Research ArticleAlzheimer’s Disease

Gain-of-Function Enhancement of IP3 Receptor Modal Gating by Familial Alzheimer’s Disease–Linked Presenilin Mutants in Human Cells and Mouse Neurons

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Science Signaling  23 Mar 2010:
Vol. 3, Issue 114, pp. ra22
DOI: 10.1126/scisignal.2000818


Familial Alzheimer’s disease (FAD) is caused by mutations in amyloid precursor protein or presenilins (PS1 and PS2). Many FAD-linked PS mutations affect intracellular calcium (Ca2+) homeostasis by mechanisms proximal to and independent of amyloid production, although the molecular details are controversial. We found that several FAD-causing PS mutants enhance gating of the inositol trisphosphate receptor (IP3R) Ca2+ release channel by a gain-of-function effect that mirrored the genetics of FAD and was independent of secretase activity. In contrast, wild-type PS or PS mutants that cause frontotemporal dementia had no such effect. FAD-causing PS mutants altered the modes in which the IP3R channel gated. Recordings of endogenous IP3R in lymphoblasts derived from individuals with FAD or cortical neurons of asymptomatic PS1-AD mice revealed that they were more likely than IP3R in cells with wild-type PS to dwell in a high open-probability burst mode, resulting in enhanced Ca2+ signaling. These results indicate that exaggerated Ca2+ signaling through IP3R-PS interaction is a disease-specific and robust proximal mechanism in FAD.


Alzheimer’s disease (AD) is a common form of dementia that involves slowly developing and ultimately fatal neurodegeneration. Most AD is sporadic and idiopathic and develops at ages older than 60, but about 5% is inherited in an autosomal dominant manner because of mutations in amyloid precursor protein (APP) or presenilins (PS1, PS2) (1). Although familial Alzheimer’s disease (FAD) develops at ages as early as the late 30s, both familial and sporadic AD share hallmark features that include accumulation of β-amyloid (Aβ) in extracellular plaques, intracellular neurofibrillary tangles composed largely of hyperphosphorylated tau, and cell atrophy and death in various brain regions (24). The consistent phenotypes suggest that both types of AD may share pathogenic origins. Nevertheless, the mechanisms by which these mutant proteins exert such devastating effects, and their roles and relationships in the two forms of AD, are still not clear. Insights into the molecular mechanisms and cellular functions of mutant proteins in FAD are likely to provide important clues into the etiology of AD pathogenesis and the identification of targets for therapeutic interventions.

Presenilins are transmembrane proteins that are synthesized on the endoplasmic reticulum (ER) and localized there (5). Together with nicastrin, APH-1 (anterior pharynx-defective 1) and PEN-2 (presenilin enhancer 2), PS forms a protein complex that is transported to the cell surface and to endosomes, where it functions as a γ-secretase that cleaves several type 1 transmembrane proteins, including APP (6, 7). γ-Secretase cleavage of APP releases Aβ peptides, a major component of amyloid plaques in the brains of patients with AD. Mutant PSs are believed to affect APP processing by enhancing either the total production of Aβ or the relative proportion of the more amyloidogenic Aβ-42 form (8). In the amyloid hypothesis of AD, accumulation of amyloidogenic Aβ aggregates or oligomers is a proximal feature that causes neural toxicity leading to brain pathology (9, 10). However, FAD mutations in PS cause loss of secretase function, in contrast with the dominant gain of function indicated by the genetics of the disease (11).

