Amyloid Beta Oligomers-Induced Ca2+ Entry Pathways: Role of Neuronal Networks, NMDA Receptors and Amyloid Channel Formation

Abstract

The molecular basis of amyloid toxicity in Alzheimer's disease (AD) remains controversial. Amyloid β (Aβ) oligomers promote Ca²⁺ influx, mitochondrial Ca²⁺ overload and apoptosis in hippocampal neurons in vivo and in vitro, but the primary Ca²⁺ entry pathways are unclear. We studied Ca²⁺ entry pathways induced by Aβ oligomers in rat hippocampal and cerebellar neurons. Aβ oligomers induce Ca²⁺ entry in neurons. Ca²⁺ responses to Aβ oligomers are large after synaptic networking and prevented by blockers of synaptic transmission. In contrast, in neurons devoid of synaptic connections, Ca²⁺ responses to Aβ oligomers are small and prevented only by blockers of amyloid channels (NA7) and NMDA receptors (MK801). A combination of NA7 and MK801 nearly abolished Ca²⁺ responses. Non-neuronal cells bearing NMDA receptors showed Ca²⁺ responses to oligomers, whereas cells without NMDA receptors did not exhibit Ca²⁺ responses. The expression of subunits of the NMDA receptor NR1/ NR2A and NR1/NR2B in HEK293 cells lacking endogenous NMDA receptors restored Ca²⁺ responses to NMDA but not to Aβ oligomers. We conclude that Aβ oligomers promote Ca²⁺ entry via amyloid channels and NMDA receptors. This may recruit distant neurons intertwisted by synaptic connections, spreading excitation and recruiting further NMDA receptors and voltage-gated Ca²⁺ channels, leading to excitotoxicity and neuron degeneration in AD.

Keywords: Alzheimer’s disease; amyloid β oligomers; calcium.

Figure 1

Aß oligomers induce Ca²⁺ entry in rat hippocampal neurons. Rat hippocampal neurons in primary culture (4–5 DIV) were loaded with the Ca²⁺ fluorescent probe fura2/AM and then used for calcium imaging experiments. (A) Traces are representative recordings of F340/F380 ratio of individual neurons in the same microscopic field stimulated with amyloid ß_1–42 oligomers (2 μM) and NMDA (100 μM). Data are representative of 563 cells studied in 46 independent experiments. (B) Traces are representative recordings of individual neurons in the same microscopic field stimulated with amyloid ß_1–42 oligomers (2 μM) in medium lacking extracellular Ca²⁺ when indicated and NMDA (100 μM). Data are representative of 76 cells studied in three independent experiments. Colour lines reflect recordings of individual cells.

Figure 2

Effects on Ca²⁺ channel antagonists on Ca²⁺ responses induced by Aß oligomers in rat hippocampal neurons. Rat hippocampal neurons in primary culture (3–5 DIV) were loaded with fura2/AM and used for calcium imaging experiments. Traces are representative recordings of F340/F380 ratio of individual neurons in the same microscopic field stimulated with Aß_1–42 oligomers (2 μM) in the presence of 10 μM NMDA receptor blocker MK801 (A), 100 nM P/Q channel antagonist ω-agatoxin (B) and 2 μM nifedipine, an L-type channel antagonist (C). (D) Bars are mean ± SEM values of Δ Ratio from three to five experiments with 25–35 individual cells in each experiment. * p < 0.05 vs. control without antagonist. Gray bar corresponds to the effect of oligomers without any antagonist. Colour lines reflect recordings of individual cells.

Figure 3

Hippocampal neurons in primary culture display synchronous Ca²⁺ oscillations that are prevented by blockers of synaptic activity. Rat hippocampal neurons in primary culture (7 DIV) were loaded with fura2/AM and used for calcium imaging experiments. (A) Traces are representative recordings of the F340/F380 ratio of individual neurons in the same microscopic field in the absence and the presence of several antagonists, including the NMDA receptor blocker MK801 (10 μM), the Na⁺ channel antagonist tetrodotoxin (TTX, 500 nM), the L-type Ca²⁺ channel antagonist nifedipine (Nif, 2 μM) and the P/Q type Ca²⁺ channel antagonist ω-agatoxin (ω-Aga, 100 nM) added before stimulating the cells with NMDA 100 μM. Data correspond to seven individual neurons studied in four independent experiments. (B) Bars are mean ± SEM values of the oscillation index obtained in resting conditions during perfusion of the cells with the antagonists shown above. Data are from three to four independent experiments with 43, 28, 15 and 28 cells studied. * p < 0.05 vs. control. Colour lines reflect recordings of individual cells.

Figure 4

Ca²⁺ responses induced by Aß oligomers are enhanced in cells showing synchronous Ca²⁺ oscillations. Rat hippocampal neurons in primary culture were loaded with fura2/AM and used for calcium imaging experiments. Traces are representative recordings of the F340/F380 ratio of individual neurons cultured for 1 DIV (A) or 7 DIV (B) stimulated with medium lacking extracellular Mg²⁺, 2 μM Aß_1–42 oligomers and 100 μM NMDA. Bright-field images representative of hippocampal neurons at 1 and 7 DIV. Recordings correspond to 4 individual cells each panel, representative of 196 and 284 cells studied in 12 and 18 independent experiments, respectively. Bars show the mean ± SEM values of percent (%) of responsive cells to Aβ oligomers and NMDA (C) at 1 DIV and 7 DIV. Bars also show mean ± SEM values of Δ Ratio of the Ca²⁺ responses to Aβ oligomers and NMDA at 1 DIV and 7 DIV (D). * p < 0.05 vs. 7 DIV. Colour lines reflect recordings of individual cells.

