Constitutive cAMP response element binding protein (CREB) activation by Alzheimer's disease presenilin-driven inositol trisphosphate receptor (InsP3R) Ca2+ signaling

Abstract

Mutations in presenilins (PS) account for most early-onset familial Alzheimer's disease (FAD). Accumulating evidence suggests that disrupted Ca(2+) signaling may play a proximal role in FAD specifically, and Alzheimer's disease (AD) more generally, but its links to the pathogenesis of AD are obscure. Here we demonstrate that expression of FAD mutant PS constitutively activates the transcription factor cAMP response element binding protein (CREB) and CREB target gene expression in cultured neuronal cells and AD mouse models. Constitutive CREB activation was associated with and dependent on constitutive activation of Ca(2+)/CaM kinase kinase β and CaM kinase IV (CaMKIV). Depletion of endoplasmic reticulum Ca(2+) stores or plasma membrane phosphatidylinositol-bisphosphate and pharmacologic inhibition or knockdown of the expression of the inositol trisphosphate receptor (InsP(3)R) Ca(2+) release channel each abolished FAD PS-associated constitutive CaMKIV and CREB phosphorylation. CREB and CaMKIV phosphorylation and CREB target gene expression, including nitric oxide synthase and c-fos, were enhanced in brains of M146V-KI and 3xTg-AD mice expressing FAD mutant PS1 knocked into the mouse locus. FAD mutant PS-expressing cells demonstrated enhanced cell death and sensitivity to Aβ toxicity, which were normalized by interfering with the InsP(3)R-CAMKIV-CREB pathway. Thus, constitutive CREB phosphorylation by exaggerated InsP(3)R Ca(2+) signaling in FAD PS-expressing cells may represent a signaling pathway involved in the pathogenesis of AD.

Fig. 1.

Constitutive CREB phosphorylation in mutant PS1-M146L–expressing cells. (A–C) Representative spontaneous [Ca²⁺]_i signals demonstrating the spectrum of signals observed among different untransfected and stable PS1-WT– and PS1-M146L–expressing SH-SY5Y cells, respectively. (D) Summary and representative Western blots showing the effects of PS1 on P-CREB in stable SH-SY5Y cells under basal conditions. P-CREB was normalized to T-CREB. n = 7 experiments. (E) Effects of PS1 RNA_i on basal P-CREB in SH-SY5Y–stable lines. CTL was exposed to scrambled siRNA.

Fig. 2.

Spontaneous Ca²⁺ release in PS1-M146L–expressing cells is responsible for constitutive CREB phosphorylation. (A–C) Representative spontaneous single-cell [Ca²⁺]_i oscillations, representing the spectrum of behaviors observed in PS1-M146L–expressing SH-SY5Y cells under basal conditions (A), after 30 min of XeB (2 μM) (B), and after 48 h of transient transfection with InsP₃R-1 siRNA (C). (D–F) Densitometric analyses and representative Western blots of P-CREB in SH-SY5Y cells after 30 min of incubation in the absence or presence of InsP₃R inhibitor XeB (XB, 2 μM; n = 3 experiments) (D), after 48 h of transient transfection with InsP₃R-1 siRNA (R) or nontargeting siRNA (NT) (E), and in the absence or presence of rapamycin (Rap, 100 nM) (F). SH-SY5Y cells stably expressing WT or FAD PS1 were transfected with pmFRB-FKBP12-5ptase. After 48 h, cells were preincubated with rap for 30 min. Some gels are digitally rearranged for presentation purposes. n = 3 experiments. Data are expressed as mean ± SEM. *^,#P < 0.05; **^,##P < 0.01.

Fig. 3.

Constitutive CaMKIV phosphorylation is upstream of enhanced CREB activation in FAD PS-expressing cells. (A) Basal CaMKIV phosphorylation in SH-SY5Y cells stably transfected with PS1-WT or PS1-M146L, and representative Western blots of T-CaMKIV and P-CaMKIV. n = 4 experiments. (B) CREB phosphorylation in stable SH-SY5Y cells preincubated for 30 min in the presence of CaMK inhibitor KN93 or inactive analog KN92 (10 μM). n = 3 experiments. (C–E) Densitometric analyses and representative Western blots of P-CaMKIV in SH-SY5Y cells after 30 min of incubation in absence or presence of InsP₃R inhibitor XeB (XB, 2 μM; n = 3 experiments) (C), after 48 h of transient transfection with InsP₃R-1 siRNA (R) or nontargeting siRNA (NT) (D), and in the absence or presence of rapamycin (rap, 100 nM) (E). Stable SH-SY5Y cells were transfected with pmFRB-FKBP12-5ptase (E). After 48 h, cells were preincubated with rapamycin for 30 min. n = 3 experiments. (F) Effects on P-CREB in the absence or presence of the CaMKKβ inhibitor STO-609 (10 μM for 30 min). Results are summarized from three experiments for each cell type. Some gels are digitally rearranged for presentation. Data are expressed as mean ± SEM. *^,#P < 0.05; **^,##P < 0.01.

Fig. 4.

CREB and CaMKIV are constitutively activated in presymptomatic 3xTg-AD mouse brain. Expression of T-and P-CREB (A; n = 5) and CaMKIV (B; n = 4), and levels of CREB target genes c-fos (C), nNOS (D), and BDNF (E) and control gene GAPDH (F) in whole-brain extracts from 4- to 6-wk-old 3xTg-AD mice. Mice of the same background strain, sex, and age were used as controls (n = 4). Protein levels were normalized to β-tubulin (β-tub). Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01.

Fig. 5.

Enhanced spontaneous and Aβ-induced cell death in FAD PS1-expressing cells is abolished by inhibition of InsP₃R-CaMKIV-CREB signaling. (A) Viability of control and stable PS1-M146L– or PS1-WT–expressing SH-SY5Y cells after 48 h in the presence of Aβ 1–40 oligomers or scrambled peptide as control (10 μM each; n = 4). (B–G) Effects of CaMKKβ inhibitor STO-609 (2.5 μM) (B and C), CREB siRNA (D and E), or InsP₃R-1 siRNA (F and G) on basal (B, D, and F) and Aβ-induced (C, E, and G) cell death (n = 3 each). Controls in D–G were exposed to nontargeting siRNA. Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01.