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Presenilin-2 dampens intracellular Ca2+ stores by increasing Ca2+ leakage and reducing Ca2+ uptake.

Brunello L, Zampese E, Florean C, Pozzan T, Pizzo P, Fasolato C - J. Cell. Mol. Med. (2009)

Bottom Line: We here examined the mechanism by which wild-type and mutant PS2 affect store Ca(2+) handling.In summary, both physiological and increased levels of wild-type and mutant PS2 reduce the Ca(2+) uptake by intracellular stores.To exert this newly described function, PS2 needs to be in its full-length form, even if it can subsequently be cleaved.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, University of Padova, Padova, Italy.

ABSTRACT
We have previously shown that familial Alzheimer's disease mutants of presenilin-2 (PS2) and, to a lesser extent, of presenilin-1 (PS1) lower the Ca(2+) concentration of intracellular stores. We here examined the mechanism by which wild-type and mutant PS2 affect store Ca(2+) handling. By using HeLa, SH-SY5Y and MEFs as model cells, and recombinant aequorins as Ca(2+) probes, we show evidence that transient expression of either wild-type or mutant PS2 increases the passive Ca(2+) leakage: both ryanodine- and IP(3)-receptors contribute to Ca(2+) exit out of the ER, whereas the ribosome translocon complex is not involved. In SH-SY5Y cells and MEFs, wild-type and mutant PS2 potently reduce the uptake of Ca(2+) inside the stores, an effect that can be counteracted by over-expression of SERCA-2B. On this line, in wild-type MEFs, lowering the endogenous level of PS2 by RNA interference, increases the Ca(2+)-loading capability of intracellular stores. Furthermore, we show that in PS double knockout MEFs, reduction of Ca(2+) stores is mimicked by the expression of PS2-D366A, a loss-of-function mutant, uncleaved because also devoid of presenilinase activity but not by co-expression of the two catalytic active fragments of PS2. In summary, both physiological and increased levels of wild-type and mutant PS2 reduce the Ca(2+) uptake by intracellular stores. To exert this newly described function, PS2 needs to be in its full-length form, even if it can subsequently be cleaved.

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Effect of PS2 variants on passive ER Ca2+ leak. (A) Representative traces of [Ca2+]ER measurements in SH-SY5Y cells transiently cotransfected with the cDNAs coding for ER-Aeq and PS2-T122R (black) or with the void vector as control (grey). After Aeq reconstitution (see Materials and Methods), cells were washed and bathed in a Ca2+-free, EGTA (0.6 mM)-containing medium before exposure to CaCl2 (1 mM). The passive ER Ca2+ leak was estimated by addition, at the plateau, of CPA (20 μM) together with EGTA (1 mM). (B) For quantitative analysis of ER Ca2+ leak, different steady-states of [Ca2+]ER were obtained by the addition of CaCl2 ranging from 0.125 to 1 mM. The single traces were averaged and aligned to CPA addition (grey and black traces for control and PS2-T122R–expressing cells, respectively, mean ± S.E.M., n= 24). (C) The rate of ER Ca2+ loss (-d[Ca2+]/dt) was plotted as a function of the instantaneous [Ca2+]ER estimated from single traces as shown in (B) (black and grey symbols, for PS2-T122R-expressing and control cells, respectively). The S.E.M. was omitted for clarity. (D–F) Experiments with HeLa cells were carried out as described in (A) and analysed as shown in (B) and (C) (black and grey symbols for PS2-expressing and control cells, respectively, n= 6–9).
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fig01: Effect of PS2 variants on passive ER Ca2+ leak. (A) Representative traces of [Ca2+]ER measurements in SH-SY5Y cells transiently cotransfected with the cDNAs coding for ER-Aeq and PS2-T122R (black) or with the void vector as control (grey). After Aeq reconstitution (see Materials and Methods), cells were washed and bathed in a Ca2+-free, EGTA (0.6 mM)-containing medium before exposure to CaCl2 (1 mM). The passive ER Ca2+ leak was estimated by addition, at the plateau, of CPA (20 μM) together with EGTA (1 mM). (B) For quantitative analysis of ER Ca2+ leak, different steady-states of [Ca2+]ER were obtained by the addition of CaCl2 ranging from 0.125 to 1 mM. The single traces were averaged and aligned to CPA addition (grey and black traces for control and PS2-T122R–expressing cells, respectively, mean ± S.E.M., n= 24). (C) The rate of ER Ca2+ loss (-d[Ca2+]/dt) was plotted as a function of the instantaneous [Ca2+]ER estimated from single traces as shown in (B) (black and grey symbols, for PS2-T122R-expressing and control cells, respectively). The S.E.M. was omitted for clarity. (D–F) Experiments with HeLa cells were carried out as described in (A) and analysed as shown in (B) and (C) (black and grey symbols for PS2-expressing and control cells, respectively, n= 6–9).

