B103 rat neuroblastoma cells were transiently transfected with APP695 (A) or the C-terminal fragment comprised of the terminal 99 amino acids of APP (C99) (D) and subsequently treated with Aβ (10µM, 24hours). Cell homogenates were purified to obtain membrane fractions which then were immunoprecipitated with the APP C-terminal specific G369 antibody, run on a SDS-PAGE gel and Go protein binding was assessed by immunoblot with a monoclonal Go antibody.
(A) Immunoprecipitation studies show a 40kDa band representing the interaction between APP695 and Go in cells transfected with APP695.
(B) Western blot analysis with the Go antibody (upper panel) demonstrates that transfection with APP695 and/or treatment with Aβ does not affect expression levels of Go or APP.
(C) Similar immunoprecipitation studies in cells that had been transfected with C99 show an interaction between the C99 region of APP and Go. As with full-length APP, this interaction was reduced in cells treated with Aβ.
(D) Western blot analysis with the G369 antibody demonstrates that expression level of APP695 is not affected by 24 hour treatment with Aβ (10µM), Pertussis toxin (PTX, 100ng/ml), or a combination of the two.

B103 rat neuroblastoma cells were transiently transfected with APP695 and subsequently treated for 24 hours with vehicle, Aβ (10µM), PTX (100ng/ml) or a combination of Aβ and PTX. Following treatment, cell death was analyzed via the lactate dehydrogenase (LDH) cell death assay.
(A) Minimal cell death was observed in B103 cells with or without APP695 transfection, treatment with Aβ significantly increased cell death in APP695 transfected cells (*). Treatment PTX alone had no significant effect upon cell death in cells with or without APP695 transfection, however when administered in combination with Aβ, PTX significantly reduced the cell death associated with Aβ treatment of APP695 transfected cells (**).
* indicates a significant difference between vehicle- and Aβ-treated APP695 transfected cells (p<0.05, by one way ANOVA and post hoc Fisher).
** indicates a significant difference between Aβ/PTX- and Aβ-treated APP695 transfected cells (p<0.05, by one way ANOVA and post hoc Fisher).
(B) Neurite outgrowth was assessed in cells transfected with APP695 and treated with Aβ. There was a significant reduction in neurite length in Aβ-treated APP695-transfected cells which was abrogated upon the addition on PTX.
* indicates a significant difference (p<0.05, by one way ANOVA and post hoc Fisher).
(C) B103 rat neuroblastoma cells were transiently transfected with APP695, APP695ΔY or APP695ΔC31 and subsequently treated for 24 hours with Aβ (10µM). Following treatment, cell death was analyzed via the lactate dehydrogenase (LDH) cell death assay.
APP-dependent Aβ-induced cell death was clearly observed in cells transfected with APP695, whilst cells transfected with the APP695ΔY or APP695ΔC31 constructs failed to display significant cell death upon Aβ treatment in comparison to untransfected cells.
* indicates a significant difference between vehicle- and Aβ-treated APP695 transfected cells (p<0.05, by one way ANOVA and post hoc Fisher).
** indicates a significant difference between Aβ-treated APP695 transfected cells and Aβ-treated APP695ΔY and APP695ΔC31 transfected cells (p<0.05, by one way ANOVA and post hoc Fisher).
(D) Neurite outgrowth was assessed in cells transfected with APP695, APP695ΔY or APP695ΔC31 and subsequently treated for 24 hours with Aβ (10µM). There was a significant reduction in neurite length in Aβ-treated APP695-transfected cells which was not evident in cells transfected with the APP695ΔY or APP695ΔC31 constructs
* indicates a significant difference (p<0.05, by one way ANOVA and post hoc Fisher).

B103 rat neuroblastoma cells were transiently transfected with APP695 or with APP695 with a substitution of alanine for aspartic acid at position 664 (APP695D664A). Cells were subsequently treated with Aβ (10µM, 24hours).
(A) For immunoprecipitation studies cell homogenates were immunoprecipitated with the APP C-terminal specific G369 antibody, run on a SDS-PAGE gel and Go protein binding assessed by immunoblot with a monoclonal Go antibody. The interaction between APP695 and Go (40kDa) is reduced in cells that have been treated with Aβ. This interaction is also present in cells transfected with APP695D664, however Aβ treatment of APP695D664 transfected cells failed to reduce the band signal intensity.
(B) Western blot analysis with the Go antibody demonstrates that transfection with APP695 or APP695D664 and/or treatment with Aβ does not affect expression levels of Go. Similar analysis with the G369 antibody demonstrates that Aβ treatment does not affect expression levels of APP695 or APP695D664.
(C) B103 rat neuroblastoma cells were transiently transfected with APP695 or APP695D664 and subsequently treated for 24 hours with Aβ (10µM) were analyzed via the lactate LDH cell death assay to obtain a measure of APP695-dependent Aβ-induced cell death.
Minimal cell death was observed in B103 cells with or without APP695 transfection, whilst treatment with Aβ significantly increased cell death in APP695 transfected cells (*). Aβ failed to induce significant levels of cell death in cells transfected with APP695D664 (**)
* indicates a significant difference between Aβ-treated APP695 transfected cells and Aβ-treated untransfected cells (p<0.05, by one way ANOVA and post hoc Fisher).
** indicates a significant difference between Aβ-treated APP695D664A transfected cells and Aβ-treated APP695 transfected cells (p<0.05, by one way ANOVA and post hoc Fisher).
(D) Measurements of intracellular calcium demonstrate a significant increase in calcium influx in cells transfected with APP695 upon Aβ-treatment in comparison to Aβ-treated untransfected cells (*). Aβ-treatment of APP695D664A transfected cells failed to elicit a significant increase in intracellular calcium levels in comparison to Aβ-treated untransfected cells (**).
* indicates a significant difference between Aβ-treated APP695 transfected cells and Aβ-treated untransfected cells (p<0.05, by one way ANOVA and post hoc Fisher).
** indicates a significant difference between Aβ-treated APP695D664A transfected cells and Aβ-treated APP695 transfected cells (p<0.05, by one way ANOVA and post hoc Fisher).

