Mechanism of Ca2+ disruption in Alzheimer's disease by presenilin regulation of InsP3 receptor channel gating

Abstract

Mutations in presenilins (PS) are the major cause of familial Alzheimer's disease (FAD) and have been associated with calcium (Ca2+) signaling abnormalities. Here, we demonstrate that FAD mutant PS1 (M146L)and PS2 (N141I) interact with the inositol 1,4,5-trisphosphate receptor (InsP3R) Ca2+ release channel and exert profound stimulatory effects on its gating activity in response to saturating and suboptimal levels of InsP3. These interactions result in exaggerated cellular Ca2+ signaling in response to agonist stimulation as well as enhanced low-level Ca2+signaling in unstimulated cells. Parallel studies in InsP3R-expressing and -deficient cells revealed that enhanced Ca2+ release from the endoplasmic reticulum as a result of the specific interaction of PS1-M146L with the InsP3R stimulates amyloid beta processing,an important feature of AD pathology. These observations provide molecular insights into the "Ca2+ dysregulation" hypothesis of AD pathogenesis and suggest novel targets for therapeutic intervention.

Figure 1. Effects of PS1 expression on InsP₃R single channel activity in Sf9 cells

(A-C) Representative current recordings in isolated nuclei from Sf9 cells infected with PS1 WT or M146L baculoviruses in absence (A) or presence of saturating (10 μM; B) or sub-saturating (33 nM; C) InsP₃ in pipette solution. Channel activity was not evoked by PS1 alone in absence of InsP₃ (A) whereas InsP₃R channels were activated in presence of InsP₃ (B,C). Pipette [Ca²⁺] was 1 μM; arrows: zero current level. Summary of effects of PS1 expression on InsP₃R channel open probability P_o (D) mean open time (τ_o) (E) and mean closed time (τ_c) (F). Asterisks: p < 0.01, unpaired t-test. Data presented in Supplementary Table 1.

Figure 2. Biochemical interaction between PS and InsP₃R

Western blot of PS1 (A) or PS2 (B) immunoprecipitates from lysates of Sf9 cells co-expressing either PS1-WT or PS1-M146L (A) or PS2-WT or PS2-N141I (B) with rat InsP₃R isoforms 1 or 3; expressed proteins shown at top and probing antibodies shown on right. (C) Western blots of PS1 (top) and PS2 (bottom) immunoprecipitates from mouse brain lysate. Top: Lanes 1 and 2: total input; lane 3: IgG immunoprecipitate control; lane 4: PS1 immunoprecipitate. Bottom: Lane 1: total input; lane 2: PS2 immunoprecipitate; lane3: rabbit serum immunoprecipitate control.

Figure 3. Effects of PS1 expression on InsP₃R single channel activity in DT40 cells

Representative InsP₃R single-channel current recordings in presence of saturating (10 μM; A) or sub-saturating (100 nM; B) InsP₃ in DT40 cells stably transfected with PS1 WT or M146L. Pipette [Ca²⁺] was 1 μM, optimal for channel activity; arrows: zero current level. Summary of effects of PS1 expression on InsP₃R P_o (C), mean open time (τ_o) (D) and mean closed time (τ_c) (E). Asterisks: p < 0.01, unpaired t-test. Data presented in Supplementary Table 2.

Figure 4. Effect of PS2 on InsP₃R single channel activity in Sf9 cells

(A,B) Representative current recordings in isolated nuclei from Sf9 cells infected with PS2 WT or N141I baculovirus in the presence of saturating (10 μM; A) or sub-saturating (33 nM; B) InsP₃. Pipette [Ca²⁺] was 1 μM; arrows: zero current level. Summary of effects of PS2 expression on InsP₃R P_o (C), channel mean open time (τ_o) (D) and channel mean closed time (τ_c) (E). Asterisks: p < 0.01, unpaired t-test. Data presented in Supplementary Table 3.

