In this work, we have evaluated the efficacy of the treatment of Epm2b-/- mice with four drugs, two of them previously used as glutamatergic modulators (riluzole and memantine) and the other two used as neuroinflammatory-modifying therapeutic agents (resveratrol and minocycline). Treatments were administered in male mice of 3 months of age (corresponding to an early stage of LD) for two months. After treatments, we performed an in vivo analysis of anxiety-like, cognitive behavior and neurodegenerative signs followed by an ex vivo histopathological analysis of PG inclusions, astrogliosis, and microgliosis in the corresponding mouse brains. For the sake of clarity, we present the results as independent treatments (although memantine and minocycline were performed at the same time since they shared the same vehicle), comparing the values of treated Epm2b-/- mice to Epm2b-/- mice that received only water, vehicle, or saline, respectively.
Decreased anxiety-like and hyperactive behavior of Epm2b-/- mice are attenuated by anti-neuroinflammatory treatments
The anxiety-like behavior was evaluated in Epm2b-/- mice at 5 months of age by carrying out Open Field and Elevated Plus maze tests. In the Open File test, the percentage of traveled distance in the center and the number of entries into the center were measured as key indicators of anxiety and hyperactivity, respectively. Untreated Epm2b-/- mice showed a tendency not only to travel more distance in the center zone (Fig. 1) [e.g., Epm2b-/- (26.17 ± 2.49) and WT (19.97 ± 2.13, p=0.073, d=-0.71 medium) (Fig. 1C)] but they also made a greater number of entries into the center [e.g., Epm2b-/- (21.92 ± 2.53) and WT (16.92 ± 2.19, p=0.239, d=-0.56 medium) (Table S1)]. Although not statistically significant, both behaviors pointed out a tendency to decrease anxiety and hyperactive behavior in Epm2b-/- mice, as already reported by other authors , . After the anti-inflammatory treatments (resveratrol and minocycline), the % traveled distance of Epm2b-/- in the center was decreased significantly by resveratrol (14.64 ± 2.15, p=0.011*, d=0.80 large) (Fig. 1D) and minocycline (18.01 ± 2.24, p=0.032*, d=0.95 large) (Fig. 1E), in comparison to the corresponding Epm2b-/- mice treated with the respective vehicle, normalizing its anxiety levels up to control levels (Fig. 1D and 1E). In contrast, riluzole (24.71 ± 2.34, p=0.896, d=0.38 small) (Fig. 1B) and memantine (24.36 ± 2.79, p=0.593, d=0.17 negligible) (Fig. 1C) had only a minor effect on this parameter. Regarding the number of entries into the center, only minocycline was capable to decrease this parameter significantly (9.63 ± 1.47, p=0.0017**, d=1.57 much large) up to control levels (Table S1).
We used the Elevated Plus Maze as an alternative method to confirm the low anxiety and hyperactivity present in Emp2b-/- mice. We observed the same abnormal behavior in Epm2b-/- mice, which showed a slight increase in the number of entries into arms (38.53 ± 4.38) than WT (32.93 ± 4.23, p=0.310, d=-0.33 small) (Table S1), confirming reduced anxiety and hyperactive behavior. None of the treatments were able to modify the Epm2b-/- mice behavior in the elevated plus maze test (Table S1), except for minocycline which significantly decreased the number of entries into arms (25.33 ± 3.33, p=0.050*, d=0.87 large) compared to Epm2b-/- treated with the corresponding vehicle. Therefore, minocycline was the only effective treatment to control the hyperactive behavior of Epm2b-/- mice in both anxiety tests, Open Field and Elevated Plus Maze.
Attention defect in Epm2b-/- mice is improved by memantine and minocycline treatments
The cognitive profile of Epm2b-/- mice was evaluated at 5 months of age by assessing working memory, and short-term location memory. To evaluate working memory, animals were tested for the % spontaneous alternations in the Y-maze (Table S1), and % incomplete alternations were quantified (Fig. 2). Regarding % spontaneous alternations, although a repeated tendency to a slight decrease of this parameter in untreated Epm2b-/- mice compared to WT was present through the trials (Table S1), we concluded that Epm2b-/- mice did not display any working memory defect due to the absence of either statistical significance (p-value) or large effect size (d) in the means, in agreement with previously published results . In contrast, % incomplete alternations were increased in untreated Epm2b-/- compared to WT mice (Fig. 2) (e.g., 40.38 ± 7.40 in Epm2b-/- mice vs 15.03 ± 5.90, in WT mice; p=0.017*, d=-0.97 large; Fig. 2C), suggesting an attention defect in ending the task. Interestingly, the % incomplete alternations in Epm2b-/- mice were significantly decreased by memantine (19.11 ± 6.18, p=0.035*, d=0.80 medium) and minocycline (20.46 ± 6.06, p=0.0780, d=0.80 medium), suggesting a positive effect of memantine and minocycline in improving the staying on-task of exploration.
