The link between the size of soluble amyloid β (Aβ) oligomers and their toxicity to rat cerebellar granule cells (CGC) was investigated. Variation in conditions during in vitro oligomerization of Aβ 1-42 resulted in peptide assemblies with different particle size as measured by atomic force microscopy and confirmed by the dynamic light scattering and fluorescence correlation spectroscopy. Small oligomers of Aβ 1-42 with a mean particle z-height of 1-2 nm exhibited propensity to bind to the phospholipid vesicles and they were the most toxic species that induced rapid neuronal necrosis at submicromolar concentrations whereas the bigger aggregates (z-height above 4-5 nm) did not bind vesicles and did not cause detectable neuronal death. Similar neurotoxic pattern was also observed in primary cultures of cortex neurons whereas Aβ 1-42 oligomers, monomers and fibrils were nontoxic to glial cells in CGC cultures or macrophage J774 cells. However, both oligomeric forms of Aβ 1-42 induced reduction of neuronal cell densities in the CGC cultures.
Prolonged heart ischaemia causes an inhibition of oxidative phosphorylation and an increase of Ca2+ in mitochondria. We investigated whether elevated Ca2+ induces changes in the oxidative phosphorylation system relevant to ischaemic damage, and whether Ca2+ and other inducers of mitochondrial permeability transition cause the release of cytochrome c from isolated heart mitochondria. We found that 5 microM free Ca2+ induced changes in oxidative phosphorylation system similar to ischaemic damage: increase in the proton leak and inhibition of the substrate oxidation system related to the release of cytochrome c from mitochondria. The phosphorylating system was not directly affected by high Ca2+ and ischaemia. The release of cytochrome c from mitochondria was caused by Ca2+ and 0.175-0.9 mM peroxynitrite but not by NO, and was prevented by cyclosporin A. Adenylate kinase and creatine kinase were also released after incubation of mitochondria with Ca2+, however, the activity of citrate synthase in the incubation medium with high and low Ca2+ did not change. The data suggest that release of cytochrome c and other proteins of intermembrane space may be due to the opening of the mitochondrial permeability transition pore, and may be partially responsible for inhibition of mitochondrial respiration induced by ischaemia, high calcium, and oxidants.
It has previously been shown that apoptosis is increased in ischaemic/reperfused heart. However, little is known about the mechanism of induction of apoptosis in myocardium during ischaemia. We investigated whether prolonged myocardial ischaemia causes activation of caspases and whether this activation is related to cytochrome c release from mitochondria to cytosol during ischaemia. Using an in vitro model of heart ischaemia, we show that 60 min ischaemia leads to a significant accumulation of cytochrome c in the cytosol and a decrease in mitochondrial content of cytochrome c but not cytochrome a. The release of cytochrome c from mitochondria was accompanied by activation of caspase-3-like proteases (measured by cleavage of fluorogenic peptide substrate DEVD-amc) and a large increase in number of cells with DNA strand breaks (measured by TUNEL staining). Caspase-1-like proteases (measured by YVAD-amc cleavage) were not activated during ischaemia. Addition of 14 microM cytochrome c to cytosolic extracts prepared from control hearts induced ATP-dependent activation of caspase-3-like protease activity. Our data suggest that extended heart ischaemia can cause apoptosis mediated by release of cytochrome c from mitochondria and subsequent activation of caspase-3.
Tau is a microtubule-associated protein, found at high levels in neurons, and the aggregation of which is associated with neurodegeneration. Recently it was found that tau can be actively secreted from neurons, but the effects of extracellular tau on neuronal viability are not clear. In this study, we investigated whether extracellular tau 2N4R can cause neurotoxicity in primary cultures of rat brain neurons and glial cells. Cell cultures were treated with recombinant fulllength tau 2N4R isoform, and neuronal numbers, death and phosphatidylserine exposure, as well as microglial phagocytosis were analysed by fluorescent microscopy. Aggregation of tau 2N4R was assests by atomic force microscopy. We found that extracellular addition of tau induced a gradual loss of neurons over 1-2 days, without neuronal necrosis or apoptosis, but accompanied by proliferation of microglia in the neuronal-glial co-cultures. Tau addition caused exposure of the "eat-me" signal phosphatidylserine on the neuronal cell surface (without apparent apoptosis or necrosis), and this was prevented by elimination of the microglia or by inhibition of neutral sphingomyelinase. Tau also increased the phagocytic activity of pure microglia, and this was blocked by inhibitors of neutral sphingomyelinase or protein kinase C. The neuronal loss induced by tau was prevented by inhibitors of neutral sphingomyelinase, protein kinase C or the phagocytic receptor MerTK, or by eliminating microglia from the cultures. The data suggest that extracellular tau induces primary phagocytosis of stressed neurons by activated microglia, and identifies multiple ways in which the neuronal loss induced by tau can be prevented.
