SERCA pump activity is physiologically regulated by presenilin and regulates amyloid (cid:2) production

pump activity is regulated by presenilin and regulates amyloid beta production. n the regulated intramembraneous proteolysis of key substrates, mutations in the presenilins alter calcium homeostasis, but the mechanism linking presenilins and calcium regulation is unresolved. At rest, cytosolic Ca 2+ is maintained at low levels by pumping Ca 2+ into stores in the endoplasmic reticulum (ER) via the sarco ER Ca 2+ -ATPase (SERCA) pumps. We show that SERCA activity is diminished in ﬁ broblasts lacking both PS1 and PS2 genes, despite elevated SERCA2b steady-state levels, and we show that presenilins and SERCA physically interact. Enhancing presenilin levels in Xenopus laevis oocytes accelerates clearance of cytosolic Ca 2+ , whereas higher levels of SERCA2b phenocopy PS1 overexpression, accelerating Ca 2+ clearance and exaggerat-ing inositol 1,4,5-trisphosphate – mediated Ca 2+ liberation. The critical role that SERCA2b plays in the pathogenesis of Alzheimer ’ s disease is underscored by our ﬁ ndings that modulating SERCA activity alters amyloid (cid:2) production. Our results point to a physiological role for the presenilins in Ca 2+ signaling via regulation of the SERCA pump.

In support of a role in Ca 2+ homeostasis, overexpression of wild-type PS1 or PS2 in Xenopus laevis oocytes causes enhanced IP 3 -mediated Ca 2+ release, an effect that is exacerbated by mutations in both genes ( Leissring et al., 1999b ). However, it remains unclear whether the exaggerated IP 3 -evoked responses result from modulation of the IP 3 signaling pathway, such as sensitization of IP 3 receptors by presenilins, or as a consequence of overfi lling of ER stores. Recently, the presenilins have been reported to be able to form ER leak channels, and it has been reported that mutations in the presenilins disrupt this function ( Tu et al., 2006 ). However, it is unclear how leak channel formation could account for the numerous reports of wild-type presenilin overexpression increasing IP 3 -mediated calcium release.
Ca 2+ pumps, along with Ca 2+ release channels, are the key components of Ca 2+ regulatory systems in neuronal and nonneuronal cells ( Berridge et al., 2000 ). The sarco ER Ca 2+ -ATPase (SERCA) pumps have the highest affi nity for Ca 2+ removal from the cytosol and, together with plasma membrane Ca 2+ -ATPases and transporters, determine the resting cytosolic Ca 2+ concentration. Three differentially expressed genes encode at least fi ve isoforms of the SERCA pump. SERCA1a and -1b are expressed in skeletal muscle, whereas SERCA2a is expressed in cardiac muscle ( Aubier and Viires, 1998 ). SERCA2b, which has a C-terminal extension, is ubiquitously expressed in smooth muscle tissues and nonmuscle tissues including neurons ( Baba-Aissa et al., 1998 ). SERCA3 has limited expression in various nonmuscle tissues ( Baba-Aissa et al., 1998 ). I n addition to disrupting the regulated intramembraneous proteolysis of key substrates, mutations in the presenilins also alter calcium homeostasis, but the mechanism linking presenilins and calcium regulation is unresolved. At rest, cytosolic Ca 2+ is maintained at low levels by pumping Ca 2+ into stores in the endoplasmic reticulum (ER) via the sarco ER Ca 2+ -ATPase (SERCA) pumps. We show that SERCA activity is diminished in fi broblasts lacking both PS1 and PS2 genes, despite elevated SERCA2b steadystate levels, and we show that presenilins and SERCA physically interact. Enhancing presenilin levels in Xenopus laevis oocytes accelerates clearance of cytosolic Ca 2+ , whereas higher levels of SERCA2b phenocopy PS1 overexpression, accelerating Ca 2+ clearance and exaggerating inositol 1,4,5-trisphosphate -mediated Ca 2+ liberation. The critical role that SERCA2b plays in the pathogenesis of Alzheimer ' s disease is underscored by our fi ndings that modulating SERCA activity alters amyloid ␤ production. Our results point to a physiological role for the presenilins in Ca 2+ signaling via regulation of the SERCA pump.