More related to hippocampal memory, we studied spatial short-term memory using the object location memory test (OLM) (Fig. 3). The discrimination index (DI) of object location and the total activity time were measured. There were no significant differences (p-value) or remarkable effect size (d) in DI among all the groups (Table S1), suggesting that short-term location memory was not affected in Epm2b-/- mice at 5 months of age, in agreement with previous results . However, we noticed a tendency to an increase in the total activity time in Emp2b-/- compared to WT in untreated animals (e.g., 196.86 ± 14.10 sec in Epm2b-/- mice vs 157.00 ± 12.22 sec in WT mice; p=0.048*, d=-0.77 medium; Fig. 3C), highlighting again the hyperactive behaviour in Epm2b-/- mice. Among the treatments, we nicely observed that only memantine treatment significantly decreased the total activity time in Epm2b-/- mice (145.26 ± 15.73 sec, p=0.016*, d=-0.89 large) (Fig. 3C), reducing hyperactive behavior.
Neurodegenerative signs detected in 5-month-old Epm2b-/- mice are ameliorated by memantine, resveratrol, and minocycline treatments
Epm2b-/- mice were evaluated for abnormal postures related to neurodegeneration by using the hindlimb clasping test. In untreated Epm2b-/- mice at 5 months of age the hindlimb clasping score was significantly and repeatedly worse in all trials compared to WT (Fig. 4A-D) (Table S2). Among the treatments, we observed a significant improvement after memantine (p=0.0007***) (Fig. 4B), resveratrol (p=1.63e-07****) (Fig. 4C), and minocycline (p=4.73e-09****) (Fig. 4D) treatments. Therefore, neurodegenerative signs present in Epm2b-/- mice were ameliorated by these pharmacological treatments. However, riluzole treatment worsened the neurodegenerative signs (p=2.14e-10****) (Fig. 4A) (Table S2).
Only glutamatergic modulators have a minor effect on the formation of polyglucosan inclusions in Epm2b-/- mice
To evaluate the presence of PGs inclusions in mice, brain slices were obtained and stained with a periodic acid-Schiff stain (PAS) which detects polysaccharides such as glycogen. The number of PGs per 10,000 µm2 was quantified by image analysis as indicated in the Materials and Methods section and the percentages of PGs number versus untreated Epm2b-/- mice were plotted on a graph. Representative pictures of PAS staining (Fig. 5A) disclosed an enormous number of PGs in Epm2b-/- compared to WT mice (e.g., 100.00 ± 17.31 in Epm2b-/- mice vs 1.18 ± 0.77 in WT mice; p=0.0099**, d=-2.75 much large; Fig. 5B), which was significantly repeated through all trials (Fig. 5B-E) (Table S1). This greater number of PGs was ameliorated by 40% in riluzole (60.23 ± 9.26, p=0.260, d=1.13 much large) and by 15% in memantine (84.76 ± 8.79, p=0.264, d=0.88 large) treated Epm2b-/- mice (Fig. 5B-C) compared to Epm2b-/- mice treated with the corresponding vehicle, albeit it was not statistically significant. Even though not being significant, the effect size (Cohen’s coefficient) of the reduction in the PGs number was much large for riluzole and large for memantine treatments, suggesting a positive reduction. Thus, we consider that two-month glutamatergic treatments might have, if any, only a minor effect preventing the formation of PG inclusions in Epm2b-/- mice. On the contrary, anti-neuroinflammatory treatments did not affect PGs accumulation (resveratrol 98.34 ± 8.58, p=0.864, d=0.05 negligible; minocycline 98.03 ± 4.51, p=0.780, d=0.17 negligible) (Fig. 5D-E) (Table S1).
Reactive astrogliosis in Epm2b-/- mice is not modulated by any of the pharmacological treatments
Since we have found that the accumulation of PGs is significantly correlated to the appearance of reactive astroglia and microglia in Epm2b-/- mice , we examined reactive astrogliosis in Epm2b-/- mice. We detected the astrocytic marker GFAP (in magenta), and the nuclear marker DAPI (in blue) by immunofluorescence (Fig. 6A). As described previously , , , untreated Epm2b-/- showed a massive GFAP+ area compared to WT mice (Fig. 6A) (e.g., 100.00 ± 9.57 in Epm2b-/- mice vs 38.98.15 ± 7.05 in WT mice; p=0.0062**, d=-2.96 much large; Fig. 6C) (Table S1), which suggests a remarkable pathological reactive astrogliosis in the brain of Epm2b-/- mice. Unfortunately, neither riluzole (Fig. 6B), nor memantine (Fig. 6C), nor resveratrol (Fig. 6D) nor minocycline (Fig. 6E) treatments significantly modified the affectation of astrogliosis in the brain of Epm2b-/- mice (Table S1).
Only riluzole treatment has a minor effect on the microgliosis in Epm2b-/- mice
Finally, we detected the microglial marker Iba1 (in gray) by immunofluorescence (Fig. 7A) and the number of Iba1+ cells with clear changes in morphology was counted as a marker of microglial activation . We confirmed an increase in activated microglia in untreated Epm2b-/- (100.00 ± 11.48) compared to WT (77.18 ± 4.49, p=0.543, d=-0.94 large) (Fig. 7) (Table S1). Riluzole was the only pharmacological treatment capable to decrease the number of altered microglia in Epm2b-/- (71.75 ± 4.82, p=0.090, d=1.21 much large) down to control levels. In contrast, memantine (Fig. 7C), resveratrol (Fig. 7D), and minocycline (Fig. 7E) did not affect the number of reactive microglia (Table S1).