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Early-stage treatment with Withaferin A reduces levels of misfolded superoxide dismutase 1 and extends lifespan in a mouse model of amyotrophic lateral sclerosis.

Patel P, Julien JP, Kriz J - Neurotherapeutics (2015)

Bottom Line: Approximately 20% of cases of familial amyotrophic lateral sclerosis (ALS) are caused by mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1).The beneficial effects of WA in the SOD1(G93A) mice model were accompanied by an alleviation of neuroinflammation, a decrease in levels of misfolded SOD1 species in the spinal cord, and a reduction in loss of motor neurons resulting in delayed disease progression and mortality.These results suggest that WA may represent a potential lead compound for drug development aiming to treat ALS.

View Article: PubMed Central - PubMed

Affiliation: Research Centre of Institut Universitaire en Santé Mentale de Québec, and Department of Psychiatry and Neuroscience, Laval University, 2601 Chemin de la Canardière, Québec, QC, G1J 2G3, Canada.

ABSTRACT
Approximately 20% of cases of familial amyotrophic lateral sclerosis (ALS) are caused by mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1). Recent studies have shown that Withaferin A (WA), an inhibitor of nuclear factor-kappa B activity, was efficient in reducing disease phenotype in a TAR DNA binding protein 43 transgenic mouse model of ALS. These findings led us to test WA in mice from 2 transgenic lines expressing different ALS-linked SOD1 mutations, SOD1(G93A) and SOD1(G37R). Intraperitoneal administration of WA at a dosage of 4 mg/kg of body weight was initiated from postnatal day 40 until end stage in SOD1(G93A) mice, and from 9 months until end stage in SOD1(G37R) mice. The beneficial effects of WA in the SOD1(G93A) mice model were accompanied by an alleviation of neuroinflammation, a decrease in levels of misfolded SOD1 species in the spinal cord, and a reduction in loss of motor neurons resulting in delayed disease progression and mortality. Interestingly, WA treatment triggered robust induction of heat shock protein 25 (a mouse ortholog of heat shock protein 27), which may explain the reduced level of misfolded SOD1 species in the spinal cord of SOD1(G93A) mice and the decrease of neuronal injury responses, as revealed by real-time imaging of biophotonic SOD1(G93A) mice expressing a luciferase transgene under the control of the growth-associated protein 43 promoter. These results suggest that WA may represent a potential lead compound for drug development aiming to treat ALS.

