An anti-diabetes agent protects the mouse brain from defective insulin signaling caused by Alzheimer's disease- associated Aβ oligomers

Abstract

Defective brain insulin signaling has been suggested to contribute to the cognitive deficits in patients with Alzheimer's disease (AD). Although a connection between AD and diabetes has been suggested, a major unknown is the mechanism(s) by which insulin resistance in the brain arises in individuals with AD. Here, we show that serine phosphorylation of IRS-1 (IRS-1pSer) is common to both diseases. Brain tissue from humans with AD had elevated levels of IRS-1pSer and activated JNK, analogous to what occurs in peripheral tissue in patients with diabetes. We found that amyloid-β peptide (Aβ) oligomers, synaptotoxins that accumulate in the brains of AD patients, activated the JNK/TNF-α pathway, induced IRS-1 phosphorylation at multiple serine residues, and inhibited physiological IRS-1pTyr in mature cultured hippocampal neurons. Impaired IRS-1 signaling was also present in the hippocampi of Tg mice with a brain condition that models AD. Importantly, intracerebroventricular injection of Aβ oligomers triggered hippocampal IRS-1pSer and JNK activation in cynomolgus monkeys. The oligomer-induced neuronal pathologies observed in vitro, including impaired axonal transport, were prevented by exposure to exendin-4 (exenatide), an anti-diabetes agent. In Tg mice, exendin-4 decreased levels of hippocampal IRS-1pSer and activated JNK and improved behavioral measures of cognition. By establishing molecular links between the dysregulated insulin signaling in AD and diabetes, our results open avenues for the investigation of new therapeutics in AD.

Figure 1. IRS-1pSer is increased in AD brain and in hippocampal neurons exposed to AβOs.

Higher density of IRS-1pSer636/639–positive neurons in CA1 hippocampal region from a 68-year-old AD patient (B) compared with a 73-year-old NCI control (A). IRS-1pSer636-639 in AD sometimes extends into apical dendrites (inset in B; scale bar: 20 μm). (C) In 22 matched pairs, AD patients presented a higher density of IRS-1pS636/639–positive cells and mean optical density (OD) of IRS-1pSer636-639 labeling/cell than NCI individuals (P = 0.0001). IRS-1pSer636 (D and E) or IRS-1pTyr465 (G and H) immunolabeling in hippocampal neurons exposed to vehicle (D and G) or 500 nM AβOs (E and H) for 3 hours (scale bar: 50 μm). Boxes under each panel show optical zoom images of selected dendrite segments (white dashed rectangles; scale bar: 10 μm). (F) Integrated IRS-1pSer and (I) IRS-1pTyr465 immunofluorescence levels determined from 4–8 experiments (independent cultures, 30 images analyzed/experimental condition/experiment). Veh, vehicle. (J and L) Representative images showing elevated IRS-1pSer636 (red, J) in a neuron targeted by AβOs (NU4 oligomer-specific antibody labeling; cyan, K, white arrow). (L) Merged image reveals a cell not attacked by AβOs (white arrowhead) with low IRS-1pSer636 levels. (M) Western blots of hippocampal cultures exposed to vehicle or 500 nM AβOs for 3 hours. Lanes were run on the same gel but were noncontiguous. Graph shows densitometric analysis for IRS-1pSer epitopes normalized by total IRS-1 level. Cyclophilin B (Cyclo B) was used as an additional loading control. *P < 0.05, ANOVA followed by Bonferroni post-hoc test, relative to vehicle-treated cultures.

Figure 2. Elevated IRS-1pSer levels in the hippocampi of cynomolgus monkeys that received i.c.v. injections of AβOs and in APP/PS1 Tg mice.

(A and B) Photomontage reconstruction of the hippocampus of a monkey that received i.c.v. injections of AβOs. The dashed rectangle indicates a region in the dentate gyrus that is shown enlarged in B, revealing IRS-1pSer636 immunoreactivity (green). DAPI staining is in blue. (C–F) IRS-1pSer636 immunoreactivities in the same segments of dentate gyri from a control (sham-operated) monkey (C) and 3 different monkeys that received injections of AβOs (D and F). Scale bars: 500 μm (A), 20 μm (B), 50 μm (C–F). (G) IRS-1pSer636 immunolabeling density (see Methods) from 20–31 images acquired in dentate gyri of sham-operated (S) or AβO-injected monkeys (M1–M3). *P < 0.001, ANOVA followed by Bonferroni post-hoc test, relative to sham-operated monkey. (H) Representative blots of IRS-1pSer636, IRS-1pSer307, and IRS-1pSer312 in hippocampi of APP/PS1 Tg mice. Lanes for IRS-1pSer636 and pSer312 were run on the same gel but were noncontiguous. Graph shows densitometric quantification of IRS-1pSer levels in Tg (n = 7) or WT mice (n = 5) normalized by total IRS-1 levels. Cyclophilin B was used as an additional loading control. *P < 0.02, Student’s t test, compared with WT.