Mentions: SH-SY5Y cells were cotransfected with the cDNAs coding for a recombinant Aeq targeted to the endoplasmic reticulum (ER-Aeq) and for PS2-T122R, a FAD-linked mutant PS2 whose effect at the Ca2+ store level was originally described in human FAD fibroblasts and HeLa cells [18, 21]; control cells were transfected with ER-Aeq and vector alone (pcDNA3). Twenty-four to 48 hrs after transfection, Ca2+ stores were depleted in a Ca2+-free, EGTA-containing medium to allow ER-Aeq reconstitution (see Materials and Methods) and subsequently the refilling process was continuously monitored upon addition of CaCl2 (1 mM) to the bathing medium. Under these conditions, the [Ca2+]ER increased in a couple of minutes up to a plateau that stabilized at a significant lower level in PS2−T122R–expressing cells, with respect to control cells (Fig. 1A). Table 1 reports the steady-state [Ca2+]ER obtained with this protocol in all the cell types here investigated: HeLa, SH-SY5Y, wt and DKO MEFs. Note that PS2-T122R was maximally effective in SH-SY5Y cells (–53 ± 3%, mean ± S.E.M., n= 29).


Presenilin-2 dampens intracellular Ca2+ stores by increasing Ca2+ leakage and reducing Ca2+ uptake.

Brunello L, Zampese E, Florean C, Pozzan T, Pizzo P, Fasolato C - J. Cell. Mol. Med. (2009)

Effect of PS2 variants on passive ER Ca2+ leak. (A) Representative traces of [Ca2+]ER measurements in SH-SY5Y cells transiently cotransfected with the cDNAs coding for ER-Aeq and PS2-T122R (black) or with the void vector as control (grey). After Aeq reconstitution (see Materials and Methods), cells were washed and bathed in a Ca2+-free, EGTA (0.6 mM)-containing medium before exposure to CaCl2 (1 mM). The passive ER Ca2+ leak was estimated by addition, at the plateau, of CPA (20 μM) together with EGTA (1 mM). (B) For quantitative analysis of ER Ca2+ leak, different steady-states of [Ca2+]ER were obtained by the addition of CaCl2 ranging from 0.125 to 1 mM. The single traces were averaged and aligned to CPA addition (grey and black traces for control and PS2-T122R–expressing cells, respectively, mean ± S.E.M., n= 24). (C) The rate of ER Ca2+ loss (-d[Ca2+]/dt) was plotted as a function of the instantaneous [Ca2+]ER estimated from single traces as shown in (B) (black and grey symbols, for PS2-T122R-expressing and control cells, respectively). The S.E.M. was omitted for clarity. (D–F) Experiments with HeLa cells were carried out as described in (A) and analysed as shown in (B) and (C) (black and grey symbols for PS2-expressing and control cells, respectively, n= 6–9).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4516491&req=5