PSDKO fi broblasts displayed elevated resting cytosolic Ca 2+ levels compared with control cells ( Fig. 1, A and B ). Application of thapsigargin in the bath perfusion promoted a transient rise of cytosolic [Ca 2+ ] signal as a consequence of constitutively active Ca 2+ leakage from the ER, providing a signal proportional to the amount of Ca 2+ sequestered in the ER, although it does not take into account differences in Ca 2+ effl ux across the plasma membrane. PSDKO fi broblasts showed reduced responses to thapsigargin as compared with control fi broblasts ( Fig. 1 B ). These results are consistent with diminished SERCA activity, as it is this ER Ca 2+ pump which helps to maintain low cytosolic resting Ca 2+ level by actively pumping Ca 2+ from the cytosol into the ER stores, thereby regulating the Ca 2+ levels between the two compartments.
To determine the respective contribution of the two presenilins, we next investigated Ca 2+ responses in PS1-defi cient (PS1KO) or PS2-defi cient (PS2KO) fi broblasts. Basal cytosolic Ca 2+ levels were elevated in the PS2KO cells at comparable levels to those observed in the PSDKO fi broblasts. Likewise, the PS2KO cells also showed diminished responses after treatment with thapsigargin. In contrast, the PS1KO cells showed small or no changes in basal Ca 2+ levels but did show diminished thapsigargin-mediated responses ( Fig. 1, A and B ). Therefore, although both PS1 and 2 appear to regulate intracellular Ca 2+ homeostasis, it appears to be PS2 that plays a larger role in maintaining low cytosolic Ca 2+ levels and appropriate fi lling of ER Ca 2+ stores. We next looked at ionomycinevoked calcium release in control and PSDKO cells ( Fig. 1 C ). Ionomycin is an ionophore that causes the release of Ca 2+ from most intracellular stores. Application of 1 μ M ionomycin in the absence of extracellular Ca 2+ evoked smaller rises and areas Given that overfi lled ER Ca 2+ stores are one consequence of most PS1 mutations, we hypothesized that presenilin may regulate SERCA pump activity. In this paper, we used both gain-of-function and loss-of-function genetic approaches to show that presenilins are required for proper functioning of SERCA activity in both mammalian cell lines and X. laevis oocytes. Notably, we fi nd that presenilins physically associate with SERCA, and modulation of SERCA function via genetic or pharmacological means results in altered A ␤ production. Furthermore, SERCA2b knockdown mimics the Ca 2+ dynamics seen in presenilin-null cells. Collectively, these results suggest that presenilins regulate and are necessary for normal functioning of the SERCA2b pump, most likely through a direct protein -protein interaction, and that SERCA activity itself impacts A ␤ generation.

Elevated cytosolic Ca 2+ levels and attenuated ER Ca 2+ stores in presenilinnull cells
We previously showed that presenilin mutations lead to enhanced fi lling of ER Ca 2+ stores ( Leissring et al., 1999a , b ). To further explore the role of endogenous presenilin in intracellular Ca 2+ signaling, we investigated ER Ca 2+ stores in immortalized mouse embryonic fi broblast (MEF) cells from presenilin double-knockout (PSDKO) mice ( Herreman et al., 1999 ). Cytosolic Ca 2+ signals were recorded in Fura-2-AMloaded cells before and during stimulation with 1 μ M thapsigargin, a potent irreversible inhibitor of the SERCA pump ( Lytton et al., 1991 ).  The observed phenotype of elevated basal cytosolic Ca 2+ and reduced ER Ca 2+ store content in presenilin-null cells is consistent with either reduced levels of SERCA2b or a reduction in functional activity of SERCA pumps ( Zhang et al., 2006 ). Surprisingly, we found that SERCA2b steady-state levels, the isoform of SERCA found in brain neurons, were elevated, rather than diminished, in the PSDKO fi broblasts ( Fig. 2, C and E ), suggesting that SERCA activity was impaired in the absence of presenilins and that the cells attempt to compensate by increasing SERCA expression. The presence of either PS1 or 2 is suffi cient to prevent this increase in SERCA2b steady-state levels as shown in PS1KO and PS2KO cell lines ( Fig. 2,D and E ). Given our hypothesis that SERCA activity was impaired in the absence of presenilin, we attempted to mimic the Ca 2+ phenotype seen in PSDKO cells via siRNA knockdown of SERCA2b in CHO cells. Knockdown of SERCA2b levels resulted in elevated basal Ca 2+ levels, and a marked decrease in thapsigargin evoked Ca 2+ release ( Fig. 2 F ). under the curve (9,425 vs. 7,394 fl uorescent units) from the PSDKO cells compared with the controls.
