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Extracellular-signal regulated kinase (ERK) signaling is critical for memory and tightly regulated by acute environmental stimuli. In Alzheimer disease transgenic models, active ERK is shown to first be increased, then later reduced, but whether these baseline changes reflect disruptions in ERK signaling is less clear. We investigated the influence of the familial Alzheimer’s disease transgene APPsw and β-amyloid peptide (Aβ) immunoneutralization on cannulation injury-associated (i.c.v. infusion) ERK activation. At both 12 and 22 months of age, the trauma-associated activation of ERK observed in Tg− mice was dramatically attenuated in Tg+. In cortices of 22-month-old non-infused mice, a reduction in ERK activation was observed in Tg+, relative to Tg− mice. Intracerebroventricular (i.c.v.) anti-Aβ infusion significantly increased phosphorylated ERK, its substrate cAMP-response element-binding protein (CREB) and a downstream target, the NMDA receptor subunit. We also demonstrated that Aβ oligomer decreased active ERK and subsequently active CREB in human neuroblastoma cells, which could be prevented by oligomer immunoneutralization. Aβ oligomers also inhibited active ERK and CREB in primary neurons, in addition to reducing the downstream post-synaptic protein NMDA receptor subunit. These effects were reversed by anti-oligomer. Our data strongly support the existence of an APPsw transgene-dependent and Aβ oligomer-mediated defect in regulation of ERK activation.
Alzheimer’s disease (AD) is clinically characterized by progressive cognitive impairment. Cognitive deficits in AD are widely believed to result from progressive synaptic dysfunction and neurodegeneration initiated by aggregated β-amyloid peptide 1–42 (Aβ42). Recently, soluble Aβ oligomers, including dimers, trimers, and dodecamers (12-mer or Aβ*56) have been proposed as key forms of Aβ causing cognitive deficits in animal models (Cleary et al. 2005; Lesne et al. 2006). Soluble Aβ concentrations predict synaptic changes (Lue et al. 1999) and correlate with the severity of dementia in AD patients (McLean et al. 1999). Antibody-mediated Aβ oligomer reduction can correct defects in synaptic plasticity (Klyubin et al. 2005) and reduce tau phosphorylation (Klyubin et al. 2005; Ma et al. 2006), another pathological hallmark of AD.
Although multiple mechanisms for Aβ peptide-induced memory deficits have been hypothesized including effects on tau phosphorylation and tangle formation, there is considerable evidence implicating a role for Aβ-induced disruption of kinases critical for memory (Zhu et al. 2002). For example, studies of human mental retardation syndromes have shown that extracellular-signal regulated kinases (ERKs) are critical for human learning (Costa et al. 2002). They are also known to contribute to molecular information processing in dendrites, to stabilize structural changes in dendritic spines and to interact with scaffolding and structural proteins at the synapse (Sweatt 2004). However, aberrant over-expression of ERK can lead to cell death (Zhuang and Schnellmann 2006). ERK shows stage-dependent abnormalities in mRNA and protein expression in AD (Webster et al. 2006) and AD models (Dineley et al. 2001). Although transient ERK activation plays important roles in memory-related processes, persistent activation can mediate NMDA-related excitotoxicity (Amadoro et al. 2006). Therefore, either hyper- or hypoactivation of ERK could contribute to disease pathways.
It has been reported that in early AD, there is extensive activation of ERK in astroglial cells in the white matter, while in advanced AD, there is reduced activation showing a strong inverse correlation with Braak stage and the Blessed score for cognition (Webster et al. 2006). However, ERK activation is not necessarily protective. For example, active ERK is a tau kinase that is elevated during the initial stages of neurofibrillary degeneration in the projecting neurons in the transentorhinal region. Nevertheless, ERKs causal role in neurofibrillary tangle formation remains unclear as many neurons with the highest amounts of ERK/mitogen-activated protein kinase immunoreactivity do not appear vulnerable to neurofibrillary tangles (Hyman et al. 1994). Interestingly, at late stages (although some ERK is still apparent in astrocytes), ERK activation is suppressed relative to early stage and normal controls in neuronal cell bodies and dystrophic neurites (Webster et al. 2006). In summary, studies in AD brain suggest stage-dependent ERK activation followed by loss of active ERK.
