Alzheimer's β-secretase (BACE1) regulates the cAMP/PKA/CREB pathway independently of β-amyloid

Abstract

β-Amyloid protein (Aβ), the major component of neuritic plaques in Alzheimer's disease (AD), is derived from proteolytic cleavages of the amyloid precursor protein (APP) by β-site APP-cleaving enzyme 1 (BACE1) and the γ-secretase complex. BACE1 is the rate-limiting enzyme for Aβ production, and an increase in BACE1 level/activity contributes to the pathogenesis of sporadic AD. In addition to cleaving APP for Aβ generation, BACE1 plays multiple physiological roles including the regulation of synaptic functions. Here, we found that overexpression of BACE1 reduces cAMP response element binding protein (CREB) phosphorylation, protein kinase A (PKA) activity, and cAMP levels, whereas downregulation of BACE1 has the opposite effect. We showed that BACE1's effect is independent of its activity for Aβ production, which is corroborated by the observation that BACE1 transgenic mice have impaired learning/memory in the absence of neurotoxic human Aβ. Furthermore, we demonstrated that BACE1 interacts via its transmembrane domain with adenylate cyclase, resulting in reduction of cellular cAMP levels and thus PKA inactivation and reduced CREB phosphorylation. Our study suggests that in addition to its function as the β-secretase to produce Aβ, BACE1 may contribute to the memory and cognitive deficits typical of AD by regulating the cAMP/PKA/CREB pathway, which is important for memory functions.

Figure 1.

BACE1 regulates CREB phosphorylation. A, N2a cells were transfected with a control or human BACE1 vector. Cell lysates were immunoblotted for total and phosphorylated CREB. AU, Arbitrary units. **p < 0.01. n = 3. B, Primary neurons were infected with AAV-EGFP or AAV-BACE1. Cell lysates were immunoblotted for total and phosphorylated CREB, PSD-95, and synaptophysin (Syn). **p < 0.01. n = 3. C, Brain lysates from BACE1 transgenic (Tg) mice (n = 3) and WT littermate controls (n = 3) were analyzed for total and phosphorylated CREB and synaptic markers. **p < 0.01. D, Brains from BACE1 Tg mice (n = 3) and WT littermate controls (n = 3) were analyzed by immunohistochemistry for phosphorylated CREB. Top, Hippocampus, 20×; bottom, cortex, 20×. *p < 0.05; **p < 0.01. E, Brains from BACE1 Tg mice (n = 3) and WT littermate controls (n = 3) were analyzed by immunofluorescence for PSD-95 (green) and synaptophysin (red). Top, Hippocampus, 10×; bottom, hippocampus, 40×. *p < 0.05; **p < 0.01. F, Brain lysates from BACE1 KO mice (n = 3) and WT littermate controls (n = 3) were analyzed for total and phosphorylated CREB and synaptic markers. *p < 0.05; **p < 0.01.

Figure 2.

BACE1 regulates cAMP levels and PKA activity. A, N2a cells were transfected with a control or human BACE1 vector at different doses. Cell lysates were analyzed for PKA activity and cAMP levels. Data were normalized to control cells (set as one arbitrary unit). *p < 0.05; **p < 0.01. n = 3. B, Primary neurons were infected with AAV-EGFP or AAV-BACE1. Cell lysates were analyzed for PKA activity and cAMP levels. *p < 0.05. n = 3. C, Brain lysates from BACE1 transgenic (Tg) mice (n = 3) and WT littermate controls (n = 3) were analyzed for PKA activity and cAMP levels. *p < 0.05. D, Brain lysates from BACE1 KO mice (n = 3) and WT littermate controls (n = 3) were analyzed for PKA activity and cAMP levels. *p < 0.05. E, N2a cells transfected with a control or BACE1 vector for 48 h were treated with forskolin at 10 μm for 2 or 4 h. Cell lysates were immunoblotted for total and phosphorylated CREB.

Figure 3.

The effect of BACE1 is independent of Aβ levels. A, WT or APP/APLP2 double KO (dKO) MEF cells were transfected with a control or human BACE1 vector. Cell lysates were immunoblotted for total and phosphorylated CREB. **p < 0.01. n = 3. B, C, N2a cells were transfected with a control, BACE1 or mutant BACE1 D93A vector. Cell lysates were either immunoblotted for total and phosphorylated CREB (B) or analyzed for PKA activity and cAMP levels (C). *p < 0.05; **p < 0.01. n = 3. D, E, Primary neurons were infected with AAV-EGFP, AAV-BACE1, or AAV-mutant BACE1 (AAV-D93A). Cell lysates were either immunoblotted for total and phosphorylated CREB (D) or analyzed for PKA activity and cAMP levels (E). *p < 0.05; **p < 0.01. n = 3. F, At 4 months of age, BACE1 transgenic (Tg) mice (n = 8) and WT controls (n = 8) were evaluated in an open-field test. The total activity upon re-exposure on day 5 was compared with that during first exposure on day 1. *p < 0.05.

Figure 4.

BACE1 interacts via its transmembrane domain with adenylate cyclase. A, N2a cells were cotransfected with adenylate cyclase (AC) and with wild-type or mutant BACE1 (D93A). Cell lysates were immunoprecipitated with mouse IgG (mIgG), rabbit IgG (rIgG), HA, or AC antibodies, and immunoblotted with HA or AC antibodies. B, Wild-type mice brain lysates were immunoprecipitated with rabbit rIgG, BACE1, or AC antibodies, and immunoblotted with BACE1 or AC antibodies. C, N2a cells were cotransfected with AC and wild-type BACE1 or BACE1 carrying the nicastrin transmembrane domain (BACE1-N). Cell lysates were immunoprecipitated with mIgG, rIgG, HA, or AC antibodies, and immunoblotted with HA or AC antibodies. D, E, N2a cells were transfected with a control, wild-type BACE1, or BACE1-N vector. Cell lysates were either immunoblotted for total and phosphorylated CREB (D) or were analyzed for PKA activity and cAMP levels (E). *p < 0.05; **p < 0.01. n = 3.