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Exogenous neuroprotective protein neuroglobin (Ngb) cannot cross the blood–brain barrier. To overcome this difficulty, we synthesized hyaluronate nanoparticles (NPs), able to deliver Ngb into the brain in an animal model of stroke (MCAO). These NPs effectively reached neurons, and were microscopically identified after 24 h of reperfusion. Compared to MCAO non-treated animals, those treated with Ngb-NPs showed survival rates up to 50% higher, and better neurological scores. Tissue damage improved with the treatment, but no changes in the infarct volume or in the oxidative/nitrosative values were detected. A proteomics approach (p-value < 0.02; fold change = 0.05) in the infarcted areas showed a total of 219 proteins that significantly changed their expression after stroke and treatment with Ngb-NPs. Of special interest, are proteins such as FBXO7 and NTRK2, which were downexpressed in stroke, but overexpressed after treatment with Ngb-NPs; and ATX2L, which was overexpressed only under the effect of Ngb. Interestingly, the proteins affected by the treatment with Ngb were involved in mitochondrial function and cell death, endocytosis, protein metabolism, cytoskeletal remodeling, or synaptic function, and in regenerative processes, such as dendritogenesis, neuritogenesis, or sinaptogenesis. Consequently, our pharmaceutical preparation may open new therapeutic scopes for stroke and possibly for other neurodegenerative pathologies. [ABSTRACT FROM AUTHOR]

Aim: Functional in vivo studies on the mitochondrial thioredoxin system are hampered by the embryonic or larval lethal phenotypes displayed by murine or Drosophila knock-out models. Thus, the access to alternative metazoan knock-out models for the mitochondrial thioredoxin system is of critical importance., Results: We report here the characterization of the mitochondrial thioredoxin system of Caenorhabditis elegans that is composed of the genes trx-2 and trxr-2. We demonstrate that the proteins thioredoxin 2 (TRX-2) and thioredoxin reductase 2 (TRXR-2) localize to the mitochondria of several cells and tissues of the nematode and that trx-2 and trxr-2 are upregulated upon induction of the mitochondrial unfolded protein response. Surprisingly, C. elegans trx-2 (lof ) and trxr-2 (null) single and double mutants are viable and display similar growth rates as wild-type controls. Moreover, the lack of the mitochondrial thioredoxin system does not affect longevity, reactive oxygen species production or the apoptotic program. Interestingly, we found a protective role of TRXR-2 in a transgenic nematode model of Alzheimer's disease (AD) that expresses human β-amyloid peptide and causes an age-dependent progressive paralysis. Hence, trxr-2 downregulation enhanced the paralysis phenotype, while a strong decrease of β-amyloid peptide and amyloid deposits occurred when TRXR-2 was overexpressed., Innovation: C. elegans provides the first viable metazoan knock-out model for the mitochondrial thioredoxin system and identifies a novel role of this system in β-amyloid peptide toxicity and AD., Conclusion: The nematode strains characterized in this work make C. elegans an ideal model organism to study the pathophysiology of the mitochondrial thioredoxin system at the level of a complete organism.

The mechanism for regeneration of the active-site "peroxidatic" cysteine in 1-Cys peroxiredoxins is a matter of debate. Saccharomyces cerevisiae Prx1 is a mitochondrial enzyme belonging to the 1-Cys Prx, whereas Grx2 is involved in antioxidant defense and localizes at the mitochondria, so we hypothesized that it could be a perfect candidate to resolve the sulfenate in Prx1 with GSH. In vitro experiments with purified Prx1p and Grx2p demonstrate that Grx2p, at concentrations <1 microM, coupled to GSH, is a very efficient thiolic intermediary for the reduction of the peroxidatic Cys in Prx1p. Prx1p forms oligomeric aggregates natively, but depolymerizes down to a dimeric state after treatment with GSH. The catalytic cycle involves glutathionylation of dimeric Prx1p and deglutathionylation by Grx2p. Dihydrolipoamide, a genuine mitochondrial dithiol, can efficiently substitute for GSH. The activity is highest at alkaline pH, consistent with the conditions of active respiring mitochondria, and the process is highly specific for 1-Cys Prx because Grx2p is totally inactive with human PRX1, a typical 2-Cys Prx, as opposed to the promiscuity of Trx. Our results suggest that although Trx is the reductant involved in the reduction of peroxides by 2-Cys-Prx, Grx might be the natural resolving partner of 1-Cys Prx through a monothiolic mechanism.