Similar to AD, the amyloid precursor protein (APP) transgenic Tg2576 model shows early stage ERK activation, while at later stages ERK is reduced (Dineley et al. 2001). Sustained activation of ERK was also observed in brains of an APP transgenic rat model with significant intraneuronal Aβ accumulation (Echeverria et al. 2004). In vitro, it has been demonstrated that sustained ERK activation is related to cell death after Aβ treatment in neurons and PC12 cells (Dineley et al. 2001; Jang and Surh 2005). Conversely, ERK activation can be reduced by γ-secretase inhibitor treatment of APPsw transfected cells (Echeverria et al. 2005). Abnormally sustained activation may contribute to cell death in other amyloid diseases like familial amyloidotic polyneuropathy (Monteiro et al. 2006) and work via multiple mechanisms, including pro-apoptotic events upstream of caspase 3 or via suppression of the anti-apoptotic signaling molecule Akt (Zhuang and Schnellmann 2006).
The known importance of ERK on dendrites and memory is consistent with the hypothesis that ERK hypoactivation in AD contributes to cognitive decline. Under certain conditions, Aβ or its fragments have been shown to inhibit ERK (or downstream cAMP-response element-binding protein; CREB) in neuroblastoma cells (Daniels et al. 2001), endothelial cells (Magrane et al. 2006), hippocampal neuron preparations (Xie 2004), or after Aβ25–35 i.c.v. injection into rats (Jin et al. 2005). Different Aβ aggregates may have different effects depending on the system, for example high levels of fibrillar Aβ in PC12 cells inhibited ERK, while other forms of Aβ increased ERK activation (Echeverria et al. 2005). In contrast, fibrillar Aβ stimulated ERK, while low (100 nmol/L) soluble oligomers initially stimulated but later down-regulated ERK in hippocampal slice cultures (Bell et al. 2004).
In view of the conflicting results with Aβ’s stimulatory or inhibitory effects on ERK in culture systems with different Aβ preparations and biphasic effects, we have focused on chronic in vivo studies. We decided to assess ERK regulation using injury which is known to stimulate ERK. Trauma or ischemia can induce ERK activation (Shackelford and Yeh 2006), possibly via growth factors that are induced by injury including transforming growth factor beta1 (Logan et al. 1992), insulin-like growth factor (IGF), and brain derived neurotrophic factor (Kazanis et al. 2004) all which are known to stimulate ERK (Willaime-Morawek et al. 2005; Johnson-Farley et al. 2006). To determine if AD models showed disruption in regulation of ERK, we examined whether trauma induction of ERK was altered by APPsw transgene and corrected by Aβ oligomer neutralization. We also investigated the impact of Aβ oligomer on downstream CREB and NMDA receptor (NMDAR) subunit (NR2B).
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We demonstrate that regardless of baseline ERK phosphorylation levels, ERK activation can be impaired by APP transgene, and this effect is mediated by Aβ oligomers. Previously age-dependent alterations in Tg2576 were evaluated in the hippocampus and baseline pERK was found to be increased as early as 4 months of age in the dentate gyrus prior to Aβ deposition (Dineley et al. 2001) and in the absence of detectable synaptic and neuronal loss (Irizarry et al. 1997). ERK hyperactivation has been linked to APP transgene-dependent up-regulation of the alpha7 nicotinic receptor and Aβ signaling to ERK (Dineley et al. 2001). We evaluated age-dependent changes in the cortex to see if parallel changes occurred. Unlike in the hippocampus, our results show that cortical pERK was not reduced or elevated at 12 months. Consistent with a previous report, we also observed a trend for increased baseline pERK in CA1 of the hippocampus (Dineley et al. 2001).
Infusion-induced pERK at both 11–12 months of age and at 16–18 months of age in wild-type (Tg−) mice, but these robust responses were either not present or blunted in APP Tg+ mice, demonstrating an APP transgene-mediated ERK dysregulation. To address the role of Aβ, we infused anti-Aβ antibodies. Infusion of antibodies against Aβ increased pERK and pCREB at both ages, essentially correcting the suppressed response to infusion/injury. Thus, the significant increase in ERK and CREB observed with anti-Aβ antibody treatment cannot be viewed simply as a correction of a pre-existing baseline deficit, but rather as a correction of a blunted ERK/CREB response to CNS infusion-induced factors. Trophic factors are one possibility because subtoxic levels of Aβ oligomers have been reported to blunt the ERK/CREB response to trophic factors including nerve growth factor (Chromy et al. 2003) and brain derived neurotrophic factor (Tong et al. 2004). IGF-1 is one example of a persistent cannulation-induced trophic factor (Garcia-Estrada et al. 1992). Our results showing that IGF induced ERK in SY5Y neuroblastoma is attenuated by Aβ oligomers further support the idea that Aβ oligomers may diminish ERK signaling. While we found the Tg+ mice had lost the ERK response to injury-induced stimulation, our results are consistent with the hypothesis that oligomers in APP Tg+ mice reduce the ERK response to injury-induced trophic factors.
