Rosmarinic Acid Alleviates Neurological Symptoms in the G93A-SOD1 Transgenic Mouse Model of Amyotrophic Lateral Sclerosis

Abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects motor neurons in the brain and spinal cord, resulting in paralysis of voluntary skeletal muscles and eventually death, usually within 2~3 years of symptom onset. The pathophysiology mechanism underlying ALS is not yet clearly understood. Moreover the available medication for treating ALS, riluzole, only modestly improves neurological symptoms and increases survival by a few months. Therefore, improved therapeutic strategies are urgently needed. In the present study, we investigated whether rosmarinic acid has a therapeutic potential to alleviate neurological deterioration in the G93A-SOD1 transgenic mouse model of ALS. Treatment of G93A-SOD1 transgenic mice with rosmarinic acid from 7 weeks of age at the dose of 400 mg/kg/day significantly extended survival, and relieved motor function deficits. Specifically, disease onset and symptom progression were delayed by more than one month. These symptomatic improvements were correlated with decreased oxidative stress and reduced neuronal loss in the ventral horns of G93A-SOD1 mice. These results support that rosmarinic acid is a potentially useful supplement for relieving ALS symptoms.

Keywords: ALS; Rosmarinic acid; antioxidant; neuroprotection.

Fig. 1. Rosmarinic acid extends the survival of G93A-SOD1 transgenic mice. (A) G93A-SOD1 transgenic mice treated with rosmarinic acid (30 or 400 mg/kg/day) from 7 weeks of age exhibited extended survival compared with untreated G93A-SOD1 mice. (B) Body weight changes of G93A-SOD1 mice during the administration of rosmarinic acid. Body weights were monitored weekly and are presented as percentages of mean body weight of each group at 7 weeks of age. Rosmarinic acid was administrated at 30 or 400 mg/kg/day to G93A-SOD1 mice from 7 weeks of age. Non-Tg, non-transgenic WT control; Tg-Con, G93A-SOD1 transgenic control; Tg+RA30 and Tg+RA400, G93A-SOD1 transgenic mice treated with rosmarinic acid of 30 mg/kg/day and 400 mg/kg/day, respectively; Tg+Rilu, G93A-SOD1 transgenic mice treated with 35 mg/kg/day of riluzole. Data are presented as means±SEM (n=6~13). ^**denotes differences from the Tg-control at 16 weeks at p<0.01. Two-way ANOVA and Bonferroni post hoc test were used.

Fig. 2. Rosmarinic acid improves motor function deficits in G93A-SOD1 transgenic mice. (A~D) Motor function levels in the limb extension reflex test (A), rota-rod test (B), PaGE test (C), and grip strength test (D). Behavioral tests were performed from 7 weeks of age, twice a week described in Materials and Methods. Tests were performed in the sequence listed above. Non-Tg, non-transgenic WT control; Tg-Con, G93A-SOD1 transgenic control; Tg+RA30 and Tg+RA400, G93A-SOD1 transgenic mice treated with 30 mg/kg/day and 400 mg/kg/day rosmarinic acid, respectively; Tg+Rilu, G93A-SOD1 transgenic mice treated with 35 mg/kg/day riluzole. Data are presented as means±SEM (n=6~13). ^* and ^** denote differences from the control at the indicated points at p<0.05 or p<0.01, respectively. Two-way ANOVA and Bonferroni post hoc test were used.

Fig. 3. Rosmarinic acid treatment suppresses oxidative stress in vitro and in the lumbar cords of G93A-SOD1 transgenic mice. (A) Rosmarinic acid treatment suppressed H₂O₂ (800 µM)-induced cytotoxicity in SH-SY5Y neuroblastoma cells. Cells were treated with 15 or 30 µg/ml rosmarinic acid (RA). Vitamin C (VitC) and trolox (TRX) were used at 200 µM. Cell viability was measured using the WST-1 assay after 24 h of treatment. Data points represent means±SEM (n=6). ^**denotes difference compared to untreated control at p<0.01. ^##denotes difference compared to H₂O₂-treated cells at p<0.01. One-way ANOVA and Newman-Keuls post hoc test were used. (B~F) Photomicrographs showing anti-HNE-stained ventral horns of the lumbar cord of non-transgenic control mice (Non-Tg; B), G93A-SOD1 transgenic control mice (Tg-Con; C), G93A-SOD1 transgenic mice treated with 400 mg/kg/day rosmarinic acid (Tg+RA; D), and G93A-SOD1 mice treated with 35 mg/kg/day riluzole (Tg+Rilu; E). (F) Fluorescence levels in the ventral horns [circled area in (B), 600 µm in diameter] of the lumbar cord of each group were quantified. All animals were examined at 16 weeks of age. Scale bar; 500 µm. Data are presented as means±SEM (n=8~13). ^* and ^** denote differences compared to non-transgenic control (NonTg) at p<0.05 and p<0.01, respectively. ^# and ^## denote difference compared to Tg-CON at p<0.05 and p<0.01, respectively. One-way ANOVA and Newman-Keuls post hoc test were used.

Fig. 4. Rosmarinic acid increases the survival of neuronal cells in the lumbar cord of G93A-SOD1 transgenic mice. (A~H) Representative photomicrographs showing cresyl violet-stained lumbar L3~5 cords (A~D) or anti-ChAT-stained lumbar L3~5 cords (E~H) of non-transgenic control mice (Non-Tg; A, E), G93A-SOD1 transgenic control (Tg-Con; B, F), G93A-SOD1 mice treated with 400 mg/kg/day rosmarinic acid (Tg+RA; C, G), and G93A-SOD1 mice treated with 35 mg/kg/day riluzole (Tg+Rilu; D, H). (I, J) Quantification of the numbers of cresyl violet-stained cells (I) or anti-ChAT-stained cells (J) in the ventral horns of non-transgenic control mice (Non-Tg), G93A-SOD1 transgenic control (Tg-Con), G93A-SOD1 mice treated with 400 mg/kg/day rosmarinic acid (Tg+RA), and G93A-SOD1 mice treated with 35 mg/kg/day riluzole (Tg+Rilu). Cresyl violet-stained cells larger than 5 µm in diameter within the circled area in (A) (600 µm in diameter) were counted in both ventral horns of each section using TOMORO ScopeEye 3.6, as described in Materials and Methods. Anti-ChAT-stained cells were counted according to the same procedure. All animals were examined at 16 weeks of age. Scale bar; 500 µm. Data are presented as means±SEM (n= 8~13). ^**denotes difference compared to non-transgenic control (NonTg) at p<0.01. ^##denotes difference compared to Tg-CON at p<0.01. One-way ANOVA and Newman-Keuls post hoc test were used.