The pathogenesis of Alzheimer's disease (AD), a slowly-developing age-related neurodegenerative disorder, is a result of the action of multiple factors including deregulation of Ca 2+ homeostasis, mitochondrial dysfunction, and dysproteostasis. Interaction of these factors in astrocytes, principal homeostatic cells in the central nervous system, is still poorly understood. Here we report that in immortalized hippocampal astrocytes from 3xTg-AD mice (3Tg-iAstro cells) bioenergetics is impaired, including reduced glycolysis and mitochondrial oxygen consumption, and increased production of reactive oxygen species. Shotgun proteomics analysis of mitochondria-ER-enriched fraction showed no alterations in the expression of mitochondrial and OxPhos proteins, while those related to the ER functions and protein synthesis were deregulated. Using ER-and mitochondria-targeted aequorin-based Ca 2+ probe we show that, in 3Tg-iAstro cells, ER was overloaded with Ca 2+ while Ca 2+ uptake by mitochondria upon ATP stimulation was reduced. This was accompanied by the increase in short distance (≈8-10 nm) contact area between mitochondria and ER, upregulation of ER-stress/unfolded protein response genes Atf4, Atf6 and Herp, and reduction of global protein synthesis rate. We suggest that familial AD mutations in 3Tg-iAstro cells induce mitochondria-ER interaction changes that deregulate astrocytic bioenergetics, Ca 2+ homeostasis and proteostasis. These factors may interact, creating a pathogenic loop compromising homeostatic and defensive functions of astroglial cells predisposing neurons to dysfunction.
The present study reports on the encapsulation of Elsholtzia ciliata ethanolic extract by freeze-drying method using skim milk, sodium caseinate, gum Arabic, maltodextrin, beta-maltodextrin, and resistant-maltodextrin alone or in mixtures of two or four encapsulants. The encapsulation ability of the final mixtures was evaluated based on their microencapsulating efficiency (EE) of total phenolic compounds (TPC) and the physicochemical properties of freeze-dried powders. Results showed that the freeze-dried powders produced using two encapsulants have a lower moisture content, but higher solubility, Carr index, and Hausner ratio than freeze-dried powders produced using only one encapsulant in the formulation. The microencapsulating efficiency of TPC also varied depending on encapsulants used. The lowest EE% of TPC was determined with maltodextrin (21.17%), and the highest with sodium caseinate (83.02%). Scanning electron microscopy revealed that freeze-drying resulted in the formation of different size, irregular shape glassy particles. This study demonstrated good mucoadhesive properties of freeze-dried powders, which could be incorporated in buccal or oral delivery dosage forms. In conclusion, the microencapsulation of E. ciliata ethanolic extract by freeze-drying is an effective method to produce new value-added pharmaceutical or food formulations with polyphenols.
We investigated to what extent different types of NO donors induce caspase activation by opening of the mitochondrial permeability transition pore (PTP) or inhibition of mitochondrial respiration. We found that nitrosothiols can directly open the PTP in isolated mitochondria and cause cytochrome c release, whereas NONOate donors can not. In macrophages nitrosothiols cause caspase activation that is blocked by cyclosporin A or calcium chelation, both of which prevent PTP opening, whereas caspase activation caused by NONOates is much less sensitive to these agents. Inhibitors of mitochondrial respiration did not promote PTP opening in isolated mitochondria, and although they cause caspase activation in macrophages, this activation was slower than that caused by NO donors, and was relatively insensitive to cyclosporin and calcium chelators suggesting that PTP opening was not involved.z 2000 Federation of European Biochemical Societies.
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