Acute Hypoxia Alters Extracellular Vesicle Signatures and the Brain Citrullinome of Naked Mole-Rats (Heterocephalus glaber)

Abstract

Peptidylarginine deiminases (PADs) and extracellular vesicles (EVs) may be indicative biomarkers of physiological and pathological status and adaptive responses, including to diseases and disorders of the central nervous system (CNS) and related to hypoxia. While these markers have been studied in hypoxia-intolerant mammals, in vivo investigations in hypoxia-tolerant species are lacking. Naked mole-rats (NMR) are among the most hypoxia-tolerant mammals and are thus a good model organism for understanding natural and beneficial adaptations to hypoxia. Thus, we aimed to reveal CNS related roles for PADs in hypoxia tolerance and identify whether circulating EV signatures may reveal a fingerprint for adaptive whole-body hypoxia responses in this species. We found that following in vivo acute hypoxia, NMR: (1) plasma-EVs were remodelled, (2) whole proteome EV cargo contained more protein hits (including citrullinated proteins) and a higher number of associated KEGG pathways relating to the total proteome of plasma-EVs Also, (3) brains had a trend for elevation in PAD1, PAD3 and PAD6 protein expression, while PAD2 and PAD4 were reduced, while (4) the brain citrullinome had a considerable increase in deiminated protein hits with hypoxia (1222 vs. 852 hits in normoxia). Our findings indicate that circulating EV signatures are modified and proteomic content is reduced in hypoxic conditions in naked mole-rats, including the circulating EV citrullinome, while the brain citrullinome is elevated and modulated in response to hypoxia. This was further reflected in elevation of some PADs in the brain tissue following acute hypoxia treatment. These findings indicate a possible selective role for PAD-isozymes in hypoxia response and tolerance.

The Extracellular Vesicle Citrullinome and Signature in a Piglet Model of Neonatal Seizures

“…This may indicate roles for citrullination in CNS immune responses against bacterial infection, and citrullination is indeed well known to contribute to anti-pathogenic and anti-bacterial functions [56,57]. For example, higher amounts of cyclic citrullinated peptides in sera have been related to infectious diseases, including viral, bacterial and parasitic infections [58], and this KEGG pathway has previously been identified as citrullinated in EV cargo of naked mole-rats under normoxic and hypoxic conditions [59]. The identification of citrullination in this pathway in neonatal seizures here in the current study may possibly be of importance in relation to meningitis and septicemia in neonates [60,61] and seizures in pediatric patients in relation to S. aureus infection [62][63][64][65].…”

“…Commonly "total EVs", comprised of small and medium-sized EVs (or 30-1000 nm) are isolated via differential and ultracentrifugation approaches and further characterization of surface markers and morphological analysis by transmission electron microscopy, to meet the minimum requirements of the society of the International Society for Extracellular Vesicles [36]. Here, the EVs were isolated from plasma and CSF according to established protocols [59,92]. Plasma and CSF aliquots (100 µL of each per individual animal) were diluted 1:4 in Dulbecco's phosphate-buffered saline (DPBS) and centrifuged at 4000× g for 20 min to remove apoptotic bodies and then ultracentrifuged at 100,000× g for 1 h at 4 • C for isolation of total EVs.…”

Differential, Stage Dependent Detection of Peptidylarginine Deiminases and Protein Deimination in Lewy Body Diseases—Findings from a Pilot Study

“…PAD6 was also less elevated than PAD1-3 in the PD brains in this study, but did though show some higher levels compared with control brains. A putative role for PAD6 in PD may be of some interest, as this isozyme has recently been linked to hypoxia responses in naked mole-rat brain [ 38 ], but otherwise not previously linked to neuronal injury and mainly been linked to developmental processes [ 39 , 40 , 41 ], while recently also suggested to have roles in some animal cancers [ 42 ]. Overall, there may be a need for more in depth investigations into the individual PAD isozymes in PD, as the identification of PAD isozyme specific roles in different neurodegenerative disorders may be of considerable importance.…”