Beta-amyloid activates the mitogen-activated protein kinase cascade via hippocampal alpha7 nicotinic acetylcholine receptors: In vitro and in vivo mechanisms related to Alzheimer's disease

Abstract

Alzheimer's Disease (AD) is the most common of the senile dementias, the prevalence of which is increasing rapidly, with a projected 14 million affected worldwide by 2025. The signal transduction mechanisms that underlie the learning and memory derangements in AD are poorly understood. beta-Amyloid (Abeta) peptides are elevated in brain tissue of AD patients and are the principal component of amyloid plaques, a major criterion for postmortem diagnosis of the disease. Using acute and organotypic hippocampal slice preparations, we demonstrate that Abeta peptide 1-42 (Abeta42) couples to the mitogen-activated protein kinase (MAPK) cascade via alpha7 nicotinic acetylcholine receptors (nAChRs). In vivo elevation of Abeta, such as that exhibited in an animal model for AD, leads to the upregulation of alpha7 nAChR protein. alpha7 nAChR upregulation occurs concomitantly with the downregulation of the 42 kDa isoform of extracellular signal-regulated kinase (ERK2) MAPK in hippocampi of aged animals. The phosphorylation state of a transcriptional mediator of long-term potentiation and a downstream target of the ERK MAPK cascade, the cAMP-regulatory element binding (CREB) protein, were affected also. These findings support the model that derangement of hippocampus signal transduction cascades in AD arises as a consequence of increased Abeta burden and chronic activation of the ERK MAPK cascade in an alpha7 nAChR-dependent manner that eventually leads to the downregulation of ERK2 MAPK and decreased phosphorylation of CREB protein.

Fig. 1.

Nicotine activation of ERK2 in hippocampal slices is mediated by α7 nAChRs. a, Quantitative immunoblot demonstrates that 500 μm nicotine stimulated ERK2 MAPK activity, which is antagonized by 1 μm MLA in hippocampal slices from mice heterozygous for the L250T α7 nAChR transgene. Basal, 1.00 ± 0.22; nicotine (nic), 2.33 ± 0.13; MLA + nicotine, 1.34 ± 0.14. *Significant difference from basal level; p < 0.01, post hocTukey multiple comparison test. Representative immunoblot results are depicted below the response histogram. ERK1 (topband) activation paralleled that of ERK2; however, absolute levels of phospho-ERK1 were far below ERK2 and were not quantified. Slices from wild-type animals did not exhibit significant ERK2 MAPK activation with nicotine treatment, likely because of the rapid desensitization kinetics of wild-type α7 nAChRs precluding biochemical detection of ERK2 MAPK activation (Seguela et al., 1995). b, Quantitative immunoblot demonstrates that ERK2 MAPK activation by nicotine in cultured rat hippocampal slices is blocked by 1 μm MLA. Basal, 1.01 ± 0.08; nicotine, 1.66 ± 0.13; MLA + nicotine, 1.22 ± 0.09. *Denotes nicotine stimulation is significantly different from basal level;p < 0.001, post hoc Tukey multiple comparison test. Representative immunoblot results are depictedbelow the response histogram.

Fig. 2.

Time course and concentration dependence of Aβ42 activation of ERK2 MAPK in cultured rat hippocampal slices.a, Aβ42 activates ERK MAPK in the picomolar to nanomolar range. Data points are as follows: 0 nm(1.00 ± 0.15), 0.01 (0.98 ± 0.16), 0.1 (1.98 ± 0.48), 1.0 (1.74 ± 0.25), 10 (2.70 ± 0.62), and 100 (2.01 ± 0.30) nm Aβ42 for 5 min. Representative immunoblot results are depicted below the response curve.b, The 100 nm Aβ42 rapidly activates ERK2 MAPK in rat hippocampal slice cultures. Peak response occurs at 5 min Aβ42 and returns to baseline within 2 hr. Data points are as follows: 2 (1.88 ± 0.30), 5 (3.92 ± 1.15), 10 (2.61 ± 0.83), 30 (1.81 ± 0.44), and 120 (1.34 ± 0.17) min. *Significant difference from basal level (1.06 ± 0.12); p< 0.05, Student's t test with Welch's correction because variances differ significantly according to Bartlett's test. Representative immunoblot results are depicted below the response curve.

Fig. 3.