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Bioluminescence imaging of astrocyte activation in the spinal cord of glial fibrillary acidic protein (GFAP)–luciferase (luc)/superoxide dismutase 1 (SOD1)G93A mice. (A) Typical sequence of images of spinal cord area obtained from GFAP–luc/SOD1G93A mice at different time points (9, 17, and 18 weeks). (B) Quantitative analysis of the total GFAP signal/bioluminescence (total flux of photon/s) in GFAP–luc/SOD1G93A control mice (blue, n =8) and GFAP–luc/SOD1G93A treated (red, n =8) at postnatal day 40 revealed that early treatment with WA reduced the GFAP signal at 8, 9, and 10 weeks. A second reduction in GFAP signal after treatment was observed at later stage of disease, at 17 and 18 weeks of age. Two-way analysis of variance revealed a statistically significant reduction in the GFAP signal between the treated and untreated group (p <0.05 at 8 and 10 weeks, and, p <0.05 at 17 and 18 weeks). Error bar represents mean ± SEM. (C) Photomicrograph of GFAP immunostaining in ventral horn of the spinal cord from wild-type (wt), vehicle, and WA-treated SOD1G93A mice at 120 days (n =3 for all groups). (D) Graph represents quantitative analysis of GFAP labeling by measure of optical density (O.D.) (p =0.05, n =3). (E) Photomicrograph of ionized calcium binding adaptor molecule 1 (Iba1) staining in ventral horn of spinal cord from wt, vehicle, and WA-treated SOD1G93A mice at 120 days. (F) Graph represents quantitative analysis of Iba1 labeling by measure of O.D. (p =0.03, n =3). (G) Lumber spinal cord lysate from vehicle- and WA-treated SOD1G93A mice at postnatal day 120 (P120) were subjected to immunoblotting (IB) against Iba-1 and Toll-like receptor 2 (TLR2; n =3). Actin was used as an internal control (*p ≤0.05; **p ≤0.01 by t test)
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Fig4: Bioluminescence imaging of astrocyte activation in the spinal cord of glial fibrillary acidic protein (GFAP)–luciferase (luc)/superoxide dismutase 1 (SOD1)G93A mice. (A) Typical sequence of images of spinal cord area obtained from GFAP–luc/SOD1G93A mice at different time points (9, 17, and 18 weeks). (B) Quantitative analysis of the total GFAP signal/bioluminescence (total flux of photon/s) in GFAP–luc/SOD1G93A control mice (blue, n =8) and GFAP–luc/SOD1G93A treated (red, n =8) at postnatal day 40 revealed that early treatment with WA reduced the GFAP signal at 8, 9, and 10 weeks. A second reduction in GFAP signal after treatment was observed at later stage of disease, at 17 and 18 weeks of age. Two-way analysis of variance revealed a statistically significant reduction in the GFAP signal between the treated and untreated group (p <0.05 at 8 and 10 weeks, and, p <0.05 at 17 and 18 weeks). Error bar represents mean ± SEM. (C) Photomicrograph of GFAP immunostaining in ventral horn of the spinal cord from wild-type (wt), vehicle, and WA-treated SOD1G93A mice at 120 days (n =3 for all groups). (D) Graph represents quantitative analysis of GFAP labeling by measure of optical density (O.D.) (p =0.05, n =3). (E) Photomicrograph of ionized calcium binding adaptor molecule 1 (Iba1) staining in ventral horn of spinal cord from wt, vehicle, and WA-treated SOD1G93A mice at 120 days. (F) Graph represents quantitative analysis of Iba1 labeling by measure of O.D. (p =0.03, n =3). (G) Lumber spinal cord lysate from vehicle- and WA-treated SOD1G93A mice at postnatal day 120 (P120) were subjected to immunoblotting (IB) against Iba-1 and Toll-like receptor 2 (TLR2; n =3). Actin was used as an internal control (*p ≤0.05; **p ≤0.01 by t test)

Mentions: Progressive increase in neuroinflammatory signals is a hallmark of chronic neurodegenerative disorders, including ALS. Namely, the substantial activation of microglial cells and astrocytes is one of the first microscopic findings in the spinal cord sections of patients with ALS and SOD1 mutant mice [52, 53]. Our previous work, using biophotonic/bioluminescence imaging, demonstrated that one of the first signs of disease in SOD1G93A mice is early induction of the biophotonic GFAP signal [24]. Here it is noteworthy that the GFAP gene promoter activity is a target of activated NF-кB and we have previously shown and validated its sensitivity to WA treatments [17]. The in vivo effect of WA treatment on astrogliosis was assessed by bioluminescence imaging of luc activity driven by the GFAP promoter in live GFAP–luc/SOD1G93A mice. We injected GFAP–luc/SOD1G93A double transgenic mice with 4 mg/kg body weight of WA twice a week , starting at postnatal day 40 until the end stage of the disease. Analysis of the signal emitted from the spinal cord revealed marked decrease in the luc signal in WA-treated GFAP–luc/SOD1G93A mice at 8–10 weeks compared with nontreated controls (p <0.05) (Fig. 4A,B). Another significant decrease in luc signal was observed at 17 and 18 weeks of age (p <0.05) in WA-treated mice (Fig. 4B). In line with the obtained in vivo imaging results, immunofluorescence analysis of the GFAP staining in spinal cord sections (ventral horn area) from WA-treated SOD1G93A mice at 17 weeks revealed a significant reduction in the signal compared with vehicle-treated SOD1G93A age-matched littermates (p <0.05) (Fig. 4C,D). In addition, fluorescence analysis of Iba-1 immunorectivity revealed a significant reduction in spinal cord sections from treated mice compared with the control group, thus suggesting a decrease in microglial activation (p <0.05) (Fig. 4E,F). This was further confirmed by Western blot analysis. As shown in Fig. 4G, WA treatment resulted in decreased levels of Iba-1 and Toll-like receptor 2 expression (Fig. 4G). Taken together, our data suggest that WA exerted marked anti-inflammatory effects in the SOD1 mutant model, resulting in decreased astrogliosis and microgliosis.Fig. 4


Early-stage treatment with Withaferin A reduces levels of misfolded superoxide dismutase 1 and extends lifespan in a mouse model of amyotrophic lateral sclerosis.

