Neurotrophins enhance CaMKII activity and rescue amyloid-β-induced deficits in hippocampal synaptic plasticity

Abstract

Amyloid-β (Aβ) peptide-induced impairment of hippocampal synaptic plasticity is considered an underlying mechanism for memory loss in the early stages of Alzheimer's disease and its animal models. We previously reported inhibition of long-term potentiation (LTP) and miniature excitatory postsynaptic currents by oligomeric Aβ(1-42) at hippocampal synapses. While multiple cellular mechanisms could be involved in Aβ-induced synaptic dysfunction, blockade of activity-dependent autophosphorylation of Ca2+ and calmodulin-dependent protein kinase II (CaMKII) appeared to be a major component of Aβ action in our studies. The present study further tested this hypothesis and examined the therapeutic potential of trkB receptor-acting neurotrophins in rescuing Aβ-induced synaptic and signaling impairments. As expected, treatment of rat hippocampal slices with Aβ(1-42) significantly reduced LTP in the Schaffer collateral-CA1 pathway and dentate medial perforant path. LTP-associated CaMKII activation and AMPA receptor phosphorylation were blocked by Aβ(1-42) at the same concentration that inhibited LTP. Aβ-induced LTP impairment, however, was prevented when slices were co-treated with neurotrophin 4 (NT4). Western blotting and immunohistochemical analyses confirmed that treatment with NT4 or brain-derived neurotrophic factor, another trkB-acting neurotrophin, could oppose Aβ action, enhancing autophosphorylation of CaMKII, and AMPA receptor phosphorylation at a CaMKII-dependent site. These findings support the view that CaMKII is a key synaptic target of Aβ toxicity as well as a potential therapeutic site of neurotrophins for Alzheimer's disease.

Fig. 1

Neurotrophin 4 (NT4) prevents amyloid-β (Aβ)-induced LTP deficits in the Schaffer collateral-CA1 pathway (A, B) and dentate medial perforant path (C) of rat hippocampal slices. Slices were pretreated with 1 μM Aβ_1–42 for 30 min in the absence or presence of NT4 (100 ng/ml) and received a single HFS for LTP induction. Note inhibition of LTP by Aβ in both pathways and reversal of such inhibition by NT4 co-treatment. NT4 treatment alone did not affect LTP. n = 9–27 for each group. Representative CA1 fEPSP recordings before (1) and after (2) HFS from the same slices are superimposed and shown in (A). Calibration bars: 1 mV/5 ms.

Fig. 2

Neurotrophins enhance CaMKII autophosphorylation (A) and phosphorylation of GluR1 subunits of AMPA receptors (B). Hippocampal slices were treated for 30 min in aCSF containing NT4 or BDNF (50 or 100 ng/ml as labeled) and processed for Western blotting. The representative images (top) and group data (bottom) show dose-dependent increases in p-CaMKII and p-GluR1 levels after neurotrophin treatment, with little changes in total CaMKII or GluR1 levels. Changes in the protein levels were calculated relative to that of control slices treated with drug-free aCSF. n = 15 for each group. ^* and ^**, p < 0.05 and 0.01, respectively, compared to the controls.

Fig. 3

Immunohistochemical analysis of neurotrophin-induced CaMKII and GluR1 phosphorylation. Hippocampal slices were treated with aCSF (Control), BDNF, or NT4 (100 ng/ml) for 30 min and processed for immunoreactive double labeling of p-CaMKII (top) and p-GluR1 (bottom) using phosphospecific antibodies. Confocal fluorescent images were obtained using a Zeiss LSM 5 Pascal laser scanning microscope with a 5× water immersion objective. Calibration bar: 400 μm.

Fig. 4

Neurotrophins prevent inhibition by Aβ_1–42 of LTP-induced CaMKII and GluR1 phosphorylation in the dentate gyrus. A) Representative western blot images. Multiple slices from the same rats were used for different treatments in a single set of experiment as labeled. Note markedly increased p-CaMKII and p-GluR1 levels in slices pretreated with BDNF or NT4 (100 ng/ml), and lack of inhibition by Aβ_1–42 (1 μM) of HFS-induced phosphorylation when slices were co-treated with neurotrophins. B and C) Phosphorylation changes in slices pretreated with Aβ, NT4, or both. LTP-induced p-CaMKII and p-GluR1 increases were measured 15 min after HFS. The basal phosphorylation levels were determined in paired slices that received matching treatment except no HFS. All data are expressed as percentages of the basal level measured in aCSF-treated slices (control). n = 19 for each group. # p < 0.05 compared to the basal level of aCSF-treated slices. ^* and ^**, p < 0.05 and 0.01, respectively, compared to the post-HFS level in Aβ-treated slices. Except in slices treated with Aβ alone, HFS induced significant increases in p-CaMKII and p-GluR1 levels from the paired basal levels.