In addition to disrupting APP processing, many FAD-linked PS mutations affect intracellular calcium (Ca2+) homeostasis (12, 13). Although extracellular Aβ influences intracellular Ca2+ homeostasis in vitro (14, 15) and in vivo (16, 17), FAD mutant PS also influences intracellular Ca2+ signaling by proximal, Aβ-independent mechanisms. Such Ca2+ signaling disruptions have manifested as attenuated capacitive Ca2+ entry (1820), but most commonly as exaggerated Ca2+ liberation from the ER (18, 2124), the major intracellular Ca2+ storage organelle. The molecular mechanisms underlying exaggerated ER Ca2+ release have been ascribed to enhanced loading of the ER lumen (23), as a result of either enhanced SERCA (sarco-endoplasmic reticulum Ca2+-ATPase) pump activity (25), or to disruption of a putative Ca2+ channel function of wild-type (WT) PS (26, 27). Alternatively, exaggerated Ca2+ release has been accounted for by enhanced Ca2+ liberation from normal stores through inositol trisphosphate receptor (IP3R) (21, 23) or ryanodine receptor (RyR) (22, 28, 29) Ca2+ release channels, both in vivo (22, 24, 28, 29) and in vitro (3033), as a consequence of either enhanced channel abundance (28, 3436) or, in the case of the IP3R, enhanced activity in response to its ligand IP3 (32, 37). Notably, enhanced agonist-induced IP3R-mediated Ca2+ signals have been used diagnostically to identify individuals with FAD (31, 32). Biochemical interactions of the IP3R with both WT and FAD mutant PS1 and PS2 have been demonstrated (37). Single-channel recordings of Sf9 insect cell IP3R demonstrated that recombinant FAD mutant PS1 and a FAD mutant PS2 could enhance IP3R Ca2+ release channel gating (37). These single-channel studies were performed in the absence of Aβ or cellular pathology, suggesting that modulation of IP3R gating is a fundamental mechanism that contributes to exaggerated Ca2+ signaling in FAD PS–expressing cells.

It is not known whether the effects of FAD PS on IP3R gating represent a gain or loss of function. Moreover, although many (>100) PS mutations (especially in PS1) that cause FAD have been identified (38), only two FAD mutant PSs have been examined for their effects on IP3R channel gating (37). In addition, some PS1 mutations result in frontotemporal dementia (FTD), a neurological disorder lacking Aβ accumulation (39, 40). If FAD PS–mediated alteration of IP3R-mediated Ca2+ signaling is proximal in AD pathogenesis, then other FAD mutant PSs might be expected to have similar enhancing effects on IP3R channel gating, whereas those associated with FTD might not. Previous studies of the effects of mutant PS on IP3R investigated endogenous insect (Sf9 ovarian cells) and chicken (DT40 B lymphocytes) IP3Rs (37), whereas AD, in which the pathological consequences are primarily in brain neurons, affects humans. Consequently, the relevance of these data in appropriate cell types with endogenous amounts of PS and IP3R are unclear. Here, we studied IP3R channel kinetics under the influence of several FAD and FTD mutant PSs in four different systems, including transgenic AD mouse neurons, B lymphoblasts derived from human FAD patient cells, and fibroblasts from PS1 and PS2 double-knockout (DKO) cells. All FAD-linked PS mutations enhanced IP3R single-channel gating, leading to exaggerated intracellular Ca2+ signaling, whereas FTD-associated PS1 mutations did not affect IP3R channel kinetics. Furthermore, the effects of FAD PS mutants were gain-of-function effects, consistent with the genetics of FAD. In contrast, the secretase activity of PS was not required. The results indicate that exaggerated Ca2+ signaling through IP3R-PS interaction is a disease-specific and robust proximal mechanism in FAD.


Multiple FAD PS mutations modulate IP3R channel gating by mode switching

To determine whether enhanced IP3R channel activity is a phenotype conserved in FAD PS–expressing cells, we recorded single IP3R channel activities in the presence of one of eight different PS mutants (PS1-L113P, -M146L, -L166P, -G183V, -D257A, -G384A, and -D385A and PS2-N141I) (41). We performed single-channel patch-clamp electrophysiology of the outer membrane of isolated Sf9 cell nuclei (42) 48 hours after infecting cells with recombinant baculovirus (fig. S1). Because enhancement of IP3R activity is more apparent at subsaturating IP3 concentrations (37), we used 100 nM IP3 and 1 μM Ca2+ to suboptimally activate channel gating. We consistently detected IP3R channels with open probability (Po) of 0.27 ± 0.04 in membrane patches from control nuclei infected with EVER1 (an irrelevant ER transmembrane protein) (Fig. 1, A and B). IP3R channels recorded in membrane patches from PS1-WT– or PS2-WT–infected cells had Po similar to those from EVER1-infected control cells (Po = 0.32 ± 0.04 and 0.25 ± 0.03, respectively; P > 0.05; Fig. 1, A and B). In contrast, IP3R channel Po was significantly enhanced by 250% in nuclei from cells infected with mutant PS1-M146L (Po = 0.81 ± 0.02; Fig. 1, A and B) to a degree similar to that achieved with saturating ligand concentrations (37). Increased Po resulted from a marked reduction of channel mean closed time (τc; Fig. 1C). FAD mutant PS2 (N141I) also markedly enhanced IP3R channel activity (Fig. 1, A and B), with Po increased by 200% (0.66 ± 0.05; Fig. 1B), also mainly resulting from a significant reduction of τc (Fig. 1C). Similar results were obtained for two other FAD-causing PS1 mutants: IP3R channel Po was increased 200% with PS1-L166P (Po = 0.63 ± 0.08) or PS1-G384A (Po = 0.61 ± 0.05; Fig. 1, A and B). Thus, all four FAD PS mutants examined had similar effects on IP3R channel activity. The γ-secretase–dead mutants PS1-D257A and PS1-D385A, which have mutations in intramembrane sites involved in PS1 catalytic activity, also significantly enhanced IP3R channel activity, although to a lesser extent than did the FAD mutants (Po = 0.50 ± 0.05 and 0.46 ± 0.08, respectively; Fig. 1, A and B). Thus, the secretase activity of PS is not required for its effects on IP3R gating. Po of channels recorded from cells infected with FTD-associated mutant PS1-L113P and PS1-G183V were 0.28 ± 0.04 and 0.29 ± 0.04, respectively, not different from controls (Fig. 1, A and B). Thus, several FAD mutant PSs have similar effects on IP3R gating, and these effects are not recapitulated in PS mutants associated with a different neurological disease.

