Brain-Specific Basal and Novelty-Induced Alternations in PI3K-Akt and MAPK/ERK Signaling in a Middle-Aged AβPP/PS1 Mouse Model of Alzheimer’s Disease

Abstract

Although it is well established that insulin/IGF and BDNF signaling are dysfunctionally regulated in Alzheimer’s disease, there are very few studies documenting changes in major target proteins in different murine models of the disease. We investigated a panel of proteins in the PI3K-Akt and MAPK/ERK cascades in parietal cortex, dentate gyrus and CA1 in 13-month-old AβPP/PS1 transgenic mice to determine whether amyloid pathology is associated with basal dysregulation of these proteins or following exposure to novelty. The most striking effect we found was that there was little common regulation of proteins either by pathology alone or exposure to novelty across the three structures, suggesting dysfunctional mechanisms that occur simultaneously have important structure specificity. CA1 shared certain dysfunctional regulation of proteins in the MAPK/ERK cascade, but shared dysfunctional regulation of the PI3K/Akt cascade with the dentate gyrus. Changes in ERK/CREB in transgenic mice did not result in coordinated dysfunction of the downstream transcription factor, Egr1, as it was overexpressed in a normal manner following exposure to novelty. In the PI3K-Akt cascade, there was a flagrant increase in the levels of proteins associated with inflammation, such as NFκB, and structure specific regulation of proteins associated with autophagy, such as mTOR and FOXO1 and lack of regulation of Beclin-1. Finally, Beclin-1 was increased by novelty in wild-type mice but deficient in transgenic mice. Results are interpreted in terms of structure-specific dysfunctional regulation of signaling mechanisms associated with Alzheimer’s disease.

Keywords

Alzheimer’s disease autophagy Beclin 1 CA1 cortex dentate gyrus inflammation mTOR NFκB transgenic mice

INTRODUCTION

Until recently consensus suggested that the cognitive deficits associated with Alzheimer’s disease (AD) were largely due to the increasing load of amyloid plaques in the brain. This view was driven principally by the amyloid cascade hypothesis [1] and supported by data of several amyloid plaque forming transgenic mouse lines that showed memory impairment with age [2 –5]. This hypothesis prevailed despite accumulating data at both clinical and experimental levels suggesting that hard-core amyloid plaques may not be the major causal effect of the cognitive deficits associated with AD. Direct imaging of brain amyloid load by amyloid binding PET-ligands suggests that the amyloid load is close to maximal levels already at the time when the clinical diagnosis of AD is made [6, 7]. More damaging to the theory are results from clinical trials using compounds to reduce production or aggregation of amyloid, or to clear existing plaques from the brain, that have had a very poor success rate in improving cognitive performance (see [8, 9]).

The classic signature pathology of AD, accumulation of amyloid-β (Aβ) peptide into amyloid plaques, is also accompanied by other pathological features such as dysfunctional glucose metabolism [10], mitochondrial failure [11], oxidative stress [12], inflammation [13, 14], a dysfunctional autophagosome [15], synaptic failure [16], and an increase in apoptosis [17]. Currently, there is a growing consensus that amyloid accumulation and plaque formation are only triggers of multiple pathogenic cascades that ultimately account for neuronal dysfunction and eventual loss of neurons [18]. An important finding in this regard is the impairment in neurotrophic signaling in AD brains, including both the ligand and its receptor for insulin, insulin like growth factor (IGF-1 and IGF-2) [19], and brain-derived neurotrophic factor (BDNF) [20, 21]. Evidence also suggests that Aβ directly interferes with signal transduction through the insulin/IGF [22] and trkB [23] receptors.

The neurotrophin receptors couple two major signaling pathways for neuronal growth, survival, and plasticity, the PI3K-Akt and the MAPK/ERK pathways. There is now an abundance of data showing that Akt signaling is involved, at least, in homeostatic regulation of apoptosis through inhibition of proapoptotic transcription factors such as FOXO1 or activation of antiapoptotic proteins such as Bcl2 (see [24]); by regulation of the autophagosome through mTOR and Beclin [15, 25]; glucose metabolisms through GSK3β [26] and inflammation via NFκB [27]. Moreover, other evidence linking PI3K-Akt signaling to AD pathologies have shown that GSK3β is capable of phosphorylating Tau at pathological sites [28, 29]. In addition, the MAPK/ERK signaling pathway and downstream transcription factors are associated with activation of genomic programs involved in synaptic plasticity and the encoding of long-term memories [30]. As yet, it is not clear at what stage these signaling pathways might be disrupted in the AD pathology and whether they contribute substantially to cognitive deficits associated with the disease.