Figure 3. IRS-1 and IRS-2 levels in mature hippocampal neuronal cultures exposed to AβOs and in the hippocampi of Tg mice.

Representative immunofluorescence images of total IRS-1 and total IRS-2 levels in hippocampal neurons exposed to vehicle (A and F) or 500 nM AβOs (B and G) for 3 hours. Graphs show integrated immunofluorescence levels of total IRS-1 (C) or IRS-2 (H). Results are from 3 experiments using independent cultures (20 images analyzed per experimental condition per culture). *P < 0.001, Student’s t test, relative to vehicle-treated cultures. Scale bars: 50 μm. (D and I) Immunoblots for total IRS-1 and IRS-2 levels in hippocampal neurons exposed to vehicle or 500 nM AβOs for 3 hours. Graphs show densitometric quantification of IRS-1 (D) and IRS-2 (I) levels normalized by cyclophilin B. (E and J) Total IRS-1 and IRS-2 levels, respectively, in hippocampal homogenates from APP/PS1 Tg (n = 7) or WT mice (n = 5). Lanes for IRS-2 (J) were run on the same gel but were noncontiguous. Graphs show densitometric quantification of IRS-1 or IRS-2 levels normalized by cyclophilin B immunoreactivity. *P < 0.05, Student’s t test, relative to vehicle-treated cultures.

Figure 4. JNK mediates AβO-induced IRS-1pSer.

(A) Representative image showing low IRS-1pSer levels (red) in a hippocampal neuron transfected with GFP-fused DN JNK (green; scale bar: 50 μm). (B) Higher-magnification image of dendrite segment (white box in A; scale bar: 10 μm). (C and D) integrated IRS-1pSer636 and IRS-1pTyr465 immunofluorescence levels, respectively. *P < 0.001 relative to vehicle-treated cultures, ^#P < 0.001 relative to AβO-exposed cultures; ANOVA followed by Bonferroni post-hoc test. (E–H) Hippocampal neurons exposed for 3 hours to vehicle (E), 500 nM AβOs (F), 10 μM SP600125 (SP) plus 500 nM AβOs (G), or 1 μg/ml infliximab (Inflix) plus 500 nM AβOs (H). Scale bar: 50 μm. (I) Integrated IRS-1pSer immunofluorescence levels determined from 4 experiments (independent cultures, 20 images analyzed/experimental condition/experiment). *P < 0.001 relative to vehicle-treated cultures, ^#P < 0.001 relative to AβO-exposed cultures; ANOVA followed by Bonferroni post-hoc test. Scr, scrambled Aβ_1–42 peptide. (J) Immunoblot of p-JNK in cultures exposed to 500 nM AβOs for 3 hours. (K) p-JNK levels in hippocampal homogenates from APP/PS1 Tg or WT mice. tJNK, total JNK. (L) TNF-α immunoblot in concentrated conditioned medium from cultures exposed to AβOs for 3 hours. (M and N) TNF-α receptor levels in cultures exposed to 500 nM AβOs for 3 hours and in hippocampal homogenates from APP/PS1 Tg (n = 7) or WT mice (n = 5), respectively. (M and N) Densitometric quantification normalized by cyclophilin B. Lanes in J and L–N were run on the same gel but were noncontiguous. *P < 0.02, Student’s t test.

Figure 5. Elevated p-JNK levels in AD brains and in hippocampi of cynomolgus monkeys that received i.c.v. injections of AβOs.

(A–D) Density of neurons in the same segment of hippocampal field CA1 with detectable JNK1/2pT183/pY185 in NCI controls (A) or AD patients (B). In control cases (A), immunoreactivity for p-JNK is generally limited to portions of cell nuclei (arrow), with only a small number of neurons showing immunoreactivity in the cytoplasm (arrowheads). Neurons with detectable cytoplasmic JNK1/2 pT183/pY185 also showed increased levels of this activated protein in AD (B and C) as indicated by higher mean OD in AD than in control samples (D). Graphs show mean values ± SD for neurons throughout CA1 in 22 matched pairs of control and AD cases. *P < 0.0001 relative to NCI individuals. (E–H) p-JNK immunoreactivities in the same segments of the dentate gyri from a sham-operated monkey (E) and 3 different monkeys that received i.c.v. injections of AβOs (F–H). (I) p-JNK immunolabeling density determined (see Methods) from 20–33 images acquired from dentate gyri of sham (S) or oligomer-injected monkeys (M1–M3). *P < 0.001 relative to the sham-operated monkey, ANOVA followed by Bonferroni post-hoc test. J, K, and L, enlarged images demonstrating both cytoplasmic and nuclear p-JNK labeling (J) of NeuN-positive cells (K). (L) p-JNK immunoreactivity was not associated with GFAP-positive cells. Scale bars: 50 μm (A, B, and E–H); 5 μm (J–L).

