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. 2007 Nov 13;104(46):18091-6.
doi: 10.1073/pnas.0708959104. Epub 2007 Nov 6.

Targeted polyphosphatase expression alters mitochondrial metabolism and inhibits calcium-dependent cell death

Andrey Y Abramov  1 , Cresson Fraley, Catherine T Diao, Robert Winkfein, Michael A Colicos, Michael R Duchen, Robert J French, Evgeny Pavlov
Affiliations

Targeted polyphosphatase expression alters mitochondrial metabolism and inhibits calcium-dependent cell death

Andrey Y Abramov et al. Proc Natl Acad Sci U S A. .

Abstract

Polyphosphate (polyP) consists of tens to hundreds of phosphates, linked by ATP-like high-energy bonds. Although polyP is present in mammalian mitochondria, its physiological roles there are obscure. Here, we examine the involvement of polyP in mitochondrial energy metabolism and ion transport. We constructed a vector to express a mitochondrially targeted polyphosphatase, along with a GFP fluorescent tag. Specific reduction of mitochondrial polyP, by polyphosphatase expression, significantly modulates mitochondrial bioenergetics, as indicated by the reduction of inner membrane potential and increased NADH levels. Furthermore, reduction of polyP levels increases mitochondrial capacity to accumulate calcium and reduces the likelihood of the calcium-induced mitochondrial permeability transition, a central event in many types of necrotic cell death. This confers protection against cell death, including that induced by beta-amyloid peptide, a pathogenic agent in Alzheimer's disease. These results demonstrate a crucial role played by polyP in mitochondrial function of mammalian cells.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Mitochondrial localization and functional activity of the MTS-GFP-PPX polyphosphatase construct (MGP). (a) Confocal images showing green signal from MGP (Upper Left), red signal from TMRM located in mitochondria (Upper Right), superposed MGP and TMRM images (Lower Left), and intensity profiles for MGP and TMRM (Lower Right), which were collected along the line of the red arrow (Lower Left). (b and c) Thin-layer chromatograms of mitochondrial lysates, from nontransfected controls and transfected cells, incubated in the presence of poly 32P. Activity is indicated by the conversion of polyP to Pi during the course of the incubation (uppermost spots in each lane). (b) Lack of endogenous PPX activity in lysates from nontransfected cells. Chromatograms showed no detectable hydrolysis of polyP until the addition of purified wild-type PPX after 45 min of incubation (last lane). (c) Polyphosphatase (PPX) activity of the MGP construct.
Fig. 2.
Fig. 2.
Mitochondrially targeted expression of polyphosphatase (MGP) reduces levels of polyP (mouse C2C12 cells; similar results were obtained with HepG2 cells). (a) In each set of four images, three separate color channels plus their superposition are shown: green, GFP fluorescence indicating MGP expression (I); red, TMRM signal reflecting inner membrane potential (II); blue, DAPI-polyP signal (III); and superposition of the three individual channels (IV). Data are shown from an MGP-expressing cell (Left) and a nonexpressing control (Right). Below the images are the intensity profile traces (green, MGP; red, TMRM; blue, DAPI) showing fluorescence intensity along the red arrows in image IV for each cell. Note the reduced DAPI emission associated with MGP expression, reflecting a reduction in polyP. (b) Differing DAPI fluorescence emission spectra recorded from nuclear (solid line) and mitochondrial (dotted line) regions, excitation wavelength, 405 nm. These spectra resemble the distinct emissions previously reported for pure DNA and for polyP, respectively, in test-tube experiments (see Results and SI Methods). (c) Relative intensity of DAPI-polyP fluorescence (integrated, >580 nm), expressed as a percentage of the signal from nontransfected control cells (100%). Cells expressing MGP show a reduction to ≈30%, whereas the signal from cells expressing only MTS-GFP is indistinguishable from nontransfected control cells. **, P < 0.001.
Fig. 3.
Fig. 3.
Influence of polyP on mitochondrial function. Cells with no identifiable green fluorescence were used as controls. (a) Reduction of mitochondrial inner membrane potential associated with reduced polyP levels. HepG2 expressing MGP showed lower TMRM fluorescence than controls. **, p < 0.001. (b) Increase of the basal level of NADH autofluorescence in cells expressing MGP. Values of fluorescence in the presence of FCCP (1 μM), or NaCN (1 mM), were taken as 0% and 100%, respectively.
Fig. 4.
Fig. 4.
Role of polyP in mitochondrial Ca2+ uptake and PT in permeabilized HepG2 cells. Cells were permeabilized by including 20 μM digitonin in the external medium, which also contained 3 mM EGTA. Calcium was added to give the free concentrations indicated. (a) Confocal image of cell loaded with the high-affinity mitochondrial calcium dye XRhod-1, showing cells before (Upper Left) and at 30, 210, and 330 s after permeabilization. The green arrows indicate a cell with MGP overexpression. Cells with no identifiable green fluorescence were used as controls. (b) Time courses of XRhod-1 fluorescence in a group of control and MGP-expressing cells. The controls, but not MGP-expressing cells, show an abrupt calcium release. (c) Fluorescence intensity from mitochondria loaded with Rhod-5n, a low-affinity dye. MGP-expressing cells were able to accumulate calcium until the uncoupler FCCP (1 μM) was added.
Fig. 5.
Fig. 5.
Protection against cell death by MGP. (a) Fluorescent images of GFP-positive HepG2 cells treated with 10 μM ionomycin for 2 h and loaded with PI. Nuclei of dead cells were stained by PI; only GFP-positive cells were counted. (b) Control cells, transfected with MTS-GFP (no PPX expression), were more vulnerable to ionomycin-induced death than cells expressing MTS-GFP-PPX (MGP). (c) βA caused mitochondrial membrane depolarization in astrocytes. (d) MGP-expressing astrocytes were protected from both fast, transient depolarizations and from the profound baseline membrane depolarization occurring in some controls, which precluded additional depolarization after FCCP application. (e and f) Cell death in, respectively, control and MGP-expressing cocultures of hippocampal neurons and astrocytes. Staining and cell counts were performed after 24-h incubation with βA. MGP expression was associated with protection of both neurons and astrocytes from βA-induced cell death. Error bars represent SEM from four independent experiments.
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References

    1. Kulaev I, Vagabov V, Kulakovskaya T. J Biosci Bioeng. 1999;88:111–129. - PubMed
    1. Kornberg A, Rao NN, ult-Riche D. Annu Rev Biochem. 1999;68:89–125. - PubMed
    1. Negoda A, Xian M, Reusch RN. Proc Natl Acad Sci USA. 2007;104:4342–4346. - PMC - PubMed
    1. Kumble KD, Kornberg A. J Biol Chem. 1995;270:5818–5822. - PubMed
    1. Reusch RN. Proc Soc Exp Biol Med. 1989;191:377–381. - PubMed

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