Figure 5

The effects of antagonists on Ca²⁺ responses to oligomers depend on the presence of synchronous Ca²⁺ oscillations. Rat hippocampal neurons cultured either 1 DIV or 7 DIV are loaded with fura2/AM and used for calcium imaging experiments. Traces shown represent typical recordings of the F340/F380 ratio of individual neurons in the same microscopic field stimulated with Aß_1–42 oligomers (2 μM) in the absence (A,F) and the presence of the NMDA receptor antagonist 10 μM MK801 (B,G), 1 μM amyloid channel inhibitor NA7 (C,H), 100 nM P/Q type Ca²⁺ channel-specific antagonist ω-agatoxin (D,I) and 500 nM Na⁺ channel antagonist TTX (E,J). Traces correspond to four to six individual neurons representative of 26201373 cells studied in three to nine independent experiments. (K,L) Bars represent the mean ± SEM values of the Ca²⁺ response (% activation corresponding to the fraction of responsive cells multiplied by the Δ Ratio) to the oligomers in the absence and the presence of the antagonists at 1 DIV and ≥7 DIV, respectively. * p < 0.05 vs. control. Colour lines reflect recordings of individual cells.

Figure 6

The combination of MK801 and NA7 nearly abolished Ca²⁺ responses to amyloid ß oligomers. Rat hippocampal neurons cultured either 1 DIV or 7 DIV are loaded with fura2/AM and used for calcium imaging experiments. (A) Traces are representative recordings of RatioF340/F380 of individual neurons in the same microscopic field stimulated with Aß_1–42 oligomers (2 μM) in the presence of 10 μM MK801 (NMDA receptor antagonist) and 1 μM NA7 (amyloid channel antagonist) before stimulating them with 100 μM NMDA. Data are representative of 72 cells studied in six independent experiments. Bars represent the mean ± SEM values of % activation (fraction of responsive cells multiplied by the Δ Ratio) of similar experiments in which neurons cultured for 1 DIV (B) or 7 DIV (C) were stimulated with Aß_1–42 oligomers in the absence of antagonists, the presence of MK801 or NA7 alone, or added in combination. * p < 0.05 vs. control. ** p < 0.05 vs. MK801 or NA7 added alone. Colour lines reflect recordings of individual cells.

Figure 7

Only cell types expressing functional NMDA receptors also display Ca²⁺ responses to Aß oligomers. Different cell types lacking expression of functional NMDA receptors (HEK293 and HT29 cells) or expressing NMDA receptors (anterior pituitary cells and Jurkat T cells) were cultured, loaded with fura2/AM and used for calcium imaging experiments. Traces are representative recordings of fluorescence F340/F380 ratios of individual HEK293 cells (A), HT29 cells (B), mouse anterior pituitary cells (C) and Jurkat T cells (D) stimulated sequentially with Aß_1–42 oligomers (2 μM), NMDA (100 μM) and ATP (100 μM). Images below recordings correspond to representative pseudo color Ca²⁺ images of each cell type before and after stimulation with each agonist. Notice that dark blue corresponds to low intracellular Ca²⁺ concentration, whereas red denotes high intracellular Ca²⁺ concentration. Data correspond to 4–8 representative cells from 126, 132, 147 and 98 cells studied of each type in six, five, five and four independent experiments, respectively. Colour lines reflect recordings of individual cells.

Figure 8

Only GT-1 neurons responsive to NMDA are responsive to amyloid oligomers. GT-1 hypothalamic neurons were loaded with fura2/AM and subjected to calcium imaging. (A) Traces are representative recordings of the F340/F380 ratio of individual GT1 neurons stimulated sequentially with Aß_1–42 oligomers (2 μM), NMDA (100 μM) and depolarizing medium containing a high concentration of K+ (75 mM) to open voltage-gated Ca²⁺ channels. Pictures show pseudo color calcium images GT1 cells before (control) and after perfusion with Aß_1–42 oligomers. (B) Bars show mean ± SEM values of the percent of cells responsive to treatments. Data are representative of 118 cells studied in four independent experiments. Colour lines reflect recordings of individual cells.

Figure 9

Expression of NMDA receptor subunits in cells lacking NMDA receptors restores Ca²⁺ responses to NMDA but not to oligomers. HEK293 cells lacking endogenous expression of NMDA receptors were transfected with NMDA receptor subunits co-expressing GFP and subjected to calcium imaging. Traces are representative recordings of the F340/F380 ratio corresponding to individual transfected cells with NR1 (A), NR2A (B), NR2B (C) and the combinations NR1/NR2A (D) and NR1/NR2B (E), stimulated sequentially with Aß_1–42 oligomers (2 μM), NMDA (100 μM) and ATP (100 μM). Data are representative of 184, 153, 169, 345 and 367 cells studied in four to eight independent experiments for each condition. Pictures show GFP fluorescence images and calcium pseudo color images during stimulation with Aß_1–42 oligomers and NMDA. Notice that dark blue pseudo color corresponds to low cytosolic Ca²⁺ concentration, whereas red pseudo color denotes high cytosolic Ca²⁺ concentration. Colour lines reflect recordings of individual cells.