Fig. 1

Effect of recombinant PS on IP3R single-channel activity in Sf9 cells. (A) Representative current recordings (+20 mV) in outer membrane patches of Sf9 cell nuclei infected with different recombinant PS baculoviruses. EVER1 served as an ER membrane protein infection control. Pipette solution contained 1 μM free Ca2+ and 100 nM IP3. Arrows indicate closed-channel current level. (B and C) Summary of effects of PS on IP3R channel Po (B), and mean open time τo (open circle) and mean closed time τc (solid circle) (C). (D) Summary of relative prevalence of channel being in a gating mode (πM) of PS on IP3R modal gating. Bars, mean ± SEM. *P < 0.05 by ANOVA compared with EVER1-infected cells.

To gain deeper insight into the mechanisms of IP3R channel activation by FAD mutant PS, we used modal gating analysis. Previous studies demonstrated that ligand (IP3, Ca2+) regulation of IP3R gating is largely mediated by altering the propensity of the channel to gate in particular modes (43). Strongly activated channels gate in a high-Po H mode characterized by long bursting activities; an intermediate-Po I mode is characterized by fast channel openings and closings; and a low-Po L mode is characterized by long closed periods containing brief openings (43). In control nuclei isolated from EVER1-infected cells, the L gating mode was dominant, with the channel spending ~60% of its time in this mode and ~25% in the H mode (Fig. 1D). In nuclei from cells infected with either wild-type or FTD PS, similar modal gating distributions were observed (Fig. 1D). In contrast, the H mode was the dominant gating mode of IP3R recorded from all of the FAD-causing mutant PS–expressing cells (Fig. 1D). Thus, FAD mutant PSs enhance IP3R channel gating by mode switching, causing the channel to spend more time in the H mode at the expense primarily of the L mode (Fig. 1D and fig. S2).

IP3R single-channel gating and IP3-mediated Ca2+ signals are enhanced in human FAD B cells

Enhancement of IP3R channel activity by heterologous expression of mutant PS has been demonstrated in both Sf9 and DT40 cells [(37) and this study], systems that use PS overexpressed in nonhuman cells. To determine the effects of endogenous PS in human cells, we studied IP3R activity in normal and FAD human B cell lymphoblasts. Currents from endogenous human IP3R single channels have never been previously recorded. Thus, we initially characterized endogenous IP3R channels from human B lymphoblasts by nuclear membrane patch-clamp electrophysiology. In the absence of IP3, no channel activity was apparent (n = 18; Fig. 2B), whereas with IP3 (10 μM) in the pipette solution, we observed heparin-sensitive single channels with brief openings and long closings (n =15; Fig. 2, A and B). These channels showed a linear current-voltage (I-V) relationship with slope conductance of ~475 pS (Fig. 2C), typical of mammalian IP3R under these ionic conditions (44). IP3R currents recorded from human B cells were long-lasting (Fig. 2A), with relatively low Po (0.18 ± 0.02, n = 20; Fig. 2D).

