Postsynaptic dysfunction is associated with spatial and object recognition memory loss in a natural model of Alzheimer's disease

Abstract

Alzheimer's disease (AD) is an age-related neurodegenerative disorder associated with progressive memory loss, severe dementia, and hallmark neuropathological markers, such as deposition of amyloid-β (Aβ) peptides in senile plaques and accumulation of hyperphosphorylated tau proteins in neurofibrillary tangles. Recent evidence obtained from transgenic mouse models suggests that soluble, nonfibrillar Aβ oligomers may induce synaptic failure early in AD. Despite their undoubted value, these transgenic models rely on genetic manipulations that represent the inherited and familial, but not the most abundant, sporadic form of AD. A nontransgenic animal model that still develops hallmarks of AD would be an important step toward understanding how sporadic AD is initiated. Here we show that starting between 12 and 36 mo of age, the rodent Octodon degus naturally develops neuropathological signs of AD, such as accumulation of Aβ oligomers and phosphorylated tau proteins. Moreover, age-related changes in Aβ oligomers and tau phosphorylation levels are correlated with decreases in spatial and object recognition memory, postsynaptic function, and synaptic plasticity. These findings validate O. degus as a suitable natural model for studying how sporadic AD may be initiated.

Fig. 1.

Age-dependent decline in cognitive performance in O. degus. (A) Average number of correct choices per day in the T-maze test. Two-way ANOVA [F_(11,33) = 34.59, *P < 0.0001], followed by the Bonferroni post hoc test (P < 0.05) in the last 3 d for aged (36–60 mo-old) vs. young (6–12 mo-old). (B) Average correct choices at the end of the experimental phase for 6-mo-old (white), 12-mo-old (gray), 36-mo-old (blue), and 60-mo-old (red) degus. One-way ANOVA [F_(3,26) = 6.509, *P = 0.002], followed by Tukey´s post hoc test (P < 0.05). (C) Preference index for object recognition. Paired two-tailed t test (*P < 0.01), novel vs. familiar objects. (D) Average exploration time for novel vs. familiar objects. Paired two-tailed t test (*P < 0.01) vs. familiar objects. The values in parentheses indicate the number of animals.

Fig. 2.

Altered synaptic transmission and postsynaptic deficits in the Schaffer collateral–CA1 pathway. Representative traces of fEPSP at different stimulus intensities from 6-mo-old (white), 12 mo-old (gray), 36-mo-old (blue), and 60-mo-old (red) degus. (Scale bars: 1 mV, 10 ms.) AMPAR-mediated input–output curves from 6-mo-old (white), 12-mo-old (gray), 36-mo-old (blue), and 60-mo-old (red) degus. One-way ANOVA [F_(3,58) = 157.3, *P < 0.0001], followed by Tukey's post hoc test (P < 0.05). (A2 and A3) Relationship between stimulus intensity and fiber volley amplitude (A2) and fEPSP slope (A3) in 6-mo-old (white), 12-mo-old (gray), 36-mo-old (blue), and 60-mo-old (red) degus. Repeated-measures ANOVA [F_(15,45) = 61.06, *P < 0.0001], followed by the Bonferroni post hoc test (P < 0.05). (B1) Normal paired-pulse facilitation between groups. (B2) Representative traces at interstimulus intervals of 50 ms are shown. (Scale bars: 1 mV, 20 ms.) (C1) Amplitudes and frequencies of AMPAR-mediated mEPSCs from young (white) and aged (red) degus. (C2) Cumulative probability plots for mEPSC amplitude size. (Inset) Representative superposed events. Calibration: 5 pA, 20 ms. (C3) Representative traces of mEPSCs. (Scale bars: 20 pA, 2 s.) Calibration: 1 mV, 10 ms. Unpaired two-tailed t test (*P < 0.01) for young vs. aged. The values in parentheses indicate the number of hippocampal slices (first number) and the number of animals (second number) used.

Fig. 3.

Postsynaptic proteins affect synapse during degus aging. (A) Representative blot of synaptic proteins from hippocampus extracts (age indicated above lanes). Arrows indicate respective migration positions. (B) Relative levels of synaptophysin (SYP), PSD-95, GluR2-AMPAR subunit, and NR2b-NMDAR subunit in the hippocampus from 6-mo-old (white), 12-mo-old (gray), 36-mo-old (blue), and 60-mo-old (red) degus. Mean values of synaptic proteins are relative to β-tubulin levels. One-way ANOVA [F_(3,20) = 6.626, *P = 0.0027 for PSD-95; F_(3,20) = 3.968, *P = 0.0227 for GluR2; F_(3,21) = 2.662, *P = 0.0745 for NR2b], followed by Tukey´s post hoc test (P < 0.05), young vs. aged.

Fig. 4.

