Memantine lowers amyloid-beta peptide levels in neuronal cultures and in APP/PS1 transgenic mice

Abstract

Memantine is a moderate-affinity, uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist that stabilizes cognitive, functional, and behavioral decline in patients with moderate to severe Alzheimer's disease (AD). In AD, the extracellular deposition of fibrillogenic amyloid-beta peptides (Abeta) occurs as a result of aberrant processing of the full-length Abeta precursor protein (APP). Memantine protects neurons from the neurotoxic effects of Abeta and improves cognition in transgenic mice with high brain levels of Abeta. However, it is unknown how memantine protects cells against neurodegeneration and affects APP processing and Abeta production. We report the effects of memantine in three different systems. In human neuroblastoma cells, memantine, at therapeutically relevant concentrations (1-4 muM), decreased levels of secreted APP and Abeta(1-40). Levels of the potentially amylodogenic Abeta(1-42) were undetectable in these cells. In primary rat cortical neuronal cultures, memantine treatment lowered Abeta(1-42) secretion. At the concentrations used, memantine treatment was not toxic to neuroblastoma or primary cultures and increased cell viability and/or metabolic activity under certain conditions. In APP/presenilin-1 (PS1) transgenic mice exhibiting high brain levels of Abeta(1-42), oral dosing of memantine (20 mg/kg/day for 8 days) produced a plasma drug concentration of 0.96 microM and significantly reduced the cortical levels of soluble Abeta(1-42). The ratio of Abeta(1-40)/Abeta(1-42) increased in treated mice, suggesting effects on the gamma-secretase complex. Thus, memantine reduces the levels of Abeta peptides at therapeutic concentrations and may inhibit the accumulation of fibrillogenic Abeta in mammalian brains. Memantine's ability to preserve neuronal cells against neurodegeneration, to increase metabolic activity, and to lower Abeta level has therapeutic implications for neurodegenerative disorders.

Figure 1

Effect of memantine on cell viability and toxicity in human neuroblastoma cells MTS, CTG and LDH assays were carried out on control and memantine-treated cells as described in the text. A: Cell viability was measured using 20μl of harvested cells from each well at day 12 of the chronic treatments. B: To measure LDH release, 30μl aliquots of the final day 12 CM samples were analyzed as previously mentioned. The levels of both endpoints showed no significant change by drug treatments from the vehicle. C: After a 48 hr treatment, cells treated with memantine (0.1-100 μM) were harvested in PBS and 30μl aliquots were subjected to the luminescent Cell Titer-Glo (CTG) assay. A small but statistically significant increase in signal was observed in the 10μM and the 100 μM conditions (both p<0.01)

Figure 2

Effect of memantine in neuroblastoma cells: Analysis of sAPP levels by western blot at different days Cells were treated with memantine at the indicated concentrations for 12 days, with media samples collected at 3 day intervals and subjected to SDS-PAGE and Western blotting. A-D: Densitometric analysis of Western blot data shows that levels of sAPP in conditioned medium samples collected at day 3 (A) was not significantly changed by memantine. However, sAPP in the day 6 samples (B) and day 9 samples (C), were significantly lower in cultures treated with 2 μM and 4 μM memantine, compared to vehicle (all p<0.05). In the day 12 samples (D), statistically significant decreases in sAPP were observed for all three memantine concentrations (all p<0.05). E: A representative Western blot of day 12 CM samples probed with anti-APP (22C11) and anti-β-actin (AC-15) monoclonal antibodies.

Figure 3

Effect of memantine on sAPP in SK-N-SH cultures over time Equal amounts of CM samples were analyzed in a second set of Western blots, one containing a time course for vehicle-treated cells and one for cells treated with 1 μM memantine. Data are represented as percent of the first media sample collection at day 3. A: A significant decrease was observed by day 12 in vehicle-treated cultures (p=0.01). B: In the 1μM memantine treatment, significant decreases were observed at day 9 and day 12 (both p<0.05). Representative blots inset; data are actin-adjusted and presented as a proportion of day 3 sAPP levels.

Figure 4

Effect of memantine on Aβ_1-40 in SK-N-SH cultures Equal amounts of CM samples of neuroblastoma cells treated with vehicle or memantine at the indicated concentrations were subjected to ELISA to detect Aβ_1-40. Memantine at the tested concentrations did not change Aβ_1-40 at day 3 (A). However, all treatment groups were significantly decreased at day 6 (B; all p≤0.02) and day 9 (C; all p≤0.03). No significant effect was observed at day 12 (D).

Figure 5

Effect of memantine on cell viability in primary cortical neurons Starting at day 7 in vitro, primary rat cortical cultures were treated with vehicle or memantine at the indicated concentrations. After 48 hours cells were collected in PBS and 20 μl aliquots of cell suspension were subjected to MTS assay (A), calcein AM labeling (C), and ethidium homodimer labeling (D), and 30μl aliquots of CM were used for LDH assay (B). Memantine treatment produced no significant change in viability (A and C) or toxicity (B and D) by any of these four assays.

Figure 6

Effect of memantine on soluble Aβ_1-40 and Aβ_1-42 in primary rat cortical cultures Aβ was measured in media samples of vehicle- or memantine-treated primary rat cortical cultures by ELISA. A: No significant effect was observed for Aβ_1-40 although there was a trend for a decrease at 18 μM (p=0.067) B: Aβ_1-42 was significantly reduced at 4 μM and 18 μM (both p<0.01) memantine with a trend toward a decrease at 9 μM memantine (p=0.085).

Figure 7

Effect of memantine on levels of soluble and insoluble Aβ_1-42 in APP/PS-1 transgenic mice. Soluble and insoluble Aβ_1-42 levels and soluble Aβ_1-40 levels in the cortex of APPswe/PS1dE9 mice receiving 2.5mg/kg by daily i.p. injections for 8 days, or 20 mg/kg/day in their drinking water (p.o.) for 8 days, or a single i.p. injection of saline were measured by ELISA. A: Soluble Aβ_1-40 levels were unchanged in all treatment groups B: Soluble Aβ_1-42 was significantly reduced in mice receiving memantine orally in drinking water (p=0.002) but not by i.p. injection. C: Upon oral administration of memantine, the Aβ_1-40 to Aβ_1-42 ratio increased significantly (p=0.003), suggesting a change in γ-secretase activity. D: Insoluble Aβ_1-42 was unchanged by memantine treatment, possibly due to the short duration of treatments.