Protein Deimination Signatures in Plasma and Plasma-EVs and Protein Deimination in the Brain Vasculature in a Rat Model of Pre-Motor Parkinson’s Disease

Abstract

The identification of biomarkers for early diagnosis of Parkinson's disease (PD) is of pivotal importance for improving approaches for clinical intervention. The use of translatable animal models of pre-motor PD therefore offers optimal opportunities for novel biomarker discovery in vivo. Peptidylarginine deiminases (PADs) are a family of calcium-activated enzymes that contribute to protein misfolding through post-translational deimination of arginine to citrulline. Furthermore, PADs are an active regulator of extracellular vesicle (EV) release. Both protein deimination and extracellular vesicles (EVs) are gaining increased attention in relation to neurodegenerative diseases, including in PD, while roles in pre-motor PD have yet to be investigated. The current study aimed at identifying protein candidates of deimination in plasma and plasma-EVs in a rat model of pre-motor PD, to assess putative contributions of such post-translational changes in the early stages of disease. EV-cargo was further assessed for deiminated proteins as well as three key micro-RNAs known to contribute to inflammation and hypoxia (miR21, miR155, and miR210) and also associated with PD. Overall, there was a significant increase in circulating plasma EVs in the PD model compared with sham animals and inflammatory and hypoxia related microRNAs were significantly increased in plasma-EVs of the pre-motor PD model. A significantly higher number of protein candidates were deiminated in the pre-motor PD model plasma and plasma-EVs, compared with those in the sham animals. KEGG (Kyoto encyclopedia of genes and genomes) pathways identified for deiminated proteins in the pre-motor PD model were linked to "Alzheimer's disease", "PD", "Huntington's disease", "prion diseases", as well as for "oxidative phosphorylation", "thermogenesis", "metabolic pathways", "Staphylococcus aureus infection", gap junction, "platelet activation", "apelin signalling", "retrograde endocannabinoid signalling", "systemic lupus erythematosus", and "non-alcoholic fatty liver disease". Furthermore, PD brains showed significantly increased staining for total deiminated proteins in the brain vasculature in cortex and hippocampus, as well as increased immunodetection of deiminated histone H3 in dentate gyrus and cortex. Our findings identify EVs and post-translational protein deimination as novel biomarkers in early pre-motor stages of PD. Figure 2. EV characterisation from rat plasma. (A) Representative Nanosight graphs showing nanoparticle tracking analysis (NTA) analysis of plasma EV profiles from sham-treated rats (Sham; n = 3). (B) Representative Nanosight graphs showing NTA analysis of plasma EV profiles from the premotor PD rat models (PD; n = 3). (C) Western blotting (WB) confirms that rat plasma-EVs are positive for the EV-specific markers CD63 and flotillin-1 (Flot-1); the molecular weight standard is indicated in kilo Daltons (kDa). (D,E) Transmission electron microscopy (TEM) images showing EVs isolated from sham (D) and pre-motor PD model (PD; (E)) rat plasma, revea...