Calcium flux-independent NMDA receptor activity is required for Aβ oligomer-induced synaptic loss

Abstract

Synaptic loss is one of the major features of Alzheimer's disease (AD) and correlates with the degree of dementia. N-methyl-D-aspartate receptors (NMDARs) have been shown to mediate downstream effects of the β-amyloid peptide (Aβ) in AD models. NMDARs can trigger intracellular cascades via Ca(2+) entry, however, also Ca(2+)-independent (metabotropic) functions of NMDARs have been described. We aimed to determine whether ionotropic or metabotropic NMDAR signaling is required for the induction of synaptic loss by Aβ. We show that endogenous Aβ as well as exogenously added synthetic Aβ oligomers induced dendritic spine loss and reductions in pre- and postsynaptic protein levels in hippocampal slice cultures. Synaptic alterations were mitigated by blocking glutamate binding to NMDARs using NMDAR antagonist APV, but not by preventing ion flux with Ca(2+) chelator BAPTA or open-channel blockers MK-801 or memantine. Aβ increased the activity of p38 MAPK, a kinase involved in long-term depression and inhibition of p38 MAPK abolished the loss of dendritic spines. Aβ-induced increase of p38 MAPK activity was prevented by APV but not by BAPTA, MK-801 or memantine treatment highlighting the role of glutamate binding to NMDARs but not Ca(2+) flux for synaptic degeneration by Aβ. We further show that treatment with the G protein inhibitor pertussis toxin (PTX) did not prevent dendritic spine loss in the presence of Aβ oligomers. Our data suggest that Aβ induces the activation of p38 MAPK and subsequent synaptic loss through Ca(2+) flux- and G protein-independent mechanisms.

Figure 1

Blocking glutamate binding to NMDARs but not Ca²⁺ influx prevents dendritic spine loss in arcAβ-transgenic slice cultures. (a) Confocal images of dendrites from CA1 neurons in the stratum radiatum of non-transgenic and arcAβ-transgenic hippocampal slice cultures treated with NMDAR antagonist APV (100 μM). Scale bar: 5 μm. (b) APV treatment reverses the dendritic spine loss in arcAβ-transgenic cultures. n=10–13. (c) Confocal images of non-transgenic and arcAβ-transgenic cultures treated with NMDAR open-channel blocker memantine (1 μM) or MK-801 (30 μM). (d) Neither memantine nor MK-801 treatment reverses spine loss. n=11–13. (e) Confocal images of cultures treated with Ca²⁺ chelator BAPTA (2 mM) or vehicle (BAPTA solvent NaHCO₃). (f) BAPTA treatment does not affect spine loss in transgenic cultures. (g) Confocal images of cultures treated with AMPAR antagonist CNQX (10 μM). (h) CNQX treatment does not affect spine loss in transgenic cultures. n=11–15. (i) Western blot of lysate from non-transgenic cultures after synaptic activation—in the presence of the reagents used above—showing phosphorylated and total ERK levels. (j) APV (100 μM), memantine (1 μM), MK-801 (30 μM) and BAPTA (2 mM) pre-treatment prevent ERK phosphorylation after synaptic activation. All values are shown as mean±S.E.M.; ***P<0.001; two-tailed unpaired Student's t-test; significances show difference to the respective non-transgenic control (b–h) or to non-activated cultures (j). non-tg, non-transgenic; tg, arcAβ transgenic; Mem, memantine; BAP, BAPTA; MK, MK-801; p-ERK, phospho-ERK

Figure 2

Blocking glutamate binding to NMDARs but not Ca²⁺ influx prevents the loss of pre- and postsynaptic markers in arcAβ-transgenic cultures. (a) Representative western blot of cell lysates from non-transgenic or arcAβ-transgenic cultures after treatment with NMDAR antagonists APV (100 μM), memantine (1 μM) or MK-801 (30 μM). (b) Quantification of western blots. PSD-95 and synaptophysin levels are strongly reduced in arcAβ-transgenic cultures. APV treatment restores PSD-95 and synaptophysin signals back to control levels, whereas memantine and MK-801 have no effect. n=6. (c) Representative western blot of cell lysates from non-transgenic or arcAβ-transgenic slices after treatment with Ca²⁺ chelator BAPTA (2 mM) or vehicle (BAPTA solvent NaHCO3). (d) BAPTA treatment does not affect loss of synaptic proteins in transgenic cultures. n=6. (e) Aβ40 levels in the supernatant of arcAβ-transgenic cultures after treatment with NMDAR antagonists measured by MSD. Aβ40 production is not influenced by any NMDAR antagonist. Aβ levels were corrected by protein levels from lysates. n=3. All values are shown as mean±S.E.M. (*P<0.05, **P<0.01, ***P<0.001; two-tailed unpaired Student's t-test; significances indicate differences to the respective non-transgenic control). non-tg, non-transgenic; tg, arcAβ-transgenic; Mem, memantine; MK, MK-801

