Memantine rescues transient cognitive impairment caused by high-molecular-weight aβ oligomers but not the persistent impairment induced by low-molecular-weight oligomers

Abstract

Brain accumulation of soluble amyloid-β oligomers (AβOs) has been implicated in synapse failure and cognitive impairment in Alzheimer's disease (AD). However, whether and how oligomers of different sizes induce synapse dysfunction is a matter of controversy. Here, we report that low-molecular-weight (LMW) and high-molecular-weight (HMW) Aβ oligomers differentially impact synapses and memory. A single intracerebroventricular injection of LMW AβOs (10 pmol) induced rapid and persistent cognitive impairment in mice. On the other hand, memory deficit induced by HMW AβOs (10 pmol) was found to be reversible. While memory impairment in LMW oligomer-injected mice was associated with decreased hippocampal synaptophysin and GluN2B immunoreactivities, synaptic pathology was not detected in the hippocampi of HMW oligomer-injected mice. On the other hand, HMW oligomers, but not LMW oligomers, induced oxidative stress in hippocampal neurons. Memantine rescued both neuronal oxidative stress and the transient memory impairment caused by HMW oligomers, but did not prevent the persistent cognitive deficit induced by LMW oligomers. Results establish that different Aβ oligomer assemblies act in an orchestrated manner, inducing different pathologies and leading to synapse dysfunction. Furthermore, results suggest a mechanistic explanation for the limited efficacy of memantine in preventing memory loss in AD.

Figure 1.

AβOs induce cognitive impairment in mice. A–C, Male Swiss mice received a single intracerebroventricular injection of 1, 10, or 50 pmol AβOs [or vehicle (VEH)] and were tested in the novel object recognition task 24 h (A), 7 d (B), or 14 d (C) after injection. Error bars represent means ± SEM of the time spent on the novel (black bars) or familiar (white bars) objects (n = 10 mice/group). *p < 0.05 (a statistically significant difference; one-sample Student's t test). D, Anti-AβOs (NU4) immunoreactivity in dot blots of hippocampal extracts from vehicle- or AβO-injected mice. Error bars represent means ± SEM (n = 3–5 mice/group). *p < 0.05 (statistically significant difference; Student's t test).

Figure 2.

LMW AβOs, but not HMW AβOs, induce persistent cognitive impairment. A, HPLC size-exclusion chromatography revealed that oligomer preparations comprised two major peaks that were isolated and characterized by dot blot (left inset) and Western immunoblotting (right inset). High-molecular-weight AβOs are the major components in fraction 1, while low-molecular-weight oligomers constitute the majority of fraction 2. B–D, Mice received a single intracerebroventricular injection of 10 pmol of fractionated HMW AβOs, LMW AβOs or vehicle (VEH) and were tested in the object recognition task 24 h (B), 7 d (C), or 14 d (D) after injection. Error bars represent means ± SEM (n = 10 mice/group) of time spent on the novel (black bars) or familiar (white bars) objects. *p < 0.05 (statistically significant difference; one-sample Student's t test).

Figure 3.

LMW AβOs induce hippocampal synapse pathology in mice. Synaptophysin immunoreactivity was examined 14 d after intracerebroventricular injection of 10 pmol HMW AβOs, LMW AβOs, or vehicle (VEH). A–L, Representative images are shown for CA1 (A–C), CA3 (E–G), and dentate gyrus (DG; I–K) hippocampal subfields. Scale bars: 50 μm. Graphs show integrated synaptophysin immunoreactivity (optical density) in CA1 (D), CA3 (H), and dentate gyrus (L). Error bars represent means ± SEM of integrated optical densities in three images per animal (n = 5–8 mice/group). M, Integrated synaptophysin immunoreactivity for the entire hippocampus (n = 5–8 mice/group). Western immunoblots for GluN2B and GluN1 subunit-containing NMDAR in mouse hippocampal extracts 14 d after intracerebroventricular injection of 10 pmol HMW AβOs, LMW AβOs, or vehicle. N, O, Graphs show densitometric quantification of GluN2B (N) and GluN1 (O) levels normalized by β-tubulin. *p < 0.05 (statistically significant difference; one-way ANOVA followed by Dunnett's test comparing means of different experimental groups with vehicle-injected mice).

Figure 4.

LMW AβOs induce a decrease in surface immunoreactivity of GluN1. A–D, Cultured hippocampal neurons were exposed for 3 h at 37°C to vehicle (VEH; A), 500 nm AβOs (unfractionated oligomers; B), 500 nm HMW AβOs (C), or 500 nm LMW AβOs (D). Immunocytochemistry in nonpermeabilized cells was performed using an antibody against an extracellular epitope of the GluN1 subunit of NMDARs. E, Integrated GluN1 immunoreactivity. Error bars show means ± SEM for data from two experiments with independent neuronal cultures (3 coverslips per experimental condition, 10 images per coverslip in each experiment). *p < 0.05 (statistically significant difference; one-way ANOVA followed by Dunnett's test comparing means of different experimental groups with vehicle-injected control animals). R.U., relative units.

Figure 5.

HMW AβOs induce excessive ROS generation in hippocampal neurons. A–F, Representative DCF fluorescence images in hippocampal cultures exposed to vehicle (VEH; A), 500 nm HMW AβOs (B), 500 nm LMW AβOs (C), memantine (MEM; D), 500 nm HMW AβOs plus memantine (E), or 500 nm LMW AβOs plus memantine (F). When present, memantine (10 μm) was added 30 min before AβOs. To allow direct comparison of ROS levels, identical conditions and exposure times for image acquisition were used for all experimental conditions. G, Integrated DCF fluorescence from experiments with two independent cultures. *p < 0.05 (statistically significant difference; one-way ANOVA followed by Dunnett's test comparing means of different experimental groups with vehicle-treated control cultures).

Figure 6.

NMDA receptor blockade rescues HMW AβO-induced memory impairment. A, Mice were treated daily by gavage with 2, 10, or 20 mg/kg memantine (MEM) for 7 d before testing in the object recognition paradigm (n = 7–9 mice/group). B, Mice received a single intracerebroventricular injection of 10 pmol HMW AβOs and were treated daily by gavage with PBS or memantine (2 or 10 mg/kg) for 7 d before testing in the object recognition task (n = 8–10/group). C, Mice received a single intracerebroventricular injection of 10 pmol LMW AβOs and were treated daily by gavage with PBS or memantine (10 mg/kg) for 7 d before testing in the object recognition task (n = 8–10/group). D, Mice received a single intracerebroventricular injection of 10 pmol HMW AβOs and one intraperitoneal injection of PBS or MK-801 (0.1 mg/kg) 24 h before testing in the object recognition task (n = 8–10/group). Error bars represent means ± SEM of time spent on the novel (black bars) or familiar (white bars) objects. *p < 0.05 (statistically significant difference; one-sample Student's t test). VEH, Vehicle.