The presenilins are an integral part of the ␥ -secretase complex that is responsible for cleavage of the C99 fragment of APP to form A ␤ and the APP intracellular domain (AICD). Cells lacking presenilins do not have ␥ -secretase activity and consequently produce no A ␤ or AICD ( Zhang et al., 2000 ;Hass and Yankner, 2005 ). Because the absence of the AICD region of APP has been shown to cause defi cits in Ca 2+ release from ER stores ( Leissring et al., 2002 ), we sought to determine whether the Ca 2+ dyshomeostasis in PSDKO cells could be attributed to lack of AICD production rather than to the absence of presenilins themselves. For this purpose, PSDKO fibroblasts were transfected with the AICD (C57) fragment and compared with control pseudo-cDNA (pcDNA) -transfected PSDKO fi broblasts. However, even after 48 h of incubation to allow expression of AICD ( Fig. 1 D ), control and AICDexpressing PSDKO fi broblasts showed no signifi cant differences in basal or thapsigargin-evoked Ca 2+ levels. Given that overexpression of AICD into PSDKO cells did not recover either basal or thapsigargin-evoked Ca 2+ levels, we then overexpressed PS1, PS2, or both together in PSDKO cells. Overexpression of either Basal cytosolic Ca 2+ levels were reduced ( ‫ف‬ 20 nM) in the presenilin-transfected fi broblasts compared with controls, whereas the peak Ca 2+ signals after application of thapsigargin were signifi cantly increased in presenilin-transfected fi broblasts. (B) Mean values of basal cytosolic Ca 2+ and thapsigargin-evoked Ca 2+ signals, derived from the experiments in A. *, signifi cance in peak rise versus pcDNA (P < 0.05); #, significance in basal levels versus pcDNA (P < 0.05). (C) Steady-state levels of SERCA2b protein are higher in PSDKO fi broblasts compared with controls based on Western blotting. The PSDKO fi broblasts also displayed C83 -C99 APP fragments that were not present in the controls because of the lack of ␥ -secretase activity. ␤ -Actin levels are shown as a loading control. (D) Steady-state levels of SERCA2b in PS1KO and PS2KO fi broblasts compared with controls based on Western blotting. (E) Densitometric analysis of the SERCA2b levels in C and D, normalized to ␤ -actin, showing elevation of SERCA2b levels in the PSDKO fi broblasts. *, signifi cance in peak rise versus pcDNA (P < 0.05; n = 3). (F) Genetic down-regulation of SERCA2b expression lowers ER Ca 2+ stores and elevates basal Ca 2+ levels. siRNA-mediated down-regulation of SERCA2b leads to lower ER Ca 2+ stores (*, P < 0.001; n = 105 cells) when compared with cells treated with a control (scrambled) siRNA. Error bars show SEM.
expression system to monitor the clearance of Ca 2+ ions from the cytosol after a transient infl ux across the plasma membrane. For this purpose, oocytes were induced to express the Ca 2+ -permeable nicotinic acetylcholine receptor (nAChR) that served as a " Ca 2+ switch, " allowing precisely controlled cytosolic Ca 2+ transients to be evoked by pulsing the membrane potential to strongly negative voltages to increase the electrochemical driving force for Ca 2+ entry. Oocytes were loaded with the Ca 2+ -sensitive dye Oregon Green BAPTA 1 and were voltage-clamped at a holding potential of 0 mV to minimize Ca 2+ infl ux. In the presence of 100 -500 nM acetylcholine, a brief (300 ms) hyperpolarizing pulse to Ϫ 150 mV produced a transient Ca 2+ fl uorescence signal because of Ca 2+ This phenotype was remarkably similar to that seen in the PS-DKO cell line, giving further credence to presenilin regulation of SERCA function. It should be noted that it is unlikely that changes in SERCA function directly modulate basal cytosolic Ca 2+ levels but that they probably do so through changes in other Ca 2+ infl ux pathways such as store-operated Ca 2+ entry.

