Because serine phosphorylation of IRS-1 is a central feature in peripheral insulin resistance (22
), we initially looked for IRS-1pSerine in human AD brain tissue. Results demonstrate that AD brains present abnormally high levels of IRS-1 phosphorylated at serine residues 636/639 (IRS-1pSer636/639) compared with brains from non–cognitively impaired (NCI) subjects (Figure , A–C, and Supplemental Table 1; supplemental material available online with this article; doi:
), in line with a recent study that examined other pSer epitopes (4
). In NCI controls, IRS-1pSer636/639 immunoreactivity was almost exclusively detectable in cell nuclei, appearing as puncta of variable sizes (Figure A). In some cases, extranuclear immunoreactivity in neuronal cell bodies was also detected, but this was rare in subjects younger than 75 years. In contrast, in AD patients a high density of neurons with IRS-1pSer636/639 labeling in cell bodies and, occasionally, in proximal dendrites was found from the earliest ages studied (i.e., 51 years). This was most conspicuous in the hippocampal CA1 region (Figure B). In 20 of 22 (91%) age- and sex-matched pairs of AD and control cases, the density of CA1 neurons with extranuclear IRS-1pSer636/639 labeling was greater in the AD case (Wilcoxon signed-ranks test; W
= 239, P
= 0.0001; Figure C and Supplemental Table 1). Control specificity tests on the IRS-1pSer636/639 antibody showed that labeling in AD brain could be fully blocked by competition with synthetic phosphorylated immunogen (Supplemental Figure 1), but not with the corresponding non-phosphorylated peptide. These findings are in harmony with peripheral mechanisms leading to type 2 diabetes and support the idea that AD is characterized by CNS insulin resistance.
IRS-1pSer is increased in AD brain and in hippocampal neurons exposed to AβOs.
Memory impairment in AD is now attributed, at least in part, to the synaptotoxicity of AβOs (12
), which accumulate in AD brains (14
) and in animal models of AD (25
). Recent studies have implicated oligomers in neuronal insulin resistance (9
). Thus, we next investigated whether pathological IRS-1pSer could develop from the neuronal impact of AβOs. Using highly differentiated hippocampal neuronal cultures, we found that AβOs induced abnormal elevation in somatodendritic IRS-1pSer636 levels (Figure , D–F). These results provide a salient pathogenic basis to account for elevated IRS-1pSer levels in AD brains. Because phosphorylation of IRS-1 at additional serine residues (other than Ser636) is also known to account for insulin resistance in peripheral tissue (23
), we searched for neuronal IRS-1 phosphorylation at other epitopes. We found that IRS-1pSer616, IRS-1pSer312, and IRS-1pSer307 levels were also increased in hippocampal neurons exposed to AβOs (Figure , F and M). In parallel, and consistent with the expected insulin resistance associated with serine phosphorylation of IRS-1, oligomers inhibited physiological IRS-1 phosphorylation at tyrosine residue 465 (IRS-1pTyr465; Figure , G–I), an essential step in the IR-stimulated signaling pathway. Neurons targeted by AβOs exhibited increased IRS-1pSer levels, whereas non-attacked neurons showed low IRS-1pSer levels, as illustrated in Figure , J–L. Dysregulation of IRS-1 signaling, which we found to be prominent in AD brains (Figure B), is thus instigated by AβOs in central neurons.
Neuronal cultures used throughout our study were maintained in Neurobasal medium supplemented with B-27, an insulin-containing supplement, considered optimal conditions to preserve synapse health and function and to grow mature hippocampal cultures (27
). In order to determine whether the increase in IRS-pSer described above might be related to insulin coming from B-27, we used cultures grown in insulin-free B-27. As shown in Supplemental Figure 2, in insulin-free medium AβOs triggered very similar increases in IRS-1pSer levels. Results thus establish that IRS-1pSer is specifically triggered by oligomers rather than by a possible physiological action of insulin present in B-27.