Patel P, Julien JP, Kriz J - Neurotherapeutics (2015)

Bioluminescence imaging of astrocyte activation in the spinal cord of glial fibrillary acidic protein (GFAP)–luciferase (luc)/superoxide dismutase 1 (SOD1)G93A mice. (A) Typical sequence of images of spinal cord area obtained from GFAP–luc/SOD1G93A mice at different time points (9, 17, and 18 weeks). (B) Quantitative analysis of the total GFAP signal/bioluminescence (total flux of photon/s) in GFAP–luc/SOD1G93A control mice (blue, n =8) and GFAP–luc/SOD1G93A treated (red, n =8) at postnatal day 40 revealed that early treatment with WA reduced the GFAP signal at 8, 9, and 10 weeks. A second reduction in GFAP signal after treatment was observed at later stage of disease, at 17 and 18 weeks of age. Two-way analysis of variance revealed a statistically significant reduction in the GFAP signal between the treated and untreated group (p <0.05 at 8 and 10 weeks, and, p <0.05 at 17 and 18 weeks). Error bar represents mean ± SEM. (C) Photomicrograph of GFAP immunostaining in ventral horn of the spinal cord from wild-type (wt), vehicle, and WA-treated SOD1G93A mice at 120 days (n =3 for all groups). (D) Graph represents quantitative analysis of GFAP labeling by measure of optical density (O.D.) (p =0.05, n =3). (E) Photomicrograph of ionized calcium binding adaptor molecule 1 (Iba1) staining in ventral horn of spinal cord from wt, vehicle, and WA-treated SOD1G93A mice at 120 days. (F) Graph represents quantitative analysis of Iba1 labeling by measure of O.D. (p =0.03, n =3). (G) Lumber spinal cord lysate from vehicle- and WA-treated SOD1G93A mice at postnatal day 120 (P120) were subjected to immunoblotting (IB) against Iba-1 and Toll-like receptor 2 (TLR2; n =3). Actin was used as an internal control (*p ≤0.05; **p ≤0.01 by t test)
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Fig4: Bioluminescence imaging of astrocyte activation in the spinal cord of glial fibrillary acidic protein (GFAP)–luciferase (luc)/superoxide dismutase 1 (SOD1)G93A mice. (A) Typical sequence of images of spinal cord area obtained from GFAP–luc/SOD1G93A mice at different time points (9, 17, and 18 weeks). (B) Quantitative analysis of the total GFAP signal/bioluminescence (total flux of photon/s) in GFAP–luc/SOD1G93A control mice (blue, n =8) and GFAP–luc/SOD1G93A treated (red, n =8) at postnatal day 40 revealed that early treatment with WA reduced the GFAP signal at 8, 9, and 10 weeks. A second reduction in GFAP signal after treatment was observed at later stage of disease, at 17 and 18 weeks of age. Two-way analysis of variance revealed a statistically significant reduction in the GFAP signal between the treated and untreated group (p <0.05 at 8 and 10 weeks, and, p <0.05 at 17 and 18 weeks). Error bar represents mean ± SEM. (C) Photomicrograph of GFAP immunostaining in ventral horn of the spinal cord from wild-type (wt), vehicle, and WA-treated SOD1G93A mice at 120 days (n =3 for all groups). (D) Graph represents quantitative analysis of GFAP labeling by measure of optical density (O.D.) (p =0.05, n =3). (E) Photomicrograph of ionized calcium binding adaptor molecule 1 (Iba1) staining in ventral horn of spinal cord from wt, vehicle, and WA-treated SOD1G93A mice at 120 days. (F) Graph represents quantitative analysis of Iba1 labeling by measure of O.D. (p =0.03, n =3). (G) Lumber spinal cord lysate from vehicle- and WA-treated SOD1G93A mice at postnatal day 120 (P120) were subjected to immunoblotting (IB) against Iba-1 and Toll-like receptor 2 (TLR2; n =3). Actin was used as an internal control (*p ≤0.05; **p ≤0.01 by t test)
Mentions: Progressive increase in neuroinflammatory signals is a hallmark of chronic neurodegenerative disorders, including ALS. Namely, the substantial activation of microglial cells and astrocytes is one of the first microscopic findings in the spinal cord sections of patients with ALS and SOD1 mutant mice [52, 53]. Our previous work, using biophotonic/bioluminescence imaging, demonstrated that one of the first signs of disease in SOD1G93A mice is early induction of the biophotonic GFAP signal [24]. Here it is noteworthy that the GFAP gene promoter activity is a target of activated NF-кB and we have previously shown and validated its sensitivity to WA treatments [17]. The in vivo effect of WA treatment on astrogliosis was assessed by bioluminescence imaging of luc activity driven by the GFAP promoter in live GFAP–luc/SOD1G93A mice. We injected GFAP–luc/SOD1G93A double transgenic mice with 4 mg/kg body weight of WA twice a week , starting at postnatal day 40 until the end stage of the disease. Analysis of the signal emitted from the spinal cord revealed marked decrease in the luc signal in WA-treated GFAP–luc/SOD1G93A mice at 8–10 weeks compared with nontreated controls (p <0.05) (Fig. 4A,B). Another significant decrease in luc signal was observed at 17 and 18 weeks of age (p <0.05) in WA-treated mice (Fig. 4B). In line with the obtained in vivo imaging results, immunofluorescence analysis of the GFAP staining in spinal cord sections (ventral horn area) from WA-treated SOD1G93A mice at 17 weeks revealed a significant reduction in the signal compared with vehicle-treated SOD1G93A age-matched littermates (p <0.05) (Fig. 4C,D). In addition, fluorescence analysis of Iba-1 immunorectivity revealed a significant reduction in spinal cord sections from treated mice compared with the control group, thus suggesting a decrease in microglial activation (p <0.05) (Fig. 4E,F). This was further confirmed by Western blot analysis. As shown in Fig. 4G, WA treatment resulted in decreased levels of Iba-1 and Toll-like receptor 2 expression (Fig. 4G). Taken together, our data suggest that WA exerted marked anti-inflammatory effects in the SOD1 mutant model, resulting in decreased astrogliosis and microgliosis.Fig. 4