PS1 and PS2 mimic SERCA2b-accelerated cytosolic Ca 2+ clearance
Based on these data from presenilin-null cell lines, we moved to a more regulatable system to directly establish whether presenilins modulate SERCA pump activity. We used the X. laevis oocyte Ca 2+ infl ux across the plasma membrane, with PS2 having the most robust effect ( Fig. 3 ). To prove that overexpression of presenilin was accelerating Ca 2+ clearance from the cytosol by increasing SERCA activity, we measured Ca 2+ clearance in the presence of thapsigargin, a specifi c inhibitor of SERCA. If our hypothesis was true, then presenilin should no longer accelerate clearance of Ca 2+ from the cytosol in the presence of thapsigargin, compared with control oocytes also in the presence of thapsigargin. We incubated control and PS2-expressing oocytes in 30 μ M thapsigargin in the bathing solution for 30 min. After a 30-min incubation, we applied a 300-ms hyperpolarization pulse, as before, to allow a controlled infl ux of Ca 2+ across the plasma membrane into the cytosol and then tracked the clearance of this Ca 2+ into the intracellular stores. In both control and PS2-expressing oocytes, thapsigargin reduced the speed of the Ca 2+ fl uorescence decay, with PS2-expressing oocytes showing the strongest effect, which is consistent with impairment of PS2 modulation of SERCA activity ( Fig. 4, A  and B ). Double-exponential curve fi tting revealed that both control and PS2-expressing oocytes had similar 1 and 2 values, despite PS2-overexpressing oocytes having markedly faster 2 values compared with control oocytes in the absence of thapsigargin ( Fig. 4 C ).

PS1 familial AD (FAD) mutation M146V accelerates cytosolic Ca 2+ sequestration
Mutations in the presenilins have been associated with enhanced IP 3 -mediated Ca 2+ release from oocytes ( Leissring et al., 1999a , b ) and a variety of mammalian cell types ( Smith et al., 2002 ). We have showed that wild-type presenilins accelerate Ca 2+ sequestration from the cytosol in a thapsigargin-sensitive pathway and that overexpression of SERCA phenocopies this. We expressed the presenilin FAD mutant PS1M146V in X. laevis infl ux through open nicotinic channels. The decay rate of the fl uorescence signals after termination of the voltage pulse was then used to quantify the rate of Ca 2+ sequestration from the cytosol. For video footage of Ca 2+ entry and subsequent clearance from the cytosol in this system please see Video 1 (available at http://www.jcb.org/cgi/content/full/jcb.200706171/DC1). Fig. 3 A shows mean traces of Oregon Green fl uorescence, illustrating differences in the decay rate of the Ca 2+ signal in control, PS1-, PS2-and SERCA2b-expressing oocytes. The decay presumably refl ects a summation of several factors (e.g., diffusion of Ca 2+ ions into the interior of the oocyte, mitochondrial uptake, and extrusion across the plasma membrane) in addition to sequestration into the ER by SERCA pumps. Consistent with this, decay kinetics were best fi t by doubleexponential processes ( Fig. 2, B -D ), with time constants of a few hundred milliseconds and a few seconds ( Fig. 2 E ). Sequestration by SERCA pumps is expected to be refl ected primarily in the slower component ( 2) and, in agreement with this interpretation, 2 was accelerated almost twofold in SERCA2boverexpressing oocytes as compared with control cells.
The acceleration of Ca 2+ sequestration was even greater in oocytes expressing PS2 than in those overexpressing SERCA2b, and a small acceleration was also evident with overexpression of PS1 ( Fig. 3 E ). These results confi rm our fi nding that PS2 plays a more important role in regulating ER store Ca 2+ refi lling than does PS1 ( Fig. 1 ), and they point to regulatory roles in both mammalian cells and X. laevis oocytes.

Pharmacological inhibition of SERCA prevents presenilin-mediated acceleration of cytosolic Ca 2+ sequestration
Overexpression of either PS1, 2, or SERCA resulted in an accelerated sequestration of the cytosolic Ca 2+ after a controlled Although both PS1 and PS2 have been localized to ER in several cell types ( Walter et al., 1996 ) and SERCA is an ER Ca 2+ pump, it is not known if they colocalize within specifi c regions of the ER. To address this question, we used confocal dual-label imaging of wild-type fi broblasts using a polyclonal antiserum against SERCA2b and a polyclonal antiserum against either PS1 or PS2 and found that SERCA2b and PS2 do colocalize, whereas colocalization between SERCA2b and PS1 was present but not as prevalent ( Fig. 7 A ).
To biochemically determine whether presenilins physically associate with SERCA2b, we conducted immunoprecipitation experiments. Fibroblast cell lysates were immunoprecipitated with either a PS1-or PS2-specifi c antibody or an irrelevant control antibody (anti-p35). The resultant pellets were fractionated on a 4 -12% Bis/Tris gel and immunoblotted for SERCA2b. Both PS1 and PS2 were found to specifi cally bind to SERCA2b, whereas SERCA2b did not coprecipitate with the control antibody ( Fig. 7 B ). Conversely, immunoprecipitating with an anti-SERCA2b antibody followed by immunoblotting for PS1 or PS2 was not feasible because the weights of the IgG heavy and light chains ( ‫ف‬ 55 and 25 kD) are the same as of presenilin holoprotein ( ‫ف‬ 55 kD) or carboxy fragment ( ‫ف‬ 22 kD), and although using different species of polyclonal antibodies revealed bands at the correct weights, we could not absolutely determine whether they were presenilin or cross-reactivity with intraspecies IgG chains.