Objective

To explore some of these mechanisms, we used an established transgenic murine model of AD, the AβPPswe/PSdE9 (AβPP/PS1) transgenic mouse [31] at an age when their spatial memory deficit become manifest [32] and characterized phosphoregulation of member proteins of the signaling pathways in wild-type and transgenic mice. Further, to compare basal and behaviorally induced levels of these proteins, we exposed half of the animals to a novel environment with novel objects to explore, while controls remained in home cages throughout the study. We chose to use novelty as a means of environmentally-induced regulation as it effectively engages the medial temporal lobe memory systems as shown by activation of immediate early genes in rodents [33, 34] and human functional imaging studies [35, 36]. Furthermore, brain activation in responses to novel environments differs between individuals with AD versus elderly controls [37].

METHODS

Animals

The AβPPswe/PS1dE9 (AβPP/PS1) founder mice were obtained from Johns Hopkins University, Baltimore, MD, USA (D. Borchelt and J. Jankowsky, Dept. Pathology) and a colony was established at the University of Eastern Finland (Kuopio, Finland). These mice were generated by co-injection of chimeric mouse/human AβPPswe (mouse AβPP695 harboring a human Aβ domain and mutations K595N and M596L linked to Swedish familial AD pedigrees) and human PS1-dE9 (deletion of exon 9) vectors controlled by independent mouse prion protein promoter elements [31]. This mouse line was originally maintained in a hybrid C3HeJ C57BL6/J F1 background, but the mice used in the present study were derived from backcrossing to C57BL6/J for 14 generations. The housing conditions (National Animal Center, Kuopio, Finland) were controlled (temperature +22°C, light from 07:00 to 19:00; humidity 50–60%), and fresh food and water were available ad libitum. All behavioral tests were conducted between 9:00–16:00. The experiments were conducted according to the Council of Europe (Directive 86/609) and Finnish guidelines, and approved by the State Provincial Office of Eastern Finland.

Behavioral protocol

Male AβPP/PS1 and wild-type littermate mice, aged 12 months either remained in their home cages as controls (n = 6 per genotype) or were exposed to two novel cage environments (n = 6 per genotype). The first novel cage was a metal rat cage (49 cm 29 cm 20 cm) covered with a perforated transparent acrylic sheet. The bedding was made of shredded white tissue paper and odorized with lemon juice. In addition, two extra male mice visited the cage a couple of hours before the experiment. There were two mouse-size plastic objects to explore (a Duplo^R cow and a thin hair curler). The second cage was an open opaque acrylic cage (50 cm 35 cm 25 cm) filled with regular clean bedding odorized with birch extract and by a visit from two different male mice. There were three small objects to explore (a plastic cloth pin, a Duplo (R) flower, and a wooden ball, 1.5 cm in diameter). The exploration protocol was as follows: The first novel cage 10 min, home cage 60 min, the second novel cage 10 min, home cage 10 min, sacrifice. The home cage group was sacrificed directly from their home cages. This protocol ensured maximum sensory stimulation and active exploration throughout the two 10-min cage visits and a time window that allows detection of both newly translated immediate early gene products and phosphorylated proteins.

Sample preparation

At the end of the experiment, all mice were deeply anesthetized with pentobarbiturate - chloralhydrate cocktail (60 mg/kg each intraperitoneally) and perfused with 50 ml heparinized ice-cold 0.9 % saline (10 ml/min) for 3 min to rinse blood from brain circulation. The brain was then removed and the dorsal parietal cortex, CA1, and dentate gyrus were dissected on ice. Brain tissue was snap frozen in liquid nitrogen and stored at –70°C for biochemical assays.

