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Figure 1. Elevated cytosolic Ca2+ and reduced ER Ca2+ stores in presenilin-null cell line. (A) Changes in cytosolic Ca2+ evoked by thapsigargin in control fibroblasts (control; n = 48 cells) and fibroblasts from PSDKO (n = 57 cells), PS1KO (n = 48), and PS2KO (n = 56) mice. ER Ca2+ stores were released into the cytosol by application of 1 μM thapsigargin, a specific blocker of SERCA activity, with 0 mM Ca2+ in the bathing solution. Basal cytosolic Ca2+ levels were elevated (â¼70 nM) in the PSDKO and PS2KO fibroblasts compared with controls and PS1KO cells (â¼50 nM; P < 0.05), whereas the peak Ca2+ signals after application of thapsigargin were substantially reduced in PSDKO and PS2KO fibroblasts. (B) Mean values of basal cytosolic [Ca2+] and thapsigargin-evoked Ca2+ signals, derived from the experiments in A. *, significance in peak rise versus pcDNA (P < 0.05); #, significance in basal levels versus pcDNA (P < 0.05). (C) Changes in cytosolic Ca2+ evoked by 1 μM ionomycin in control fibroblasts (control; n = 12 cells) and fibroblasts from PSDKO mice (n = 37 cells), with 0 mM Ca2+ in the bathing solution. (D) Cytosolic Ca2+ signals in PSDKO fibroblasts transfected either with pcDNA (n = 63 cells) or AICD (n = 54 cells) 48 h earlier. No significant differences were apparent in either basal cytosolic Ca2+ levels or the peak response after application of 1 μM thapsigargin. Error bars show SEM.
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Figure 2. Ca2+ deficits in presenilin-null fibroblasts are not caused by reduced SERCA2b expression. (A) Changes in cytosolic Ca2+ evoked by thapsigargin in PSDKO fibroblasts transfected 48 h earlier with pcDNA (n = 25 cells), PS1 (n = 40 cells), PS2 (n = 48 cells), or both PS1 and 2 (n = 47 cells), with 0 mM Ca2+ in the bathing solution. Basal cytosolic Ca2+ levels were reduced (â¼20 nM) in the presenilin-transfected fibroblasts compared with controls, whereas the peak Ca2+ signals after application of thapsigargin were significantly increased in presenilin-transfected fibroblasts. (B) Mean values of basal cytosolic Ca2+ and thapsigargin-evoked Ca2+ signals, derived from the experiments in A. *, significance 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 fibroblasts compared with controls based on Western blotting. The PSDKO fibroblasts also displayed C83âC99 APP fragments that were not present in the controls because of the lack of γ-secretase activity. β-Actin levels are shown as a loading control. (D) Steady-state levels of SERCA2b in PS1KO and PS2KO fibroblasts compared with controls based on Western blotting. (E) Densitometric analysis of the SERCA2b levels in C and D, normalized to β-actin, showing elevation of SERCA2b levels in the PSDKO fibroblasts. *, significance in peak rise versus pcDNA (P < 0.05; n = 3). (F) Genetic down-regulation of SERCA2b expression lowers ER Ca2+ stores and elevates basal Ca2+ levels. siRNA-mediated down-regulation of SERCA2b leads to lower ER Ca2+ stores (*, P < 0.001; n = 105 cells) when compared with cells treated with a control (scrambled) siRNA. Error bars show SEM.
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Figure 3. PS1 and PS2 accelerate the clearance of cytosolic Ca2+ after entry via muscle nAChR channels. (A) Ca2+-dependent fluorescence changes (ÎF/Fo) after 300-ms hyperpolarizing pulses to induce Ca2+ entry through nAChR expressed in the plasma membrane. Traces show mean data from >35 recordings in multiple oocytes injected 3 d earlier with αβδγ nAChR subunits alone (control) or together with PS1, PS2, or SERCA2b cRNA. The inset shows the same data on a semilogarithmic scale. (BâD) Data from, respectively, control, PS2, and SERCA2b oocytes, fitted by biexponential decays (superimposed red traces). (E) Table listing fast (T1) and slow (T2) time constants of double-exponential fits to the Ca2+ decay data.
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Figure 4. Thapsigargin-sensitive cytosolic Ca2+ sequestration is bigger in PS2-expressing oocytes as compared with control oocytes. (A) Traces show Ca2+-dependent fluorescent signals obtained in control and PS2-expressing oocytes in the absence of (control) and during application of 30 μM thapsigargin (Tg) in the bathing solution. (B) Comparison of thapsigargin-sensitive component of cytosolic Ca2+ clearance in control oocytes versus PS2-expressing oocytes. Traces were obtained by direct subtraction of records obtained in the presence of 30 μM thapsigargin minus records obtained in the absence of thapsigargin. (C) Table listing fast (T1) and slow (T2) time constants of double-exponential fits to the Ca2+ decay data.
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Figure 5. PS1 FAD mutation M146V accelerates cytosolic Ca2+ sequestration. (A) Traces show Ca2+-dependent fluorescence changes (ÎF/Fo) after 300-ms hyperpolarizing pulses to induce Ca2+ entry through nAChR expressed in the plasma membrane obtained from control and PS1 M146V expressing oocytes. (B) Table listing fast (T1) and slow (T2) time constants of double-exponential fits to the Ca2+ decay data.
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Figure 6. Overexpression of SERCA2b phenocopies the enhanced IP3-evoked signals and accelerated Ca2+ decay observed with presenilin overexpression. (A) Representative linescan images showing Ca2+ signals evoked by photolysis flashes of 10-, 40-, 70-, and 100-ms durations. In each panel, distance along the scan line is depicted vertically, time runs from left to right, and increasing fluorescence ratio (increasing Ca2+) corresponds to increasingly âwarmâ colors. Note the enhanced response in the oocyte overexpressing SERCA2b. (B and C) B and C show, respectively, Ca2+-dependent fluorescence signals evoked by photoreleased IP3 in a control oocyte and an oocyte expressing SERCA2b. In each panel, superimposed traces show responses (fluorescence ratio change averaged across the scan line) evoked by UV light flash durations in 10-ms increments between 10 and 110 ms. (D) Mean peak Ca2+ concentration plotted as a function of flash duration (linearly proportional to IP3) in control oocytes (n = 6) and in oocytes expressing SERCA2b (n = 5) from at least six recordings from multiple oocytes. Error bars show SEM.
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Figure 7. Colocalization of presenilin and SERCA2b. (A) Confocal images of fibroblasts 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 fibroblast 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 μm.
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Figure 8. SERCA activity regulates Aβ production. (A) SERCA2b overexpression leads to higher Aβ levels. Aβ40 and Aβ42 levels were measured by sandwich ELISA. Note that Aβ40 levels are significantly increased in SERCA2b-transfected cells, compared with control pcDNA vector-treated cells (*, P < 0.05; n = 5). (B) Genetic down-regulation of SERCA2b expression lowers Aβ levels. siRNA-mediated down-regulation of SERCA2b leads to lower steady-state Aβ 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 Aβ levels. Inhibition of SERCA2b with 1 μM thapsigargin in the presence of extracellular Ca2+ decreases Aβ40 levels within 4.5 h (n = 4) and also decreases Aβ42 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.
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