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. 2011 Aug 12;286(32):27875-81.
doi: 10.1074/jbc.C110.216580. Epub 2011 May 24.

Nicotinic acid adenine dinucleotide phosphate (NAADP) regulates autophagy in cultured astrocytes

Gustavo J S Pereira  1 , Hanako Hirata, Gian M Fimia, Lúcia G do Carmo, Claudia Bincoletto, Sang W Han, Roberta S Stilhano, Rodrigo P Ureshino, Duncan Bloor-Young, Grant Churchill, Mauro Piacentini, Sandip Patel, Soraya S Smaili
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Nicotinic acid adenine dinucleotide phosphate (NAADP) regulates autophagy in cultured astrocytes

Gustavo J S Pereira et al. J Biol Chem. .

Abstract

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca(2+)-mobilizing messenger that in many cells releases Ca(2+) from the endolysosomal system. Recent studies have shown that NAADP-induced Ca(2+) mobilization is mediated by the two-pore channels (TPCs). Whether NAADP acts as a messenger in astrocytes is unclear, and downstream functional consequences have yet to be defined. Here, we show that intracellular delivery of NAADP evokes Ca(2+) signals from acidic organelles in rat astrocytes and that these signals are potentiated upon overexpression of TPCs. We also show that NAADP increases acidic vesicular organelle formation and levels of the autophagic markers, LC3II and beclin-1. NAADP-mediated increases in LC3II levels were reduced in cells expressing a dominant-negative TPC2 construct. Our data provide evidence that NAADP-evoked Ca(2+) signals mediated by TPCs regulate autophagy.

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Figures

FIGURE 1.
FIGURE 1.
NAADP-mediated Ca2+ signaling in astrocytes. A, typical cytosolic Ca2+ responses of individual Fura-2-loaded cells microinjected with 100 nm NAADP. Cells were from untreated cultures (black) or cultures pretreated with bafilomycin A1 (100 nm, 1 h) or NED-19 (10 μm, 10 min). B, summary data quantifying the amplitude of the Ca2+ signals in response to the indicated concentration of NAADP. + Baf., bafilomycin A1. C, confocal images of cells transfected with either mRFP-tagged TPC1 or mRFP-tagged TPC2 (red) and labeled with LysoTracker Green. Merged images are shown to the right. Line plots are the relative intensities across the dashed lines. Scale bar = 2 μm. D, typical cytosolic Ca2+ responses of individual Fura-2-loaded cells expressing GFP, GFP-tagged TPC1 (gray), or GFP-tagged TPC2 (black) and microinjected with 100 nm NAADP. E, summary data quantifying the amplitude of the Ca2+ signals in the indicated cell types. F, end-point RT-PCR analysis of TPCs. The expected sizes of the amplicons were 500 bp (TPC1) and 650 bp (TPC2). G, quantitative RT-PCR analysis of TPCs. Data were normalized to the expression level of the housekeeping gene β-actin. Data in B and E are expressed as mean ± S.E. of the indicated number of cells (in brackets). All data are from at least three different cultures. **, p < 0.01; ***, p < 0.001 (ANOVA one-way test, followed by Dunnett's post hoc test).
FIGURE 2.
FIGURE 2.
Effect of NAADP on autophagic markers. In A and B, the main panels show the density plots from flow cytometry analysis of cells loaded with acridine orange. Insets are confocal images of cells transfected with GFP-LC3. A, effect of NAADP-AM (1 μm, middle) and rapamycin (1 μm, right) as compared with control untreated cells (left). B, effect of NAADP-AM in the presence of NED-19 (10 μm, left) and 3-MA (10 mm, right).Treatments were for either 24 h (flow cytometry) or 4 h (confocal imaging). Scale bar = 10 μm. C and D, summary data (mean ± S.E. from at least three experiments) quantifying the flow cytometry (C) and GFP-LC3 (D) analyses under the various conditions. CTRL, control; RAP, rapamycin. E and F, Western blot analysis of LC3 (E) and beclin-1 (F). Expression of β-actin is shown for comparison. Results of densitometry are shown below each panel. *, p < 0.05 (ANOVA one-way test, followed by Bonferroni's post hoc test).
FIGURE 3.
FIGURE 3.
Effect of TPC2 overexpression on autophagy. A, overlaid confocal images of astrocytes co-transfected with mRFP-TPC2 and GFP-LC3 (left) or GFP-TPC2 L265P and mCherry-LC3 (right). Cells are from untreated cultures (−) or cultures treated for 4 h with 1 μm NAADP-AM (+). Scale bar = 10 μm. B, summary data (mean ± S.E. from at least three independent cultures) quantifying the number of LC3 puncta per cell from the indicated cultures before and after treatment with NAADP-AM. *, p < 0.05 (ANOVA one-way test followed by Bonferroni's post hoc test).
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References

    1. Berridge M. J. (1998) Neuron 21, 13–26 - PubMed
    1. Berridge M. J., Lipp P., Bootman M. D. (2000) Nat. Rev. Mol. Cell Biol. 1, 11–21 - PubMed
    1. Verkhratsky A., Petersen O. H. (2002) Eur. J. Pharmacol. 447, 141–154 - PubMed
    1. Patel S., Docampo R. (2010) Trends Cell Biol. 20, 277–286 - PMC - PubMed
    1. Guse A. H., Lee H. C. (2008) Sci. Signal. 1, re10. - PubMed

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