Fig. 2

Characterization of endogenous IP3R single channels in human B lymphoblasts. (A) Continuous single IP3R channel current trace (300 s) recorded from outer membrane of nucleus isolated from human B lymphoblasts at +20 mV with 10 μM IP3 and 1 μM free Ca2+ in pipette solution. Arrows indicate closed-channel current levels. (B) Representative current traces (+20 mV) in nuclei isolated from human B lymphoblasts. Channel activity required the presence of IP3 (n = 20) and was inhibited by heparin (n = 15). (C) I-V relationship obtained by ramping holding potential from −60 to +60 mV. (D) Summary of IP3R channel Po, and mean open τo (open circle) and closed τc (solid circle) durations.

We compared IP3R gating in B lymphoblasts derived from three individuals with FAD, harboring PS1-M146L, PS1-A246E, or PS2-N141I (FAD lymphoblasts), with that in B lymphoblasts derived from two different age-matched individuals without FAD or FAD-associated PS mutations (control lymphoblasts CTL1 and CTL2) (Table 1). IP3R in control lymphoblasts derived from the two individuals without FAD had low channel Po (0.18 ± 0.02 and 0.23 ± 0.03, respectively; Fig. 3, A and B), with channel activities characterized by brief openings and relatively long closings (Fig. 3, A and C). IP3R Po recorded from lymphoblasts derived from all three individuals with FAD were increased 200 to 300% relative to those from control lymphoblasts (PS1-M146L: 0.62 ± 0.05; PS1-A246E: 0.67 ± 0.06; PS2-N141I: 0.50 ± 0.04; Fig. 3, A and B), mainly because of a marked decrease in τc (Fig. 3C), with many channels bursting for extended periods (Fig. 3E). In control lymphoblasts, the L and I gating modes dominated channel kinetics, whereas IP3R analyzed in FAD lymphoblasts spent 50 to 75% of the time in the high-Po H mode (Fig. 3, D and E). Analogous results were obtained with low (100 nM) IP3. IP3R Po was 0.04 ± 0.01 in control lymphoblasts derived from an individual without FAD, whereas Po was 0.22 ± 0.05 in PS1-A246E FAD lymphoblasts (Fig. 3, F and G). These observations in human B lymphoblasts with endogenous PS and IP3R are similar to those made in Sf9 and DT40 cells. FAD-linked PS mutations therefore have a robust, common effect to enhance IP3R single-channel activity in insect, avian, and human cells.

Table 1

Human FAD and control B lymphoblast lines.

View this table:
Fig. 3

Effect of FAD PS on IP3R gating in human FAD B lymphoblasts. (A) Representative IP3R currents (+20 mV) in nuclei isolated from human FAD B lymphoblasts and control lymphoblasts from age-matched individuals without FAD activated with 10 μM IP3 and 1 μM Ca2+ in pipette solution. (B to D) Summary of channel Po (B), τo (open circles) and τc (solid circles) (C), and modal gating analysis (D). *P < 0.05, ANOVA compared with CTL1. (E) Modal gating analyses. Each section shows continuous recording with gating mode assignment in color code below. In cells from normal individuals, low Po is associated with switching between the L and I modes. In cells from all three individuals with FAD, enhanced gating is manifested by increased occupancy of the H mode at the expense of the L mode. (F to H) Single IP3R channel current traces from human B cells activated by suboptimal IP3. (F) Representative currents (+20 mV) in isolated nuclei from human FAD lymphoblasts and age-matched control B lymphoblasts activated by suboptimal 100 nM IP3 and 1 μM Ca2+. Summary of IP3R Po (G) and τo (open circles) and τc (solid circles) (H) from aged-matched control and FAD human B lymphoblasts. *P < 0.05 by Student’s t test.

