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Cu II (atsm) improves the neurological phenotype and survival of SOD1 G93A mice and selectively increases enzymatically active SOD1 in the spinal cord

View Article: PubMed Central - PubMed

ABSTRACT

Ubiquitous expression of mutant Cu/Zn-superoxide dismutase (SOD1) selectively affects motor neurons in the central nervous system (CNS), causing the adult-onset degenerative disease amyotrophic lateral sclerosis (ALS). The CNS-specific impact of ubiquitous mutant SOD1 expression is recapitulated in transgenic mouse models of the disease. Here we present outcomes for the metallo-complex CuII(atsm) tested for therapeutic efficacy in mice expressing SOD1G93A on a mixed genetic background. Oral administration of CuII(atsm) delayed the onset of neurological symptoms, improved locomotive capacity and extended overall survival. Although the ALS-like phenotype of SOD1G93A mice is instigated by expression of the mutant SOD1, we show the improved phenotype of the CuII(atsm)-treated animals involves an increase in mature mutant SOD1 protein in the disease-affected spinal cord, where concomitant increases in copper and SOD1 activity are also evident. In contrast to these effects in the spinal cord, treating with CuII(atsm) had no effect in liver on either mutant SOD1 protein levels or its activity, indicating a CNS-selective SOD1 response to the drug. These data provide support for CuII(atsm) as a treatment option for ALS as well as insight to the CNS-selective effects of mutant SOD1.

No MeSH data available.


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The effect of orally administered CuII(atsm) on mutant SOD1 and Cu levels in spinal cords and livers of SOD1G93A mice.Relative abundance of mutant SOD1 protein in spinal cord (A) and liver (D) samples determined via western blot using an antibody that detects only human SOD1. Mutant SOD1 protein levels are expressed relative to the loading control GAPDH. Representative western blot images are shown. SOD1 activity in TBS-soluble extracts from mouse spinal cords (B) and livers (E) presented as pmol superoxide decay min−1 mg−1 tissue protein. The amount of Cu g−1 protein in spinal cord (C) and liver (F) tissue. Treatments were administered twice daily by gavage and commenced when the mice were 50 days old. CuII(atsm) administered per dose was 50 mg kg−1 mouse body weight. Mice were killed at 120 days old to collect tissues for analysis. Graphed data are box (median ± 95% CI) and whisker (maximum and minimum) plots and P value represents statistically significant treatment effect on mean values (unpaired t-test in (A and D) or one-way ANOVA with Tukey’s multiple comparisons test in (B,C,E and F)). NS = not statistically different. For all data shown, n = 6 mice per treatment group.
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f2: The effect of orally administered CuII(atsm) on mutant SOD1 and Cu levels in spinal cords and livers of SOD1G93A mice.Relative abundance of mutant SOD1 protein in spinal cord (A) and liver (D) samples determined via western blot using an antibody that detects only human SOD1. Mutant SOD1 protein levels are expressed relative to the loading control GAPDH. Representative western blot images are shown. SOD1 activity in TBS-soluble extracts from mouse spinal cords (B) and livers (E) presented as pmol superoxide decay min−1 mg−1 tissue protein. The amount of Cu g−1 protein in spinal cord (C) and liver (F) tissue. Treatments were administered twice daily by gavage and commenced when the mice were 50 days old. CuII(atsm) administered per dose was 50 mg kg−1 mouse body weight. Mice were killed at 120 days old to collect tissues for analysis. Graphed data are box (median ± 95% CI) and whisker (maximum and minimum) plots and P value represents statistically significant treatment effect on mean values (unpaired t-test in (A and D) or one-way ANOVA with Tukey’s multiple comparisons test in (B,C,E and F)). NS = not statistically different. For all data shown, n = 6 mice per treatment group.

Mentions: Assessing the influence of CuII(atsm) on levels of mutant SOD1 protein in spinal cord tissue from SOD1G93A mice at the mid-stages of symptom progression (indicated via vertical dashed lines in Fig. 1A and B) demonstrated that treating with CuII(atsm) increased levels of mutant SOD1 in the disease-affected CNS tissue (Fig. 2A). Catalytic activity of SOD1 is dependent upon the protein binding Cu18. Thus, we measured SOD1 activity in spinal cord extracts from sham- and CuII(atsm)-treated mice to assess whether the increase in mutant SOD1 protein in response to the CuII(atsm) translated to an increase in SOD1 activity. Reflecting overall differences in SOD1 protein levels between non-transgenic mice and the over-expressing SOD1G93A mice19, SOD1 activity was relatively low in extracts collected from non-transgenic mice and this was unchanged by the CuII(atsm) treatment (Fig. 2B). As a result of human SOD1 overexpression, and because the G93A mutation does not affect the enzyme’s dismutase activity320, SOD1 activity was relatively high in the spinal cords of the sham-treated SOD1G93A mice (Fig. 2B). This was further increased by the CuII(atsm) treatment (Fig. 2B). Moreover, analysing the Cu content of spinal cords supported outcomes from the SOD1G37R model8; elevated spinal cord Cu in CuII(atsm)-treated non-transgenic mice confirmed that oral administration of the compound affects Cu levels in the CNS, and the same dose administered to mice expressing mutant SOD1 elicits a greater response (Fig. 2C). In contrast to these effects in the spinal cord, administering CuII(atsm) to SOD1G93A mice had no influence on mutant SOD1 protein levels or activity in the liver (Fig. 2D,E), nor was there any statistically significant difference between non-transgenic and SOD1G93A mice with respect to liver Cu levels in response to the CuII(atsm) treatment (Fig. 2F, P = 0.99).


