The AMPA receptor antagonist perampanel robustly rescues amyotrophic lateral sclerosis (ALS) pathology in sporadic ALS model mice

Abstract

Both TDP-43 pathology and failure of RNA editing of AMPA receptor subunit GluA2, are etiology-linked molecular abnormalities that concomitantly occur in the motor neurons of the majority of patients with amyotrophic lateral sclerosis (ALS). AR2 mice, in which an RNA editing enzyme adenosine deaminase acting on RNA 2 (ADAR2) is conditionally knocked out in the motor neurons, exhibit a progressive ALS phenotype with TDP-43 pathology in the motor neurons through a Ca(2+)-permeable AMPA receptor-mediated mechanism. Therefore, amelioration of the increased Ca(2+) influx by AMPA receptor antagonists may be a potential ALS therapy. Here, we showed that orally administered perampanel, a selective, non-competitive AMPA receptor antagonist significantly prevented the progression of the ALS phenotype and normalized the TDP-43 pathology-associated death of motor neurons in the AR2 mice. Given that perampanel is an approved anti-epileptic drug, perampanel is a potential candidate ALS drug worthy of a clinical trial.

Figure 1. Perampanel administration for 14 days prevented the death of motor neurons and normalized TDP-43 subcellular localization in AR2H (heterozygous) and AR2 (homozygous) mice.

(a) Number of TO-PRO-3-positive anterior horn cells in the spinal cord (AHCs; ≥20 μm in diameter). Data for the WT, AR2H and AR2 groups are indicated in different colors (white, red, and blue, respectively), and the vehicle groups (V; methyl cellulose) are represented as hatched columns in the corresponding colors. NT, no treatment. *p < 0.05 and ***p < 0.0001 against the AR2H-V group; ^#p < 0.05 against the AR2H-V group; and ^‡p < 0.0001 against the WT group. ns, not statistically significant. (b) Number of TDP-43-positive (black columns) and TDP-43-negative (white columns) AHCs. Hatched columns indicate the results on vehicle-treated mice. *p < 0.05, **p < 0.001, and ***p < 0.0001 against the AR2H-V group; and ^#p < 0.05 against the AR2-V group. (c) Representative AHCs with different TDP-43 subcellular localization patterns: predominantly nuclear (N), nucleo-cytoplasmic (N/C) or cytoplasmic (C). The vertical axis indicates the intensity (gray value) evaluated with ImageJ. The threshold for TDP-43 positivity was set at a level 3-fold higher (60 gray) than the background intensity (20 gray). (d) The numbers of AHCs with different subcellular TDP-43 localization patterns are indicated. Hatched columns indicate the results on vehicle-treated mice. ***p < 0.0001 against the AR2H-V with N/C group; ^†p < 0.05 and ^‡p < 0.0001 against the AR2H-V with C group; and ^#p < 0.05 against the AR2-V with C group, Wilcoxon rank sum test. (a,b,d) Data are presented as the mean ± s.e.m. Statistics were based on one-way ANOVA and Tukey post hoc tests unless indicated otherwise. In the WT group, 5 (NT) and 3 (V) mice were included; in the AR2H group, 7 (V), 5 (3.3 mg/kg/day), 8 (6.6), 5 (13.2) and 6 (20) mice were included; and in the AR2 group, 5 in both the V and 13.2 mg/kg/day groups were included.

Figure 2. Perampanel administration for 90 days rescued motor dysfunction in AR2 mice.

(a) The time schedule for performance testing and drug treatment. Body weight (b), latency to fall on the rotarod task (c) and grip strength (d) were examined every week throughout the experiment in the perampanel- (n = 8; n = 1 of male, n = 7 of female) and vehicle- (n = 7; n = 3 of male, n = 4 of female) treated AR2 mice. Weekly performance scores are indicated in the upper panels (red bars indicate the first day of administration). Mean values for the four trials before perampanel treatment (pre), initial four trials (1), from the 5th to the 8th trials (2) and from the 9th to the last trails (3) after perampanel treatment are indicated in the lower panels. All data are presented as the mean ± s.e.m. *p < 0.05, **p < 0.001 against the “pre” time point at each time point, one-way ANOVA and Tukey post hoc tests. ^#p < 0.05 perampanel against the vehicle group at each time point, Wilcoxon rank sum test.

Figure 3. Perampanel administration for 90 days rescued AHCs from death and normalized TDP-43 subcellular localization.

(a) Number of AHCs in L5. V, vehicle (methyl cellulose)-treated AR2 mice (n = 7); P, perampanel-treated AR2 mice (n = 8). Three sections were measured for each mouse. (b) Frequency histogram of AHCs in the perampanel group (P, blue columns) and the vehicle group (V, white columns). The vertical axis indicates the proportion of the total number of AHCs with a diameter within each range. (c) Immunostaining of the lumbar spinal cord for TDP-43 (green). White dotted lines indicate the margin of the ventral gray matter. Asterisk: TDP-43-positive cytoplasmic aggregates. TO-PRO-3 (blue) was used as a cell body marker. The scale bar indicates 100 μm (upper panels) or 20 μm (lower panels). (d) Number of TDP-43-positive (black columns) and TDP-43-negative (white columns) AHCs. Hatched columns indicate the results on vehicle-treated mice. ***p < 0.0001 against the vehicle (V) group, Wilcoxon rank sum test. (e) The number of AHCs showing TDP-43 immunoreactivity in the nucleus (N), cytoplasm (C), and both the nucleus and cytoplasm (N/C) are indicated. Hatched columns indicate the results on vehicle-treated mice. (a,b,d,e) All error bars represent the s.e.m.; *p < 0.05, **p < 0.001, and ***p < 0.0001 against the vehicle (V) group, Wilcoxon rank sum test.