Regulation of intracellular calcium in cortical neurons transgenic for human Abeta40 and Abeta42 following nutritive challenge

Abstract

The pathogenesis of Alzheimer's Disease (AD) is not fully understood. Amyloid plaques could be causally linked to neuronal loss in AD. Two proteolytic products of the Amyloid Precursor Protein (APP), Amyloid beta40 (Abeta40) and Amyloid beta42 (Abeta42), are considered to be critical in the neurodegeneration seen in AD. However, in transgenic mice that overexpress human Abeta40 or Abeta42, it was shown that Abeta42 was much more amyloidogenic than Abeta40. In contrast to this observation, we have found that cultured cortical neurons from mice transgenic for human Abeta40 and for Abeta42 are both and statistically equally vulnerable to nutritive challenge induced by trophic factor withdrawal (TFW). Aberrant regulation of InsP(3)R (Inositol triphosphate receptor)-mediated calcium release has been implicated in neuronal cell death. It is however not clear whether this pathway plays a critical role in cortical neurons transgenic for different species of human Abeta. We now report that Abeta40 and Abeta42 equally exacerbated intracellular calcium response to TFW in cortical neurons following TFW. When bradykinin (BK), a potent stimulant of InsP(3)R-mediated calcium release from ER, was applied to these cells, wild-type (WT) neurons exhibited a steep rise in [Ca(2+)](i) but this was not observed in either Abeta transgenic type. Similarly, when 1 muM Xestopongin C (XeC), a specific blocker of InsP(3)R, was applied to these neurons, WT cells showed a significant attenuation of increase in [Ca(2+)](i) following TFW, while elevation in [Ca(2+)](i) induced by TFW remained largely unchanged in Abeta40 and Abeta42 cells. Finally, when we treated these cells with a Ca(2+) chelator (BAPTA; 10 muM), all three cell types had a marked attenuation of [Ca(2+)](i). These findings indicate that the exacerbated calcium dysregulation following TFW in Abeta transgenic neurons are likely to be mediated by calcium channels other than ER InsP3R receptors. Overall, our results also suggest that a highly amyloidogenic Abeta species, such as Abeta42, might not necessarily be significantly more neurotoxic than a less or non-amyloidogenic Abeta species, such as Abeta40.

Keywords: Amyloid β-peptide; InsP3R; endoplasmic reticulum; intracellular calcium; transgenic mice.

Figure 1

Calcium response to trophic factor withdrawal (TFW) was exacerbated in cortical neurons transgenic for both species of Amyloid β peptide. The cell permeant, Ca²⁺-specific probe, Fluo-3 was used to capture confocal images of 1-week old cortical neuronal cultures. Samples were excited at 488 nm with a Ar/Kr laser. Emissions were recorded with filters chosen for 510 nm. TFW was achieved with incubations in glucose-free medium. PANEL A: Wild type (WT) neurons under basal conditions. PANEL B: WT neurons after 24 hours of TFW. PANEL C: Amyloid beta 1-40 transgenic neurons (Aβ40) under basal conditions. PANEL D: Aβ40 neurons after 24 hours TFW. PANEL E: Amyloid β 1-42 (Aβ42) neurons under basal conditions. PANEL F: Aβ42 neurons after 24 hours TFW. PANEL G: Bar plot of mean Fura-3 fluorescence intensity per cell (in semi-quntitative arbitrary values ±SE). WT Ctrl: corresponds to panel A; WT TFW: Panel B; Aβ40 Ctrl: Panel C; Aβ40 TFW: Panel D; Aβ42 Ctrl: Panel E; Aβ42 TFW: Panel F. One-way ANOVA with a Tukey post-hoc analysis was performed for statistical testing: * and # indicates statistical significance at p values of <0.05. Statistical significance was reached between Aβ40 TFW and WT TFW, Aβ40 TFW and Aβ40 Ctrl, Aβ42 TFW and WT TFW and Aβ42 TFW and Aβ42 Ctrl.