Cu II (atsm) improves the neurological phenotype and survival of SOD1 G93A mice and selectively increases enzymatically active SOD1 in the spinal cord
The effect of orally administered CuII(atsm) on mutant SOD1 and Cu levels in spinal cords and livers of SOD1G93A mice.Relative abundance of mutant SOD1 protein in spinal cord (A) and liver (D) samples determined via western blot using an antibody that detects only human SOD1. Mutant SOD1 protein levels are expressed relative to the loading control GAPDH. Representative western blot images are shown. SOD1 activity in TBS-soluble extracts from mouse spinal cords (B) and livers (E) presented as pmol superoxide decay min−1 mg−1 tissue protein. The amount of Cu g−1 protein in spinal cord (C) and liver (F) tissue. Treatments were administered twice daily by gavage and commenced when the mice were 50 days old. CuII(atsm) administered per dose was 50 mg kg−1 mouse body weight. Mice were killed at 120 days old to collect tissues for analysis. Graphed data are box (median ± 95% CI) and whisker (maximum and minimum) plots and P value represents statistically significant treatment effect on mean values (unpaired t-test in (A and D) or one-way ANOVA with Tukey’s multiple comparisons test in (B,C,E and F)). NS = not statistically different. For all data shown, n = 6 mice per treatment group.
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f2: The effect of orally administered CuII(atsm) on mutant SOD1 and Cu levels in spinal cords and livers of SOD1G93A mice.Relative abundance of mutant SOD1 protein in spinal cord (A) and liver (D) samples determined via western blot using an antibody that detects only human SOD1. Mutant SOD1 protein levels are expressed relative to the loading control GAPDH. Representative western blot images are shown. SOD1 activity in TBS-soluble extracts from mouse spinal cords (B) and livers (E) presented as pmol superoxide decay min−1 mg−1 tissue protein. The amount of Cu g−1 protein in spinal cord (C) and liver (F) tissue. Treatments were administered twice daily by gavage and commenced when the mice were 50 days old. CuII(atsm) administered per dose was 50 mg kg−1 mouse body weight. Mice were killed at 120 days old to collect tissues for analysis. Graphed data are box (median ± 95% CI) and whisker (maximum and minimum) plots and P value represents statistically significant treatment effect on mean values (unpaired t-test in (A and D) or one-way ANOVA with Tukey’s multiple comparisons test in (B,C,E and F)). NS = not statistically different. For all data shown, n = 6 mice per treatment group.
Mentions: Assessing the influence of CuII(atsm) on levels of mutant SOD1 protein in spinal cord tissue from SOD1G93A mice at the mid-stages of symptom progression (indicated via vertical dashed lines in Fig. 1A and B) demonstrated that treating with CuII(atsm) increased levels of mutant SOD1 in the disease-affected CNS tissue (Fig. 2A). Catalytic activity of SOD1 is dependent upon the protein binding Cu18. Thus, we measured SOD1 activity in spinal cord extracts from sham- and CuII(atsm)-treated mice to assess whether the increase in mutant SOD1 protein in response to the CuII(atsm) translated to an increase in SOD1 activity. Reflecting overall differences in SOD1 protein levels between non-transgenic mice and the over-expressing SOD1G93A mice19, SOD1 activity was relatively low in extracts collected from non-transgenic mice and this was unchanged by the CuII(atsm) treatment (Fig. 2B). As a result of human SOD1 overexpression, and because the G93A mutation does not affect the enzyme’s dismutase activity320, SOD1 activity was relatively high in the spinal cords of the sham-treated SOD1G93A mice (Fig. 2B). This was further increased by the CuII(atsm) treatment (Fig. 2B). Moreover, analysing the Cu content of spinal cords supported outcomes from the SOD1G37R model8; elevated spinal cord Cu in CuII(atsm)-treated non-transgenic mice confirmed that oral administration of the compound affects Cu levels in the CNS, and the same dose administered to mice expressing mutant SOD1 elicits a greater response (Fig. 2C). In contrast to these effects in the spinal cord, administering CuII(atsm) to SOD1G93A mice had no influence on mutant SOD1 protein levels or activity in the liver (Fig. 2D,E), nor was there any statistically significant difference between non-transgenic and SOD1G93A mice with respect to liver Cu levels in response to the CuII(atsm) treatment (Fig. 2F, P = 0.99).

View Article: PubMed Central - PubMed

ABSTRACT

Ubiquitous expression of mutant Cu/Zn-superoxide dismutase (SOD1) selectively affects motor neurons in the central nervous system (CNS), causing the adult-onset degenerative disease amyotrophic lateral sclerosis (ALS). The CNS-specific impact of ubiquitous mutant SOD1 expression is recapitulated in transgenic mouse models of the disease. Here we present outcomes for the metallo-complex CuII(atsm) tested for therapeutic efficacy in mice expressing SOD1G93A on a mixed genetic background. Oral administration of CuII(atsm) delayed the onset of neurological symptoms, improved locomotive capacity and extended overall survival. Although the ALS-like phenotype of SOD1G93A mice is instigated by expression of the mutant SOD1, we show the improved phenotype of the CuII(atsm)-treated animals involves an increase in mature mutant SOD1 protein in the disease-affected spinal cord, where concomitant increases in copper and SOD1 activity are also evident. In contrast to these effects in the spinal cord, treating with CuII(atsm) had no effect in liver on either mutant SOD1 protein levels or its activity, indicating a CNS-selective SOD1 response to the drug. These data provide support for CuII(atsm) as a treatment option for ALS as well as insight to the CNS-selective effects of mutant SOD1.

No MeSH data available.


Related in: MedlinePlus