Impaired hippocampal synaptic plasticity in aged O. degus. (A) TBS-induced LTP in the Schaffer collateral–CA1 synapse. (Left) Representative fEPSPs recorded 1 min before TBS (1) and 60 min after TBS (2). LTP protocol was delivered at the time indicated by the arrow. Averaged LTP magnitudes during the last 10 min of recording in 6-mo-old (white), 12-mo-old (gray), 36-mo-old (blue), and 60-mo-old (red) degus are shown as well. Two-way ANOVA [F_(1,19) = 1841, *P < 0.0001], followed by the Bonferroni post hoc test (P < 0.05) in the last 10 min, for aged vs. young. (B) ppLFS-induced LTD in the Schaffer collateral–CA1 synapse. (Left) Representative fEPSPs recorded 1 min before ppLFS (1) and 60 min after ppLFS (2). LTD protocol was delivered at the time indicated by the horizontal open bar. Averaged LTD magnitudes during the last 10 min of recording for 6-mo-old (white), 12-mo-old (gray), 36-mo-old (blue), and 60-mo-old (red) degus are also as well. Two-way ANOVA [F_(1,19) = 435.6, *P < 0.0001], followed by the Bonferroni post hoc test (P < 0.05) in the last 10 min for aged vs. young. (Scale bars: 1 mV, 10 ms.) The values in parentheses indicate the number of hippocampal slices (first number) and the number of animals (second number) used.

Fig. 5.

Accumulation of large soluble Aβ oligomers and tau phosphorylation during aging. (A1) Representative blot for amyloid oligomers using anti-Aβ peptide antibody 4G8. Arrows indicate respective migration positions of hexamers (6-mer), nonamers (9-mer), and dodecamers (12-mer). Synthetic Aβ42 peptide was used as size marker and positive control (right lane). (A2) Identification and relative levels of different Aβ oligomeric associations in hippocampal extracts from 6-mo-old (white), 12-mo-old (gray), 36-mo-old (blue), and 60-mo-old (red) degus. One-way ANOVA [F_(3,21) = 17.21, *P < 0.0001 for 6-mer; F_(3,19) = 5.439, *P = 0.0026 for 12-mer], followed by Tukey's post hoc test (P < 0.05) for aged vs. young. (B) Relative levels of soluble Aβ oligomers. (Inset) Representative slot blot from hippocampal extracts using antioligomeric antibody A11. One-way ANOVA [F_(3,19) = 5.439, P = 0.0072], followed by Tukey's post hoc test (P < 0.05) for aged vs. young. (C) Determination of phosphorylated tau protein levels using PHF-1 and AT8 antibodies. (Inset) Representative blot for PHF-1, AT8, and β-actin. One-way ANOVA [F_(3,20) = 23.21, *P < 0.0001 for PHF-1; F_(3,20) = 5.95, *P = 0.0059 for AT8], followed by Tukey´s post hoc test (P < 0.05) for aged vs. young. The values in parentheses indicate the number of animals used.

Fig. 7.

Amyloid deposition and tau phosphorylation begins in the cerebral cortex of O. degus. (A–D) Immunoreactivity for Aβ peptides using the specific antibody 6E10 showing extracellular (black arrows) staining in the hippocampus and the cerebral cortex in degus. A greater number of extracellular insoluble deposits were observed in aged degus (72 mo-old; C and D) compared with young degus (12 mo-old; A and B). (E–H) Immunodetection of pathological phosphorylated tau using the specific antibody AT8 in the hippocampus and cerebral cortex from degus. A greater number of positively stained somas (black arrows) were observed in aged degus (G and H) compared with young degus (E and F). (I and J) Quantification of Aβ burden (I; percentage of area occupied by 6E10-positive plaques) and the number of AT8-positive cells (J; number of neurons per area) in 6-mo-old (white, n = 2), 12-mo-old (gray, n = 2), 36-mo-old (blue, n = 2), 60-mo-old (red, n = 2), and 72-mo-old (black, n = 2) degus showing a significant increase after 36 mo in cortex and after 60 mo in hippocampus. One-way ANOVA [F_(4,88) = 238.8, *P < 0.0001 for 6E10-Hip; F_(4,88) = 646.4, *P < 0.0001 for 6E10-Cx; F_(4,88) = 266.0, *P < 0.0001 for AT8-Hip; F_(4,88) = 134.9, *P < 0.0001 for AT8-Cx], followed by Tukey's post hoc test (P < 0.05) compared with 6 mo-old.

Fig. 6.

Large Aβ oligomers and tau phosphorylation correlate with LTP and memory impairments. (A–C) Relationships between soluble dodecamer (Aβ*56) level and LTP magnitude (A), T-maze (B), and ORM (C) in young and aged degus. (D–F) Relationships between PHF-1 epitope tau phosphorylated level and LTP magnitude (D), T-maze (E), and ORM (F) in young and aged degus. High correlation for LTP and ORM can be seen. The values in parentheses indicate the number of animals used.