Western blotting

Proteins were extracted from the frozen tissue in homogenizing buffer (10 mM Tris-HCl, 50 mM NaCl, 1% Triton X-100, 100 mM sodium pyrophosphate, 50 mM NaF, 5 mM ZnCl₂, 1 mM DTT, Complete Protease Inhibitor Cocktail (Roche)) using an automated sample lyser (Precellys, Bertin Technologies, France). Homogenized samples were incubated on ice for 30 min and insoluble material was removed by centrifugation (13,000 rpm for 15 min at 4°C). The proteins were quantified using a Bio-Rad Protein Assay based on the Bradford method and equalized. Samples were then denatured in the Laemmli’s loading buffer and boiled at 95°C for 5 min. Samples were separated on 8 to 12% (w/v) acrylamide gels, transferred onto nitrocellulose membranes (Bio-Rad) using a Transblot system (BioRad). Blots were blocked for 1 h in Tris-buffered saline (Euromedex) supplemented with 0.1% Tween 20 (TBS-T) and containing 2.5% BSA (Sigma). Incubation in the primary antibody (see Table 1 for the list of antibodies and dilutions) was performed overnight at 4°C. Following washing, the blots were incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit immunoglobulins for 1 h at room temperature. Blots were developed by hand using an enhanced chemiluminescence system (GE Healthcare) and exposed to Hyperfilm ECL (GE Healthcare). When needed, membranes were stripped of antibodies using Re-blot buffer (Millipore) and washed twice with TBS-T before reprocessing with another antibody. Phosphorylated forms of proteins were probed first, followed by the total protein. The efficacy of the stripping step was assessed by omitting the first antibody and verifying the lack of signals on the blot. Films were scanned and optical density of the bands was quantified using ImageJ or Genetools analyses. Total proteins were normalized to Actin and phosphoproteins were normalized to total proteins.

Immunohistochemistry

A separate group of four 12-month-old AβPP/PS1 male mice were used to assess the extent of amyloid pathology in the brain. The mice were deeply anesthetized with pentobarbiturate-chloral hydrate cocktail (60 mg/kg each) and perfused transcardially with 50 ml heparinized ice-cold 0.9% saline (10 ml/min) followed by 4% paraformaldehyde. Brains were transferred to a 30% sucrose solution overnight and finally stored in a cryoprotectant in –20°C for later immunohistology. The brains were cut on a sliding/freezing microtome into 35μm coronal sections. All sections were pretreated with sodium citrate solution at 80°C for 30 min. To visualize amyloid plaques select sections at the level of the hippocampus were stained for monoclonal mouse anti-human antibody W0-2 (Aβ4–10, 1:30000, Genetics, Schlieren, Switzerland). The sections were incubated overnight at room temperature on a shaker table. Following incubation, the sections were rinsed thoroughly with TBS-T and transferred to a solution containing the secondary antibody, biotinylated goat– anti mouse (1:1500; Vector Laboratories, Peterborough, UK). After 2 h of incubation the sections were rinsed three times and transferred to a solution containing Streptavidin (1:1000; GE Healthcare, Buckinghamshire, UK) for another 2 h. Visualization of Aβ plaques was achieved by incubation with DAB– Ni solution. Stained sections were mounted on gelatin-coated slides and dehydrated in alcohol series, cleared with xylene, and mounted in Depex.

To further localize novelty-induced changes in protein expression, a separate group of 8-month-old wild-type male mice were exposed to the two novel environments described above (n = 4) while a control group (n = 4) remained in their home cage. Under pentobarbiturate-chloralhydrate anesthesia, the mice were transcardially perfused with 50 mL of 10 mM NaF, a Ser/Thr phosphatase inhibitor [38]. The brains were then immersion fixed with 4% PFA, followed by 30% sucrose overnight and prepared for sections as described above.

For Egr1 and pCREB stainings, section were pretreated with hot (80°C) 0.05 M sodium citrate solution (pH 6.0). Thereafter the sections for Egr1, pCREB, and Beclin-1 stainings were treated the same. They were rinsed three times in TBS-T pH 7.6 and then treated with primary solution: Rabbit anti- Egr-1 (C-19) (1:1000, Santa Cruz Biotechnology, USA); rabbit anti-phospho-Creb, (ser133) (1:2500, Millipore, USA); rabbit anti- Beclin 1 (1:500, Novus Biologicals, USA) overnight at room temperature. Next day they were incubated with biotinylated goat anti-rabbit (1:500; Vector Laboratories, USA). After 2 h, sections were rinsed three times with TBS-T and transferred to tertiary antibody with avidin, StreptAvidin (GE Healthcare, 1:1000). After rinsing in TBS-T the sections were incubated for approximately 3 min in Ni-enhanced DAB solution.

