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

MTN-EYFP expression in Drosophila.Drosophila expressing a copper-inducible MTN-EYFP construct (green) were reared until third instar on basal media (A, B) and media supplemented with 0.5 mM copper (C, D). Gut tissue incorporating the gastric caecum and anterior midgut (left) to the posterior midgut (right) was dissected from control (A, C) and midgut PSN knockdown (B, D) larvae. MTN-EYFP expression was greater in the gut of control larvae reared on copper-supplemented media(C) than those on basal media (A), indicative of increased copper levels. Under basal condition, MTN-EYFP expression in the gut of PSN knockdown (B) and control (A) larvae was comparable. When reared on copper-supplemented media MTN-EYFP levels were lower in the gut of PSN knockdown larvae (D) than control (C), consistent with reduced copper levels. Western immunoblotting with a GFP antibody was used to confirm MTN-EYFP levels in pooled samples of five larvae reared on basal media and media supplemented with 0.5 mM copper (E). α-Tubulin was used as a loading control. When reared on copper-supplemented media MTN-EYFP levels were lower in the gut of PSN knockdown larvae than control.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3646984&req=5

pone-0062811-g004: MTN-EYFP expression in Drosophila.Drosophila expressing a copper-inducible MTN-EYFP construct (green) were reared until third instar on basal media (A, B) and media supplemented with 0.5 mM copper (C, D). Gut tissue incorporating the gastric caecum and anterior midgut (left) to the posterior midgut (right) was dissected from control (A, C) and midgut PSN knockdown (B, D) larvae. MTN-EYFP expression was greater in the gut of control larvae reared on copper-supplemented media(C) than those on basal media (A), indicative of increased copper levels. Under basal condition, MTN-EYFP expression in the gut of PSN knockdown (B) and control (A) larvae was comparable. When reared on copper-supplemented media MTN-EYFP levels were lower in the gut of PSN knockdown larvae (D) than control (C), consistent with reduced copper levels. Western immunoblotting with a GFP antibody was used to confirm MTN-EYFP levels in pooled samples of five larvae reared on basal media and media supplemented with 0.5 mM copper (E). α-Tubulin was used as a loading control. When reared on copper-supplemented media MTN-EYFP levels were lower in the gut of PSN knockdown larvae than control.

Mentions: Copper distribution within Drosophila larvae was assessed using flies expressing an EYFP construct containing a copper-responsive metallothionein promoter element (Figure 4). Control (Figure 4A, C) and midgut PSN knockdown (Figure 4B, D) Drosophila were reared until third instar on either basal media (Figure 4A, B) or media supplemented with 0.5 mM copper (Figure 4C, D). The midgut was dissected to include the gastric caecum and proventriculus (left) and the entire midgut terminating at the junction with the hindgut (right). When reared on basal media, EYFP expression was comparable between control (Figure 4A) and midgut PSN knockdown (Figure 4B) larvae, with strongest expression detected in the proventriculus and copper cell region of the midgut. When reared on copper-supplemented media, EYFP expression was markedly increased throughout the entire midgut of control larvae (Figure 4C), consistent with increased copper uptake relative to basal conditions (Figure 4A). EYFP expression was also increased in the midgut of PSN knockdown larvae reared on copper-supplemented media (Figure 4D), relative to those raised on basal media (Figure 4B). However, this increase in EYFP expression was not as large in the PSN knockdown larvae (Figure 4D) when compared to control (Figure 4C). Analysis of MTN-EYFP levels in third instar larvae, by western immunoblotting with an anti-GFP antibody, confirmed that copper-induced expression of MTN-EYFP was reduced by midgut PSN knockdown (Figure 4E). These results are indicative of reduced dietary copper uptake into the midgut of PSN knockdown larvae, which is consistent with their overall lower copper levels (Figure 3A) and increased copper tolerance (Figure 1A) when reared on copper-supplemented media.