To determine whether these effects observed at the single-channel level are associated with altered intracellular calcium concentration ([Ca2+]i) signaling, we measured IP3R-mediated Ca2+ signals in B lymphoblasts derived from the same individuals with FAD who were used for single-channel studies. IP3R-mediated Ca2+ signals were elicited by cross-linking the B cell receptor (BCR) with immunoglobulin M (IgM) antibody. At high IgM concentration (5 μg/ml), 20% of cells responded with similar Ca2+ oscillations and spiking in both control and PS1-A246E FAD lymphoblasts (Fig. 4, B and D), whereas a further 27% of the FAD lymphoblasts responded with exaggerated high-amplitude transient responses (Fig. 4, A to C). With low-dose anti-IgM stimulation (50 ng/ml), Ca2+ oscillations and spiking were triggered in 19 ± 2% of control cells (Fig. 4, E and G). Perfusion with xestospongin B, a membrane-permeable specific IP3R inhibitor (45), reversibly inhibited these effects, indicating that they were due to periodic Ca2+ release through the IP3R (fig. S3). In FAD lymphoblasts, both the percentage of responding cells and the oscillation and spiking frequency were increased (Fig. 4, E, G, and H). Perfusion with culture medium containing 10% fetal bovine serum (FBS), which generates ongoing low IP3 production (46), induced spontaneous Ca2+ oscillations and spiking in 25 ± 5% of control lymphoblasts (Fig. 4, F and G). In contrast, the percentage of PS1 FAD lymphocytes displaying spontaneous Ca2+ oscillations was increased by 100% and the oscillation and spiking frequency doubled (Fig. 4, F to H). The percentage of spontaneously oscillating PS2-N141I FAD cells was similar to that in control lymphoblasts; however, the oscillation frequency was increased (Fig. 4, F to H). These responses are consistent with an enhanced sensitivity and activity of IP3-mediated Ca2+ release in human FAD lymphoblasts, consistent with the enhanced IP3R channel activity recorded in these cells.

Fig. 4

Exaggerated Ca2+ signaling in human FAD B lymphoblasts. (A) Representative single-cell Ca2+ responses to strong IgM stimulation (5 μg/ml; arrow) in control human B lymphoblasts (CTL) or FAD lymphoblasts carrying the PS1-A246E mutation. Dark lines below and to the left of each trace indicate zero Ca2+. (B) Responses to IgM stimulation. Percentages responding with Ca2+ oscillations (red) or large-amplitude Ca2+ transients (blue). (C) Summary of peak amplitudes of high-amplitude transient Ca2+ responses triggered by antibody against IgM (5 μg/ml). (D) Ca2+ oscillation frequency in response to antibody against IgM. n = 3 experiments with 30 cells in each. *P < 0.05; Student’s t test. (E and F) Representative single-cell Ca2+ responses to weak IgM stimulation (50 ng/ml; arrow) (E) and spontaneous oscillations (F) during perfusion with serum-containing medium in lymphoblasts derived from unaffected (CTL) and FAD individuals. Dark lines: zero Ca2+ level. (G) Percentage of cells responding to IgM (black) or undergoing spontaneous Ca2+ oscillations in complete medium (blue). (H) Summaries of Ca2+ oscillation (osc) frequency in response to IgM (black) or spontaneous Ca2+ oscillations observed in complete medium (blue). Data in each group were summarized from four experiments with 30 cells in each. *,#P < 0.05 by ANOVA compared with respective controls.

IP3R channel gating is enhanced in FAD mouse cortical neurons

Ca2+ signaling disruption has been observed in fibroblast or lymphoblast lines derived from human FAD cells [this study and (30, 32, 47)]. Our results above implicate mutant PS–enhanced IP3R channel gating as the underlying mechanism. To determine whether this molecular mechanism also operates in brain neurons, we isolated cortical neurons from embryonic day 14 to 16 (E14 to E16) wild-type C57BL/6 and 3xTg-AD mice and recorded single IP3R channel activities in nuclear envelopes from isolated nuclei. 3xTg-AD mice contain PS1-M146V knocked into the PS1 locus and exhibit age-dependent amyloid plaques, neurofibrillary tangles, and cognitive decline starting at 3 to 6 months of age (3, 48). In nuclei isolated from control C57BL/6 mice, channel currents were not observed in the absence of IP3 (Fig. 5B). With 10 μM IP3 and 1 μM Ca2+, heparin-sensitive (Fig. 5B) channels with a linear slope conductance of ~375 pS (Fig. 5C) were recorded (Fig. 5, A and B), with gating characterized by short openings (τo = 2.25 ± 0.11 ms) and relatively long closures (τc = 52.7 ± 12.7 ms) with Po = 0.06 ± 0.01 (Fig. 5D). Po was enhanced by 700% (0.43 ± 0.05; Fig. 5, B and D) in nuclei isolated from 3xTg-AD mice. Increased Po was caused by markedly prolonged τo (10.22 ± 1.57 ms) together with shortened τc (14.61 ± 3.04 ms). The I and L modes dominated channel gating in control C57BL/6 neurons, whereas the H mode was the major gating mode in 3xTg-AD neurons (Fig. 5, B, E, and F).