To determine whether the insulin signaling defect found in cell culture experiments also occurs in vivo, we investigated the effect of AβOs on IRS-1pSer in the brains of non-human primates. To this end, 3 adult cynomolgus monkeys (Macaca fascicularis) received intracerebroventricular (i.c.v.) injections of oligomers. A sham-operated monkey was used as a control. Remarkably, we found that the monkeys that received i.c.v. oligomer injections presented elevated levels of neuronal IRS-1pSer636 in the hippocampus compared with the control monkey (Figure , A–G). Interestingly, IRS-1pSer636 levels were also increased in the temporal cortex of monkeys that received injections of AβOs, indicating that the impact of oligomers on IRS-1 signaling extends to other brain regions in addition to the hippocampus (Supplemental Figure 3). These results demonstrate that AβOs instigate elevated serine phosphorylation of IRS-1 in the brains of monkeys, establishing the in vivo and in situ relevance of our findings.
Elevated IRS-1pSer levels in the hippocampi of cynomolgus monkeys that received i.c.v. injections of AβOs and in APP/PS1 Tg mice.
We next examined IRS-1pSer levels in the brains of APPSwe,PS1ΔE9 (APP/PS1) mice, which express transgenes for human amyloid precursor protein (APP) bearing the Swedish mutation and a deletion mutant form of presenilin 1. IRS-1pSer636 and IRS-1pSer312 levels, but not IRS-1pSer307 levels, were increased in hippocampi of APP/PS1 Tg mice compared with WT mice (Figure H).
Non-phosphopeptide antibodies were used to detect total levels of IRS-1 and IRS-2 in our experimental models. We noted that distinct patterns of dendritic labeling were obtained for IRS-1 and IRS-2 (Figure , A, B, F, and G). No differences in total IRS-1 levels were observed in oligomer-treated cultures or in hippocampi of APP/PS1 mice (Figure , A–E). In contrast, total IRS-2 levels increased in hippocampal cultures exposed to AβOs, as revealed by both immunocytochemistry and Western blot analysis (Figure , F–I). This impact of oligomers may be related to the fact that IRS-2 is a negative regulator of memory formation, as shown by recent studies (28
). However, IRS-2 levels were significantly decreased in APP/PS1 Tg mice compared with WT mice (Figure J). Decreased levels of IRS-2 have also been found in AD brains (4
), suggesting that chronic exposure to AβOs may give rise to a compensatory mechanism aimed to decrease the negative impact of brain IRS-2 signaling on memory.
IRS-1 and IRS-2 levels in mature hippocampal neuronal cultures exposed to AβOs and in the hippocampi of Tg mice.
Previous studies have linked IRS-1 serine phosphorylation to JNK activation in diabetes and in obesity-related insulin resistance (22
). In peripheral tissue, IRS-1 is phosphorylated at Ser636 by p-JNK (30
). This prompted us to investigate the involvement of JNK in oligomer-induced IRS-1pSer in cell culture experiments. AβOs failed to induce IRS-1pSer in hippocampal neurons transfected with GFP-fused dominant negative JNK (DN JNK; Figure , A–D), indicating a role for JNK in neuronal insulin resistance. As a control, mock transfection with a plasmid containing only GFP had no protective effect (Figure C). Oligomer-induced accumulation of IRS-1pSer636 was also blocked by the pharmacological JNK inhibitor SP600125 (Figure , E–G and I). Moreover, oligomer-induced JNK activation was directly observed in hippocampal neuronal cultures (Figure J). Consistent with the involvement of JNK indicated by our results, a recent study showed that AβOs induce tau hyperphosphorylation and IRS-1 inactivation via JNK activation (31
JNK mediates AβO-induced IRS-1pSer.
Subsequently, we sought to determine whether p-JNK levels were elevated in the brains of APP/PS1 mice. We found a 4-fold increase in p-JNK levels in hippocampi of Tg mice compared to WT animals (Figure K), demonstrating that activation of JNK, first detected in cell culture experiments, occurs in vivo. No changes in levels of total JNK were found in hippocampal cultures exposed to oligomers or in hippocampi of APPS/PS1 Tg mice (Supplemental Figure 4). Future studies employing mice with knockout of IRS-1, JNK1/2, or JNK3 may provide additional insight into the mechanistic links between insulin resistance and AD.