Presenilin promotes dietary copper uptake.

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

MTN-EYFP expression in Drosophila.Drosophila expressing a copper-inducible MTN-EYFP construct (green) were reared until third instar on basal media (A, B) and media supplemented with 0.5 mM copper (C, D). Gut tissue incorporating the gastric caecum and anterior midgut (left) to the posterior midgut (right) was dissected from control (A, C) and midgut PSN knockdown (B, D) larvae. MTN-EYFP expression was greater in the gut of control larvae reared on copper-supplemented media(C) than those on basal media (A), indicative of increased copper levels. Under basal condition, MTN-EYFP expression in the gut of PSN knockdown (B) and control (A) larvae was comparable. When reared on copper-supplemented media MTN-EYFP levels were lower in the gut of PSN knockdown larvae (D) than control (C), consistent with reduced copper levels. Western immunoblotting with a GFP antibody was used to confirm MTN-EYFP levels in pooled samples of five larvae reared on basal media and media supplemented with 0.5 mM copper (E). α-Tubulin was used as a loading control. When reared on copper-supplemented media MTN-EYFP levels were lower in the gut of PSN knockdown larvae than control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0062811-g004: MTN-EYFP expression in Drosophila.Drosophila expressing a copper-inducible MTN-EYFP construct (green) were reared until third instar on basal media (A, B) and media supplemented with 0.5 mM copper (C, D). Gut tissue incorporating the gastric caecum and anterior midgut (left) to the posterior midgut (right) was dissected from control (A, C) and midgut PSN knockdown (B, D) larvae. MTN-EYFP expression was greater in the gut of control larvae reared on copper-supplemented media(C) than those on basal media (A), indicative of increased copper levels. Under basal condition, MTN-EYFP expression in the gut of PSN knockdown (B) and control (A) larvae was comparable. When reared on copper-supplemented media MTN-EYFP levels were lower in the gut of PSN knockdown larvae (D) than control (C), consistent with reduced copper levels. Western immunoblotting with a GFP antibody was used to confirm MTN-EYFP levels in pooled samples of five larvae reared on basal media and media supplemented with 0.5 mM copper (E). α-Tubulin was used as a loading control. When reared on copper-supplemented media MTN-EYFP levels were lower in the gut of PSN knockdown larvae than control.
Mentions: Copper distribution within Drosophila larvae was assessed using flies expressing an EYFP construct containing a copper-responsive metallothionein promoter element (Figure 4). Control (Figure 4A, C) and midgut PSN knockdown (Figure 4B, D) Drosophila were reared until third instar on either basal media (Figure 4A, B) or media supplemented with 0.5 mM copper (Figure 4C, D). The midgut was dissected to include the gastric caecum and proventriculus (left) and the entire midgut terminating at the junction with the hindgut (right). When reared on basal media, EYFP expression was comparable between control (Figure 4A) and midgut PSN knockdown (Figure 4B) larvae, with strongest expression detected in the proventriculus and copper cell region of the midgut. When reared on copper-supplemented media, EYFP expression was markedly increased throughout the entire midgut of control larvae (Figure 4C), consistent with increased copper uptake relative to basal conditions (Figure 4A). EYFP expression was also increased in the midgut of PSN knockdown larvae reared on copper-supplemented media (Figure 4D), relative to those raised on basal media (Figure 4B). However, this increase in EYFP expression was not as large in the PSN knockdown larvae (Figure 4D) when compared to control (Figure 4C). Analysis of MTN-EYFP levels in third instar larvae, by western immunoblotting with an anti-GFP antibody, confirmed that copper-induced expression of MTN-EYFP was reduced by midgut PSN knockdown (Figure 4E). These results are indicative of reduced dietary copper uptake into the midgut of PSN knockdown larvae, which is consistent with their overall lower copper levels (Figure 3A) and increased copper tolerance (Figure 1A) when reared on copper-supplemented media.

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