Identification of molecular network of gut-brain axis associated with neuroprotective effects of PPARδ-ligand erucic acid in rotenone-induced Parkinson's disease model in zebrafish.

Disruption of the gut-brain axis in Parkinson's disease (PD) may lead to motor symptoms and PD pathogenesis. Recently, the neuroprotective potential of different PPARδ-agonists has been shown. We aimed to reveal the effects of erucic acid, peroxisome proliferator-activated receptors (PPARs)-ligand in rotenone-induced PD model in zebrafish, focusing on the gut-brain axis. Adult zebrafish were exposed to rotenone and erucic acid for 30 days. Liquid chromatography-mass spectrometry and tandem mass spectrometry (LC-MS/MS) analysis was performed. Raw files were analysed by Proteome Discoverer 2.4 software; peptide lists were searched against Danio rerio proteins. STRING database was used for protein annotations or interactions. Lipid peroxidation (LPO), nitric oxide (No), alkaline phosphatase, superoxide dismutase, glutathione S-transferase (GST), acetylcholinesterase and the expressions of PD-related genes were determined. Immunohistochemical tyrosine hydroxylase (TH) staining was performed. LC-MS/MS analyses allowed identification of over 2000 proteins in each sample. The 2502 and 2707 proteins overlapped for intestine and brain. The 196 and 243 significantly dysregulated proteins in the brain and intestines were found in rotenone groups. Erucic acid treatment corrected the changes in the expression of proteins associated with cytoskeletal organisation, transport and localisation and improved locomotor activity, expressions of TH, PD-related genes (lrrk2, park2, park7, pink1) and oxidant-damage in brain and intestines in the rotenone group as evidenced by decreased LPO, No and increased GST. Our results showed beneficial effects of erucic acid as a PPARδ-ligand in neurotoxin-induced PD model in zebrafish. We believe that our study will shed light on the mechanism of the effects of PPARδ agonists and ω9-fatty acids in the gut-brain axis of PD.

Quantitative phosphoproteomics to resolve the cellular responses to octanoic acid in rotenone exposed zebrafish.

Ketosis is a potentially beneficial metabolic state for health especially in neurological conditions including Parkinson's disease (PD). Medium-chain-triglycerides (MCT) have specific metabolic properties and they are described as ketogenic even without restriction of carbohydrate. Octanoic acid (C8) is the main MCT showing this effect. Rotenone is a neurotoxin that is used to induce experimental PD model. Rotenone inhibits mitochondrial respiratory complex 1 (MRC1) and causes reactive oxygen species formation. Mass spectrometry (MS)-based phosphoproteomic methods enable discovering specific signaling events in special molecular pathways through identification and quantification of phosphoproteins. Signaling networks involved in rotenone-mediated biological processes and beneficial effects of MCTs on neurodegenerative diseases are not well understood. We aimed to gain comprehensive molecular perspective on the global phosphoproteome differences in rotenone-exposed zebrafish treated with octanoic acid. Raw files obtained from MS analysis were processed and searched against the Danio rerio protein database using SEQUEST-HT algorithm to identify and quantify phosphopeptides with 2,569 unique phosphoproteins and 4,161 unique phosphopeptides corresponding to 2005 proteins. Microtubule-associated protein (MAP) family members were significantly lower in rotenone group. Phosphoproteins involved in ion binding (calcium, magnesium, zinc ion), oxygen binding, microtubule binding, ATP- and GTP-binding were among differentially expressed 347 proteins in rotenone group and they were reversed after octanoic acid treatments. Phosphoproteins and phosphorylation sites were identified for future exploration of signaling pathways involved in rotenone toxicity. We believe our findings might help in the formulation of effective therapeutic strategies for the treatment of PD using ketogenic formulations involving MCTs. PRACTICAL APPLICATIONS: Ketosis is a potentially beneficial metabolic state for health especially in neurological conditions including Parkinson's disease (PD). Medium-chain-triglycerides (MCT) (C6-C12) have specific metabolic properties making them described as ketogenic even without restriction of carbohydrate. Octanoic acid (caprylic acid, C8) is the main MCT showing this effect. Our findings might help in the formulation of effective therapeutic strategies for the treatment of Parkinson's disease using ketogenic formulations involving Medium-chain-triglycerides.

Phosphoproteomic strategies in cancer research: a minireview.