Analysis and statistics

We wished to determine whether (a) the pathology induced in transgenic mice alone differentially regulated proteins and their phosphorylation in the two signaling pathways; and (b) the effect of exposure to novelty on protein levels and/or phosphorylation, as ability to detect novelty is essential for the formation of a number environmental and spatial memories associated with at least, the hippocampus. To test the effect of pathology, we normalized protein levels and their phosphorylation in all home caged mice to the mean of that of the wild-type mice and conducted t-test analyses. To test the effect of novelty we normalized proteins in wild-type and transgenic mice to their respective home caged counterparts and conducted t-test analyses. Finally to determine whether there was concerted regulation of proteins within the two signaling pathways to suggest a functional or dysfunctional link, we conducted correlational analyses. Protein levels were tested for normalcy using Kolmogorov-Smirnov; as the majority of proteins conformed we conducted Pearsons R correlations on those that did.

RESULTS

Non-quantitative observation of the mice during exploration suggested no discernable differences between the groups in terms of motor behavior and levels of exploration. Figure 1 shows a coronal section with typical amyloid load in the hippocampus and overlying cortex from a 12-month-old AβPP/PS1 transgenic mouse. In the hippocampus, there is substantial amyloid pathology in the molecular layer of DG and stratums oriens and lacunosum moleculare of CA1. Although the cell-body layers show little plaque deposition, this is typical, as early plaque deposition follows axonal terminal zones, in this case those from the entorhinal cortex. In contrast, CA3 is largely devoid of amyloid plaques.

Sample western blots for all proteins analyzed in the MAPK/ERK and PI3K-Akt signaling pathway, transcription factors, amyloid-β protein precursor (AβPP) and pTau-PHF in the three structures examined are presented in Figure 2 and all values in summary Table 2.

Regulation of signaling pathways in the parietal cortex

To assess the potential role of the AD pathology alone we analyzed protein levels and their activation in home caged control and transgenic mice. In the MAPK/ERK signaling pathway that leads to regulation of the transcription factors Egr1 and c-Fos, we found an increase in total ERK44 levels compared with wild-type mice (p < 0.01) with a corresponding reduction of basal phosphorylation. No change in ERK42 or CREB was observed (see Fig. 3A); however, there was a significant increase in the transcription factor, c-Fos (p < 0.05) but not Egr1. A substantial increase in AβPP (p < 0.001) and pTau-PHF (p < 0.05) was also observed but none of these increases in protein levels were regulated in a coordinated manner, except for a negative correlation between pERK44 and pTau (r = –0.89; p < 0.05), suggesting an inability of ERK44 to maintain normal basal phosphorylation may be associated with the increase in pTau.

Exposure to novelty had no effect on ERK44/42 basal levels or phosphorylation in either wild-type or transgenic mice. There was, however, a significant increase in CREB in both wild-type (p < 0.05) and transgenic mice (p < 0.05) with no discernible change in phosphorylation compared in either group. Both Egr1 (p < 0.05) and cFos (p < 0.001) were increased in wild-type mice, with a level that correlated with the increase in CREB (r = 0.75; p < 0.01). Although there was a slight increase in the transcription factors in transgenic mice, it was not significant (both p values > 0.05; see Fig. 3C). This suggests that the increased levels of CREB in transgenic mice was not significant enough to impact on the transcription factors as with the wild-type mice.

In the PI3K/Akt signaling pathway, despite no change in basal FOXO1 or BAD there was a substantial elevation of phosphorylation (pFOXO1: p < 0.05; pBAD: p = 0.05) as well as an increase in mTOR levels (p = 0.05) compared with wild-type mice (see Fig. 3B). In addition pFOXO1 showed a negative correlation with pTau (r = –0.854; p < 0.05) showing the less the phosphorylation of FOXO1 the greater the phosphorylation of Tau. In contrast a number of proteins were regulated by exposure to novelty in both wild-type and transgenic mice (Fig. 3D). The main results in wild-type mice show a significant substantial increase in phosphorylation of FOXO1 (p < 0.001) and BAD (p < 0.01) in the absence of change in total levels. In addition there was a substantial increase in both total NFκB (p < 0.05) and pNFκB (p < 0.05) and total mTOR (p < 0.01) and Beclin (p < 0.001) compared with home caged mice (see Fig. 2D), where there was significant positive correlated regulation between NFκB and mTOR (r = 0.81; p < 0.05); between pFOXO1 and mTOR (r = 0.99; p < 0.05) and pNFκB and the transcription factor, cFos (r = 0.97; p < 0.05), suggesting a potential convergence on novelty induced regulation of FOXO1, mTOR, and NFκB that could lead to an increase in cFos that did not occur in transgenic mice despite a significant increase in pNFκB (p < 0.01) compared to wild-type mice. On the contrary, apart from a similar increase in pNFκB, there was a decrease in BAD (p < 0.01) and mTOR (p < 0.05) proteins compared with home caged counterparts; and although there was a slight trend toward and increase in phosphorylation in both proteins it did not reach a level of significance.