In peripheral insulin resistance, JNK activity is known to be stimulated by TNF-α signaling (22
), and TNF-α levels are elevated in AD (32
). Interestingly, we found that abnormal IRS-1pSer636 triggered by AβOs was completely blocked by infliximab, a TNF-α neutralizing antibody (Figure , H and I). We further detected an increase in TNF-α levels in concentrated conditioned medium from hippocampal cultures exposed to AβOs (Figure L). No differences were found in levels of TNF-α receptor in cultured neurons exposed to oligomers or in hippocampi of Tg mice (Figure , M and N). The results suggest that oligomer-induced elevation in proinflammatory TNF-α levels triggers aberrant activation of JNK and, ultimately, serine phosphorylation of IRS-1.
We next analyzed levels of p-JNK in AD brains and found that the density of neurons with detectable levels of activated JNK was significantly increased in AD hippocampi (Figure , A–D), giving strong support to our proposal that activation of the JNK pathway plays a key role in AD pathology. Finally, we examined JNK activation in the brains of cynomolgus monkeys. Consistent with elevated IRS-1pSer levels, the 3 monkeys that received i.c.v. injections of AβOs presented elevated neuronal p-JNK levels in their hippocampi compared with the sham-operated monkey (Figure , E–I). Both cytoplasmic and nuclear p-JNK labeling were detected in NeuN-positive cells, but not in GFAP-positive cells (Figure , J–L), demonstrating neuronal specificity of JNK activation induced by oligomers. These results establish that abnormal activation of neuronal JNK is triggered by AβOs in the brains of non-human primates and support a key role of JNK in neuronal insulin resistance in AD.
Elevated p-JNK levels in AD brains and in hippocampi of cynomolgus monkeys that received i.c.v. injections of AβOs.
Aberrant activation of JNK has been linked to impaired axonal transport in neurological disorders (33
). Several neurodegenerative diseases, including AD, display axonal pathologies comprising defective transport and abnormal accumulation of proteins and organelles (34
). Because AβOs were recently shown to impair axonal transport in hippocampal neurons (35
), we further asked whether oligomer-induced JNK activation might be responsible for defects in axonal transport of dense core vesicles (DCVs) (see Supplemental Figure 5 for a scheme describing axonal transport measurements). Significantly, the JNK inhibitor SP600125 blocked axonal transport alterations induced by oligomers (Figure , Supplemental Table 2, and Supplemental Video 1), implicating JNK activation in impaired axonal transport in AD.
JNK mediates oligomer-induced impairment of axonal transport of DCVs in hippocampal neurons.
Double-stranded RNA-dependent protein kinase (PKR) and IκB kinase (IKK) are two stress-sensitive kinases that mediate serine phosphorylation of IRS-1 and are critical regulators of peripheral insulin resistance (36
). In an additional set of experiments, we examined whether PKR and/or IKK were also activated by AβOs. A selective PKR inhibitor completely blocked oligomer-induced IRS-1pSer636, IRS-1pSer312, and IRS-1pSer307 in hippocampal cultures (Figure , A–L). IKK was also found to be involved in oligomer-induced IRS-1pSer, as acetylsalicylic acid completely prevented abnormal IRS-1pSer636 (Figure , M–O and Q). Abnormally activated mTOR signaling has also been implicated in peripheral insulin resistance (40
). However, the mTOR inhibitor rapamycin had no effect on IRS-1pSer636 triggered by oligomers (Figure , P and Q), suggesting that mTOR is not involved in oligomer-induced serine phosphorylation of IRS-1. The involvement of PKR and IKK in AβO-induced IRS-1pSer provides additional evidence for a close parallelism between inflammation-associated brain insulin resistance in AD and chronic inflammation-induced insulin resistance in peripheral tissues in type 2 diabetes.
PKR and IKK, but not mTOR, mediate AβO-induced IRS-1pSer.