Fig. 5

IP3R single-channel activity in mouse primary embryonic cortical neurons. (A) Continuous single IP3R current trace (200 s) in outer membrane of nucleus isolated from embryonic cortical neuron (+40 mV with 10 μM IP3 and 1 μM free Ca2+ in pipette solution). Arrows indicate closed-channel current level. (B) Representative current traces (+40 mV) in nuclei from C57BL/6 (wild type) or 3xTg-AD mice (E14 to E16). Channel activities in both mouse lines required IP3 and were inhibited by heparin. (C) IP3-activated currents from C57BL/6 (blue) or 3xTg-AD (red) mice were linear with a slope conductance of 375 pS. (D) Summary of IP3R channel Po, τo (open circles), and τc (solid circles) in cortical neuron nuclei. Bars, mean ± SEM. *P < 0.05 by unpaired Student’s t test. (E) Summary of IP3R modal gating analysis. Colors for gating modes are the same as in Figs. 1 and 3. (F) Modal gating analysis of IP3R from cortical neurons. Each section is a continuous single-channel current record with modal assignment indicated by color code. In cells from C57BL/6 mouse, channel gating alternates between the L and I modes, whereas in 3xTg-AD mouse, IP3R gating alternates between the H and I modes.

FAD PS enhancement of IP3R channel gating is a gain-of-function effect

Our results reveal that FAD mutant PS consistently enhances IP3R channel gating. To explore the mechanisms involved, we recorded endogenous IP3R channels in nuclei from embryonic fibroblasts (MEFs) derived from PS DKO mice (49, 50). In the absence of PS, the endogenous MEF IP3R Po was 0.30 ± 0.03 (Fig. 6). Stable expression of human PS1 by retroviral transduction was without effect on IP3R Po (0.27 ± 0.05), whereas FAD mutant PS1-M146L approximately doubled channel gating activity (0.54 ± 0.05) by enhancing H mode gating (Fig. 6). Similar results were obtained in independently derived MEF clones (fig. S4). These results indicate that the effects of FAD mutant PSs on IP3R channel involve a gain of function. As shown above in Sf9 cells, this function is independent of PS secretase activity, because the secretase-dead PS1-D257A also enhanced channel activity (Fig. 6).

Fig. 6

FAD PS enhances IP3R channel gating by gain-of-function effect. (A) Continuous single IP3R channel current traces recorded from outer membranes of nuclei isolated from PS-deficient MEFs (PS DKO) and PS DKO stably expressing human PS1-WT, FAD PS1-M146L, or secretase-dead PS1-D257A (+40 mV; 10 μM IP3 and 1 μM free Ca2+ in pipette solution). Arrows indicate closed-channel current levels. (B) Western blot for PS1 in PS-deficient and stably transduced MEF cells. (C to E) Summary of IP3R channel Po (C), τo (open circles) and τc (solid circles) (D), and modal gating analysis (E). *P < 0.05 by ANOVA compared with PS DKO.


Our results demonstrate a consistent and robust phenotype associated with the presence of mutant PS linked to FAD. In five different cell systems (four here and DT40 cells previously) from four species, FAD-causing mutant PSs resulted in exaggerated responses of IP3R Ca2+ release channels and exaggerated Ca2+ signals in response to agonist stimulation, as well as a small degree of constitutive Ca2+ signaling. The FAD mutant PS phenotype involves gain-of-function effects, consistent with disease genetics, and is independent of the secretase function of PS. Moreover, the FAD mutant PS phenotype is not observed in cells harboring either wild-type PS or PS mutants associated with a different disease, FTD. The FAD mutant PS phenotype is manifested independently of any pathology associated with AD and, in the mouse model, precedes such pathology. Moreover, it is apparent in physiologically relevant cell types (cells derived from humans with FAD and AD mouse neurons) with all proteins present in endogenous amounts. We propose that exaggerated Ca2+ signaling through an IP3R-PS interaction is a robust proximal gain-of-function molecular mechanism in FAD.

Our single-channel analyses demonstrate that FAD mutant PS enhances single-channel activity of the IP3R by affecting modal gating kinetics, the major mechanism by which IP3 and Ca2+ regulate the channel (43). That FAD mutant PS drives the channel into the H mode may have important physiological implications. The channel open time when it is in the L gating mode (~10 ms) is short enough that it may not increase local [Ca2+] sufficiently to recruit additional IP3R- or RyR-mediated Ca2+ release by Ca2+-induced Ca2+ release (CICR). In contrast, the much longer activity bursts of the channel in the H mode (>200 ms) will provide a sufficiently large flux of Ca2+ to enable a normally local Ca2+ signal to be amplified and propagated by CICR (51). Because IP3R and RyR are clustered and spatially localized to different regions of cells to provide local [Ca2+]i signals as a critical element of physiological specificity, mode shifting by mutant PS–induced FAD may result not only in exaggerated local Ca2+ signaling, but also a disruption of spatial specificity by enabling CICR to transmit the signals more globally (43, 51). Exaggerated and spatially disrupted Ca2+ signaling may in turn impinge on APP processing (16, 5255), calpain activation (16, 55), and tau phosphorylation (56, 57), linking our findings here to the amyloid hypothesis of AD (Fig. 7).