(A) Brain samples were homogenized and immunoprecipitated with the APP C-terminal specific CT15 antibody, run on a SDS-PAGE gel and Go protein binding was assessed by immunoblot with a monoclonal Go antibody. Signal intensity of the 40kDa band (corresponding to the interaction between APP and Go) was analyzed for each sample at each Braak stage. A decrease in the signal intensity of the 40kDa band was observed with increasing disease severity (mean ± SEM).
* indicates a significant difference between expression levels at Braak stage 0 and Braak stage V–VI (p<0.05, by one way ANOVA and post hoc Fisher).
(B) In order to assess the activation capability of G-proteins in AD, G-protein activation at different Braak stages was assessed using the classic G-protein activator NE (10µM). Results from this assay demonstrate that G-proteins in purified membrane fractions from human frontal cortex from each Braak stage are equally capable of being activated by NE (10µM).
(C) G-protein activation was assessed at different Braak stages and showed that G-protein activation increases with increasing disease severity.
* indicates a significant difference between activity levels at Braak stages I–II, III–IV and V–VI and Braak stage 0 (p<0.05, by one way ANOVA and post hoc Fisher).

Immunoreactivity for Go and APP in untreated control B103 cells (A,B), Aβ-treated B103 cells (D,E) APP-transfected B103 cells (G,H) and Aβ-treated APP transfected B103 cells (J,K) and colocalization of signals in cells under the different conditions (in C, F, I and L respectively). Scale bar = 20µm.

B103 rat neuroblastoma cells were transiently transfected with full length APP (APP695), APP695 lacking the YENPTY domain (APP695ΔY) or APP695 lacking the C-terminal C31 domain (APP695ΔC31) and subsequently treated with Aβ (10µM, 24hours).
(A) For immunoprecipitation studies cell homogenates were immunoprecipitated with the APP C-terminal specific G369 antibody, run on a SDS-PAGE gel and Go protein binding was assessed by immunoblot with a monoclonal Go antibody. The interaction between APP695 and Go (40kDa) is reduced in cells that have been treated with Aβ. This interaction is present in cells transfected with APP695ΔY and APP695ΔC31 however, in these cells, Aβ failed to reduce the level of interaction observed.
(B) Western blot analysis with G369 antibody demonstrates that treatment with Aβ did not affect the expression levels of APP695, APP695ΔY or APP695ΔC31.
(C) B103 rat neuroblastoma cells were transiently transfected with APP695 and subsequently treated for 24 hours with, vehicle, Aβ (10µM), PTX (100ng/ml), or a combination of Aβ and PTX. Following treatment, levels of intracellular calcium were analyzed using the FLIPR calcium assay.
Aβ-treatment of APP695 transfected cells resulted in a significant increase in intracellular calcium levels as compared to vehicle-treated APP695 transfected cells (*). Treatment PTX alone had no significant effect upon calcium influx in cells with or without APP695 transfection, however when administered in combination with Aβ, PTX significantly reduced the calcium influx associated with Aβ treatment of APP695 transfected cells (**).
* indicates a significant difference between vehicle- and Aβ-treated APP695 transfected cells (p<0.05, by one way ANOVA and post hoc Fisher).
** indicates a significant difference between Aβ/PTX- and Aβ-treated APP695 transfected cells (p<0.05, by one way ANOVA and post hoc Fisher).
(D) B103 rat neuroblastoma cells were transiently transfected with APP695, APP695ΔY or APP695ΔC31 and subsequently treated for 24 hours with Aβ (10µM). Following treatment, intracellular calcium levels were analyzed via FLIRP calcium assay.
APP-dependent Aβ-induced calcium influx was clearly observed in cells transfected with APP695, whilst cells transfected with the APP695ΔY or APP695ΔC31 constructs failed to display significant calcium influx upon Aβ treatment in comparison to APP695 transfected cells.
* indicates a significant difference between vehicle- and Aβ-treated APP695 transfected cells (p<0.05, by one way ANOVA and post hoc Fisher).
** indicates a significant difference between Aβ-treated APP695 transfected cells and Aβ-treated APP695ΔY and APP695ΔC31 transfected cells (p<0.05, by one way ANOVA and post hoc Fisher).

Membrane fractions were obtained from human brains at various Braak stages of AD (I–II n=10, III–IV n=10 and V–VI n=10) and 9 non-demented age-matched control brains (designated Braak stage 0). 10µg of each sample were used for immunoblot analysis of various AD-related proteins. In each case signal intensity was measured (mean ± SEM).
(A) Levels of synaptophysin immunoreactivity show a decrease upon disease progression
(B) Levels of APP immunoreactivity remain stable across the different Braak stages
(C) Levels of GO immunoreactivity remain stable across the different Braak stages
(D) Levels of Aβ immunoreactivity increase as the disease progresses.
* indicates a significant difference between expression levels at Braak stage 0 and Braak stage V–VI (p<0.05, by one way ANOVA and post hoc Fisher).

APP binding to Go under basal conditions (A) allows to the G-protein to remain in ans inactive state. Increasing levels of Aβ, such as those found in AD (B), disrupt APP:Go binding, this Aβ-responsiveness requires an intact C31/-YENPTY- region. The Go is liberated and goes on to achieve an active conformation and activate downstream pathways including the influx of calcium from ER stores or from the extracellular space. Elevated levels of intracellular calcium lead to eventual cell death.