD brains compared to those of healthy donors. Findings are relevant to the distribution of pathological αsyn and indicate that Coro1a protects microglia from lysosomal overload, inflammasome activation, and pyroptotic demise.
Significance Statement Microglia are responsible for clearing toxic protein aggregates such as alpha synuclein (αsyn) in Parkinson’s Disease (PD). PD is slowly progressive, implying that microglia are under proteinaceous stress for an extended time, maintaining some level of homeostasis while attempting to clear pathologically aggregated proteins. Pathological proteins can overload the lysosomes resulting in rupture, decreasing the ability of microglia to clear protein aggregates, and contributing to a hyperreactive inflammatory state. We determined that the protein Coronin1A functions in microglia to attenuate αsyn-induced lysosomal stress, preventing Nlrp3-inflammasome activation, and cell death. These findings identify a protective cellular mechanism operating in microglia that may contribute to the distribution of pathological proteins into the microenvironment.
Footnotes
The authors declare no conflicts of interest
This work was funded by the NIH, National Institute for Environmental Health Sciences [R01 ES033462 and R01ES024745 (MH)] and grants to Arminja Kettenbach from NIH (R35 GM119455). The Life Sciences Light Microscopy Facility at Dartmouth is supported by NIDDK P30/DartCF (P30-DK117469), NCI Cancer Center Support Grant (P30 CA023108), and bioMT COBRE (P20-GM113132). The super-resolution spinning disk confocal (SoRa) is supported through NIH award S10OD032310 to Dr. Yashi Ahmed (PD). Work would not have been possible without infrastructure and technical support available within the Dartmouth Cancer Center (National Cancer Institute (NCI) Cancer Center Support Grant 5P30 CA023108-44).
We would like to thank Dr. Chris Shoemaker for his valuable insights on autophagic flux and his generous sharing of key reagents for the autophagic flux experiments. We would like to thank Dr. Ashley Mason for her critical commentary and feedback on the manuscript.
We would like to thank Ann Lavanway of the Dartmouth Life Sciences Light Microscopy Facility and Scott Palisoul of the Dartmouth Pathology Shared Resource. We would like to thank Dr. Bruce Stanton, Roxana Barnaby, and Carolyn Winston for their expertise and making available the NanoSight NS300 instrument. We would like to thank Drs. Stephen Lee and Mary Feldman for ongoing support and clinical insights. We are indebted to Drs. William Hickey and Harker Rhodes for their work in establishing and maintaining a biorepository critical for all post-mortem tissue analyses at Dartmouth. We would like to thank Dr. Richard Flavell (Yale School of Medicine) for supplying the Nlrp3-/- mice used in the study.
↵*Co-Corresponding Authors
↵†Current address, Merck, Boston, MA USA.