α7 couples Aβ42 activation of ERK2 MAPK in cultured rat hippocampal slices. a, Quantitative immunoblot demonstrates that ERK2 MAPK activation by 100 nm Aβ42 is blocked by 1 μm MLA and 100 μm BTX. Basal, 1.00 ± 0.04; Aβ42 (light-shaded bar), 1.58 ± 0.09; MLA + Aβ42, 1.19 ± 0.07; Aβ42 (dark-shaded bar), 1.67 ± 0.11; BTX + Aβ42, 0.94 ± 0.14. *Significant difference from basal ERK2 MAPK activity; p < 0.0001, Student'st test with Welch's correction because variances differ significantly according to Bartlett's test. Representative immunoblot results are depicted below the response histogram.b, Aβ42 (100 nm) desensitizes the nicotine-induced (500 μm) activation of ERK2 MAPK. Basal, 1.05 ± 0.14; nicotine (nic), 1.94 ± 0.22; Aβ42 (at 2 hr), 1.14 ± 0.24; Aβ42 + nicotine (at 2 hr), 1.03 ± 0.13. *Significant difference from basal ERK2 MAPK activity; p < 0.001, post hoc Tukey multiple comparison test. Representative immunoblot results are depicted below the response histogram. c, Aβ42 (100 nm) does not desensitize the PDA-induced (10 μm) activation of ERK2 MAPK. Basal, 1.00 ± 0.08; Aβ42 (at 2 hr), 0.78 ± 0.11; Aβ42 + PDA (at 2 hr), 2.15 ± 0.54; PDA, 2.25 ± 0.13. *Significant difference from basal ERK2 MAPK activity; p < 0.01, post hoc Tukey multiple comparison test. Representative immunoblot results are depicted below the response histogram.d, The 100 nm Aβ42 activation of ERK2 MAPK is not blocked by TTX and exhibits Ca²⁺ dependency. Basal, 1.00 ± 0.06; Aβ42, 1.30 ± 0.07; TTX, 0.67 ± 0.10; Aβ42 + TTX, 1.02 ± 0.05; EGTA, 0.59 ± 0.04; Aβ42 + EGTA, 0.65 ± 0.05. *Significant difference from basal-induced ERK2 MAPK activity. #, Significant difference from TTX-induced ERK2 MAPK activity; p < 0.001, post hocTukey multiple comparison test.

Fig. 4.

α7 nAChR is upregulated in hippocampus and DG of Tg2576 mice. a, Quantitative immunoblot of area CA1 and DG from 4, 13, and ∼20 month Tg2576 hippocampus reveals upregulation of α7 nAChR protein as early as 4 months. *Significantly higher α7 nAChR level than age-matched control animals (p < 0.03) by Student's ttest. Dashed line represents normalized control animal level; note the change in scale for ∼20 month animals. Filled bar, CA1; shaded bar, DG. b, Representative immunoblot for α7 nAChR level in Tg2576 and age-matched control animals.

Fig. 5.

Downstream targets of α7 nAChR activation in Tg2576 hippocampus exhibit dysregulation. ERK2 and CREB proteins undergo hyperactivation, followed by downregulation, as compared with age-matched control animals. Differences between Tg2576 and age-matched control animals were detected by quantitative immunoblot of area CA1 and DG from 4, 13, and ∼20 month Tg2576 hippocampus. Samples were evaluated for total and phospho-ERK2 MAPK (a, c) and total and phospho-CREB (b, d) levels in CA1 and DG, respectively. *Significant difference from age-matched control animal level (p ≤ 0.05) by Student'st test. Dashed line represents normalized control animal level. Filled bar, Total; shaded bar, phospho.

Fig. 6.

Animals with elevated α7 nAChR level fail to perform to criteria in the Morris water maze. Scatter plot of the third probe trial performance of individual ∼20-month-old Tg2576 and control animals versus α7 nAChR protein levels in CA1 and DG.Dashed line indicates performance criterion.Filled squares, Control; filled diamonds, Tg.

Fig. 7.

Chronic exposure to Aβ42 leads to increased α7 nAChR protein in hippocampal slice cultures. Cultured rat hippocampal slices were exposed to 100 pm Aβ42 for 144 hr, and α7 nAChR protein was quantified by immunoblot. The data that are expressed are normalized to the α7 nAChR protein level in cultures that were left untreated. Representative immunoblot results are shownbelow the histogram. *Significant difference from control level (p < 0.0001) by Student'st test. Basal, 1.00 ± 0.11; Aβ42-treated, 2.35 ± 0.13.