Regulation of signaling pathways in CA1

Basal proteins levels in the MAPK/ERK signaling pathway in CA1 in transgenic mice were more susceptible to regulation than in the parietal cortex (Fig. 4B). An increase in total protein levels with a corresponding decrease in phosphorylation was observed in ERK44 (both p values < 0.05); ERK42, albeit the levels did not reach significance; and CREB (p < 0.05; pCREB: p < 0.0001), suggesting that the capacity for phosphorylation of these proteins is compromised. Moreover, there was no change in basal levels of Egr1 and c-Fos (see Fig. 4A) but negative correlations between ERK44 (r = –0.90, p < 0.05), ERK42 (r = –0.89, p < 0.05), and CREB (r = –0.84; p < 0.05) with Egr1, may impeach their ability to regulate Egr1 under basal conditions. As with the parietal cortex, both AβPP (p < 0.01) and pTAU-PHF (p < 0.05) were elevated compared with wild-type mice (see Fig. 4A). Exposure to novelty had no substantial effect in either wild-type or transgenic mice (Fig. 4C,D). As expected, exposure to novelty induced an increase in Egr1 (p < 0.001) in wild-type mice, but it also induced an increase in both Egr1 (p = 0.05) and c-Fos (p < 0.05) in transgenic mice, suggesting the transcription factors may be regulated via other means. Immunohistochemistry also confirms the increase in both wild-type and transgenic mice (Fig. 6). As in the parietal cortex novelty elicited no change in pTau-PHF in either wild-type or transgenic mice, however, it did increase AβPP levels (p < 0.05) in wild-type but not transgenic mice (see Fig. 4C).

In the PI3K-Akt signaling pathway basal FOXO1 (p < 0.05) and mTOR (p < 0.05) were elevated in transgenic mice with no significant change in levels of phosphorylation. In the absence of any change in total protein, there was a significant increase in pNFκB (p < 0.05). Exposure to novelty induced similar regulation of FOXO1, GSK3β, and NFκB in both wild-type and transgenic mice; where both showed an increase in total FOXO1 (Wt: p < 0.01; Tg; p < 0.05) with no change in phosphorylation (Fig. 4D). In contrast, in the absence of a change in total GSK3β there was a significant decrease in phosphorylation compared with home caged mice (Wt: p = 0.05; Tg: p < 0.001). Both groups showed a significant increase in pNFκB compared with home caged mice (Wt: p < 0.001; Tg: p < 0.05), although in transgenic mice pNFκB was substantially less than in wild-type mice. Finally, total level of BAD (p < 0.01) was increased in transgenic mice only, and as with the parietal cortex there was a significant increase in Beclin (p = 0.01) specific to wild-type mice (Fig. 4D), which was confirmed by immunohistochemistry (Fig. 6). Unlike the parietal cortex, novelty induced a significant increase in AβPP in wild-type mice (p < 0.05) that did not occur in transgenic mice (see Fig. 3B).

Regulation of signaling pathways in the dentate gyrus

In the MAPK/ERK signaling pathway (Fig. 5A), in contrast to both parietal cortex and CA1, there was no substantial change of either ERK isoforms or the transcription factors Egr1 and c-Fos; however, there was an increase in phosphorylation of CREB (p < 0.05). In keeping with regulation in the parietal cortex and CA1 there was also a significant increase in basal AβPP (p < 0.05) and pTau (p < 0.05). Exposure to novelty induced an increase in pERK44 in wild-type mice (p < 0.01), with no change in ERK42 or CREB (Fig. 5C, Fig. 6), but as expected Egr1 levels were increased (p < 0.001; Fig. 6), and confirmed with immunohistochemistry (Fig. 6). As with CA1, novelty also induced a significant increase in AβPP in wild-type mice (p < 0.05) that did not occur in transgenic mice (Fig. 5C). Interestingly, novelty induced an increase in total levels of ERKs (ERK42: p < 0.05), albeit ERK44 did not reach a level of significance in transgenic mice, with no change in phosphorylation. Despite the lack of phosphorylation of either ERK isoforms or CREB, the transcription factor Egr1 was increased in a parallel manner to that in the wild-type mice (Tg: p < 0.05), and c-Fos was increased abnormally in transgenic mice (p < 0.05).