Figure 6. JNK mediates oligomer-induced impairment of axonal transport of DCVs in hippocampal neurons.

(A) Representative kymographs comparing axonal DCV transport in neurons exposed to vehicle, 500 nM AβOs, or 10 μM SP600125 plus 500 nM oligomers for 18 hours. (B) Quantification of total DCV transport flux. A minimum of 15 cells per condition from at least 2 independent cultures were analyzed. Transport parameters (organelle flux, velocity, run length) of DCVs were extracted from quantitative analysis of kymograph traces (see Methods). *P < 0.001 relative to vehicle-treated cultures; ^#P < 0.001 relative to cultures exposed to AβOs.

Figure 7. PKR and IKK, but not mTOR, mediate AβO-induced IRS-1pSer.

(A–Q) Hippocampal neurons were exposed for 3 hours to vehicle (A, E, I, and M), 500 nM AβOs (B, F, J, and N), 1 μM PKR inhibitor (inh) plus 500 nM AβOs (C, G, and K), 5 mM acetylsalicylic acid (ASA) plus 500 nM AβOs (O), or 0.1 μM rapamycin (Rap) plus 500 nM AβOs (P). Scale bars: 50 μm. Integrated IRS-1pSer636 (D and Q), IRS-1pSer307 (H), and IRS-1pSer312 (L) immunofluorescence levels determined from 4 experiments using independent neuronal cultures (20 images analyzed per experimental condition per experiment). *P < 0.001 relative to vehicle-treated cultures; ^#P < 0.001 relative to cultures exposed to AβOs; ANOVA followed by Bonferroni post-hoc test.

Figure 8. Exendin-4 prevents AβO-induced IRS-1pSer and p-JNK pathology and improves cognition in Tg mice.

IRS-1pSer636 or IRS-1pTyr465 immunofluorescence of hippocampal neurons exposed for 3 hours to vehicle (A and E), 500 nM AβOs (B and F), 300 nM exendin-4 plus AβOs (C and G). Scale bar: 50 μm. Integrated IRS-1pSer636 (D) and IRS-1pTyr465 (H) immunofluorescence (4 independent experiments; 20 images analyzed/experimental condition/culture). *P < 0.001, relative to vehicle-treated cultures; ^#P < 0.001, relative to AβO-exposed cultures; **P < 0.001, relative to cultures treated with exendin-4 plus AβOs. (I and J) Total DCV (I) and mitochondria (Mito; J) transport in neurons exposed to 500 nM AβOs, 300 nM exendin-4 plus AβOs, or 1 μM insulin plus AβOs for 18 hours (15 neurons/condition from ≥2 cultures were analyzed). *P < 0.001 relative to vehicle-exposed cultures; ^#P < 0.001 relative to AβO-exposed cultures. (K and L) IRS-1pSer636, IRS-1pSer312, and p-JNK levels in hippocampi from 13-month-old WT mice (n = 5), vehicle-treated Tg mice (n = 7), or exendin-4–treated Tg mice (n = 5). Lanes were run on the same gel but were noncontiguous. Graphs show densitometric quantification of IRS-1pSer and p-JNK levels (normalized by total IRS-1 and JNK, respectively). *P < 0.05 relative to WT; ^#P < 0.001 relative to vehicle-treated Tg mice; ANOVA followed by Bonferroni post-hoc test. (M–O) Exendin-4 improves spatial memory. (M and N) and memory retention (O) in the Morris water maze (10-month-old vehicle- or exendin-4–treated Tg mice were used; n = 12 in both groups). (P and Q) Brain amyloid plaque load (scale bar: 50 μm) and soluble Aβ levels (R) in vehicle- or exendin-4–treated APP/PS1 mice (n = 6, both groups; *P < 0.05, Student’s t test).

Figure 9. Proposed mechanism underlying disrupted brain insulin signaling in AD.

(A) AβOs stimulate TNF-α signaling, which activates the JNK pathway and, possibly, PKR and IKK pathways. Activation of these stress-sensitive kinases, which can also be triggered by endoplasmic reticulum stress (36), results in serine phosphorylation of IRS-1, blocking downstream insulin signaling. (B) Stimulation of insulin and GLP1 receptors blocks AβO-induced defects in insulin signaling. Binding of exendin-4 to GLP1 receptors and of insulin to IRs prevented activation of JNK, allowing physiological tyrosine phosphorylation of IRS-1 and stimulating downstream insulin signaling. In both panels, red arrows indicate inhibitory pathways and green arrows indicate stimulatory pathways of insulin signaling. I, insulin.