Bottom Line: Approximately 20% of cases of familial amyotrophic lateral sclerosis (ALS) are caused by mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1).The beneficial effects of WA in the SOD1(G93A) mice model were accompanied by an alleviation of neuroinflammation, a decrease in levels of misfolded SOD1 species in the spinal cord, and a reduction in loss of motor neurons resulting in delayed disease progression and mortality.These results suggest that WA may represent a potential lead compound for drug development aiming to treat ALS.

View Article: PubMed Central - PubMed

Affiliation: Research Centre of Institut Universitaire en Santé Mentale de Québec, and Department of Psychiatry and Neuroscience, Laval University, 2601 Chemin de la Canardière, Québec, QC, G1J 2G3, Canada.

ABSTRACT
Approximately 20% of cases of familial amyotrophic lateral sclerosis (ALS) are caused by mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1). Recent studies have shown that Withaferin A (WA), an inhibitor of nuclear factor-kappa B activity, was efficient in reducing disease phenotype in a TAR DNA binding protein 43 transgenic mouse model of ALS. These findings led us to test WA in mice from 2 transgenic lines expressing different ALS-linked SOD1 mutations, SOD1(G93A) and SOD1(G37R). Intraperitoneal administration of WA at a dosage of 4 mg/kg of body weight was initiated from postnatal day 40 until end stage in SOD1(G93A) mice, and from 9 months until end stage in SOD1(G37R) mice. The beneficial effects of WA in the SOD1(G93A) mice model were accompanied by an alleviation of neuroinflammation, a decrease in levels of misfolded SOD1 species in the spinal cord, and a reduction in loss of motor neurons resulting in delayed disease progression and mortality. Interestingly, WA treatment triggered robust induction of heat shock protein 25 (a mouse ortholog of heat shock protein 27), which may explain the reduced level of misfolded SOD1 species in the spinal cord of SOD1(G93A) mice and the decrease of neuronal injury responses, as revealed by real-time imaging of biophotonic SOD1(G93A) mice expressing a luciferase transgene under the control of the growth-associated protein 43 promoter. These results suggest that WA may represent a potential lead compound for drug development aiming to treat ALS.

Show MeSH
Related in: MedlinePlus