Sodium selenate treatment improves symptoms and seizure susceptibility in a malin-deficient mouse model of Lafora disease

Abstract

Objective: To search for new therapies aimed at ameliorating the neurologic symptoms and epilepsy developing in patients with Lafora disease.

Methods: Lafora disease is caused by loss-of-function mutations in either the EPM2A or EPM2B genes. Epm2a^-/- and Epm2b^-/- mice display neurologic and behavioral abnormalities similar to those found in patients. Selenium is a potent antioxidant and its deficiency has been related to the development of certain diseases, including epilepsy. In this study, we investigated whether sodium selenate treatment improved the neurologic alterations and the hyperexcitability present in the Epm2b^-/- mouse model.

Results: Sodium selenate ameliorates some of the motor and memory deficits and the sensitivity observed with pentylenetetrazol (PTZ) treatments in Epm2b^-/- mice. Neuronal degeneration and gliosis were also diminished after sodium selenate treatment.

Significance: Sodium selenate could be beneficial for ameliorating some symptoms that present in patients with Lafora disease.

Keywords: Epilepsy; Epm2b−/− mouse; Lafora bodies; Oxidative stress; Sodium selenate.

Figure 1

Analysis of motor coordination and abnormal postures in Epm2b^−/− mutant mice after treatment with sodium selenate. (A) Rotarod-based analysis of motor coordination in controls (n = 23) and Epm2b^−/− mice (n = 24), in Epm2b^−/− mice with selenate treatment for 4 weeks (n = 22), and in Epm2b^−/− mice with selenate treatment over 10 weeks (n = 15) (trials 1–4). The mean latencies (time to fall from the rotarod) were significantly lower for Epm2b^−/− mice than for controls. Sodium selenate treatment for 4 weeks in Epm2b^−/− mice slightly increased the latency period, whereas a longer treatment significantly improved performance. Student’s t-test was performed for statistical evaluation. (B) Percentage of animals showing normal posture (0), partially altered (1) or high abnormal (2) stereotypical clasping of the hind limbs upon tail suspension. The frequent hind-limb clasping of Epm2b^−/− mice improved progressively with sodium selenate treatment (n = 23 for wild-type, n = 24 for ML, n = 20 for ML + Sel [4 weeks], and n = 15 for ML + Sel [10 weeks] mouse groups). A chi-square test was performed for statistical analysis. *Indicates p < 0.05, **indicates p < 0.01, and ***indicates p < 0.001 when control mice were compared to Epm2b^−/− mice; #indicates p < 0.05 and ###indicates p < 0.001 when Epm2b^−/− mice were compared to Epm2b^−/− mice with selenate treatment for 10 weeks. ML, malin-deficient mouse; Sel, sodium selenate.

Figure 2

Motor and mood abnormalities in Epm2b^−/− mice after sodium selenate treatments. Patterns of accumulated motor activity in controls, Epm2b^−/− mice, and Epm2b^−/− mice with selenate treatment for 4 and 10 weeks, measured as (A) accumulated, (B) rearing, and (C) stereotyped movements (n = 25 for wild type, n = 25 for ML, n = 25 for ML + Sel [4 weeks], and n = 15 for ML + Sel [10 weeks] mouse groups). Quantitative data represent mean + SEM. Student’s t-test was performed for statistical evaluation. (D) Percentage of time spent by all the four groups in the central zone of arena as a measure of anxiety-like behavior (n = 12 for wild type, n = 12 for ML, n = 12 for ML + Sel [4 weeks], and n = 12 for ML + Sel [10 weeks] mouse groups). Statistical analysis was performed with one-way ANOVA or the chi-square test. *p < 0.05; **p < 0.01; ***p < 0.001. Note that after 4 weeks of treatment with selenate, motor performance and anxiety improved, whereas a longer treatment eliminated this advance in motor performance and reduced mood improvement. ML, malin-deficient mouse; Sel, sodium selenate.

Figure 3

Assessment of memory performance with the object recognition task. The ORT was performed in control mice (n = 20), Epm2b^−/− mice (n = 19), and Epm2b^−/− mice with sodium selenate treatment for 4 (n = 20) and 10 (n = 12) weeks. The discrimination index (DI) was calculated as the ratio of the difference between the exploration time of the new (tC) and the familiar object (tA), and the total (tT = tA + tC) exploration time (DI = tC−tA/tT). The DI of Epm2b^−/− mice was poor, indicating deficits in the retention of episodic memory. Treatments with sodium selenate significantly and gradually improved the memory performance of malin-deficient mice. Values are expressed as mean + SEM. One-way ANOVA was performed.*p < 0.05; **p < 0.01. ML, malin-deficient mouse; Sel, sodium selenate.

Figure 4

Sensitivity of Epm2b^−/− mice to the chemoconvulsant agent PTZ after treatment with sodium selenate. Percentage of Epm2b^−/− mice with myoclonic jerks after intraperitoneal injection of PTZ at doses of (A) 30 mg/kg and (B) 50 mg/kg significantly decreased after treatment with selenate for 10 weeks. Sodium selenate administration also reduced PTZ-induced (C) generalized seizures, and (D) lethality after 4 week of treatment, whereas at 10 weeks, generalized seizures and lethality disappeared (n = 25 for wild-type, n = 25 for ML, n = 15 for ML + Sel [4 weeks], and n = 12 for ML + Sel [10 weeks] mouse groups). Chi-square was performed for statistical analysis. *p < 0.05; **p < 0.01; ***p < 0.001. ML, malin-deficient mouse; Sel, sodium selenate.

Figure 5

Neuronal loss, gliosis and Lafora bodies in the hippocampus of Epm2b^−/− mice after treatment with sodium selenate. Images of representative immunostained sections from dentate gyrus with GFAP (A) and from CA1 with NeuN (B) and PAS staining (C) are shown from wild-type, Epm2b^−/−, and Epm2b^−/− after selenate treatment for 4 and 10 weeks (scale bars 25 μm). Graphs show the quantification of GFAP-positive cells (D), NeuN-positive cells (E), and PAS+ inclusions (F) in wild-type, Epm2b^−/−, and Epm2b^−/− after sodium selenate treatment for 4 or 10 weeks. Visual blinded quantification was performed in the dentate gyrus and CA1 regions of the hippocampus. Both hemispheres of the brain were quantified in three mice of each group. One-way ANOVA was performed for statistical analysis. *p < 0.05; **p < 0.01; ***p < 0.001. ML, malin-deficient mouse; Sel, sodium selenate.