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NADH shuttles mediate the transfer of the reducing equivalents of cytosolic NADH into mitochondria. Cumulating evidence has suggested that malate-aspartate shuttle (MAS), one of the two types of NADH shuttles, plays significant roles in such biological processes as glutamate synthesis in neurons. However, there has been no information regarding the roles of NADH shuttle in the survival and energy metabolism of microglia. In current study, using microglial BV2 cells as a cellular model, we determined the roles of MAS in the survival and energy metabolism of microglia by using aminooxyacetate acid (AOAA)-a widely used MAS inhibitor. Our study has suggested that AOAA can effectively inhibit the MAS activity of the cells. We also found that AOAA can induce both early- and late-stage apoptosis of resting microglia and lipopolysaccharides (LPS)-activated microglia. AOAA also induced mitochondrial depolarization, increases in the cytosolic Ca(2+) concentrations, and decreases in the intracellular ATP levels. Moreover, our study has excluded the possibility that the major nonspecific effect of AOAA-inhibition of GABA transaminase-is involved in theses effects of AOAA. Collectively, our study has provided first information suggesting significant roles of MAS in the survival and energy metabolism in both resting microglia and LPS-activated microglia.

Nicotinamide adenine dinucleotide (NAD(+)) plays critical roles in not only energy metabolism and mitochondrial functions, but also calcium homeostasis and immunological functions. It has been reported that NAD(+) administration can reduce ischemic brain damage. However, the mechanisms underlying the protective effects remain unclear. Because mitochondrial impairments play a key role in the cell death in cerebral ischemia, in this study we tested our hypothesis that NAD(+) can decrease mitochondrial damage-induced cell death using differentiated PC12 cells as a cellular model. We found that NAD(+) can decrease both early-stage and late-stage apoptosis, as well as necrosis of rotenone-treated PC12 cells, as assessed by FACS-based Annexin V/AAD assay. We also found that NAD(+) treatment can restore the intracellular NAD(+) levels of the rotenone-treated cells. Moreover, NAD(+) treatment can prevent rotenone-induced mitochondria depolarization. In summary, our study has provided first direct evidence that NAD(+) treatment can prevent rotenone-induced apoptosis and necrosis. Our study has also indicated that NAD(+) treatment can prevent mitochondrial damage-induced cell death, which may at least partially result from its protective effects on rotenone-induced mitochondrial depolarization. Because both mitochondrial damage and apoptosis play key roles in multiple neurological disorders, our study has highlighted the therapeutic potential of NAD(+) for brain ischemia and other neurological diseases., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Sirtuin 2 (SIRT2), a sirtuin family protein, is a tubulin deacetylase. Recent studies have indicated that SIRT2 plays a key role in programmed necrosis, and the SIRT2 inhibitor AGK2 can decrease the cell death both in a cellular model of Parkinson's disease and in an animal model of myocardial ischemia-reperfusion. However, there has been little information regarding the role of SIRT2 in microglial survival and functions, which play critical roles in multiple neurological disorders. Our current study found that AGK2 at 10 μM - a widely used AGK2 concentration - can induce both late-stage apoptosis and necrosis, as well as a decrease in the intracellular ATP levels of microglial BV2 cells. Our study also showed that both the AGK2-induced cell death and the AGK2-induced ATP decline are mediated by poly(ADP-ribose) polymerase (PARP) activation. Collectively, our study has provided the first evidence suggesting a significant role of SIRT2 in the basal survival of microglia, as well as a mechanism accounting for the effects of SIRT2 on intracellular ATP levels., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Laser irradiation has been shown to trigger cellular proliferation and apoptosis in various cell types. Studying the signaling pathways involved in the laser irradiation is important for understanding these processes. In present study, to monitor the protein kinase Cs (PKCs) activity in living cells in real time, we transfected and screened human lung adenocarcinoma cells (ASTC-a-1) stably expressing C kinase activity reporter (CKAR) constructed based on fluorescence resonance energy transfer (FRET) technique. The CKAR is a specific, reversible reporter of phosphorylation by PKCs and it can monitor the ongoing balance between PKCs and phosphatases. The increasing dynamics of PKCs activity is monitored during cell proliferation induced by low-power laser irradiation (LPLI) (0.8 J/cm2) in serum-starved ASTC-a-1 cells stably expressing CKAR reporter using FRET imaging on laser scanning confocal microscope and using spectrofluorometric analysis on a luminescence spectrometer, respectively. However, the decreasing dynamics of PKCs activity has been monitored in real time using FRET imaging for the cells treated with high fluence LPLI (60 J/cm2), which was previously found to induce cell apoptosis. Taken together, LPLI induces the ASTC-a-1 cell proliferation by specifically activating PKCs. However, PKCs activity decreases during cell apoptosis induced by high fluence LPLI. Our results indicate that PKCs play an important role in the laser irradiation-induced biological effects., (Copyright (c) 2005 Wiley-Liss, Inc.)