SERCA activity regulates A ␤ production
Our data indicate that presenilins are required for normal SERCA function and physiological maintenance of cellular calcium homeostasis. Moreover, given that presenilins are integral for the production of A ␤ , we investigated whether SERCA function infl uenced A ␤ production. To address this issue, we used pharmacological and gain-of-function and loss-of-function genetic approaches. First, we considered the consequences of overexpressing SERCA2b in CHO cells stably expressing APP. After 48 h, we found that higher SERCA2b levels caused a marked increase in A ␤ 40 production ( Fig. 8 A ). Conversely, reducing SERCA2b via siRNA-mediated knockdown caused a signifi cant decrease in both A ␤ 40 and A ␤ 42 levels ( Fig. 8 B ). Pharmacological inhibition of SERCA2b with thapsigargin rapidly reduced A ␤ 40 and A ␤ 42 production ( Fig. 8, C and D ), which is in agreement with SERCA2b knockdown. The sum of these fi ndings indicates that SERCA pump activity impacts A ␤ production.

Discussion
Cellular Ca 2+ dyshomeostasis has been consistently observed in numerous experimental systems harboring presenilin mutations, including X. laevis oocytes, transfected cell lines, genetically altered mice, and human fi broblasts from FAD patients ( LaFerla, 2002 ). These pathological disruptions in Ca 2+ signaling suggest that the presenilins may play a physiological role in cellular Ca 2+ regulation, although this has not been fi rmly established or characterized. In this paper, we examined the role of wild-type presenilins oocyte and recorded cytosolic Ca 2+ decay after a 300-ms Ca 2+ infl ux through the nAChR as before. The Ca 2+ decay was substantially accelerated in oocytes expressing PS1M146V compared with control ( Fig. 5 A ). Double-exponential curve fi tting revealed substantial acceleration in both 1 and 2 components with PS1M146V over control ( Fig. 5 B ). Rates of decay were faster than wild-type PS1 expression alone ( 1, 0.373 vs. 0.276; 2, 1.919 vs. 1.114), suggesting that this mutation impacts Ca 2+ cytosolic sequestration more effectively than wildtype PS1 protein.

SERCA2b overexpression increases IP 3 -mediated calcium release
We previously showed that overexpression of either PS1 or PS2 leads to an increase in IP 3 -mediated Ca 2+ release in X. laevis oocytes ( Leissring et al., 1999a , b ), but it was unclear whether this arose through increased activation or conductance of IP 3 receptor/channels or from enhanced store fi lling. To discriminate between these possibilities, we examined whether enhanced SERCA pump activity could replicate the increased IP 3 response. We monitored Ca 2+ liberation by linescan confocal microscopy in response to photorelease of IP 3 from a caged precursor evoked by UV fl ashes of varying duration as previously described ( Leissring et al., 1999a , b ). Similar to the effects of presenilin overexpression, mean Ca 2+ responses evoked by high IP 3 were enhanced by ‫ف‬ 50% ( Fig. 6 D ), and the decay of IP 3 -evoked Ca 2+ transients was accelerated ( Figs. 6, B and C ). These fi ndings show that an increase in SERCA pump activity mimics the phenotype previously seen with presenilin overexpression. Ca 2+ leak channels in the ER ( Tu et al., 2006 ) and are an integral part of intracellular Ca 2+ homeostasis via passive leak from the ER stores. The prospect of presenilins forming ER leak channels is, however, at odds with the observation that overexpression of presenilins leads to enhanced IP 3 -mediated Ca 2+ release ( Leissring et al., 1999a , b ;Smith et al., 2002 ). Wild-type presenilin should increase passive leak from the ER stores, resulting in diminished IP 3 -mediated Ca 2+ release, whereas the opposite actually occurs, with FAD-linked presenilin mutations augmenting IP 3 -mediated Ca 2+ release even further ( Leissring et al., 1999a , b ). This paradox suggests that presenilins also have an impact on Ca 2+ homeostasis through other means, which should be suffi cient to explain how wild-type presenilin overexpression enhances IP 3mediated responses, whereas presenilin knockout cells have reduced IP 3 -mediated responses ( Leissring et al., 2002 ). Presenilin regulating SERCA function is consistent with both of these observations, as we have shown in both mammalian cell lines and X. laevis oocyte expression systems, with overexpression of SERCA resulting in enhanced IP 3 -mediated Ca 2+ release mimicking the phenotype seen with overexpression of presenilin ( Fig. 5 ). Combining this with our data, it appears that presenilins contribute to both the active fi lling of the ER stores and the passive emptying and helps explain why mutations in these proteins have such widespread effects on global Ca 2+ signaling.