fig01: Effect of PS2 variants on passive ER Ca2+ leak. (A) Representative traces of [Ca2+]ER measurements in SH-SY5Y cells transiently cotransfected with the cDNAs coding for ER-Aeq and PS2-T122R (black) or with the void vector as control (grey). After Aeq reconstitution (see Materials and Methods), cells were washed and bathed in a Ca2+-free, EGTA (0.6 mM)-containing medium before exposure to CaCl2 (1 mM). The passive ER Ca2+ leak was estimated by addition, at the plateau, of CPA (20 μM) together with EGTA (1 mM). (B) For quantitative analysis of ER Ca2+ leak, different steady-states of [Ca2+]ER were obtained by the addition of CaCl2 ranging from 0.125 to 1 mM. The single traces were averaged and aligned to CPA addition (grey and black traces for control and PS2-T122R–expressing cells, respectively, mean ± S.E.M., n= 24). (C) The rate of ER Ca2+ loss (-d[Ca2+]/dt) was plotted as a function of the instantaneous [Ca2+]ER estimated from single traces as shown in (B) (black and grey symbols, for PS2-T122R-expressing and control cells, respectively). The S.E.M. was omitted for clarity. (D–F) Experiments with HeLa cells were carried out as described in (A) and analysed as shown in (B) and (C) (black and grey symbols for PS2-expressing and control cells, respectively, n= 6–9).
Mentions: SH-SY5Y cells were cotransfected with the cDNAs coding for a recombinant Aeq targeted to the endoplasmic reticulum (ER-Aeq) and for PS2-T122R, a FAD-linked mutant PS2 whose effect at the Ca2+ store level was originally described in human FAD fibroblasts and HeLa cells [18, 21]; control cells were transfected with ER-Aeq and vector alone (pcDNA3). Twenty-four to 48 hrs after transfection, Ca2+ stores were depleted in a Ca2+-free, EGTA-containing medium to allow ER-Aeq reconstitution (see Materials and Methods) and subsequently the refilling process was continuously monitored upon addition of CaCl2 (1 mM) to the bathing medium. Under these conditions, the [Ca2+]ER increased in a couple of minutes up to a plateau that stabilized at a significant lower level in PS2−T122R–expressing cells, with respect to control cells (Fig. 1A). Table 1 reports the steady-state [Ca2+]ER obtained with this protocol in all the cell types here investigated: HeLa, SH-SY5Y, wt and DKO MEFs. Note that PS2-T122R was maximally effective in SH-SY5Y cells (–53 ± 3%, mean ± S.E.M., n= 29).

Bottom Line: We here examined the mechanism by which wild-type and mutant PS2 affect store Ca(2+) handling.In summary, both physiological and increased levels of wild-type and mutant PS2 reduce the Ca(2+) uptake by intracellular stores.To exert this newly described function, PS2 needs to be in its full-length form, even if it can subsequently be cleaved.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, University of Padova, Padova, Italy.

ABSTRACT
We have previously shown that familial Alzheimer's disease mutants of presenilin-2 (PS2) and, to a lesser extent, of presenilin-1 (PS1) lower the Ca(2+) concentration of intracellular stores. We here examined the mechanism by which wild-type and mutant PS2 affect store Ca(2+) handling. By using HeLa, SH-SY5Y and MEFs as model cells, and recombinant aequorins as Ca(2+) probes, we show evidence that transient expression of either wild-type or mutant PS2 increases the passive Ca(2+) leakage: both ryanodine- and IP(3)-receptors contribute to Ca(2+) exit out of the ER, whereas the ribosome translocon complex is not involved. In SH-SY5Y cells and MEFs, wild-type and mutant PS2 potently reduce the uptake of Ca(2+) inside the stores, an effect that can be counteracted by over-expression of SERCA-2B. On this line, in wild-type MEFs, lowering the endogenous level of PS2 by RNA interference, increases the Ca(2+)-loading capability of intracellular stores. Furthermore, we show that in PS double knockout MEFs, reduction of Ca(2+) stores is mimicked by the expression of PS2-D366A, a loss-of-function mutant, uncleaved because also devoid of presenilinase activity but not by co-expression of the two catalytic active fragments of PS2. In summary, both physiological and increased levels of wild-type and mutant PS2 reduce the Ca(2+) uptake by intracellular stores. To exert this newly described function, PS2 needs to be in its full-length form, even if it can subsequently be cleaved.

Show MeSH
Related in: MedlinePlus