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An in vitro screening cascade to identify neuroprotective antioxidants in ALS.

Barber SC, Higginbottom A, Mead RJ, Barber S, Shaw PJ - Free Radic. Biol. Med. (2009)

Bottom Line: Analysis of the in silico properties of hit compounds and a review of published literature on their in vivo effectiveness have enabled us to systematically identify molecules with antioxidant activity combined with chemical properties necessary to penetrate the central nervous system.These compounds were tested for their ability to rescue primary motor neuron cultures after trophic factor withdrawal, and the mechanisms of action of their antioxidant effects were investigated.Subsequent in vivo studies can be targeted using molecules with the greatest probability of success.

View Article: PubMed Central - PubMed

Affiliation: Academic Neurology Unit and Sheffield Care and Research Centre for Motor Neuron Disorders, University of Sheffield, Sheffield, UK.

ABSTRACT
Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease, characterized by progressive dysfunction and death of motor neurons. Although evidence for oxidative stress in ALS pathogenesis is well described, antioxidants have generally shown poor efficacy in animal models and human clinical trials. We have developed an in vitro screening cascade to identify antioxidant molecules capable of rescuing NSC34 motor neuron cells expressing an ALS-associated mutation of superoxide dismutase 1. We have tested known antioxidants and screened a library of 2000 small molecules. The library screen identified 164 antioxidant molecules, which were refined to the 9 most promising molecules in subsequent experiments. Analysis of the in silico properties of hit compounds and a review of published literature on their in vivo effectiveness have enabled us to systematically identify molecules with antioxidant activity combined with chemical properties necessary to penetrate the central nervous system. The top-performing molecules identified include caffeic acid phenethyl ester, esculetin, and resveratrol. These compounds were tested for their ability to rescue primary motor neuron cultures after trophic factor withdrawal, and the mechanisms of action of their antioxidant effects were investigated. Subsequent in vivo studies can be targeted using molecules with the greatest probability of success.

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Related in: MedlinePlus

Effects of the best-hit molecules on survival of primary mouse motor neurons deprived of growth factors. Results show the mean numbers of motor neurons ± SEM counted in 20 random fields from four cultures. All conditions used medium without growth factors or antioxidants, except “Complete,” which contained complete motor neuron medium, as defined under Materials and methods. ⁎p = 0.020, ⁎⁎p = 0.014 (Wilcoxon test, adjusted for false discovery rate, after significant results from a Kruskal–Wallis test (p = 0.0005)).
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fig6: Effects of the best-hit molecules on survival of primary mouse motor neurons deprived of growth factors. Results show the mean numbers of motor neurons ± SEM counted in 20 random fields from four cultures. All conditions used medium without growth factors or antioxidants, except “Complete,” which contained complete motor neuron medium, as defined under Materials and methods. ⁎p = 0.020, ⁎⁎p = 0.014 (Wilcoxon test, adjusted for false discovery rate, after significant results from a Kruskal–Wallis test (p = 0.0005)).

Mentions: Having identified molecules capable of rescuing oxidative stress associated with G93A-SOD1 in a motor neuron cell line, we then tested the molecules for their ability to support survival of primary MN cultures in which oxidative stress had been induced by trophic factor withdrawal [51]. Primary MNs were cultured from mouse embryos for 24 h, after which trophic factors and antioxidants were withdrawn for a further 24 h. Cultures deprived of trophic factors and antioxidants showed significantly decreased viability, as assessed by counting the number of surviving MNs in 20 random fields (42.75 ± 6.25% of control cultures (mean ± SEM), p = 0.007, Wilcoxon test). Addition of either CAPE or esculetin at 10 μM significantly increased MN viability (p = 0.014 and 0.020, respectively; Fig. 6). Neither resveratrol nor ebselen was capable of increasing MN viability.


An in vitro screening cascade to identify neuroprotective antioxidants in ALS.

Barber SC, Higginbottom A, Mead RJ, Barber S, Shaw PJ - Free Radic. Biol. Med. (2009)

Effects of the best-hit molecules on survival of primary mouse motor neurons deprived of growth factors. Results show the mean numbers of motor neurons ± SEM counted in 20 random fields from four cultures. All conditions used medium without growth factors or antioxidants, except “Complete,” which contained complete motor neuron medium, as defined under Materials and methods. ⁎p = 0.020, ⁎⁎p = 0.014 (Wilcoxon test, adjusted for false discovery rate, after significant results from a Kruskal–Wallis test (p = 0.0005)).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2742740&req=5

fig6: Effects of the best-hit molecules on survival of primary mouse motor neurons deprived of growth factors. Results show the mean numbers of motor neurons ± SEM counted in 20 random fields from four cultures. All conditions used medium without growth factors or antioxidants, except “Complete,” which contained complete motor neuron medium, as defined under Materials and methods. ⁎p = 0.020, ⁎⁎p = 0.014 (Wilcoxon test, adjusted for false discovery rate, after significant results from a Kruskal–Wallis test (p = 0.0005)).
Mentions: Having identified molecules capable of rescuing oxidative stress associated with G93A-SOD1 in a motor neuron cell line, we then tested the molecules for their ability to support survival of primary MN cultures in which oxidative stress had been induced by trophic factor withdrawal [51]. Primary MNs were cultured from mouse embryos for 24 h, after which trophic factors and antioxidants were withdrawn for a further 24 h. Cultures deprived of trophic factors and antioxidants showed significantly decreased viability, as assessed by counting the number of surviving MNs in 20 random fields (42.75 ± 6.25% of control cultures (mean ± SEM), p = 0.007, Wilcoxon test). Addition of either CAPE or esculetin at 10 μM significantly increased MN viability (p = 0.014 and 0.020, respectively; Fig. 6). Neither resveratrol nor ebselen was capable of increasing MN viability.

Bottom Line: Analysis of the in silico properties of hit compounds and a review of published literature on their in vivo effectiveness have enabled us to systematically identify molecules with antioxidant activity combined with chemical properties necessary to penetrate the central nervous system.These compounds were tested for their ability to rescue primary motor neuron cultures after trophic factor withdrawal, and the mechanisms of action of their antioxidant effects were investigated.Subsequent in vivo studies can be targeted using molecules with the greatest probability of success.

View Article: PubMed Central - PubMed

Affiliation: Academic Neurology Unit and Sheffield Care and Research Centre for Motor Neuron Disorders, University of Sheffield, Sheffield, UK.

ABSTRACT
Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease, characterized by progressive dysfunction and death of motor neurons. Although evidence for oxidative stress in ALS pathogenesis is well described, antioxidants have generally shown poor efficacy in animal models and human clinical trials. We have developed an in vitro screening cascade to identify antioxidant molecules capable of rescuing NSC34 motor neuron cells expressing an ALS-associated mutation of superoxide dismutase 1. We have tested known antioxidants and screened a library of 2000 small molecules. The library screen identified 164 antioxidant molecules, which were refined to the 9 most promising molecules in subsequent experiments. Analysis of the in silico properties of hit compounds and a review of published literature on their in vivo effectiveness have enabled us to systematically identify molecules with antioxidant activity combined with chemical properties necessary to penetrate the central nervous system. The top-performing molecules identified include caffeic acid phenethyl ester, esculetin, and resveratrol. These compounds were tested for their ability to rescue primary motor neuron cultures after trophic factor withdrawal, and the mechanisms of action of their antioxidant effects were investigated. Subsequent in vivo studies can be targeted using molecules with the greatest probability of success.

Show MeSH
Related in: MedlinePlus