Limits...
Presenilin promotes dietary copper uptake.

Southon A, Greenough MA, Ganio G, Bush AI, Burke R, Camakaris J - PLoS ONE (2013)

Bottom Line: Expression of a copper-responsive EYFP construct was also lower in the midgut of these larvae, indicative of reduced dietary copper uptake.SOD activity was reduced by midgut PSN knockdown, and these flies were sensitive to the superoxide-inducing chemical paraquat.These results are consistent with previous studies of mammalian presenilins, supporting a conserved role for these proteins in mediating copper uptake.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, The University of Melbourne, Victoria, Australia.

ABSTRACT
Dietary copper is essential for multicellular organisms. Copper is redox active and required as a cofactor for enzymes such as the antioxidant Superoxide Dismutase 1 (SOD1). Copper dyshomeostasis has been implicated in Alzheimer's disease. Mutations in the presenilin genes encoding PS1 and PS2 are major causes of early-onset familial Alzheimer's disease. PS1 and PS2 are required for efficient copper uptake in mammalian systems. Here we demonstrate a conserved role for presenilin in dietary copper uptake in the fly Drosophila melanogaster. Ubiquitous RNA interference-mediated knockdown of the single Drosophila presenilin (PSN) gene is lethal. However, PSN knockdown in the midgut produces viable flies. These flies have reduced copper levels and are more tolerant to excess dietary copper. Expression of a copper-responsive EYFP construct was also lower in the midgut of these larvae, indicative of reduced dietary copper uptake. SOD activity was reduced by midgut PSN knockdown, and these flies were sensitive to the superoxide-inducing chemical paraquat. These data support presenilin being needed for dietary copper uptake in the gut and so impacting on SOD activity and tolerance to oxidative stress. These results are consistent with previous studies of mammalian presenilins, supporting a conserved role for these proteins in mediating copper uptake.

Show MeSH

Related in: MedlinePlus

SOD activity in Drosophila.SOD activity (A) and SOD1 protein levels (B) were assessed for control (RNAi control, GAL4 control) and midgut PSN knockdown (PSN RNAi) third instar larvae. (A) SOD activity was measured colorimetrically and expressed relative to activity in RNAi control larvae. Values are mean activity with S.E.M. from twenty replicates of 10 larvae. PSN knockdown Drosophila had reduced SOD activity. aSignificant difference from RNAi control, bsignificant difference from GAL4 control, as determined by one-way ANOVA with Tukey’s post-hoc test (P<0.05). (B) SOD1 monomer and dimer were identified by western immunoblotting with a SOD1 antibody. α-Tubulin used as a loading control. SOD1 protein levels were not reduced by PSN knockdown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3646984&req=5

pone-0062811-g005: SOD activity in Drosophila.SOD activity (A) and SOD1 protein levels (B) were assessed for control (RNAi control, GAL4 control) and midgut PSN knockdown (PSN RNAi) third instar larvae. (A) SOD activity was measured colorimetrically and expressed relative to activity in RNAi control larvae. Values are mean activity with S.E.M. from twenty replicates of 10 larvae. PSN knockdown Drosophila had reduced SOD activity. aSignificant difference from RNAi control, bsignificant difference from GAL4 control, as determined by one-way ANOVA with Tukey’s post-hoc test (P<0.05). (B) SOD1 monomer and dimer were identified by western immunoblotting with a SOD1 antibody. α-Tubulin used as a loading control. SOD1 protein levels were not reduced by PSN knockdown.

Mentions: We next sought to determine whether the reduced dietary copper uptake associated with midgut PSN knockdown had any effect on activity of the cupro-enzyme SOD1 (Figure 5). Relative to each control strain, SOD activity was significantly reduced in midgut PSN knockdown larvae (Figure 5A). This effect was not due to reduced SOD1 protein levels as western immunoblotting demonstrated comparable levels of SOD1 monomer and dimer in each strain (Figure 5B). The reduction in SOD activity is consistent with a functional copper deficiency associated with midgut PSN knockdown. Given SOD is an antioxidant that functions in the dismutation of superoxide into hydrogen peroxide, we sought to determine whether midgut PSN knockdown Drosophila had any effect on tolerance to oxidative stress. Drosophila were reared on basal media or media supplemented with paraquat, a potent inducer of superoxide radicals, and survival to adulthood was measured (Figure 6). Midgut PSN knockdown significantly reduced survival on paraquat-supplemented media, compared to each control strain. This result is indicative of greater sensitivity to superoxide and is consistent with reduced SOD activity (Figure 5A). These results support the model whereby reduced PSN levels inhibit dietary copper uptake and bioavailability in Drosophila.


