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Deletion of the Homocysteine Thiolactone Detoxifying Enzyme Bleomycin Hydrolase, in Mice, Causes Memory and Neurological Deficits and Worsens Alzheimer’s Disease-Related Behavioral and Biochemical Traits in the 5xFAD Model of Alzheimer’s Disease
Background: Bleomycin hydrolase (BLMH), a homocysteine (Hcy)-thiolactone detoxifying enzyme, is attenuated in Alzheimer’s disease (AD) brains. Blmh loss causes astrogliosis in mice while the loss of histone demethylase Phf8, which controls mTOR signaling, causes neuropathy in mice and humans. Objective: To examine how Blmh gene deletion affects the Phf8/H4K20me1/mTOR/autophagy pathway, amyloid-β (Aβ) accumulation, and cognitive/neuromotor performance in mice. Methods: We generated a new mouse model of AD, the Blmh-/-5xFAD mouse. Behavioral assessments were conducted by cognitive/neuromotor testing. Blmh and Phf8 genes were silenced in mouse neuroblastoma N2a-APPswe cells by RNA interference. mTOR- and autophagy-related proteins, and AβPP were quantified by western blotting and the corresponding mRNAs by RT-qPCR. Aβ was quantified by western blotting (brains) and by confocal microscopy (cells). Results: Behavioral testing showed cognitive/neuromotor deficits in Blmh-/- and Blmh-/-5xFAD mice. Phf8 was transcriptionally downregulated in Blmh-/- and Blmh-/-5xFAD brains. H4K20me1, mTOR, phospho-mTOR, and AβPP were upregulated while autophagy markers Becn1, Atg5, and Atg7 were downregulated in Blmh-/- and Blmh-/-5xFAD brains. Aβ was elevated in Blmh-/-5xFAD brains. These biochemical changes were recapitulated in Blmh-silenced N2a-APPswe cells, which also showed increased H4K20me1-mTOR promoter binding and impaired autophagy flux (Lc3-I, Lc3-II, p62). Phf8-silencing or treatments with Hcy-thiolactone or N-Hcy-protein, metabolites elevated in Blmh-/- mice, induced biochemical changes in N2a-APPswe cells like those induced by the Blmh-silencing. However, Phf8-silencing elevated Aβ without affecting AβPP. Conclusions: Our findings show that Blmh interacts with AβPP and the Phf8/H4K20me1/mTOR/autophagy pathway, and that disruption of those interactions causes Aβ accumulation and cognitive/neuromotor deficits.
Metabolizm kwasów tłuszczowych i glukozy podczas dojrzewania in vitro oocytów bydła i jego wpływ na jakość oocytów oraz zarodków pochodzących z zapłodnienia in vitro.
Follicular-fluid extracellular vesicles support energy metabolism of bovine oocytes, improving blastocyst development and quality
Abstract Extracellular vesicles (EVs) from follicular fluid (FF) seem to play a significant role in communication within ovarian follicles in several species. The present study aimed to examine the supporting effect of FF-derived small EVs (FF-sEVs) during in vitro maturation (IVM) of bovine cumulus–oocyte complexes (COCs) under conditions of disturbed energy metabolism. Bovine COCs were matured in vitro with inhibitors targeting lipid metabolism (etomoxir) or glucose metabolism (iodoacetate combined with dehydroepiandrosterone), in the presence or absence of FF-sEVs. Following maturation, oocytes and cumulus cells were analyzed by real-time quantitative polymerase chain reaction (qPCR) and stained to visualize lipid droplets. The uptake of FF-sEVs was visualized by fluorescent labeling. In vitro fertilization and embryo culture were followed by mass spectrometry analysis of hatched blastocysts. We demonstrate for the first time that FF-sEVs are transported from the medium into the oocytes, via the cumulus cells and through transzonal projections into the perivitelline space and ooplasm. Cumulus cells under metabolic stress conditions exhibit an increased FF-sEV uptake from the maturation medium. FF-sEV supplementation during metabolic stress conditions enhances the MII rate in oocytes and positively affects subsequent embryo development and quality revealed by altered metabolic activity. Lipid droplet parameters and gene expression in cumulus cells and oocytes are affected by FF-sEV supplementation, which is more pronounced in cumulus cells. Our findings show that FF-sEV supplementation during IVM under metabolic stress conditions significantly affects COCs, with a positive effect on further blastocyst quality. We provide novel insights into the role of FF-sEVs in oocyte maturation and blastocyst development.