Glutamate receptor delocalization in postsynaptic membrane and reduced hippocampal synaptic plasticity in the early stage of Alzheimer's disease

Abstract

Mounting evidence suggests that synaptic plasticity provides the cellular biological basis of learning and memory, and plasticity deficits play a key role in dementia caused by Alzheimer's disease. However, the mechanisms by which synaptic dysfunction contributes to the pathogenesis of Alzheimer's disease remain unclear. In the present study, Alzheimer's disease transgenic mice were used to determine the relationship between decreased hippocampal synaptic plasticity and pathological changes and cognitive-behavioral deterioration, as well as possible mechanisms underlying decreased synaptic plasticity in the early stages of Alzheimer's disease-like diseases. APP/PS1 double transgenic (5XFAD; Jackson Laboratory) mice and their littermates (wild-type, controls) were used in this study. Additional 6-week-old and 10-week-old 5XFAD mice and wild-type mice were used for electrophysiological recording of hippocampal dentate gyrus. For 10-week-old 5XFAD mice and wild-type mice, the left hippocampus was used for electrophysiological recording, and the right hippocampus was used for biochemical experiments or immunohistochemical staining to observe synaptophysin levels and amyloid beta deposition levels. The results revealed that, compared with wild-type mice, 6-week-old 5XFAD mice exhibited unaltered long-term potentiation in the hippocampal dentate gyrus. Another set of 5XFAD mice began to show attenuation at the age of 10 weeks, and a large quantity of amyloid beta protein was accumulated in hippocampal cells. The location of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor and N-methyl-D-aspartic acid receptor subunits in synaptosomes was decreased. These findings indicate that the delocalization of postsynaptic glutamate receptors and an associated decline in synaptic plasticity may be key mechanisms in the early onset of Alzheimer's disease. The use and care of animals were in strict accordance with the ethical standards of the Animal Ethics Committee of Capital Medical University, China on December 17, 2015 (approval No. AEEI-2015-182).

Keywords: Alzheimer's disease; glutamate receptor; hippocampus; learning and memory; long-term potentiation; nerve regeneration; neural regeneration; synaptic plasticity; synaptic strength; βamyloid.

Figure 1

Behavioral impairment in 5XFAD mouse models began at 12–14 weeks of age. (A) Schematic diagram of two stages of the Morris water maze; (B) 12-week-old 5XFAD and WT mice were subjected to acquisition training and reversal training sequentially, and the time spent finding the hidden platform (escape latency, seconds) was recorded and used in statistical analysis (repeated- measures analysis of variance); (C) probe trials were conducted on day 12, and the percentage of swimming time spent in the target quadrant of total time (%) was recorded. *P < 0.05, vs. WT (mean ± SEM, Student’s t-test). WT: Wild-type; FAD: family Alzheimer’s disease; NW: northwest; NE: northeast; SE: southeast; SW: southwest.

Figure 2

Hippocampal LTP is not damaged in 5XFAD mice at 6 weeks old. (A) Summary plots of normalized fEPSP amplitude (%) in recording time. The fEPSPs were induced by 5XTBS (arrow). Inset traces: Representative traces of fEPSP before (black curve) and after (red curve) the induction of potentiation. (B) Normalized fEPSP amplitude (%) for PTP (1–2 minutes after theta-burst stimulation). (C) Normalized fEPSP amplitude (%) for E-LTP (15–20 minutes after theta-burst stimulation). (D) Normalized fEPSP amplitude (%) for the last 10 minutes. (E–H) Results of fEPSP slope (%) in the dentate gyrus of 6-week-old mice. All data are presented as the mean ± SEM (Student’s t-test). (B–D, F–H) or repeated-measures analysis of variance (A, E). WT: Wild-type; FAD: family Alzheimer’s disease; PTP: post-tetanic potentiation; LTP: long-term potentiation; E-LTP: early-LTP; fEPSP: field excitatory postsynaptic potential; TBS: theta-burst stimulation; 5XTBS: 5 theta-burst stimulation.

Figure 3

LTP declines in the hippocampus of 5XFAD mice at 10 weeks old. (A) Summary plots of normalized fEPSP amplitude (%) in recording time. fEPSPs were induced by 5XTBS (arrow). Inset traces: Representative traces of fEPSP before (black curve) and after (red curve) the induction of potentiation. (B) Normalized fEPSP amplitude (%) for post-tetanic potentiation (1–2 minutes after theta-burst stimulation). (C) Normalized fEPSP amplitude (%) for E-LTP (15–20 minutes after theta-burst stimulation). (D) Normalized fEPSP amplitude (%) for the last 10 minutes. (E–H) Statistical analysis of fEPSP slope (%) of LTP in the dentate gyrus of 5XFAD and WT mice. After 5XTBS, the fEPSP amplitude and slope of both groups increased over time. The increments of both amplitude and slope of fEPSP were slower in 5XAFD mice than WT controls (*P < 0.05). All data are presented as the mean ± SEM (t-test for B–D, F–H or repeated-measures analysis of variance for A, E). *P < 0.05, vs. WT (mean ± SEM; Student’s t-test). WT: Wild-type; FAD: family Alzheimer’s disease; LTP: long-term potentiation; E-LTP: early-LTP; fEPSP: field excitatory postsynaptic potential; TBS: theta-burst stimulation; 5XTBS: 5 theta-burst stimulation.

Figure 4

Basal synaptic transmission is intact in the hippocampus of 5XFAD mouse at 6 and 10 weeks of age. (A, B) Input-output curves of fEPSP amplitude and slope in mice at 6 weeks of age. (C, D) Input-output curves of fEPSP amplitude and slope in mice at 10 weeks old. All data are presented as the mean ± SEM (repeated-measures analysis of variance). WT: Wild-type; FAD: family Alzheimer’s disease; fEPSP: field excitatory postsynaptic potential.

Figure 5

Decreased subcellular distribution of glutamate receptors in the hippocampus of 5XFAD mice at 10 weeks old. (A) Western blots showing the expression of glutamate receptors in different subcellular fractions in the hippocampus from wild-type and 5XFAD mice at 10 weeks of age. The α-tubulin was considered the internal control to normalize total protein level. (B) Normalization of AMPA-selective glutamate receptor 1 (GluA1) in different subcellular fractions in the hippocampus from 10-week-old WT and 5XFAD mice. (C) Normalization of NMDA receptor 1 (NR1) in different subcellular fractions in the hippocampus from 10-week-old WT and 5XFAD mice. *P < 0.05, vs. WT (mean ± SEM; n = 3 mice/genotype for GluA1, n = 4 mice/genotype for NR1; Student’s t-test). T: Total protein in tissue; S: cytosolic fraction; P1: triton-soluble fraction in the crude synaptosome fraction, which mainly includes cytosolic proteins in synapses; P2: triton-insoluble fraction in the crude synaptosome fraction, which mainly includes membrane-associated proteins in synapses. WT: Wild-type; FAD: family Alzheimer’s disease; AMPA: α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid; NMDA: N-methyl-D-aspartic acid.

Figure 6

Amyloid beta intracellularly accumulates in the hippocampal CA1 region of 5XFAD mice at 10 weeks of age. (A, B) Brain slices of wild-type and FAD mice were subjected to immunostaining with anti-amyloid beta specific antibody, 6E10 under the optical microscope (original magnification, 20×). Scale bar: 100 μm for A and B, 50 μm for B’. Arrows in B’ indicate neurons with intracellular amyloid beta-positive signals. FAD: Family Alzheimer’s disease.