on Ca 2+ regulation in a mammalian cell system by using genetic knockout models and also in X. laevis oocytes through protein overexpression. Our results indicate that the presenilins regulate the activity of the SERCA pump and are necessary for its proper functioning. Indeed, siRNA knockdown of SERCA leads to a phenotype resembling presenilin-null cells ( Fig. 1 A vs. Fig. 2 F ), including elevated basal Ca 2+ levels (also seen in Zhang et al. [2006] ) and diminished thapsigargin-mediated Ca 2+ release. Altered cytosolic Ca 2+ levels probably arise because of changes in other Ca 2+ infl ux pathways as a result of SERCA down-regulation, as it is unlikely that SERCA directly controls basal cytosolic Ca 2+ . However, both of these phenotypes in presenilin-null cells are rescued by overexpression of presenilin ( Fig. 2 A ). Although interpretation of our functional data in cell lines is dependent on Ca 2+ rises evoked by thapsigargin, which has pitfalls associated with it, the data obtained in oocytes confi rm presenilin as a positive modulator of SERCA function. Overexpression of presenilins accelerates the clearance of a known quantity of Ca 2+ from the cytosol with similar dynamics to SERCA overexpression. Crucially, accelerated Ca 2+ clearance with presenilin overexpression is prevented by pharmacological inhibition of SERCA, providing direct functional evidence for an interaction between presenilin and SERCA. Supporting a physiological role for the presenilins, a recent study suggested that they can form In each panel, distance along the scan line is depicted vertically, time runs from left to right, and increasing fl uorescence ratio (increasing Ca 2+ ) corresponds to increasingly " warm " colors. Note the enhanced response in the oocyte overexpressing SERCA2b. (B and C) B and C show, respectively, Ca 2+ -dependent fl uorescence signals evoked by photoreleased IP 3 in a control oocyte and an oocyte expressing SERCA2b. In each panel, superimposed traces show responses (fl uorescence ratio change averaged across the scan line) evoked by UV light fl ash durations in 10-ms increments between 10 and 110 ms. (D) Mean peak Ca 2+ concentration plotted as a function of fl ash duration (linearly proportional to IP 3 ) in control oocytes ( n = 6) and in oocytes expressing SERCA2b ( n = 5) from at least six recordings from multiple oocytes. Error bars show SEM.
per, we demonstrate through both pharmacological and genetic means that modulation of SERCA2b function results in altered A ␤ production with decreased SERCA function leading to decreased A ␤ and increased SERCA function leading to increased A ␤ . Therefore, it seems possible that SERCA activity plays a modulatory role in ␥ -secretase function or at least in APP processing leading to further generation of the A ␤ peptide. Curiously, it has been reported that either stimulation ( Pierrot et al., 2004 ) or inhibition ( Yoo et al., 2000 ) of capacitative Ca 2+ entrance (CCE) leads to increased production of A ␤ 42 . Our results show that thapsigargin treatment, which depletes ER stores, diminishes As both presenilin and SERCA have activities that can be modulated by several proteins as well as by one another, this presents an interesting question: if presenilins regulate SERCA function, then can SERCA also regulate ␥ -secretase activity? Several studies have illustrated how modulating Ca 2+ infl ux can modulate production of the A ␤ peptide ( Querfurth and Selkoe, 1994 ;Pierrot et al., 2004 ), as well as increasing intracellular store release ( Querfurth et al., 1997 ). Indeed, any regimen that affects intracellular Ca 2+ will invariably alter the activity of SERCA, as it is itself modulated by cytosolic and ER Ca 2+ concentration as models have shown ( Yano et al., 2004 ). In this pa- showing dual staining for PS1 or PS2 (red) together with SERCA2b (green). Merged images with sites of colocalization in yellow are shown on the right. (B) Western blots show coimmunoprecipitation of SERCA2b with PS1 and PS2 in fi broblast cell lysates. Polyclonal rabbit anti-PS1 or -PS2 antibodies were used to immunoprecipitate SERCA2b, which was then detected using anti-SERCA2b antibody. No SERCA2b immunoprecipitation was apparent with a control (rabbit polyclonal anti-p35) antibody. Bar, 10 μ m. (B) Genetic down-regulation of SERCA2b expression lowers A ␤ levels. siRNA-mediated down-regulation of SERCA2b leads to lower steady-state A ␤ levels when compared with cells treated with a control (scrambled) siRNA (*, P < 0.05; n = 6). (C and D) Pharmacological inhibition of SERCA activity lowers steady-state A ␤ levels. Inhibition of SERCA2b with 1 μ M thapsigargin in the presence of extracellular Ca 2+ decreases A ␤ 40 levels within 4.5 h ( n = 4) and also decreases A ␤ 42 levels within 11.5 h ( n = 4). 