Understanding the cellular processes is central to comprehend disease conditions and is also true for cancer research. Proteomic studies provide significant insight into cancer mechanisms and aid in the diagnosis and prognosis of the disease. Phosphoproteome is one of the most studied complements of the whole proteome given its importance in the understanding of cellular processes such as signaling and regulations. Over the last decade, several new methods have been developed for phosphoproteome analysis. A significant amount of these efforts pertains to cancer research. The current use of powerful analytical instruments in phosphoproteomic approaches has paved the way for deeper and sensitive investigations. However, these methods and techniques need further improvements to deal with challenges posed by the complexity of samples and scarcity of phosphoproteins in the whole proteome, throughput and reproducibility. This review aims to provide a comprehensive summary of the variety of steps used in phosphoproteomic methods applied in cancer research including the enrichment and fractionation strategies. This will allow researchers to evaluate and choose a better combination of steps for their phosphoproteome studies.

Clinical importance of toxin concentration in Amanita verna mushroom.

Poisoning from Amanita group of mushrooms comprises approximately 3% of all poisonings in our country and their being responsible for nearly the entire fatal mushroom poisonings makes them important. These mushrooms contain primarily two types of toxins, amatoxins and phallotoxins. Phallotoxins have a more limited toxicity potential and they primarily consist of phalloidin (PHN) and phallacidin (PCN). Amatoxins, on the other hand, are very toxic and they primarily consist of alpha-amanitin (AA), beta-amanitin (BA) and gamma-amanitin (GA). Toxin levels can vary among various species, even among varieties of the same species, of Amanita mushroom family. Revealing the differences between the toxin compositions of the Amanita species that grow in our region may contribute to the clinics of poisonings. Our study aims at showing in detail the toxin levels in various parts of Amanita verna mushroom. A. verna mushrooms needed for toxin analysis were collected from Kozak Plateau near Ayvalik county of Balikesir, Turkey in April 2013. The mushrooms were divided into their parts as pileus, gills, stripe and volva. Following the procedures required before the analysis, the AA, BA, GA, PHN and PCN levels were measured using the RP-HPLC method. While the lowest level of amatoxin was in the volva of the mushroom, the highest was measured in the gills. This was followed by pileus and stripe where the levels were close to each other. Similarly, the highest level of phallotoxin was measured in the gills. Gamma toxin and phalloidin were at lower amounts than the other toxins. A. verna is frequently confused with edible mushrooms with white caps due to its macroscopic similarity. If just one of them is eaten by mistake by an adult person with no mushroom experience, it can easily poison them. The amount of amatoxin is more as compared to Amanita phalloides and A. phalloides var. alba. Particularly, the AA and BA levels are approximately three times higher, whereas GA levels are lower. Similarly, the level of PCN is approximately four times higher as compared to A. phalloides and A. phalloides var. alba; by contrast, the level of PNH is about a half of theirs. In summary, it can be said that A. verna is a more toxic mushroom than A. phalloides and has a higher rate of mortality. With our study, the amatoxin and phallotoxin concentrations and distribution in A. verna mushrooms were shown in detail for the first time and it would be useful to carry out more similar studies with other members of Amanita family growing in various parts of the world.

Dermal absorption and toxicity of alpha amanitin in mice.

The fungus Amanita phalloides is known to contain two main groups of toxins: amanitins and phallotoxins. The amanitins group effectively blocks the RNA polymerase II enzyme found in eukaryotic cells. As alpha amanitin has a lethal effect on the majority of eukaryotic cells, it can be valuable as an antiparasitic or antifungal drug. It can be used externally against ectoparasites. It is critical that percutaneous applications of the alpha amanitin toxin are not harmful to the recipient. In this study, the absorption and the toxicity of percutaneous and intraperitoneal (ip) applications of 1 mg/kg alpha amanitin to mice were compared. Potential skin, liver and kidney toxicities were investigated through pathological examination. HPLC analysis was used to determine the amount of the toxin. No toxicity or toxin were found in the skin, liver, or kidneys of the mice in the control group. Interestingly, the percutaneous application group also showed no toxicity, and the toxin was not present in this group. After 24 h, Councilman-like bodies and pyknotic cells were observed in the mice in which alpha amanitin was applied intraperitoneally, demonstrating the presence of toxicity. Peak levels of alpha amanitin (µg/mL) in the liver, kidney, and blood in the ip application group were measured at 3.3 (6 h), 0.2 (6 h) and 1.2 (1 h), respectively. The results demonstrated that the toxin was not absorbed through the skin of the mice and that the percutaneous application of alpha amanitin did not have any toxic effects. Thus, alpha amanitin may be administered percutaneously for therapeutic purposes.