In the PI3K-Akt signaling pathway (Fig. 5B), FOXO1 levels are increased (p < 0.01) with no significant change in phosphorylation in transgenic mice, in a similar manner to that observed in CA1. In contrast to both parietal cortex and CA1, where GSK3β were not affected by the pathology in transgenic mice, expression of GSK3β was significantly reduced (p < 0.05) whereas phosphorylation was significantly increased (p < 0.05). Finally, NFκB was highly phosphorylated in transgenic mice (p < 0.0001), as in CA1, albeit the increase was much greater in the dentate gyrus. Exposure to novelty induced a decrease in pBAD (p < 0.05) in wild-type mice, but an increase in both total NFκB (p < 0.01) and pNFκB (p < 0.05), pGSK3β (p < 0.05) and Beclin (p < 0.01) that did not occur in transgenic mice (Fig. 5D). In fact exposure to novelty induced opposing changes in transgenic mice; with a significant decrease in expression of BAD (p < 0.05) with no substantial change in phosphorylation; increased expression of GSK3β (p < 0.05) with no change in phosphorylation and; in the absence of regulation of expression of NFκB novelty induced a significant reduction in its phosphorylation (p < 0.05).

DISCUSSION

Despite well-documented decline in insulin/IGF and BNDF signaling in AD [19 –21], surprisingly few studies have investigated changes in the main downstream target proteins in the PI3K-Akt and MAPK/ERK pathways in various amyloid plaque producing mouse models. The ERK/MAPK signaling pathway constitutes a major conduit to the activation of the genomic response necessary for the encoding of long lasting memories [39], whereas the PI3K-Akt signaling pathway, besides being linked with memory consolidation [40], is also associated with a number of metabolic functions essential for neuronal viability that are dysfunctional in AD [41]. Dysfunction of both of these pathways may thus contribute to the cognitive deterioration associated with AD.

Our principle aim in analyzing a wide panel of proteins was (a) to use them to target potential deregulation of specific functions associated with amyloid pathology; and (b) to explore whether these were or were not dysfunctional in response to environmental induced regulation. We observed differential regulation of proteins associated with the pathology alone, changes in protein levels and their phosphorylation status upon exposure to novelty, but more importantly, also a number of highly specific effects of exposure to novelty on the pathology that may underlie impaired memory encoding in this model of AD.

Firstly, we were surprised to find very few changes associated with the pathology alone and few that were common to all three structures examined, only an increase in AβPP and pTau, most likely reflecting the pathology in all three structures. CA1 was the most severely effected region and shared more common changes in ERK/CREB signaling with the parietal cortex than the dentate gyrus, but more common change in the PI3K-Akt signaling pathway with the dentate gyrus. The plaque distribution in our AβPP/PS1 mouse model, however, show earliest and most extensive amyloid pathology occurs throughout the neocortex [42]; and in the hippocampus, the dentate gyrus shows the most severe pathology, followed by CA3 and CA1 [43]. Therefore we can only speculate that the results we find may not be determined by the insoluble plaque distribution but may reflect the toxic effects of soluble oligomeric species of amyloid that have been shown to disrupt synaptic function and are considered to be more detrimental to cognitive function [44 –47].