4.5 h: *, P < 0.005 for thapsigargin and control; *, P < 0.05 for thapsigargin and DMSO. 11.5 h: *, P < 0.005 for thapsigargin and control; *, P < 0.05 for thapsigargin and DMSO. Error bars show SEM. partial sequence. The cells were then incubated in 5% CO 2 atmosphere at 37 ° C for 12 h. Subsequently, cells were rescued with DME containing 10% FBS for an additional 12 h in 5% CO 2 atmosphere at 37 ° C. Thereafter, cells were given the same siRNA treatment for an additional 12 h and rescued for the next 12 h with DME containing 10% FBS in 5% CO 2 atmosphere at 37 ° C. Cells were then lysed and the lysates were stored at Ϫ 80 ° C for further immunoblotting analysis. SERCA2b siRNA was acquired as a smartpool containing the following partial sequences: GUC-AAUGUCGGUUU, CAAAGUUCCUGCUG, GAUCAUGUCUGUCA, and GAUAUAAGGUUAAC.
Oocyte calcium imaging and photolysis of caged IP 3 Plasmids containing cDNA clones encoding for human PS1, PS2, SER-CA2b, and the mouse muscle ␣ , ␤ , ␥ , and ␦ nAChR subunits (S.F. Heinemann, Salk Institute, La Jolla, CA) were linearized and transcribed in vitro with SP6 or T3 RNA polymerases as previously described ( Leissring et al. 1999b ). The RNA transcripts were extracted with phenol-chloroform, precipitated with ethanol, and suspended in RNase-free water at a concentration of 1 μ g/ μ l.
For experiments imaging the clearance of Ca 2+ after plasma membrane infl ux ( Fig. 3 ), oocytes were voltage clamped using a conventional two-microelectrodes technique. The membrane potential was held at 0 mV during superfusion with ACh (100 -500 nM) in Ringer ' s solution and was briefl y (300 ms) stepped to Ϫ 150 mV to strongly increase the electrical driving force for Ca 2+ infl ux. Oocytes were imaged at room temperature by wide-fi eld fl uorescence microscopy using an inverted microscope (IX 71; Olympus) equipped with a 60 × oil-immersion objective, a 488-nm argonion laser for fl uorescence excitation, and a charge-coupled device camera (Cascade 128+; Roper Scientifi c) for imaging fl uorescence emission (510 -600 nm) at frame rates of up to 500 s −1 . Fluorescence signals were monitored from a 40 × 40-μ m region within the animal hemisphere of the oocyte and are expressed as a ratio ( ⌬ F/F o ) of the mean change in fl uorescence ( ⌬ F) relative to the resting fl uorescence before stimulation (F o ) using Meta-Morph software (MDS Analytical Technologies). Mean values of F o were obtained by averaging over several frame before stimulation.
Experiments measuring Ca 2+ liberation in response to photolysis of caged IP 3 ( Fig. 4 ) were performed using a linescan confocal microscope (IX-70 inverted microscope with a 40 × oil-immersion fl uor objective lens, using a 488-nm beam from a 100-mW argon laser [ Callamaras and Parker, 1999 ]) to image fl uorescence signals evoked by fl ashes of UV light (340 -400 nm) from a mercury arc lamp, illuminating a disc of 100-μ m diameter surrounding the 50-μ m scan line in the animal hemisphere of the oocyte.

Protein immunoblotting
Protein extracts were prepared from cells using M-per (Thermo Fisher Scientifi c) extraction buffer and Complete Mini Protease Inhibitor Tablets (Roche). Protein concentrations were determined by the Bradford method. Equal amounts of protein (10 μ g) were separated by SDS/PAGE on a 4 -12% Bis/Tris gel (Invitrogen), transferred to PDVF membranes, blocked for 1 h in 5% vol/vol nonfat milk in Tris-buffered saline, pH 7.5, supplemented with 0.2% Tween 20, and incubated overnight at 4 ° C with primary antibody. Antibodies and dilutions used in this study include ␣ -SERCA2b (1:20,000; F. Wuytack, Katholieke Universiteit Leuven, Leuven, Netherlands), CTF20 (1:5,000; EMD), and ␣ -Actin (1:10,000; Sigma-Aldrich). Membranes were washed fi ve times and then incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Quantitative densitometric analyses were performed on digitized images of immunoblots with Scion Image 4.0 (Scion Corporation).