Figure 5. Exaggerated [Ca²⁺]_i signaling in mutant PS-expressing DT40 cells

(A,B) Responses to strong stimulation by BCR antibody of DT40 cell [Ca²⁺]_i. (A) Representative single cell responses to 5 μg/ml anti-IgM (added at arrow) in untransfected (blue) and PS1-WT (red) and PS1-M146L (green) stably-transfected DT40 cells. (B) Summary of peak [Ca²⁺]_i responses triggered by 5 μg/ml anti-IgM (n = 90). Asterisk: p < 0.01 compared with WT and PS1-WT (C-F) Responses to weak stimulation by BCR antibody of DT40 cell [Ca²⁺]_i. (C) Representative single cell [Ca²⁺]_i responses to 50 ng/ml anti-IgM (IgM; added at arrow) stimulation of BCR in control (blue), PS1-WT (red) and PS1-M146L (green and pink) stably-transfected DT40 cells. (D) Summary of percentage of cells responding to 50 ng/ml anti-IgM (n = 90). ~ 30% (purple) of PS1-M146L expressing cells exhibited a different, exaggerated [Ca²⁺]_i response. (E) [Ca²⁺]_i oscillation frequency triggered by anti-IgM in WT DT40, PS1-WT expressing and PS1-M146L expressing cells. (F) Summary of latencies to first response in WT DT40, PS1-WT expressing and PS1-M146L expressing cells. The 30% of PS1-M146L expressing cells that exhibited the exaggerated response had nearly no latency (purple). (G-I) Spontaneous [Ca²⁺]_i oscillations in PS1-expressing DT40 cells. (G) Representative spontaneous single cell [Ca²⁺]_i oscillations in control (blue) and PS1-WT (red) and PS1-M146L (green and purple) stably-transfected DT40 cells. Some PS1-M146L expressing cells (~ 4%, purple) displayed a distinct, exaggerated spontaneous [Ca²⁺]_i signal (bottom). (H) Percentage of cells displaying spontaneous [Ca²⁺]_i oscillations (n = 90). (I) Spontaneous [Ca²⁺]_i oscillation frequency observed in WT DT40 cells and PS1-WT- and PS1-M146L-expressing DT40 cells. Asterisks: p < 0.01 compared with WT DT40 cells. Asterisks with bars: p < 0.01 PS1-Wt vs PS1-M146L.

Figure 6. The amount of Ca²⁺ in the ER store is not increased by PS1-M146L expression, due to FAD mutant PS1- and InsP₃R-dependent enhanced Ca²⁺ leak permeability of the ER membrane

(A) ER [Ca²⁺], expressed as Mag-Fura2 ratio normalized to ratio at t=0 before and then during filling upon addition of MgATP (1.5 mM) and subsequent emptying by addition of InsP₃ (10 μM) in control (blue) and PS1-WT (red) and PS1-M146L (green) stable DT40 cells. Insert: Steady state ER [Ca²⁺] in presence of MgATP. Asterisks: p <0.01 compared with control cells. Cross: p<0.01 compared with PS1-WT. (B) Similar experiment with ER Ca²⁺ loading performed in presence of heparin (100 μg/ml). Asterisk: p < 0.01 and < 0.05 compared with control and PS1-WT cells, respectively. (C) ER [Ca²⁺] during filling upon addition of MgATP (1.5 mM) in presence of heparin (100 μg/ml), and then after removal of MgATP and addition of thapsigargin (1 μM), in control (blue) and PS1-WT (red) and PS1-M146L (green) stable DT40 cells. (D) Summary of ER Ca²⁺ leak rate, calculated as initial rate of decline of the Mag-Fura2 ratio. Asterisk: enhanced leak observed in M146L-expressing cells compared with control and PS1-WT (p < 0.01). (E) Similar experiments as in (D), except that heparin (100 μg/ml) was present during measurements of the Ca²⁺ leak rate. (F). Summary of ER Ca²⁺ leak rate in presence of heparin. (G) Similar experiment as in (D) performed in cells lacking InsP₃R expression (KO). Insert: steady-state ER [Ca²⁺] after MgATP-induced loading. (H) Summary of ER Ca²⁺ leak rate in PS1 expressing InsP₃R KO cells.

Figure 7. FAD mutant PS1 enhances InsP₃R-mediated ER Ca²⁺ permeability in brain neurons

(A) ER [Ca²⁺], expressed as Mag-Fura2 ratio normalized to ratio at t=0, before and then during filling upon addition of MgATP (1.5 mM), and subsequent exposure to 1 μM thapsigargin in control (blue) and PS1-WT (red) and PS1-M146L (green) transfected mouse cortical neurons. (B) Summary of steady state ER [Ca²⁺] following MgATP induced loading in (A). (C) Summary of Ca²⁺ leak rate measured in presence of thapsigargin in (A). Asterisk: p = 0.01. (D) ER [Ca²⁺] during filling upon addition of MgATP (1.5 mM) in control (black) and EGFP (blue), PS1-WT (red) and PS1-M146L (green) transfected mouse cortical neurons. (E) Response of ER [Ca²⁺] to 33 nM InsP₃ in cells from (D) and in control cells in response to 10 μM InsP₃ (black trace). (F) Summary of initial Ca²⁺ release rate, expressed as Mag-Fura2 ratio, from (E). Asterisks: p < 0.01 compared with control and PS1-WT.

Figure 8. APP processing is dependent on InsP₃R

(A) Stable expression of PS1-WT and PS1-M146L proteins in wild-type (WT) and InsP₃R-deficient (KO) DT40 cell lines that stably expressed APP_SWE. Actin probed as loading control. (B) ELISA measurements of Aβ₄₀ (top), Aβ₄₂ (middle) and Aβ₄₂/Aβ₄₀ ratio (bottom) secreted over 48 h by InsP₃R-expressing wild-type (WT; left) or InsP₃R-deficient (KO DT40 cells; right) DT40 cells stably expressing APP_SWE alone (blue) or APP_SWE with PS1-WT (red) or PS1-M146L (green). Asterisks: p < 0.01 compared with control WT cells. Cross: p < 0.01 compared with control WT cells; double cross: p < 0.01 compared with PS1-WT cells.