Fig. 7

Hypothetical molecular mechanism of enhanced Aβ production due to Ca2+ disruption in FAD PS cells. APP is processed by either α-secretase or β-secretase, the latter leading to Aβ generation after subsequent cleavage by γ-secretase. Stimulation of G protein (heterotrimeric guanosine triphosphate–binding protein)–coupled receptors (GPCRs) or other cell surface receptors by extracellular ligands activates phospholipase C (PLC), which cleaves phosphatidylinositol bisphosphate (PIP2) to produce IP3. IP3 binds to and activates the IP3R to release Ca2+ from ER stores, increasing cytoplasmic Ca2+ concentration. In normal cells, these Ca2+ signals are tightly regulated in time, space, and amplitude. In FAD cells, mutant PS exerts stimulatory effects on IP3R gating by modal switching to the H mode associated with prolonged channel openings. H mode gating generates exaggerated Ca2+ signaling by promoting additional release channel recruitment by CICR. Increased cytosolic Ca2+ concentration promotes β-secretase activity (53) and Aβ production (52, 55), which, together with mutant PS–enhanced production of amyloidogenic Aβ, results in plaque formation.

Materials and Methods

Cell culture

Spodoptera frugiperda cells (Sf9; BD Biosciences) were maintained as described (37, 42). Human PS baculovirus constructs (PS1-WT, PS1-L113P, PS1-M146L, PS1-L166P, PS1-G183V, PS1-D257A, PS1-G384A, PS1-D384A, PS2-WT, and PS2-N141I) were subcloned into pFastBac1, and baculoviruses were generated with the Bac-to-Bac system (Invitrogen). Expression was confirmed by Western blotting with antibodies directed against PS1 or PS2 as described (37). B lymphoblast lines derived from human FAD patients and normal individuals (Table 1; Coriell Institute, Camden, NJ) were maintained at 37°C (95% air and 5% CO2) in RPMI 1640 (Invitrogen) supplemented with 15% FBS (Hyclone), 2 mM l-glutamine, penicillin (100 U/ml), and streptomycin (100 μg/ml). PS−/− (genetically deficient in PS1 and PS2), stable human PS1-WT, mutant PS1-M146L, and PS1-D257A MEF cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS (58, 59). To generate stable lines expressing comparable amounts of PS1 proteins, we introduced human PS1 complementary DNAs into pMX-IRES-EGFP retroviral vector, and PS retroviruses generated with Retro-X system (Clontech) were added to the parental PS−/− MEF cells; green fluorescent protein (GFP)–positive cells were sorted by fluorescence-activated cell sorting. PS expression was confirmed by Western blot.

Cortical neuron isolation

Primary cortical neurons were prepared from E14 to E16 3xTg-AD mice as described (37). Neurons from C57BL/6 mice (Charles River) served as controls. In brief, dams were killed with CO2, and embryos were removed by cesarean section. Brains from littermates were removed and placed into phosphate-buffered saline (PBS). After the meninges were removed, cerebral cortices were dissected, minced, and digested with 0.25% trypsin in PBS at 37°C for 20 min. Dissociated cells were washed twice with DMEM supplemented with 10% FBS, triturated with a fire-polished Pasteur pipette, and resuspended in Neurobasal medium supplemented with 1× B27 (Invitrogen). All animal procedures were approved by the University of Pennsylvania Institutional Animal Care and Use Committee.

