Protection of synapses against Alzheimer's-linked toxins: insulin signaling prevents the pathogenic binding of Abeta oligomers

Abstract

Synapse deterioration underlying severe memory loss in early Alzheimer's disease (AD) is thought to be caused by soluble amyloid beta (Abeta) oligomers. Mechanistically, soluble Abeta oligomers, also referred to as Abeta-derived diffusible ligands (ADDLs), act as highly specific pathogenic ligands, binding to sites localized at particular synapses. This binding triggers oxidative stress, loss of synaptic spines, and ectopic redistribution of receptors critical to plasticity and memory. We report here the existence of a protective mechanism that naturally shields synapses against ADDL-induced deterioration. Synapse pathology was investigated in mature cultures of hippocampal neurons. Before spine loss, ADDLs caused major downregulation of plasma membrane insulin receptors (IRs), via a mechanism sensitive to calcium calmodulin-dependent kinase II (CaMKII) and casein kinase II (CK2) inhibition. Most significantly, this loss of surface IRs, and ADDL-induced oxidative stress and synaptic spine deterioration, could be completely prevented by insulin. At submaximal insulin doses, protection was potentiated by rosiglitazone, an insulin-sensitizing drug used to treat type 2 diabetes. The mechanism of insulin protection entailed a marked reduction in pathogenic ADDL binding. Surprisingly, insulin failed to block ADDL binding when IR tyrosine kinase activity was inhibited; in fact, a significant increase in binding was caused by IR inhibition. The protective role of insulin thus derives from IR signaling-dependent downregulation of ADDL binding sites rather than ligand competition. The finding that synapse vulnerability to ADDLs can be mitigated by insulin suggests that bolstering brain insulin signaling, which can decline with aging and diabetes, could have significant potential to slow or deter AD pathogenesis.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

ADDLs induce the removal of IRs from dendritic plasma membranes. Cultured hippocampal neurons were exposed to 100 nM ADDLs at 37 °C for 3 h followed by immunolabeling with anti-IRα (yellow). Nuclear staining (DAPI) is shown in blue. A and B show representative images from vehicle- and ADDL-treated cultures, respectively. C and D show high-magnification images of dendrites contained in the dotted rectangles indicated in A and B, respectively. (E) Quantification of IR immunofluorescence levels (see SI Methods) for cultures treated with vehicle (V), ADDLs (A), or scrambled Aβ peptide (Scr). (F) A representative image showing double labeling for ADDL binding (NU4 oligomer antibody; green) and IRα (red). (G) Surface abundance of IRs in hippocampal neurons exposed to vehicle or 100 nM ADDLs for 0.5 or 3 h, assessed by surface biotinylation (see SI Methods). Asterisk indicates statistically significant (*, P < 0.0002) decrease compared to vehicle-treated cultures.

Fig. 2.

CK2 and CaMKII mediate ADDL-induced loss of insulin and NMDA receptors. (A–D) Representative high magnification images of IRα labeling in dendrites from hippocampal neurons treated for 3 h with vehicle (A), 100 nM ADDLs (B), 100 nM ADDLs + 5 μM KN93 (C), or 100 nM ADDLs + 10 μM DMAT (D). (E) Quantification of IR (black bars) and NMDAR (white bars) immunofluorescence. Bars correspond to integrated immunofluorescence intensities (see SI Methods) from 3 experiments using independent cultures (30 images analyzed per experimental condition per culture). Asterisks indicate statistically significant (*, P < 0.05; **, P < 0.001) increases compared to ADDL-treated cultures.

Fig. 3.

Insulin prevents ADDL-induced pathological trafficking of IRs. (A–D) Representative IR immunofluorescence images from hippocampal neurons treated with vehicle (A), 100 nM ADDLs (B), 100 nM ADDLs + 100 nM insulin (C), and 100 nM ADDLs + 1 μM insulin (D). (E) Integrated IR immunofluorescence from 6 experiments using independent cultures (30 images analyzed per experimental condition per culture). Asterisk indicates statistically significant (*, P < 0.005) increase compared to ADDL-treated cultures. (F–K) Neurons treated for 3 h with 100 nM ADDLs alone (F–H) or with 100 nM ADDLs + 100 nM insulin + 10 μM rosiglitazone (I–K) followed by double labeling for IR (red) and ADDLs (green). (H and K) Merged images of IR and ADDL immunolabeling. Note the inverse correlation between ADDL binding and dendritic IRα immunoreactivities on dendritic process.

Fig. 4.

Insulin blocks neuronal ADDL binding and ADDL-induced oxidative stress. (A–D) Representative images from hippocampal neurons treated with vehicle (A), 100 nM ADDLs (B), 100 nM ADDLs + 100 nM insulin (C), and 100 nM ADDLs + 1 μM insulin (D). ADDL binding was detected using NU4 antibody. (E) Integrated ADDL immunofluorescence intensities from 6 experiments using independent cultures (25 images analyzed per experimental condition per culture). Asterisk indicates statistically significant (*, P < 0.01) decrease compared to ADDL-treated cultures. (F–H) Representative DHE fluorescence images in hippocampal cultures treated with vehicle (F), 1 μM ADDLs (G), or 1 μM insulin + 1 μM ADDLs (H). (I) Integrated DHE fluorescence. Asterisk indicates statistically significant (*, P < 0.007) differences relative to ADDL-treated cultures.

Fig. 5.

Insulin blocks ADDL-induced synapse loss. (A–C) Representative images from hippocampal neurons treated with vehicle (A), 100 nM ADDLs (B), or 100 nM ADDLs + 1 μM insulin (C) for 24 h. Spines were labeled using phalloidin (green). (D–F) Double-labeling high-magnification images of dendrites from neurons treated with vehicle (D), ADDLs (E), or ADDLs + insulin (F). Spines were labeled by phalloidin (green) and ADDLs were detected using the NU4 antibody (red). (G) Quantification of spine number per unit dendrite length. Asterisk indicates statistically significant difference (*, P < 0.001) relative to vehicle-treated neurons.

Fig. 6.

Protection by insulin requires IR tyrosine kinase activity. (A–C) Representative images from hippocampal neurons treated with 100 nM ADDLs (A), 100 nM ADDLs + 1 μM insulin (B), or 100 nM ADDLs + 1 μM insulin + 5 μM AG1024 (C). ADDL binding was detected using the NU4 anti-ADDL antibody. (D) Integrated ADDL immunofluorescence from 3 experiments using independent neuronal cultures (25 images analyzed per experimental condition per culture). Pound sign indicates statistically significant (#, P < 0.001) difference relative to ADDL-treated cultures. Asterisk indicates statistically significant (*, P < 0.007) differences relative to cultures treated with ADDLs + insulin.