Stimulation of brain insulin signaling has been suggested as a promising approach to prevent synapse deterioration and memory decline in AD (41
). We thus next tested whether bolstering insulin signaling might also protect neurons from aberrant activation of the JNK/IRS-1pSer pathway triggered by AβOs. We examined the effects of insulin and exendin-4 (exenatide), an incretin hormone analog that activates the insulin signaling pathway through GLP1R stimulation (43
) and has been recently approved for treatment of diabetes. GLP1Rs are present and functional in cultured neurons as well as in rodent and human brains, and emerging evidence indicates that their stimulation regulates neuronal plasticity and cell survival (44
). Significantly, we found that both insulin and exendin-4 prevented the increase in IRS-1pSer636 (Figure , A–D) and the decrease in IRS-1pTyr465 levels (Figure , E–H) induced by oligomers. Exendin 9-39, a potent GLP1R antagonist and a competitive inhibitor of exendin-4, blocked the protective action of exendin-4, demonstrating that protection was specifically mediated by activation of GLP1Rs (Figure D). Because neuronal cultures used in our study were maintained in Neurobasal B-27, an insulin-containing supplement, it is possible that these results reflect to some extent crosstalk between exendin signaling and signaling initiated by insulin. Control experiments showed that exendin-4 or insulin alone (i.e, in the absence of oligomers) had no significant effects on IRS-1pSer levels (Figure D). Interestingly, insulin and exendin-4 also protected neurons from the above-described oligomer-induced impairment of axonal transport (Figure , I and J, Supplemental Tables 2 and 3, and Supplemental Videos 2 and 3). Because JNK dysregulation appears to underlie axonal transport defects in a number of neurodegenerative disorders in addition to AD (33
), this raises the possibility that prevention of aberrant JNK activation by bolstering insulin signaling might be beneficial in such disorders.
Exendin-4 prevents AβO-induced IRS-1pSer and p-JNK pathology and improves cognition in Tg mice.
Protection by insulin against AβO-induced neuronal damage has been shown to involve downregulation of oligomer binding sites (13
). Additional experiments thus aimed to determine whether exendin-4 also interferes with oligomer binding to neurons. Results showed that exendin-4 did not block oligomer binding (Supplemental Figure 6). Along with the results presented above, this indicates that GLP1R activation by exendin-4 prevents oligomer-induced impairment in IRS-1 signaling even when oligomers are attached to neurons. We also determined GLP1R levels in AβO-treated neuronal cultures and in hippocampi of AD Tg mice. No changes in levels of GLP1R were found in hippocampal cultures exposed to oligomers or in hippocampi of APPS/PS1 Tg mice (Supplemental Figure 7). Because brain insulin signaling may decline with aging and in AD (45
), exendin-4 may thus be more efficient than insulin in protecting neurons from the toxic impact of oligomers.
Finally, since exendin-4 readily crosses the blood-brain barrier and has been shown to facilitate hippocampal synaptic plasticity and cognition (44
), we asked whether systemic administration of a GLP1R agonist could enhance brain insulin signaling in APP/PS1 mice. Mice (13–14 months of age) were treated for 3 weeks with a daily intraperitoneal injection of exendin-4 (48
). Exendin-4–treated mice exhibited significant reductions in brain levels of IRS-1pSer636, IRS-1pSer312, and p-JNK compared with vehicle-treated animals (Figure , K and L). Interestingly, spatial memory in the Morris water maze task was improved by chronic exendin-4 administration to Tg mice. Exendin-4–treated APP/PS1 mice learned the task faster, with significantly reduced escape latencies observed on days 3 and 4 of training, compared with saline-treated mice (Figure , M and N). Furthermore, exendin-4–treated mice had improved memory retention, as indicated by a significantly longer time spent in the target quadrant during the probe trial conducted 24 hours after the last training session (Figure O). These data demonstrate the beneficial effect of exendin-4 on cognition in AD Tg mice. Interestingly, we further found that treatment with exendin-4 entailed reductions in brain levels of amyloid plaque load and soluble Aβ in the cerebral cortices of AD Tg mice (Figure , P–R).
Reported effects of peripheral exendin-4 administration include reduced plasma glucose levels and decreased food intake and body weight, and these could mimic the anti-aging effects of caloric restriction. We note that, for the duration of the behavioral experiments described above, comparable and slight weight losses were observed in all groups of mice analyzed, regardless of whether they received daily intraperitoneal injections of exendin-4 or saline (Supplemental Figure 8). This may be due to the exercise regime to which the animals were subjected during training and trials in the Morris water maze. We also note that, in insulin-secreting cells, exendin-4 inhibits JNK activation (49
), counteracts TNF-α–mediated apoptosis, and reverses inhibition of the IRS-1 pathway (50
). Taken together, our results suggest that exendin-4 restored impaired brain insulin signaling, decreased plaque load and soluble Aβ levels, and improved learning and memory.