Figure 2

Stimulation of InsP₃R-mediated calcium release with bradykinin does not alter calcium response to TFW in cortical neurons transgenic for Aβ40 or Aβ42. Effect of 1-hour pretreatment with 10nM Bradykinin on intraneuronal Ca²⁺ ([Ca²⁺]_i. The cell permeant, Ca²⁺-specific probe, Fluo-3 was used to capture confocal images of 1-week old cortical neuronal cultures. Samples were excited at 488 nm with a Ar/Kr laser. Emissions were recorded with filters chosen for 510 nm. TFW was achieved with 24-hour incubations in glucose-free medium. PANEL A: Wild type (WT) neurons under TFW only conditions. PANEL B: WT neurons after 24 hours of TFW with 10nM Bradykinin. PANEL C: Amyloid β-40 transgenic neurons (Aβ40) under TFW conditions. PANEL D: Aβ40 neurons after 24 hours TFW with 10nM Bradykinin. PANEL E: Amyloid β 1-42 (Aβ42) neurons under TFW conditions. PANEL F: Aβ42 neurons after 24 hours TFW with Bradykinin. PANEL G: Bar plot of mean Fura-3 fluorescence intensity per cell (in semi-quntitative arbitrary values ±SE). WT (TFW): corresponds to panel A; WT (TFW+Brady.): Panel B; Aβ40 (TFW): Panel C; Aβ40 (TFW+Brady.): Panel D; Aβ42 (TFW): Panel E; Aβ42 (TFW+Brady.): Panel F. one-way ANOVA with Tukey post-hoc analysis was performed for statistical testing: * indicates statistical significance.

Figure 3

Blockade of InsP₃R-mediated calcium release from ER has no significant effects on calcium response to TFW in cortical neurons transgenic for Aβ40 or Aβ42. The cell permeant, Ca²⁺-specific probe, Fluo-3 was used to capture confocal images of 1-week old cortical neuronal cultures. Samples were excited at 488 nm with a Ar/Kr laser. Emissions were recorded with filters chosen for 510 nm. TFW was achieved with incubations in glucose-free medium. PANEL A: Wild type (WT) neurons under TFW only conditions. PANEL B: WT neurons after 24 hours of TFW with 1 μM XeC. PANEL C: Amyloid β 1-40 transgenic neurons (Aβ40) under TFW conditions. PANEL D: Aβ40 neurons after 24 hours TFW with 1 μM XeC. PANEL E: Amyloid β 1-42 (Aβ42) neurons under TFW conditions. PANEL F: Aβ42 neurons after 24 hours TFW with 45-minute incubation with 1 μM XeC. PANEL G: Bar plot of mean Fura-3 fluorescence intensity per cell (in semi-quntitative arbitrary values ±SE). WT (TFW): corresponds to panel A; WT (TFW+XeC): Panel B; Aβ40 (TFW): Panel C; Aβ40 (TFW+XeC): Panel D; Aβ42 (TFW): Panel E; Aβ42 (TFW+XeC): Panel F. One-way ANOVA with a Tukey post-hoc analysis was performed for statistical testing: * and ** indicate statistical significance at p values of <0.05. Statistical significance was reached between WT (TFW) and Aβ40TFW, Aβ40TFW+XeC, Aβ42TFW and Aβ42TFW+XeC, as well as WT TFW+XeC and Aβ40 TFW, Aβ40 TFW+XeC, Aβ42TFW and Aβ42 TFW+XeC.

Figure 4

Chelation of intracellular free Ca²⁺ in WT, Aβ40 and Aβ42 expressing neurons. The cell permeant, Ca²⁺-specific probe, Fura-3 was used to capture confocal images of 1-week old cortical neuronal cultures. Samples were excited at 488 nm with a Ar/Kr laser. Emissions were recorded with filters chosen for 510 nm. TFW was achieved with incubations in glucose-free medium. PANEL A: Wild type (WT) neurons under TFW only conditions. PANEL B: WT neurons after 24 hours of TFW and 45 minute incubation with 10 μM BAPTA. PANEL C: Amyloid β 1-40 transgenic neurons (Aβ40) under TFW conditions. PANEL D: Aβ40 neurons after 24 hours TFW and 10 μM BAPTA. PANEL E: Amyloid β 1-42 (Aβ42) neurons under TFW conditions. PANEL F: Aβ42 neurons after 24 hours TFW with 10 μM BAPTA. PANEL G: Bar plot of mean Fura-3 fluorescence intensity per cell (in semi-quntitative arbitrary values ±SE). WT +TFW: corresponds to panel A; WT +TFW+BAPTA: Panel B; AB40+TFW: Panel C; AB40+TFW+BAPTA: Panel D; Aβ42+ TFW: Panel E; Aβ42+TFW+BAPTA: Panel F. 1-way ANOVA with a Tukey post-hoc analysis was performed for statistical testing: * and ** indicate statistical significance at p values of <0.05. Statistical significance was reached between Aβ40+TFW and Aβ40+TFW+BAPTA; Aβ42+TFW and Aβ42+TFW+BAPTA.