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Abstract Background Protein aggregates can be found in peripheral organs, such as the heart, kidney, and pancreas, but little is known about the impact of peripherally misfolded proteins on neuroinflammation and brain functional recovery following ischemic stroke. Methods Here, we studied the ischemia/reperfusion (I/R) induced brain injury in mice with cardiomyocyte-restricted overexpression of a missense (R120G) mutant small heat shock protein, αB-crystallin (CryAB R120G ), by examining neuroinflammation and brain functional recovery following I/R in comparison to their non-transgenic (Ntg) littermates. To understand how peripherally misfolded proteins influence brain functionality, exosomes were isolated from CryAB R120G and Ntg mouse blood and were used to treat wild-type (WT) mice and primary cortical neuron-glia mix cultures. Additionally, isolated protein aggregates from the brain following I/R were isolated and subjected to mass-spectrometric analysis to assess whether the aggregates contained the mutant protein, CryAB R120G . To determine whether the CryAB R120G misfolding can self-propagate, a misfolded protein seeding assay was performed in cell cultures. Results Our results showed that CryAB R120G mice exhibited dramatically increased infarct volume, delayed brain functional recovery, and enhanced neuroinflammation and protein aggregation in the brain following I/R when compared to the Ntg mice. Intriguingly, mass-spectrometric analysis of the protein aggregates isolated from CryAB R120G mouse brains confirmed presence of the mutant CryAB R120G protein in the brain. Importantly, intravenous administration of WT mice with the exosomes isolated from CryAB R120G mouse blood exacerbated I/R-induced cerebral injury in WT mice. Moreover, incubation of the CryAB R120G mouse exosomes with primary neuronal cultures induced pronounced protein aggregation. Transduction of CryAB R120G aggregate seeds into cell cultures caused normal CryAB proteins to undergo dramatic aggregation and form large aggregates, suggesting self-propagation of CryAB R120G misfolding in cells. Conclusions These results suggest that peripherally misfolded proteins in the heart remotely enhance neuroinflammation and exacerbate brain injury following I/R likely through exosomes, which may represent an underappreciated mechanism underlying heart-brain crosstalk.
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Intra-FCY1: a novel system to identify mutations that cause protein misfolding
Protein misfolding is a common intracellular occurrence. Most mutations to coding sequences increase the propensity of the encoded protein to misfold. These misfolded molecules can have devastating effects on cells. Despite the importance of protein misfolding in human disease and protein evolution, there are fundamental questions that remain unanswered, such as, which mutations cause the most misfolding? These questions are difficult to answer partially because we lack high-throughput methods to compare the destabilizing effects of different mutations. Commonly used systems to assess the stability of mutant proteinsin vivooften rely upon essential proteins as sensors, but misfolded proteins can disrupt the function of the essential protein enough to kill the cell. This makes it difficult to identify and compare mutations that cause protein misfolding using these systems. Here, we present a novelin vivosystem named Intra-FCY1that we use to identify mutations that cause misfolding of a model protein [yellow fluorescent protein (YFP)] inSaccharomyces cerevisiae. The Intra-FCY1system utilizes two complementary fragments of the yeast cytosine deaminase Fcy1, a toxic protein, into which YFP is inserted. When YFP folds, the Fcy1 fragments associate together to reconstitute their function, conferring toxicity in media containing 5-fluorocytosine and hindering growth. But mutations that make YFP misfold abrogate Fcy1 toxicity, thus strains possessing misfolded YFP variants rise to high frequency in growth competition experiments. This makes such strains easier to study. The Intra-FCY1system cancels localization of the protein of interest, thus can be applied to study the relative stability of mutant versions of diverse cellular proteins. Here, we confirm this method can identify novel mutations that cause misfolding, highlighting the potential for Intra-FCY1to illuminate the relationship between protein sequence and stability.
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- Award ID(s):
- 2119963
- PAR ID:
- 10508441
- Publisher / Repository:
- frontiers
- Date Published:
- Journal Name:
- Frontiers in Genetics
- Volume:
- 14
- ISSN:
- 1664-8021
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3389/fgene.2023.1198203