A ␤ ELISA MaxiSorp immunoplates (Thermo Fisher Scientifi c) were coated with BAN50 at a concentration of 5 μ g/ml in 0.1 M NaCO3 buffer, pH 9.6, and blocked with 1% Block Ace (Snow Brand Milk Products, Ltd.). Synthetic A ␤ standards, internal controls, and samples were run at least in duplicate. After overnight incubation at 4 ° C, wells were probed with either HRPconjugated BA27 (for A ␤ 1-40) or BC05 (for A ␤ 1-42) for 2 -3 h at 37 ° C. 3,3 Ј ,5,5 Ј -tetramethylbenzidine was used as the chromogen, and the reaction A ␤ production, yet depletion of ER stores leads to increased Ca 2+ infl ux via CCE. This suggests that depleting ER stores of Ca 2+ has a stronger effect on preventing A ␤ production than CCE activation has on inhibiting it.
Numerous proteins, such as calsenilin, ryanodine receptor, and calmyrin (for review see Chen and Schubert, 2002 ), have been shown to interact with and bind the presenilins as well as the known components of the ␥ -secretase complex (nicastrin, PEN2, and Aph1), making the presenilins part of a very large multiprotein complex with multiple functions. Our results suggest that the interaction between SERCA and presenilin is an additional, but very important, interaction that serves to regulate sequestration of calcium into the ER stores, making presenilin a key component of cellular calcium homeostasis. A puzzling aspect is how mutations in the presenilins that modulate ␥ -secretase activity could cause the increases in intracellular Ca 2+ signaling reported ( Leissring et al., 1999a , b ). Conversely, inhibitors of ␥ -secretase, which bind to the active site of presenilin in the ␥ -secretase complex, completely diminish ER Ca 2+ release ( Leissring et al., 2002 ;Kasri et al., 2006 ). Mutations in presenilin have been reported to disrupt the formation of ER leak channels, thereby preventing passive Ca 2+ leak, which then leads to store overfi lling ( Tu et al., 2006 ) and, hence, exaggerated IP 3 -mediated release. Whether these same mutations also affect presenilins ' modulation of SERCA function remains to be seen, but many of these FAD-linked mutations are associated with an increase, or at least change, in ␥ -secretase activity, making the prospect plausible. Indeed, we have shown here that the FAD-linked PS1 mutation M146V does show enhanced clearance of cytosolic Ca 2+ compared with wild-type PS1. The requirement of SERCA activity to have presenilins present may provide problems for drugs that inhibit the actions of the ␥ -secretase complex to therapeutically reduced A ␤ levels in AD. Such drugs have already been shown to abolish ER Ca 2+ release in cell cultures ( Leissring et al., 2002 ;Kasri et al., 2006 ) but their chronic in vivo use has yet to be documented. Certainly, reducing or abolishing ER Ca 2+ release in neurons will have massive implications on memory and behavior, which suggests that BACE may be a more prudent drug target to reduce A ␤ in AD.

Materials and methods
Cell culture and calcium imaging MEFs (Bart de Strooper, Katholieke Universiteit Leuven, Leuven, Netherlands) and CHO cells were maintained with DME and 10% FBS. Calcium imaging was performed as previously described ( Leissring et al., 2002 ). In brief, measurements of intracellular calcium were obtained using the InCyt Im2 Ration imaging system (Intracellular Imaging, Inc.) using excitation at 340 and 380 nm. cDNA transfection was achieved using the Nucleofection technique (Amaxa) as per the manufacturer ' s instructions. 5 μ g cDNA was used alongside 1 μ g GFP cDNA to assess transfection effi ciency and for selecting cells that had been successfully transfected for Ca 2+ imaging. Typically, 70% transfection effi ciency was seen.

RNA interference
A 20:1 stock solution of opti-MEM (Invitrogen) to Mirus TransIT-LT1 transfection reagent (Mirus Bio) was incubated at room temperature for 20 min. siRNAs were added and the solution was incubated for an additional 20 min at room temperature. This mixture was then added to cells, already in opti-MEM, to a fi nal siRNA concentration of 25 nM of each