We describe a new method for selective cell targeting based on the use of light-absorbing microparticles and nanoparticles that are heated by short laser pulses to create highly localized cell damage. The method is closely related to chromophore-assisted laser inactivation and photodynamic therapy, but is driven solely by light absorption, without the need for photochemical intermediates (particularly singlet oxygen). The mechanism of light-particle interaction was investigated by nanosecond time-resolved microscopy and by thermal modeling. The extent of light-induced damage was investigated by cell lethality, by cell membrane permeability, and by protein inactivation. Strong particle size dependence was found for these interactions. A technique based on light to target endogenous particles is already being exploited to treat pigmented cells in dermatology and ophthalmology. With exogenous particles, phamacokinetics and biodistribution studies are needed before the method can be evaluated against photodynamic therapy for cancer treatment. However, particles are unique, unlike photosensitizers, in that they can remain stable and inert in cells for extended periods. Thus they may be particularly useful for prelabeling cells in engineered tissue before implantation. Subsequent irradiation with laser pulses will allow control of the implanted cells (inactivation or modulation) in a noninvasive manner.

NADH shuttles, including malate-aspartate shuttle (MAS) and glycerol-3-phosphate shuttle, can shuttle the reducing equivalents of cytosolic NADH into mitochondria. It is widely accepted that the major function of NADH shuttles is to increase mitochondrial energy production. Our study tested the hypothesis that the novel major function of NADH shuttles in cancer cells is to maintain glycolysis by decreasing cytosolic NADH/NAD(+) ratios. We found that AOAA, a widely used MAS inhibitor, led to decreased intracellular ATP levels, altered cell cycle and increased apoptosis and necrosis of C6 glioma cells, without affecting the survival of primary astrocyte cultures. AOAA also decreased the glycolytic rate and the levels of extracellular lactate and pyruvate, without affecting the mitochondrial membrane potential of C6 cells. Moreover, the toxic effects of AOAA were completely prevented by pyruvate treatment. Collectively, our study has suggested that AOAA may be used to selectively decrease glioma cell survival, and the major function of MAS in cancer cells may be profoundly different from its major function in normal cells: The major function of MAS in cancer cells is to maintain glycolysis, instead of increasing mitochondrial energy metabolism. [ABSTRACT FROM AUTHOR]

Highlights: [•] NAD+ treatment can decrease rotenone-induced early-stage and late-stage apoptosis of PC12 cells. [•] NAD+ treatment can decrease rotenone-induced necrosis of PC12 cells. [•] NAD+ treatment can restore the intracellular NAD+ levels of rotenone-treated PC12 cells. [•] NAD+ treatment can prevent rotenone-induced mitochondrial depolarization of PC12 cells. [Copyright &y& Elsevier]

Abstract: Confocal fluorescence imaging and fluorescence resonance energy transfer (FRET) technology have been widely used to study protein–protein interactions in living cells. However, it is very difficult to quantitatively analyze FRET efficiency due to the excitation spectral crosstalk and emission spectral crosstalk between donor and acceptor. In this study, we developed a novel method to quantitatively obtain the FRET efficiency by fitting the emission spectra (FES) of donor–acceptor pair, and this method is free from both excitation and emission spectral crosstalk. We used the FES method to quantitatively monitor the FRET efficiency of SCAT3, a caspase-3 indicator based on FRET, inside living cells stably expressing SCAT3 during STS-induced apoptosis. At 0, 6 and 12h after STS treatment, the FRET efficiency of SCAT3 obtained by FES are consistent with that by two-photon excitation (TPE) fluorescence lifetime imaging microscopy (FLIM) in living cells stably expressing SCAT3. In this study, the FES was also used to analyze the caspase-3 activation in living cells during anti-cancer drug such as taxol, Artesunate (ART) or Dihydroartemisinin (DHA) treatment. Our results showed that ART or DHA induced apoptosis by a caspase-3-dependent manner, while caspase-3 was not involved in taxol-induced cell death. [Copyright &y& Elsevier]