The most robust and consistent changes induced by the pathology alone, apart from AβPP and pTau (PHF-1) were an increased expression of proteins such as ERK44, CREB, mTOR, and FOXO1 that did not result in a corresponding increase in basal phosphorylation levels compared with wild-type littermates. This suggests that despite the increase in protein levels the capacity of the proteins to be activated is reduced. The increase in ERK/CREB finds some support from previous studies in a 3xTg-AD mouse model [48] and in humans during the early stages of the disease [49]; however, in stark contrast to our results, they showed an increase in phosphorylation of the proteins also. Although this kinase-signaling pathway is known to be regulated during learning, environmental enrichment, and memory encoding, we found little consistent regulation of proteins in any of the three structures; only protein that was globally regulated by novelty was Egr1. The lack of regulation of ERK or CREB in transgenic mice induced by novelty, most notably in CA1, may be due to them already having reached a saturated level such that no further regulation by environmental stimulation is possible. Together, this suggests that despite the dysfunctional increase in ERK44 levels in CA1 and parietal cortex, downstream regulation of the genomic response can be activated in a normal manner under the present conditions, potentially via other signaling mechanisms. Interestingly, c-Fos was abnormally upregulated by novelty in the dentate gyrus and CA1 of transgenic mice compared with wild-type mice. To date, evidence suggests that increased c-Fos in AD is associated with apoptotic mechanisms [50]. Moreover, it has been shown that abnormal regulation of c-Fos is induced by CREB in a 3xTg-AD mouse model of AD [51]. As regulation of cFos in transgenic mice is massively induced by exposure to novelty and overexpression of pCREB associated with the pathology alone only occurs in the dentate gyrus in our experiments, this may suggest the dentate gyrus is more susceptible to mechanisms that may be associated with the increase in death of newborn neurons shown in mouse models of AD [52 –54].

Altered regulation of proteins in the PI3K-Akt signaling pathway in AD is more complex as downstream targets of Akt are major mediators of energy and glucose homeostasis and apoptosis and are associated with autophagy. As well as being phosphorylated by Akt to inhibit their activation, proteins in this pathway can also be phosphorylated by other proteins to activate them under different cellular conditions; but they can also interact with each other in a feedforward or feedback manner. In the absence of any substantial change in Akt or its phosphorylation, we found differential regulation of proteins downstream of Akt either in association with the pathology alone or following exposure to novelty, changes that were specific to the three structures. For example, GSK3β was neither regulated by pathology or novelty in the parietal cortex; phosphorylation was decreased in CA1 by novelty, but in an identical manner to that of the wild-type mice. The fact that phosphorylation is downregulated in all mice in response to novelty suggests that the neuroprotective effect exerted by Akt is dysfunctional, and the protein is active; an effect that has been observed in older AβPP/PS1 transgenic mice but not young ones [55]. As proteins in the PI3-Akt signaling pathway are also dysfunctionally regulated during ageing, the decrease in phosphorylation may reflect an aging process. However, in the dentate gyrus expression of the protein was decreased by pathology, but upregulated by exposure to novelty; whereas phosphorylation was increased by pathology, but was incapable of undergoing further regulation by exposure to novelty as it was with the wild-type mice. The role of GSK3β in AD pathology is well established [41] and has been shown to undergo biphasic regulation, where during the early phases of the disease the proteins are hyperphosphorylated, reflecting neurotoxicity, and during the later stages of the disease they are hypophosphorylated, reflecting neuronal dysfunction and/or death [55]. Based on this, it would suggest our results reflect an early stage of neurotoxicity in the dentate gyrus and a potential age-related deficit in CA1.

We also found abnormal expression of FOXO1 proteins in CA1 and the dentate gyrus induced by the pathology alone; whereas there was an increase in pFOXO1 in the parietal cortex. When phosphorylated by Akt, it is extruded from the nucleus by chaperone 14-3-3 proteins for degradation by the proteosome in order to promote cell survival; however, when phosphorylated by stress-inducible kinases such as JNK or MST1, FOXO1 levels are increased, and this takes a more prominent effect over inhibition induced by Akt [56]. In AD the gene has been shown to be upregulated with increasing severity of tau pathology [57]. Therefore the increase in pFOXO1 in parietal cortex may reflect a protective mechanism that cannot be further regulated by novelty, but in CA1 and the dentate gyrus the increase in protein levels may be associated with cellular stress, which would be supported by the parallel increase in NFκB we observed.

NFκBp65 is routinely associated with inflammatory reaction and its activation can trigger both pro- and anti-inflammatory mechanisms [58, 59]. We found the protein was hyperphosphorylated in CA1 and the dentate gyrus in the transgenic mice. However, more recent data suggest that it also responds to plasticity inducing stimuli [60] and subserves cellular mechanisms for encoding certain forms of memory [60, 61], which would support the increased state of phosphorylation observed upon exposure to novelty in wild-type mice across all three structures. In CA1, although NFκB is hyperphosphorylated by novelty in the transgenic mice, it is significantly reduced in comparison to that observed in wild-type mice and is, in fact significantly reduced compared with home-caged mice, suggesting that the increase occurring in home-caged transgenic mice may compromise the potential for novelty-induced regulation as it is seen in the parietal cortex. However, the lack of change in CA1 and the decreased levels in the dentate gyrus might be determined by the pathological state in which it is regulated such that it can no longer respond to normal stimuli.