Figure 3

p38 MAPK is activated in arcAβ-transgenic cultures and mediates spine loss. (a) Representative western blot showing phosphorylated (active) and total p38 MAPK in lysates non-transgenic or arcAβ-transgenic slices after treatment with different NMDAR antagonists. (b) Quantification shows increased levels of phosphorylated p38 in arcAβ-transgenic cultures. The increased amounts of phospho-p38 were reduced to control levels by APV (100 μM) but not by memantine (1 μM) or MK-801 (30 μM) treatment. The non-transgenic untreated control was set to 1. n=6. (c) Confocal images of dendrites from CA1 neurons in the stratum radiatum of non-transgenic and arcAβ-transgenic hippocampal slice cultures treated with p38 MAP kinase inhibitor SB239063 (20 μM). Scale bar: 5 μM. (d) SB239063 treatment reverses the dendritic spine loss in arcAβ-transgenic cultures. n=14-16. (e) Representative western blot showing activated ERK (p-ERK) and activated p38 (p-p38) in non-transgenic or arcAβ-transgenic slices after synaptic activation with bicuculline and 4-aminopyridine. (f) Quantification shows increased p-ERK levels after synaptic activation, independent of transgenic background. Synaptic activation does not affect p-p38 levels. n=5. All values are shown as mean±S.E.M. (*P<0.05, **P<0.01, ***P<0.001; two-tailed unpaired Student's t-test; significances indicate differences to the respective non-tg control; for p-ERK/total ERK significances indicate differences to the respective non-activated culture). non-tg, non-transgenic; tg, arcAβ transgenic; Mem, memantine; MK, MK-801; p-ERK, phospho-ERK; p-p38, phospho-p38 MAPK; syn, synaptic activation

Figure 4

Oligomeric Aβ-induced synaptic loss is prevented by APV but not by memantine, MK-801 or BAPTA treatment. (a) Confocal images of dendrites from CA1 neurons in the stratum radiatum of non-transgenic slice cultures treated with oligomeric Aβ (500 nM) or scrambled Aβ (500 nM) and NMDAR antagonist APV (100 μM). Scale bar: 5 μM. (b) APV treatment prevents Aβ oligomer-induced dendritic spine loss. n=13–17. (c) Confocal images of non-transgenic cultures treated with oligomeric Aβ (500 nM) and NMDAR antagonist memantine (1 μM). (d) Memantine treatment does not prevent spine loss. n=12–17. (e) Confocal images of cultures treated with oligomeric Aβ (500 nM) and NMDAR antagonist MK-801 (30 μM). (f) MK-801 treatment does not prevent spine loss. n=13–15. (g) Non-transgenic cultures treated with Aβ oligomers (500 nM) and Ca²⁺ chelator BAPTA (2 mM) or vehicle (BAPTA solvent NaHCO₃). (h) BAPTA does not prevent spine loss caused by oligomeric Aβ. n=11–15. (i) Representative western blot of cell lysates from slices after treatment with Aβ oligomers (500 nM) and Ca²⁺ chelator BAPTA (2 mM). (j) BAPTA does not prevent reduction in PSD-95 or synaptophysin levels after Aβ oligomer treatment. n=6. (k) SDS gel showing oligomeric Aβ preparations and scrambled Aβ after silver staining (right panel) and western blot stained with 6E10 antibody (left panel). Monomers, tri- and tetramers are observed in the oligomeric preparation, whereas scrambled Aβ only shows monomers. (l) LDH assay showing no toxicity of Aβ oligomer treatment (500 nM) compared with scrambled Aβ. n=6. Values are shown as mean±S.E.M. (*P<0.05, **P<0.01, ***P<0.001; two-tailed unpaired Student's t-test). Scr. Aβ, scrambled Aβ; AβO, oligomeric Aβ; Mem, memantine

Figure 5

Oligomeric Aβ-induced synaptic loss is not prevented by treatment with PTX. (a) Confocal images of dendrites from CA1 neurons in the stratum radiatum of non-transgenic slice cultures treated with oligomeric Aβ (500 nM) or scrambled Aβ (500 nM) and G protein inhibitor PTX (500 ng/ml). (b) PTX treatment does not prevent Aβ oligomer-induced dendritic spine loss. n=12. Values are shown as mean±S.E.M. (**P<0.01, ***P<0.001; two-tailed unpaired Student's t-test). Scr. Aβ, scrambled Aβ; AβO, oligomeric Aβ; PTX, pertussis toxin