Calcium imaging

Human B lymphoblasts (Coriell Institute) were plated onto a CellTek (BD Biosciences)–coated glass-bottomed perfusion chamber mounted on the stage of an inverted microscope (Eclipse TE2000; Nikon) and incubated with fura-2 AM (2 μM; Invitrogen) for 30 min at room temperature in Hanks’ balanced salt solution (HBSS; Sigma) containing 1% bovine serum albumin. Cells were then continuously perfused with HBSS containing 1.8 mM CaCl2 and 0.8 mM MgCl2 (pH 7.4). Ca2+ signals were elicited by cross-linking the BCR with antibody against human IgM (50 ng/ml; SouthernBiotech). In some experiments, cells were perfused with complete culture medium containing 10% FBS. Fura-2 was alternately excited at 340 and 380 nm, and the emitted fluorescence filtered at 510 nm was collected and recorded (37, 46) with a charge-coupled device–based imaging system running Ultraview software (PerkinElmer). Dye calibration was achieved by applying experimentally determined constants to the standard equation [Ca2+] = Kdβ(RRmin)/(RmaxR).


Preparation of isolated nuclei from cells was performed as described (37, 42, 46). In brief, cells were washed twice with PBS and suspended in nuclear isolation solution containing 150 mM KCl, 250 mM sucrose, 1.5 mM β-mercaptoethanol, 10 mM tris-HCl, 0.05 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail (Complete, Roche Diagnosis) (pH 7.3). Nuclei were isolated with a Dounce glass homogenizer and plated onto a 1-ml glass-bottomed dish containing standard bath solution: 140 mM KCl, 10 mM Hepes, 0.5 mM BAPTA [1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid], and 0.192 mM CaCl2 (free [Ca2+] = 90 nM). The pipette solution contained 140 mM KCl, 10 mM Hepes, 0.5 mM dibromo-BAPTA, and 0.001 mM free Ca2+ (pH 7.3). Free [Ca2+] in solutions was adjusted by Ca2+ chelators with appropriate affinities and confirmed by fluorometry as described (42). Data were recorded at room temperature and acquired with an Axopatch 200A amplifier (Axon Instruments), filtered at 1 kHz, and digitized at 5 kHz with an ITC-16 interface (Instrutech) and Pulse software (HEKA Electronik).

Data analysis

Segments of current records exhibiting current levels for a single IP3R channel were idealized with QuB software (University of Buffalo) and SKM algorithm (60, 61). Channel gating kinetics and modal gating behaviors were characterized as described (43). In brief, short closing events (<10 ms), presumably caused by ligand-independent transitions, were removed by burst analysis (62) after idealization with QuB. Modal gating assignment was then achieved by plotting and examining durations of channel burst (tb) and burst-terminating gaps (tg) as described (43). In Sf9 cells, we set Tb = 100 ms and Tg = 200 ms for the detection of modal transitions. In both human B lymphocytes and mouse cortical neurons, we set Tb = 50 ms and Tg = 100 ms for the detection of modal transitions. Data were summarized as the mean ± SEM, and the statistical significance of differences between means was assessed with unpaired t tests or one-way analysis of variance (ANOVA) with Dunnett’s post hoc comparison test. Differences between means were accepted as statistically significant at the 95% level (P < 0.05).


Acknowledgments: We thank R. Eckenhoff and H. Wei for supplying mice and D. Shilling for assistance with the culturing of the PS DKO MEF cells. Funding: Acknowledgment is made to the donors of Alzheimer’s Disease Research, a program of the American Health Assistance Foundation (A2008-137 to J.K.F.), to the Alzheimer’s Association (IIRG-08-91662 to D.E.K), and to the Japan Science and Technology Agency (T.I.). Author contributions: K.-H.C. designed and performed the experiments, analyzed data, and wrote the manuscript. L.M. developed recombinant baculoviruses and performed infections, transfections, and cell culture. D.-O.D.M. developed software for modal gating and single-cell Ca2+ analyses and assisted in the analyses. I.H. and T.I. developed recombinant baculoviral PS constructs. D.E.K. developed DKO MEF cells. J.K.F. designed and analyzed experiments and wrote the manuscript. Competing interests: None of the authors have competing interests.

Supplementary Materials

Fig. S1. Recombinant presenilins in baculovirus-infected Sf9 cells.

Fig. S2. Modal gating analyses of IP3R channels under the influence of FAD-linked mutant PS.

Fig. S3. Effect of xestospongin B on IgM-activated Ca2+ oscillations in human B lymphoblasts.

Fig. S4. FAD PS enhances IP3R channel gating by gain-of-function effect.


References and Notes

  1. These are amino acid substitutions at particular residues. Abbreviations for the amino acid residues are as follows: A, Ala; D, Asp; G, Gly; I, Ile; L, Leu; M, Met; N, Asn; P, Pro; and V, Val.
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