In contrast to 12 month mice, significant cortical ERK and CREB deficits develop in older Tg2576 mice. These were further exacerbated by an omega-3 fatty acid depleted diet (not shown) that also increased selected synaptic deficits (Calon et al. 2005). Our in vivo and in vitro immunoneutralization data with anti-Aβ and anti-oligomer antibodies data suggest that these deficits are due to Aβ oligomers. Preparations enriched in Aβ oligomers, including trimer, 12-mer, and 24-mer, significantly down-regulated the level of active ERK (1.5 and 24 h) and active CREB (1.5 and 12 h). The conformation-specific anti-oligomer antibody, A11, ameliorated this Aβ-induced inhibition of active ERK and CREB in human SH-SY5Y cells and rat primary hippocampal neurons. As A11 antibody fails to detect monomer, low molecular weight oligomers, or fibrils (Kayed et al. 2003), active ERK and CREB losses may be attributed to one or more high molecular weight Aβ oligomer species.
Our findings of Aβ-dependent ERK suppression are distinct from data obtained with chronic incubation with aged or fibrillar Aβ which caused persistent ERK activation in hippocampal slice cultures (Bell et al. 2004) and aged hippocampal neurons (Anderson and Ferreira 2004). However, they are consistent with experiments using freshly prepared Aβ42 containing predominantly hexameric oligomer species that resulted in an initial ERK activation followed by ERK down-regulation at 16 h (Bell et al. 2004). Aβ or Aβ25–35 induced suppression of ERK or its downstream intermediate CREB have also been observed in neuroblastoma cells (Daniels et al. 2001), endothelial cells (Magrane et al. 2006), hippocampal preparations (Xie 2004), and in rats after injection of Aβ25–35 (Jin et al. 2005). In vitro, Aβ effects on ERK activation may reflect dose, aggregation status and treatment duration. Because we used 250–500 nmol/L of pre-aggregated Aβ42 with a predominant 12-mer (dodecamer) species to treat cells, we may have obtained a more rapid ERK down-regulation, consistent with the hypothesis that the Aβ 12-mer is a very potent inhibitor of mechanisms underlying cognitive function.
Large (> 50%) pCREB deficits have been reported in hippocampus of AD patients (Yamamoto-Sasaki et al. 1999). Although neither pERK nor pCREB show early hippocampal deficits in Tg2576 mice (Dineley et al. 2001), pCREB deficits occur in other regions. For example, we observed pCREB deficits in 16-month-old Tg2576 mouse entorhinal cortex where it was restored by treatment with curcumin (Cole et al. 2003), a compound that blocks Aβ oligomer and fibril formation (Yang et al. 2005) and reduces cognitive deficits induced by chronic soluble Aβ infusion (Frautschy et al. 2001). Defects in CREB signaling are likely Aβ-dependent. They are relevant to cognitive function because in bigenic mutant APP × presenlin 1 transgenic mice, increasing cAMP/protein kinase A (PKA)/CREB signaling with rolipram ameliorated deficits in LTP and cognitive function (Gong et al. 2004). PKA activity has been reported to be reduced by Aβ aggregates in vitro and in bigenic APP × presenlin 1 mice secondary to a 30–50% decrease in Uch-L1 hydrolase, but 9–12 month Tg2576 (used in our experiments) have only about a 15% decrease in Uch-L1 and no reported change in PKA activity or protein (Gong et al. 2006). Whether involving ERK or PKA or both, data from three very different compounds in three model systems show correction of Aβ-induced cognitive deficits involving concomitant increases in pCREB. Given the known role of CREB in plasticity, small molecules capable of increasing pCREB may be a useful downstream therapeutic approach.
Aberrantly increased or decreased, but dysregulated ERK signaling is likely to impact human cognition. ERK signaling events have been studied extensively in recent years for involvement in synaptic plasticity and memory function (English and Sweatt 1997). ERK alterations are involved in a human mental retardation syndrome, neurofibromatosis 1, in which defects in the ras/ERK signaling cascade contribute to hippocampal-dependent learning and memory deficits (Costa et al. 2002). Normally, ERK participates in regulating local dendritic protein synthesis (Ying et al. 2002), processing molecular information in dendrites (Watanabe et al. 2002), and stabilizing structural changes in dendritic spines (Goldin and Segal 2003).