Autophagy, the degradation of organelles to recycle proteins under conditions of cellular malnutrition or for the removal of noxious and misfolded proteins, is regulated by a number of proteins, including Beclin-1 and mTOR. These proteins act to regulate autophagy where Beclin-1 is a key initiator of the process and mTOR terminates the process in a feedback manner under normal conditions [62 –64]. In AD, the autophagic response is reduced in association with reduced clearance of aggregated amyloid [65], coupled with a sustained increased expression of mTOR [66 –69] and a decrease in Beclin-1 in later stages of AD in humans, but not in mouse models overexpressing AβPP [70]. In keeping with these results, we found an increase in mTOR and no regulation of Beclin-1 in transgenic home-caged mice. Further evidence also shows that FOXO1 and FOXO3 work in concert with Beclin-1 to activate autophagy; whereas mTOR and NFκB inhibits it (see [71]), suggesting more widespread and complex network of proteins regulated by other dysfunctional mechanisms may be involved, particularly as we find a novelty-induced increase in Beclin in wild-type mice in all three structures that does not occur in transgenic mice. This may suggest an age-related adaptive mechanism to produce more proteins in response to environmental stimuli that is dysfunctional in AD.

Although the present report is not the first to examine the potential role of these two signaling pathways in AD, to our knowledge it is the first comprehensive study addressing changes in proteins in these pathways associated with the disease and how they are modulated during acute natural stimulation by exposure to novelty in three key structures associated with cognitive processing. Several studies using postmortem tissue from patients with AD have shown there is differential regulation of components of the PI3K-Akt signaling pathway, most importantly, dysfunction in the Akt-GSK3-mTOR axis [72 –75]; however, it is not clear whether dysfunctional regulation occurs during the very early stages of AD [75] or whether it is associated with late Braak stages [72 –74].

In summary, our results point to a number of important findings, albeit an obvious limitation of our study is that we were limited to only one age point, an age when brain amyloid pathology and associated neuroinflammation was fully developed. To be able to distinguish between direct effect of amyloid plaques versus other metabolic alterations due to AβPP and PS1 overexpression, further studies with young and aged AβPP/PS1 versus control mice would be helpful. Also further studies focusing on the location of protein regulation in relation to the distribution of amyloid would constitute an important next step. Nonetheless, we found in this study that deregulation of proteins by the pathology or in response to novelty showed little common pattern across the three structures examined in transgenic mice. In general, we found that regulation of proteins in the parietal cortex by pathology alone appeared to preclude their ability to be regulated by novelty. In CA1, although posing the most susceptible structure to the pathology alone, proteins were either little affected by exposure to novelty or showed similar change in both wild-type and transgenic mice. In contrast, the dentate gyrus appeared to be the most affected structure in transgenic mice in response to exposure to novelty. This may indicate that parallel dysfunctional processes may be occurring at the same time in different brain regions that lead to the ultimate neurodegeneration associated with AD as suggested by Herrup [18].

In addition, we found that most detrimental changes in terms of known functional attributes were associated with the PI3K-Akt signaling pathway rather than the MAPK/ERK signaling pathway, as deregulation of ERK/CREB in the parietal cortex and CA1 had little impact on the transcription factors, classically associated with initial activation of the genomic response. We cannot rule out, however, the possibility they are regulated via other signals. Deregulation of certain proteins in the PI3K-Akt signaling pathway are also known to be dysfunctional with age, and as age is the major risk factor for AD, it would be important to consider whether these changes are triggered first by age and then exacerbated by the pathology. Finally, a number of proteins differentially regulated in the PI3K-Akt signaling pathway in transgenic mice are associated with regulation of the autophagasome, either directly or indirectly, that may underpin an adaptive or dysfunctional mechanism associated with encoding environmental information. How these proteins act in concert to regulate the maintenance of normal autophagic function in an ever-increasing pathological state that is exacerbated by ageing will be a key function to investigate in future studies.

Footnotes

ACKNOWLEDGMENTS

This study was supported by EU 7th Framework Programme Grant HEALTH-F2-2007-201159.

Authors’ disclosures available online ().

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