Presenilin promotes dietary copper uptake.

Southon A, Greenough MA, Ganio G, Bush AI, Burke R, Camakaris J - PLoS ONE (2013)

SOD activity in Drosophila.SOD activity (A) and SOD1 protein levels (B) were assessed for control (RNAi control, GAL4 control) and midgut PSN knockdown (PSN RNAi) third instar larvae. (A) SOD activity was measured colorimetrically and expressed relative to activity in RNAi control larvae. Values are mean activity with S.E.M. from twenty replicates of 10 larvae. PSN knockdown Drosophila had reduced SOD activity. aSignificant difference from RNAi control, bsignificant difference from GAL4 control, as determined by one-way ANOVA with Tukey’s post-hoc test (P<0.05). (B) SOD1 monomer and dimer were identified by western immunoblotting with a SOD1 antibody. α-Tubulin used as a loading control. SOD1 protein levels were not reduced by PSN knockdown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646984&req=5

pone-0062811-g005: SOD activity in Drosophila.SOD activity (A) and SOD1 protein levels (B) were assessed for control (RNAi control, GAL4 control) and midgut PSN knockdown (PSN RNAi) third instar larvae. (A) SOD activity was measured colorimetrically and expressed relative to activity in RNAi control larvae. Values are mean activity with S.E.M. from twenty replicates of 10 larvae. PSN knockdown Drosophila had reduced SOD activity. aSignificant difference from RNAi control, bsignificant difference from GAL4 control, as determined by one-way ANOVA with Tukey’s post-hoc test (P<0.05). (B) SOD1 monomer and dimer were identified by western immunoblotting with a SOD1 antibody. α-Tubulin used as a loading control. SOD1 protein levels were not reduced by PSN knockdown.
Mentions: We next sought to determine whether the reduced dietary copper uptake associated with midgut PSN knockdown had any effect on activity of the cupro-enzyme SOD1 (Figure 5). Relative to each control strain, SOD activity was significantly reduced in midgut PSN knockdown larvae (Figure 5A). This effect was not due to reduced SOD1 protein levels as western immunoblotting demonstrated comparable levels of SOD1 monomer and dimer in each strain (Figure 5B). The reduction in SOD activity is consistent with a functional copper deficiency associated with midgut PSN knockdown. Given SOD is an antioxidant that functions in the dismutation of superoxide into hydrogen peroxide, we sought to determine whether midgut PSN knockdown Drosophila had any effect on tolerance to oxidative stress. Drosophila were reared on basal media or media supplemented with paraquat, a potent inducer of superoxide radicals, and survival to adulthood was measured (Figure 6). Midgut PSN knockdown significantly reduced survival on paraquat-supplemented media, compared to each control strain. This result is indicative of greater sensitivity to superoxide and is consistent with reduced SOD activity (Figure 5A). These results support the model whereby reduced PSN levels inhibit dietary copper uptake and bioavailability in Drosophila.

Bottom Line: Expression of a copper-responsive EYFP construct was also lower in the midgut of these larvae, indicative of reduced dietary copper uptake.SOD activity was reduced by midgut PSN knockdown, and these flies were sensitive to the superoxide-inducing chemical paraquat.These results are consistent with previous studies of mammalian presenilins, supporting a conserved role for these proteins in mediating copper uptake.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, The University of Melbourne, Victoria, Australia.

ABSTRACT
Dietary copper is essential for multicellular organisms. Copper is redox active and required as a cofactor for enzymes such as the antioxidant Superoxide Dismutase 1 (SOD1). Copper dyshomeostasis has been implicated in Alzheimer's disease. Mutations in the presenilin genes encoding PS1 and PS2 are major causes of early-onset familial Alzheimer's disease. PS1 and PS2 are required for efficient copper uptake in mammalian systems. Here we demonstrate a conserved role for presenilin in dietary copper uptake in the fly Drosophila melanogaster. Ubiquitous RNA interference-mediated knockdown of the single Drosophila presenilin (PSN) gene is lethal. However, PSN knockdown in the midgut produces viable flies. These flies have reduced copper levels and are more tolerant to excess dietary copper. Expression of a copper-responsive EYFP construct was also lower in the midgut of these larvae, indicative of reduced dietary copper uptake. SOD activity was reduced by midgut PSN knockdown, and these flies were sensitive to the superoxide-inducing chemical paraquat. These data support presenilin being needed for dietary copper uptake in the gut and so impacting on SOD activity and tolerance to oxidative stress. These results are consistent with previous studies of mammalian presenilins, supporting a conserved role for these proteins in mediating copper uptake.

Show MeSH
Related in: MedlinePlus