Extracellular-signal regulated kinase activation occurs in neurons in response to excitatory glutamatergic signaling (Kurino et al. 1995; Xia et al. 1996). Glutamate receptors including NMDARs activate multiple biochemical pathways that transduce post-synaptic signals (Sheng et al. 2002). A reduction in specific NMDAR subunits, notably NR2B, occurs in AD patients and correlates with cognitive deficits (Greenamyre et al. 1987; Mishizen-Eberz et al. 2004). In Tg2576 mice, omega-3 fatty acid deficiency aggravates APP transgene-dependent decreases in NR2B, the developmentally regulated brain protein, drebrin, and PSD-95, all indices of a defect in excitatory neurons (Calon et al. 2004). In the present study, we found that anti-Aβ-infused Tg2576 mice had a significantly increased level of NR2B. In primary hippocampal neuron culture, Aβ oligomers specifically bound to neurons and neurites as puncta associated with decreased NR2B, consistent with a selective effect on NMDARs trafficking by Aβ (Snyder et al. 2005).
Considerable evidence indicates that immunotherapeutic approaches against Aβ reverse cognitive deficits (Morgan et al. 2000; Klyubin et al. 2005) and promote the rapid recovery of amyloid-associated neuritic dystrophy in APP mutant transgenic mice (Brendza et al. 2005). However, the mechanisms to explain why Aβ antibodies acutely improve learning and memory deficits even without clearance of Aβ-related pathology are not well understood. This study provides initial evidence that Aβ antibody treatment of 11–12 month Tg2576 mice has rapid effects on Aβ oligomer-modulated signal transduction, increasing NR2B and downstream active ERK and CREB, all critical components of signal transduction pathways involved in learning and memory. While insoluble deposits were not significantly reduced in our acute study, they may not play a central role in cognitive deficits. Some AD transgenic mouse models show significant cognitive dysfunction and synaptic damage but have no insoluble Aβ deposits (Kumar-Singh et al. 2000; Mucke et al. 2000). Insoluble Aβ deposits do not predict the degree of dementia in AD patients (McLean et al. 1999) and evidence has emerged suggesting that soluble Aβ oligomer may be a key toxic form of Aβ implicated in AD pathogenesis. For example, soluble oligomeric forms of Aβ caused selective synaptic protein loss and cognitive decline in Aβ oligomer-infused rats (Frautschy et al. 2001) and inhibited LTP and specifically disrupted cognitive function (Cleary et al. 2005). In particular, Aβ 12-mer has been detected as a highly stable, soluble extracellular aggregate in Tg2576 mice that is sufficient to induce cognitive deficits (Lesne et al. 2006). Our previous study demonstrated that an acute treatment with anti-Aβ antibody not only neutralized the level of ∼56 kDa oligomer (12-mer) of Aβ, but also counteracted the effect of Aβ oligomer-induced activation of the GSK3β/ptau signaling cascade without a significant reduction of insoluble Aβ in Tg2576 mice (Ma et al. 2006). Aβ vaccine or anti-Aβ antibodies are still among the most promising treatments for AD. A clinical trial report from one of the groups that participated in the ELAN/Wyeth-Ayerst AN1792 (QS-21) Phase 2A multicenter trial found benefits in patients who both received a primary and a booster immunization of Aβ42 and generated anti-Aβ plaque immunoreactivity (tissue amyloid plaque immunoreactivity assay). Compared with patients who are without such antibodies, the responders, including several with inflammatory side effects appeared to show significantly slower rates of decline of cognitive decline (Hoek et al. 2003).
In summary, this study shows that soluble Aβ oligomers can blunt the ERK and CREB response in vivo and provides direct evidence that passive immunization with anti-Aβ antibodies can increase ERK/CREB activation and NMDAR (NR2B) in Tg2576 mice and in oligomer-treated neuronal cultures. These data also strongly support therapeutic strategies targeting Aβ oligomer by highly specific anti-Aβ oligomer antibodies or drugs to limit dysregulation of multiple memory-related signaling pathways, including oligomer sensitive NMDAR/ERK/CREB, Akt/GSK3β/ptau (Ma et al. 2006), and PAK/LIMK/cofilin/drebrin (Zhao et al. 2006).